Developments and Applications of Enzymes From Thermophilic Microorganisms 0443191972, 9780443191978

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Progress in Biochemistry and Biotechnology

DEVELOPMENTS AND APPLICATIONS OF ENZYMES FROM THERMOPHILIC MICROORGANISMS

PRATIMA BAJPAI Consultant-Pulp and Paper, Kanpur, India

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2023 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

Notices

Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-443-19197-8 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals Publisher: Stacy Masucci Acquisitions Editor: Michelle Fisher Editorial Project Manager: Barbara Makinster Production Project Manager: Punithavathy Govindaradjane Cover Designer: Mark Rogers Typeset by TNQ Technologies

List of tables Table 1.1 Table 1.2 Table 1.3 Table 2.1 Table 3.1 Table 3.2 Table 4.1 Table 5.1 Table 5.2 Table 5.3 Table 6.1 Table 6.2 Table 6.3 Table 7.1 Table 8.1.1 Table 8.1.2 Table 8.1.3 Table 8.1.4 Table 8.1.5 Table 8.2.1 Table 8.2.2 Table 8.3.1 Table 8.3.2 Table 8.4.1 Table 8.5.1 Table 8.5.2 Table 8.6.1

Classification of thermophilic microorganisms. Thermophilic enzymes and their potential roles. Recent patents on thermophiles and their potential applications. Thermotoga species. Hypothermophile diversity. Thermophilic and hyperthermophilic archaea isolated from thermal springs of Vulcano Island. Hyperthermostable enzymes with commercial interest and optimal activity over 100 C in aqueous media. Vectors constructed for thermophilic expression system. Benefits of using thermophilic microorganisms in secondgeneration biorefineries. Use of thermophiles for production of bioethanol using industrially relevant feedstocks. Steps involved in enzyme production. Example of thermophilic enzymes. Examples of some commercially available hyperthermophilic enzymes. Advantages of using E. coli as the host for heterogeneous protein expression. Classification of amylases and their mode of action. Applications of a-amylases. Characteristics of bacterial and archaeal a-amylases. Characteristics of fungal a-amylases. Commercially available bacterial a-amylases. Microorganisms producing glucoamylase. Manufacturers of glucoamylases. Microorganisms producing b-glucosidases. Sources and hosts of b-glucosidases genes and properties of the recombinant b-glucosidases. Reaction specificities of pullulan-degrading enzymes. Characteristics of thermophilic bacterial amylopullulanases. Characteristics of hyperthermophilic archaeal amylopullulanases. Cyclodextrin glycosyltransferase (CGTase) sources and optimum growth conditions.

3 6 7 19 33 38 53 64 67 68 77 80 82 96 109 111 112 115 116 133 137 144 145 154 165 166 175

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List of tables

Table 8.7.1 Thermostable cellulases from various thermophilic microorganism and their characteristics. Table 8.8.1 Thermostable xylanases from various thermophilic microorganism. Table 8.9.1 Enzymes involved in the degradation of pectin. Table 8.10.1 Chitinase-producing bacteria. Table 8.10.2 Chitinase-producing fungi. Table 8.12.1 Glucose isomeraseeproducing microorganisms. Table 8.13.1 Thermostable lipaseeproducing microorganisms and their properties. Table 8.13.2 Lipase applications in the food industry. Table 8.14.1 Laccase-producing fungi. Table 8.17.1 Characteristics of esterases and lipases. Table 8.17.2 Classification of esterases on the basis of their mechanism of action. Table 8.17.3 Characteristics of esterases/lipases from (hyper)thermophilic microorganisms. Table 8.19.1 Thermostable DNA ligases for archaea and bacteria characterized to date and their cofactor utilities. Table 8.19.2 Application of DNA ligase.

184 201 213 224 225 240 244 245 252 268 269 271 281 283

List of figures Figure 1.1 Temperature-dependent growth for the categories of microorganisms. 4 Figure 1.2 Research related to thermophiles and hypothermophiles. 11 Figure 2.1 Bright orange thermophiles, microorganisms in hot springs thermal environments, Yellowstone National Park, USA. 24 Figure 2.2 Bright orange thermophiles, microorganisms in Black Warrior Lake, Yellowstone National Park, USA. 24 Figure 3.1 Photographs of hot springs in the form of a pool or basin (AeC), a stream (D), heated mud (EeF), and a pot (G). Various colors of biomats and sediments that can formin hot springs (HeL). 30 Figure 7.1 Chemical modification versus genetic modification. 91 Figure 7.2 Site-directed modification of proteins. 92 Figure 7.3 Advantages of chemical modification of proteins on solid phases. 93 Figure 7.4 Structure of an expression plasmid. 97 Figure 8.1.1 Scheme for the hydrolysis of starch by amylase. Starch is a polysaccharide made up of simple sugars (glucose). Upon the action of amylase, either glucose (a monosaccharide) or maltose (a disaccharide with two glucose molecules) is released. 107 Figure 8.1.2 Three-dimensional structures of amylases. (A) a-Amylase (RCSB PDB accession code 1SMD; the calcium-binding regions are indicated). (B) b-Amylase (RCSB PDB accession code PDB 2xfr). 108 Figure 8.1.3 (1A, 1B) Crystal structure of Bacillus amyloliquefaciens a-amylase PDB ID 3BH4 [207]; resolution: 1.40 Å. It consists of 483 amino acids. The A domain is shown in green, the B domain is shown in cyan, and the C domain is shown by reddish orange color. (1C) The blue sphere represents the single Naþ ion, and gray spheres represent the Ca2þ ions (front view). (1D) Blue spheres represent the Ca2þ ion (front view). (2A, 2B) Crystal structure of calcium (Ca2þ)-independent Bacillus sp. a-amylase KSM-K38 PDB ID 1UD4; resolution: 2.15 Å [208]. It consists of 480 amino acids. The A domain is shown in green, the B domain is shown in cyan, and C domain is shown by reddish orange color. (2C) Blue spheres represent the three Naþ ions. 109

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List of figures

Figure 8.1.4 Three steps of starch conversion: 1 gelatinization, 2 liquefaction, and 3 saccharification. 117 Figure 8.2.1 Different modular arrangements found in glucoamylases. Representative species of each arrangement are indicated: CD GH15 catalytic domain, CBM20 carbohydrate-binding domain family 20, CBM21 carbohydrate-binding domain family 21, STRD Ser/Thr-rich domain found in Sta1 from Saccharomyces cerevisiae var. diastaticus, bS super-b-sandwich domain present in prokaryotic enzymes. 133 Figure 8.2.2 A catalytic domain of Aspergillus niger glucoamylase (PDB code 3EQA), with the (a/a)6-barrel-fold characteristic of GH15 enzymes. Catalytic residues acting as acid and base in the mechanism of reaction are highlighted in red. 134 Figure 8.3.1 Hydrolysis of cellulose by the synergistic action of cellulases. 142 Figure 8.4.1 Schematic presentation of the action of amylolytic and pullulytic enzymes. Pullulanase type I also attacks a-1,6-glycosidic linkages in oligosaccharides and polysaccharides. Pullulanase type II also attacks a-1,4-linkages in various oligosaccharides and polysaccharides [3•]. Black circles indicate reducing sugars. 156 Figure 8.5.1 Scanning electron micrographs of the untreated and treated raw rice starch granules with the amylopullulanase of G. thermoleovorans NP33. (A) Untreated raw rice starch granule. (B) Hydrolyzed granules in 30 min of reaction with the enzymes; (C) hydrolyzed portion of the granule in 1 h; and (D) almost completely hydrolyzed starch granules in 2 h. 167 Figure 8.6.1 Chemical structure of a-CD, b-CD, and g-CD. CD, Cyclodextrin. 174 Figure 8.6.2 Schematic view of cyclodextrin (CD) formation by cyclodextrin glycosyltransferase (CGTase). 175 Figure 8.6.3 Flow scheme of cyclodextrin (CD) production. Highlighted are the steps where protein engineers and process controllers can influence the process efficiency. 177 Figure 8.7.1 Application of cellulases in the food industry. 190 Figure 8.9.1 Various applications of pectinases. 218 Figure 8.10.1 Action of chitinase enzyme. 223 Figure 8.10.2 Various applications of chitinase. 226 Figure 8.11.1 Food-protein-derived peptides and their roles. 234 Figure 8.15.1 Significance of phytase. (A) Phytase in preventing environmental pollution. (B) Phytase in preventing antinutritional properties of phytate in body. 260

List of figures

Figure 8.18.1 Structural overview of DNA polymerases. Ribbon diagrams of the three DNA polymerases from family A, B, and Y are shown. Taq DNA polymerase (PDB code 1TAQ), Pfu DNA polymerase (PDB code 3A2F), and Sso DinB DNA polymerase (PDB code 1K1S) represented from each family. The three distinct domains of the DNA polymerases are shown in different colors. A unique C-terminal domain in Sso DinB is called the “little finger” or “wrist” domain. 277

xi

Preface

Enzymes and microorganisms have been topics for much research during the last two decades, but the interest in thermophiles/hyperthermophiles and how their proteins are able to function at elevated temperatures actually started as early as in the 1960s. Organisms with an optimum temperature for growth between 60 C and 80 C are generally designated as thermophiles, while those growing optimally above 80 C are referred to as hyperthermophiles. Thermophilic bacteria are microbes that mostly inhabit hot springs and live and survive in temperatures above 70 C. As a consequence of growth at high temperatures and unique macromolecular properties, thermophiles can possess high metabolism, physically and chemically stable enzymes, and lower growth but higher end-product yields than similar mesophilic species. In today’s world, there is an increasing trend toward the use of renewable, cheap, and readily available biomass in the production of a wide variety of fine and bulk chemicals in different biorefineries. Biorefineries utilize the activities of microbial cells and their enzymes to convert biomass into target products. Many of these processes require enzymes that are operationally stable at high temperatures thus allowing, for example, easy mixing, better substrate solubility, high mass transfer rate, and lowered risk of contamination. Thermophiles/hypothermophiles have often been proposed as sources of industrially relevant thermostable enzymes. In this book, existing and potential applications of thermophiles/hypothermophilic enzymes are discussed. Their importance in biorefineries is explained using examples of lignocellulose and starch conversions to desired products. Furthermore, this book deals with thermophile/hyperthermophile biodiversity; cultivation of extremophilic microorganisms; enzyme production by thermophiles/hypothermophiles; biochemical and molecular properties of thermophiles/hyperthermophilic enzymes; and current status and enhancement of thermophilic/hyperthermophilic enzymes production/activity.

xv

Acknowledgments

I am grateful for the help received from many people and companies/organizations for providing information. I am also thankful to various publishers for allowing me to use their material. My deepest appreciation is extended to Elsevier, Springer, RSC, ASM Publications, John Wiley & Sons, De Gruyter, Hindawi, MDPI, IntechOpen, SpringerOpen, and other open-access journals and publications. My special thanks to Dr. Paula Monteiro de Souza, University of Brasília, Brazil, Dr. Garo Antranikian, Hamburg University of Technology, and Dr. Tulasi Satyanarayana, University of Delhi to use their material.

xvii

CHAPTER 1

General background 1.1 Historical background and commercial prospects of enzymes Enzymes are playing a significant role in many aspects of our life. These are crucially essential to the existence of life itself. Enzymes have been used for a very long time without understanding what they were and how they function. Over the past many generations, science has opened the mystery of enzymes and has used this information for making better use of these incredible substances in constantly rising applications (Bajpai, 2018). Enzymes are a class of biomolecules generally proteins which increase the rate of virtually all the chemical reactions within the cell and are characterized by remarkable efficiency and specificity (Adrio and Demain, 2014; Binod et al., 2013; Choi et al., 2015; Godfrey and West, 1996; Gurung et al., 2013; Novozymes, 2011; OECD, 1998; Panke and Wubbolts, 2002; Schafer et al., 2002; Schmid et al., 2001; van Beilen and Li, 2002). These reactions are the basis of the metabolism of all living things and offer tremendous opportunities for industry for conducting effective and economic bioconversions (Kirk et al., 2002). According to Sarmiento et al. (2015), the seventeenth and eighteenth centuries saw the emergence of preliminary theories about enzymes and bio-catalytic reactions; however, it was not until the 19th century that the first significant advances were made. The first enzyme, diastase, now known as amylase, was discovered in 1833 (Payen and Persoz, 1833). It took the German scientist K€ uhne 44 years, in 1877, to coin the term “enzyme” (1877). Otto Rohm created the first enzymatic preparation for a commercial application in 1914, almost 40 years later. To break down proteins, Otto isolated trypsin from animal pancreases and added it to laundry detergents. Enzymatic biocatalysis did not become a practical industrial option until the 1960s when microbial proteases for use in washing powders were mass-produced. Since then, the market for industrial enzymes has grown to be worth many billions of dollars. In the year 2020, the worldwide enzymes market was valued at almost USD 10 billion. The enzymes market is further expected to grow at a compound annual growth rate of 7.5% during the period 2022e27 (https://www.expertmarketresearch.com/reports/ enzymes-market). This market is expected to grow in the near future due to the developments in the biotechnology industry, the continuous requirement for economical manufacturing processes, and calls for environment-friendly technologies.

Developments and Applications of Enzymes From Thermophilic Microorganisms ISBN 978-0-443-19197-8, https://doi.org/10.1016/B978-0-443-19197-8.00008-6

© 2023 Elsevier Inc. All rights reserved.

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Developments and Applications of Enzymes From Thermophilic Microorganisms

The enzyme market is getting more and more active. The application of enzymes in the industry has expanded as a result of recent enzyme discoveries and advances in genetics and protein engineering. In fact, the use of enzymes in industrial catalysis is rising. Enzymes have developed into crucial tools for a variety of industrial markets, including biofuels, leather, pulp and paper, textiles, animal feed, food and beverage, animal feed, detergents, and technical enzymes. The use of specialty enzymes is expanding in industries like pharmaceuticals, research and development, and diagnostics (Sarmiento et al., 2015).

1.2 Thermophiles/hyperthermophiles and their enzymes Thermostable microorganisms and their enzymes have been researched extensively during the last 20 years. However, the interest in thermophilic microorganisms and how their proteins are able to function at high temperatures in fact started as early as in the 1960s by the revolutionary research of Brock and his associates (Brock and Freeze, 1969; Turner et al., 2007). Thermophiles are gaining importance worldwide because of their incredible potential to produce thermostable enzymes having a large number of industrial and biotechnological applications. Thermophilic microorganisms can be used for the production of renewable energy directly and indirectly (McClendon et al., 2012; Parmar et al., 2011; Bergquist et al., 2014; Elleuche et al., 2014; Ilyas, 2014; Bhandiwad et al., 2013; Bhalla et al., 2013; Goh et al., 2013). Thermostable enzymes are able to often resist the relatively severe conditions of industrial processing. Microorganisms are divided into following groups on the basis of their optimal growth temperatures (Brock, 1986): ➢ Psychrophiles (grow below 20
C) ➢ Mesophiles (grow at moderate temperatures) ➢ Thermophiles (grow at higher temperatures, above 55
C) Only a few eukaryotic microorganisms are found to grow above this temperature. However, some fungi are able to grow in the temperature range of 50e55
C (Maheshwari et al., 2000). The word “thermophile” is derived from two Greek words “thermotita” (means heat) and “philia” (means love). Thermophiles are a group of heat-loving microbes, which are able to withstand high temperatures and also typically need these for their growth and survival. Brock (1978) has defined thermophiles as “an organism capable of living at temperatures at or near the maximum for the taxonomic group of which it is a part” Temperatures for the growth of thermophiles range from 50
C to as high as 121
C, the temperature which is used for sterilization in autoclaves. These types of microbes have been classified into moderate thermophiles, extreme thermophiles, and hyperthermophiles (Table 1.1).

General background

Table 1.1 Classification of thermophilic microorganisms. Category

Temperature optima (8C)

Examples

Moderate thermophile

40e60
C

Extreme thermophile

60e85
C

Tepidibacter, Clostridium, Exiguobacterium, Caminibacter, Lebetimonas, Hydrogenimonas, Nautilia, Desulfonauticus, Sulfurivirga, Caminicella, Vulcanibacillus, Marinotoga, Caldithrix, Sulfobacillus, Acidimicrobium, Hydrogenobacter, Thermoplasma, Mahella, Thermoanaerobacter, Desulfovibrio Methanocaldococcus, Thermococcus, Palaeococcus, Methanotorris, Aeropyrum, Thermovibrio, Methanothermococcus, Thermosipho, Caloranaerobacter, Thermodesulfobacterium, Thermodesulfatator, Deferribacter, Thermosipho, Desulfurobacterium, Persephonella, Kosmotoga, Rhodothermus, Desulfurobacterium, Balnearium, Acidianus, Thermovibrio, Marinithermus, Oceanithermus, Petrotoga, Vulcanithermus, Carboxydobrachium, Thermaerobacter, Thermosul fi dibacter, Metallosphaera Geogemma, Archaeoglobus, Methanopyrus, Pyrococcus, Sulfolobus, Thermoproteus, Methanothermus, Acidianus, Ignisphaera, Ignicoccus, Geoglobus

Hyperthermophile >85
C

Reproduced with Permission from Mehta, D., Satyanarayana, T., 2013. Diversity of hot environments and thermophilic microbes. In: Satyanarayana, T., Littlechild, J., Kawarabayasi, Y. (Eds.), Thermophilic Microbes in Environmental and Industrial Biotechnology. Springer, Dordrecht.

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Developments and Applications of Enzymes From Thermophilic Microorganisms

Fig. 1.1 shows the temperature-dependent growth for psychrophiles, psychrotrophs, mesophiles, thermophiles, and hypothermophiles. Kristjansson and Stetter in 1992 put forward a further division of the thermophiles and a hyperthermophile boundarydgrowth at and higher than 80
C (Kristjansson and Stetter, 1992). Today, this has reached general acceptance. Most of the thermophilic bacteria characterized nowadays grow below the hyperthermophilic boundary with some exceptions, like Thermotoga and Aquifex (Stetter, 1996) whereas hyperthermophilic microorganisms are dominated by the Archaea (Turner et al., 2007). Thermophiles are organisms having affinity or liking toward high temperatures. These creatures have special adaptations that allow them to live comfortably in extreme heat. According to Santos and Da Costa (2002), microorganisms having an optimum temperature for growth between 60 and 80
C are in general termed as thermophiles. They include the prokaryotic domains Archaea and Bacteria and also eukaryotic organisms in domain Eukarya, which is represented mostly by filamentous fungi. They are not able to multiply at room temperature. Hyperthermophiles are particularly extreme thermophiles that optimally grow at temperatures higher than 80
C with some representatives flourishing even at 113
C and higher (Stetter, 2013) or that can grow at temperatures higher than 90
C (Adams and Kelly, 1998). Some hyperthermophiles are able to survive temperatures higher than 121
C, which is the average temperature of an autoclave. These are typically Archaebacteria and are found in volcanic and ocean vents. The record is presently held by a hyperthermophilic archea, close to Pyrodictium occultum, known as strain 121 that is able to grow at 121
C. This extends the earlier record that was held for a long time by other archaea, Pyrolobus fumarii, exhibiting an upper growth temperature of 113
C. The higher growth temperature of these microorganisms also suggests that the

Figure 1.1 Temperature-dependent growth for the categories of microorganisms. (https://ecampusontario. pressbooks.pub/microbio/chapter/temperature-and-microbial-growth/. Creative Commons Attribution 4.0 International License.)

General background

enzymes produced by these microorganisms are able to work at higher temperatures (Berlemont and Gerday, 2011). Hyperthermophilic Archaea are generally restricted to environments in which geothermal energy is available. Thermophiles and hyperthermophiles are found as normal occupants of continental and submarine volcanic areas, hydrothermal vents, and geothermally heated sea-sediments and thus are considered extremophiles. Thermophiles are found in the air, the soil of temperate and tropical regions, and salt and fresh water, both cold and thermal. These are extensively distributed in geothermal soils, hot springs, and manmade environments like garden compost piles where the microorganisms degrade the kitchen scraps and vegetal material (Benignetm, 1905; Blau, 1906; Catterina, 1904; De Kruyff, 1910; Falcioni, 1907; Georgevitch, 1910a,b; Gilbert, 1904; Golikowa, 1926; MacFadyen and Blaxall, 1896; Negre, 1913; Sames, 1900; Setchell, 1903; Tirelli, 1907). Thermophilic bacteria are microscopic organisms that typically inhabit hot springs and can endure temperatures of more than 700
C. Natural habitats for anaerobic thermophiles include subterranean sites like oil reservoirs, terrestrial volcanic sites like solfatara fields with temperatures up to 650
C, and submarine hydrothermal systems like sediments, submarine volcanoes, fumaroles, and vents with temperatures over 300
C. Additionally, there are hot environments created by humans, such as water heaters, slag heaps, compost piles, and industrial processes, which can range in temperature from 60 to 100
C (Mehta et al., 2016; Oshima and Moriya, 2008). Thermophiles are classified into groups such as obligate, facultative thermophiles, and hyperthermophiles: - Obligate thermophiles are also called “extreme thermophiles”. These microorganisms need high temperatures to grow and thrive in their environment. - Facultative thermophiles are also called “moderate thermophiles”. These microorganisms can flourish at high temperatures and also at relatively lower temperatures (below 50
C). - Hyperthermophiles prefer temperatures above 80
C for optimal growth. Hyperthermophilic microorganisms are found in the three domains of life, archaea, bacteria, and eukarya, but most of them are archaea and bacteria. Enzymes from hyperthermophilic microorganisms have unique structure-function properties of higher thermostability and show optimal activity at temperatures higher than 70
C. Some of these enzymes are active at temperatures of 110
C and even higher (Vieille et al., 1996; Vieille and Zeikus, 2001; Mojsov et al., 2014). Thermophilic enzymes show optimum activity between 60 and 80
C. As thermophilic and hyperthermophilic enzymes are active at high temperatures, these enzymes usually do not perform well below 40
C. As a result of growth at higher temperatures and exceptional macromolecular properties, thermophilic microorganisms possess higher metabolism, chemically and physically stable enzymes, and reduced growth but higher end-product yields than similar mesophilic microorganisms (Haki and Rakshit, 2003) (Tables 1.2 and 1.3).

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Developments and Applications of Enzymes From Thermophilic Microorganisms

Table 1.2 Thermophilic enzymes and their potential roles. Microorganisms

Enzymes

Temperature of activity

Pyrococcus woesei

alpha-Amylases

Topt. ¼ 100
C

Thennococcus profundus DT5432 Staphylothermus marines

alpha-Amylases

Applications

References

Topt. ¼ 80
C

Sugar industry and starch processing Sugar industry and starch processing

Pullulanases

Topt. ¼ 90 e105
C

Sugar industry and starch processing

Thennoplasma acidophilum

Glucoamylases

Topt. ¼ 90
C

Sugar industry and starch processing

Pyrococcus woesei

BGalactosidases

Topt. ¼ 93
C

Pyrococcus furiosus Sulfolobales sp. Pyrodictium abyssi

Cellulases

Topt. ¼ 103
C

Production of milk with low lactose content Production of alcohol, fruit industry

Alqueres et al. (2007) Eichler (2001), Antranikian et al. (2005) Eichler (2001), Antranikian et al. (2005) Eichler (2001), Antranikian et al. (2005) Dabrowski et al. (1998)

Xylanases

Topt. ¼ 100 e110
C

Paper industrydbleaching of pulp

Humicola lanuginosa strain Y-38 Myceliophthora thermophila

Lipases

Topt. ¼ 65
C

Laundry detergents

Laccases

Topt. ¼ 60
C

Myceliophthora thermophila Penicillium duponti

Phytases

Topt. ¼ 42 e45
C Topt. ¼ 50
C

Polymerization of phenolic compounds to humic substances Animal feed

Bacillus lichnifonnis

Glucose-6phosphate dehydrogenase Alcalase

Topt. ¼ 60
C

Generation of NADPH for biosynthetic reactions Component of protein-fortified soft drinks and dietetic food, helps in protein recovery from meat, fish and crustacean shell waste

Antranikian et al. (2005)

Egorova and Antranikian (2005), Eichler (2001) Arima et al. (1972) Chefetz et al. (1998) Wyss et al. (1999) Broad and Shepherd (1970) Synowiecki (2008)

Mehta, R., Singhal, P., Singh, H., Damle, D., Sharma, A.K., 2016. Insight into thermophiles and their wide-spectrum applications. 3 Biotech. 6 (1): 81. https://doi.org/10.1007/s13205-016-0368-z. Distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/).

General background

Table 1.3 Recent patents on thermophiles and their potential applications. Topic

Patent number and date

Application

References

Single step bioconversion of lignocellulosic biomass to biofuels using extreme thermophilic bacteria Thermophilic bacterium and uses of extracellular proteins there from Fermentation of moderately thermophilic Bacilli on sucrose

US 2014/0363869 A1 December 11, 2014

Bioconversion of lignocellulosic biomass to biofuels

Curvers et al. (2014)

US 8828238 B2 September 9, 2014

Excellent metal ion binding ability

Han et al. (2014)

US 8663954 B2 March 4, 2014

Kranenburg et al. (2014)

Bioremediation of persistent organic pollutants using thermophilic bacteria Phytase-producing bacteria, phytase and production method of phytase

US 2014/0042087 A1 February 13, 2014

Genetic modification of moderately thermophilic Bacillus strain to utilize sucrose as a carbon source Degradation of organic pollutants

Chu et al. (2001)

Process for producing modified microorganisms for oil treatment at high temperatures, pressures and salinity

US 5492828A February 20, 1996

Role in animal feeding, environmental protection, human nutrition and health and industrial applications. Used in microbial enhanced oil recovery

US 6180390 B1 January 30, 2001

O’Driscoll et al. (2014)

Eugene et al. (1996)

Mehta, R., Singhal, P., Singh, H., Damle, D., Sharma, A.K., 2016. Insight into thermophiles and their wide-spectrum applications. 3 Biotech. 6 (1): 81. https://doi.org/10.1007/s13205-016-0368-z. Distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/).

The majority of the hyperthermophile-derived enzymes show correspondingly improved thermostability. Both the development of technological applications that require high-temperature protein stability and the study of fundamental biological questions pertaining to protein structure and stability have made use of this property. The use

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Developments and Applications of Enzymes From Thermophilic Microorganisms

of thermostable proteins in biocatalysts, various materials, and crystallization techniques are a few examples of these applications. Another more applicable example of the recent influence of enzymes isolated from hyperthermophilic organisms on biochemistry and molecular biology is the use of DNA polymerases from hyperthermophilic microorganisms in the polymerase chain reaction (PCR) (Rees and Adams, 1995). Thermophilic and hyperthermophilic enzymes are actually part of another enzyme category called extremozymes, which evolved in extremophiles. Extremozymes are able to function under highly alkaline conditions, at higher salt levels, and in other extreme conditions of acidity, pressure, etc. (Adams et al., 1995). Thermophilic and hyperthermophilic enzymes are intrinsically stable and active at higher temperatures and offer several biotechnological advantages. Operating biotechnological processes at higher temperatures has several benefits. Higher temperature has a considerable effect on the solubility and bioavailability of organic compounds. The viscosity reduces and the diffusion coefficient of organic compounds increases when the temperature is increased. Therefore, high reaction rates because of smaller boundary layers are anticipated (Becker et al., 1997; Krahe et al., 1996). Reactions involving less soluble hydrophobic substrates like fats, polyaromatic, aliphatic hydrocarbons and polymeric compounds like proteins, cellulose, hemicelluloses, and starch are of special interest. The bioavailability of hardly biodegradable and insoluble environmental pollutants can also be enhanced significantly at higher temperatures permitting effective bio-remediation. Moreover, by performing biological processes at temperatures higher than 60
C, the chance of contamination is reduced and controlled processes under stringent conditions can be conducted. The number of genes from thermophilic microorganisms that have been cloned and expressed in mesophilic microorganisms has increased significantly (Ciaramella et al., 1995). Most of the proteins produced by mesophilic microorganisms can maintain their thermostability, are correctly folded at a lower temperature, are not hydrolyzed by host proteases, and can be purified by using thermal denaturation of the mesophilic host proteins. The degree of enzyme purity achieved is mostly sufficient for many applications. The interest shown by researchers in hyperthermophiles has continuously increased over the past decades (Asgher et al., 2007; Henne et al., 2004; Bergquist et al., 2014; Schiraldi and De Rosa, 2002). Microorganisms obtained from the vents are able to grow at temperatures higher than 100
C. Important examples are Pyrodictium and Pyrobolus archaea, which grow at 105
C and survive autoclaving. P. fumarii, the organism that is known to be the most thermophilic, can grow between 90 and 113
C. Although the maximum temperature at which life can exist is still unknown, it is most likely not much higher than 113
C. The Crenarchaeota and Euryarchaeota contain hyperthermophiles. Hyperthermophile communities are intricate networks of primary producers and organic matter decomposers. The majority of enzymes isolated from hyperthermophiles are most active between 70 and 125
C, which

General background

is close to the host organism’s ideal growth temperature. Two types of protein stability (thermodynamic and long term) are of interest from an applied viewpoint. Enzymes that are in an active state as opposed to one that is irreversibly inactive are what industrialists require. For other enzymes, like diagnostics enzymes, improving long-term stability is frequently necessary. Long-term stability may be beneficial while improving thermodynamic thermal stability. A potent technique called directed evolution is now being used to create enzymes with improved thermostability. This technique has also been applied to the creation of thermostable enzymes with high activity between 20 and 37
C and enzymes active in solvents. Enzymes improved by directed evolution have been commercialized (Hicks et al., 1999a,b; Adams and Kelly, 1995; Mozhaev, 1993; Mojsov et al., 2014; Bhattacharya and Pletschke, 2014). Thermophiles as well as hyperthermophiles need special heat-stable enzymes, which can resist denaturation and unfolding. Quite the reverse to their psychrophilic homologs, they are more firmly folded making them less bendable with a lesser available catalytic site. Furthermore, these microorganisms express protective chaperone proteins for helping with protein folding and maintaining their native structure. The enzymes of these microorganisms are of special interest to biotechnology. Biochemist Kary Mullis invented the PCR technique using the Thermus aquaticus’s heat-active DNA polymerase. PCR technique is being used in all fields of biology and has revolutionized the field of microbiology in combination with sequencing advances and the development of metagenomics. In the year 1993, the Nobel Prize in Chemistry was awarded to Dr. Mullis (https://ecampusontario.pressbooks.pub/microbio/chapter/temperatureand-microbial-growth/). In comparison to mesophilic microorganisms, macromolecules in thermophilic and hyperthermophilic microorganisms show some noteworthy structural differences. The ratio of saturated to polyunsaturated lipids is higher. This limits the fluidity of the cell membranes. DNA sequences show a higher proportion of guanineecytosine nitrogen bases. These are held together by three hydrogen bonds quite the opposite of adenine and thymine, which are connected in the double helix by two hydrogen bonds. The replacement of key amino acids to stabilize folding and additional secondary ionic and covalent bonds contribute to the resistance of proteins to denaturation. The thermoenzymes obtained from thermophilic microorganisms have important applications. For instance, the amplification of nucleic acids in PCR reaction depends upon the thermal stability of Taq polymerase, obtained from T. aquaticus. Mehta et al. (2016) have reported factors affecting the heat tolerance of thermophilic microorganisms: “Permeability: Cell membranes effectively function as a permeability barrier, controlling the in-flow and out-flow of low-molecular-weight compounds. The permeability of fatty acyl ester lipid membranes is highly temperature dependent and their phase-transition temperature

9

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Developments and Applications of Enzymes From Thermophilic Microorganisms

is dependent on the fatty acid composition, so when the growth temperature shifts, the fatty acid composition of membrane lipids is quickly regulated (Koga, 2012). Chemical stability: Thermophilic organisms are able to grow at high temperatures due to the chemical stability of their membrane lipids (Koga, 2012). Temperature: Lipids that increase in proportion to an increase in growth temperature may be designated as thermophilic lipids. In the extremely thermophilic environment, methanoarchaea Methanocaldococcus jannaschii have been reported. When the growth temperature increases from 45 to 65
C, the diether lipids (archaeol-based lipids) decrease from 80% to 20%, while the standard caldarchaeol-based and cyclic archaeol-based lipids increase from 10% to 40%, respectively (Sprott et al., 1991). G þ C content: rRNA and tRNA molecules of thermophilic bacteria have higher G þ C contents than mesophiles (Galtier and Lobry, 1997). Because the GC base pair forms more hydrogen bonds than the AT base pair, higher G þ C contents in the double-stranded stem region improve the thermostability of the RNA molecules (Lao and Forsdyke, 2000; Paz et al., 2004). Proteins: The surface regions of thermophilic proteins have fewer (noncharged) polar amino acids and more charged amino acids, and these charged residues result in an increased number of intramolecular salt bridges (Thompson and Eisenberg, 1999). The ability of microorganisms to survive under harsh conditions has prompted researchers to study these organisms to better understand their characteristics and eventually utilize them in various applications.” The structural proteins for instance transport proteins (permeases), ribosomal proteins, and enzymes of thermophilic and hyperthermophilic microorganisms are very thermostable in comparison with the mesophilic microorganisms. The proteins are modified in several ways including dehydration and through small changes in their prime structure, which is responsible for their thermal stability. For maintaining the stability and functionality of the plasma membrane at a high temperature, the membrane lipids of thermophiles contain a higher content of saturated fatty acids. Because of their linear structure, saturated lipids are able to pack more firmly which gives a more organized membrane and increases its melting temperature. Thermophilic microorganisms express protective chaperone proteins. These help to fold protein and maintain their native structure. Furthermore, the ether linkage of the thermophilic and hyperthermophilic archaea is more thermostable as compared to the ester linkage of phospholipids. The membranes of hyperthermophiles, almost all of which are Archaea, are not composed of repeating subunits of the C5 compound, phytane which is a branched

General background

Figure 1.2 Research related to thermophiles and hypothermophiles. (Reproduced with Permission from Urbieta, M.S., Donati, E.R., Chan, K.G., Shahar, S., Sin, L.L., Goh, K.M., 2015. Thermophiles in the genomic era: biodiversity, science, and applications. Biotechnol. Adv. 33 (6 Pt 1): 633e647.)

saturated, “isoprenoid” substance. This contributes a lot to the ability of these bacteria to thrive in superheated environments. The higher thermal stability of the hyperthermophilic archaeal membranes is also due to their tetraether monolayer structure, as the inner and outer layers of a membrane bilayer will separate at higher temperatures (https://microbeonline.com/psychrophilesmesophiles-thermophiles). Fig. 1.2 shows research related to thermophiles and hypothermophiles (Urbieta et al., 2015).

References Adams, M.W.W., Kelly, R.M., 1998. Finding and using hyperthermophilic enzymes. Trends Biotechnol. 16, 329e332. Adams, M.W.W., Perler, F.B., Kelly, R.M., 1995. Extremozymes: expanding the limits of biocatalysis. Nat. Biotechnol. 13, 662e668. Adams, M.W.W., Kelly, R.M., 1995. Enzymes from microorganisms from extreme environments. C & E News. 73, 32e42. Adrio, J.L., Demain, A.L., 2014. Microbial enzymes: tools for biotechnological processes. Biomolecules 4, 117e139.

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Alqueres, S.M.C., Almeida, R.V., Clementino, M.M., Vieira, R.P., Almeida, W.I., Cardoso, A.M., Martins, O.B., 2007. Exploring the biotechnological applications in the archeal domain. Braz. J. Microbiol. 38, 398e405. Antranikian, G., Vorgias, C.E., Bertoldo, C., 2005. Extreme environments as a resource for microorganisms and novel biocatalysts. Adv. Biochem. Eng. Biotechnol. 96, 219e262. Arima, K., Liu, W.H., Beppu, T., 1972. Studies on the lipase of thermophilic fungus Humicola lanuginosa. Agric. Biol. Chem. 36, 893e895. Asgher, M., Asad, M.J., Rahman, S.U., Legge, R.L., 2007. A thermostable a-amylase. from a moderately thermophilic Bacillus subtilis strain for starch processing. J. Food Eng. 79, 950e955. Bajpai, P., 2018. Industrial enzymesean update. In: 1 ed. Bookboon, UK, p. 118. ISBN:978-87-403-2129-6. Benignetm, D., 1905. Di un germe termofflo isolato dai fanghi d’Acqui. Riv d’igiene santa pubbl 16, 449e455. Bergquist, L., Peter, W., Hugh, M., David, S., 2014. Selected enzymes from extreme thermophiles with applications in biotechnology. Curr. Biotechnol. 3, 45e59. Becker, P., AbuReesh, I., Markossian, S., Antranikian, G., Markl, H., 1997. Determination of the kinetic parameters during continuous cultivation of the lipase producing thermophile Bacillus sp. IHI-91 on olive oil. Appl. Microbiol. Biotechnol. 48, 184e190. Berlemont, R., Gerday, C., 2011. Extremophiles. In: Comprehensive Biotechnology. Elsevier. Bhalla, A., Bansal, N., Kumar, S., Bischoff, K.M., Sani, R.K., 2013. Improved lignocellulose conversion to biofuels with thermophilic bacteria and thermostable enzymes. Bioresour. Technol. 128, 751e759. Bhandiwad, A., Guseva, A., Lynd, L., 2013. Metabolic engineering of Thermoanaerobacterium thermosaccharolyticum for increased n-Butanol production. Adv. Microbiol. 3, 46e51, 909. Bhattacharya, A., Pletschke, B.I., 2014. Review of the enzymatic machinery of Halothermothrix orenii with special reference to industrial applications. Enzym. Microb. Technol. 55, 159e169. Binod, P., Palkhiwala, P., Gaikaiwari, R., Namppthiri, K.M., Duggal, A., Dey, K., Pandey, A., 2013. Industrial enzymesdpresent status and future perspectives for India. J. Sci. Ind. Res. 72, 271e286. Blau, O., 1906. Ueber die Temperaturmaxidma der Sporenkeimung und der Sporenbildung,sowie die supramaximalen Totungszeiten der Sporen der Bakterien, auch derjenigen mit hohen Temperaturmaxima Zentr Bakt Parasitenk Infek II 15, 97e143. Broad, T.E., Shepherd, M.G., 1970. Purification and properties of glucose-6-phosphate dehydrogenase from the thermophilic fungus Penicillium duponti. Biochim. Biophys. Acta 198, 407e414. Brock, T.D., 1978. Thermophilic microorganisms and life at high temperatures. Springer Series in Microbiology. Springer, New York, NY. https://doi.org/10.1007/978-1-4612-6284-8_3. Brock, T.D., Freeze, H., 1969. Thermus aquaticus gen. n. and sp. n., a non-sporulating extreme thermophile. J. Bacteriol. 98, 289e297. Brock, T.D., 1986. Introduction, an overview of the thermophiles. In: Brock, T.D. (Ed.), Thermophiles: General, Molecular and Applied Microbiology. John Wiley & Sons, New York, pp. 1e16. Catterina, G., 1904. Beitrag zum Studium der thermophilen Bakterien. Zentr Bakt Parasitenk Infek Il 12, 353e355. Chefetz, B., Chen, Y., Hadar, Y., 1998. Purification and characterization of laccase from Chaetomium thermophilium and its role in humification. Appl. Environ. Microbiol. 64, 3175e3179. Choi, J.M., Han, S.S., Kim, H.S., 2015. Industrial applications of enzyme biocatalysis: current status and future aspect. Biotechnol. Adv. 33, 1443e1454. Chu, J.S., Chung, S.F., Tseng, M., Wen, C.Y., Chu, W.S., 2001. Phytase Producing Bacteria, Phytase and Production Method of Phytase. US 6180390 B1. Ciaramella, M., Cannio, R., Moracci, M., Pisani, F.M., Rossi, M., 1995. Molecular biology of extremophiles. World J. Microbiol. Biotechnol. 11, 71e84. Curvers, S., Koln, D.E., Svetlichnyi, V., 2014. Single Step Bioconversion of Lignocellulosic Biomass to Biofuels Using Extreme Thermophilic Bacteria. US Patent 0363869 A1. Dabrowski, S., Maciu nska, J., Synowiecki, J., 1998. Cloning and nucleotide sequence of the thermostable beta-galactosidase gene from Pyrococcus woesei in Escherichia coli and some properties of the isolated enzyme. Mol. Biotechnol. 10 (3), 217e222. De kruyff, E., 1910. Les bact6ries thermophiles dans le tropiques Zentr Bakt Parasitenk Infek II 26, 65e74. Egorova, K., Antranikian, G., 2005. Industrial relevance of thermophilic. Archaea Curr. Opin. Microbiol. 8, 649e655. Eichler, J., 2001. Biotechnological uses of archaeal extremozymes. Biotechnol. Adv. 19, 261e278.

General background

Elleuche, S., Schröder, C., Sahm, K., Antranikian, G., 2014. Extremozymesebiocatalysts with unique properties from extremophilic microorganisms. Curr. Opin. Biotechnol. 29, 116e123. Eugene, T., Premuzic, E.M., Lin, M., Point, R., 1996. Process for Producing Modified Microorganisms for Oil Treatment at High Temperatures, Pressures and Salinity. US 005492828A. Falcioni, D., 1907. I germi termofili nelle acque del Bullicame. Arch. Farmacol. Speri. 6, 1e5. Galtier, N., Lobry, J.R., 1997. Relationships between genomic G? Ccontent, RNA secondary structures, and optimal growth temperature in prokaryotes. J. Mol. Evol. 44 (6), 632e636. Georgevitch, P., 1910a. Bacillus thermophilus Jivoini nov spec and Bacillus thermophilus Losanitchi nov spec Zentr Bakt Parasitenk Infek II 27, 150e167. Georgevitch, P., 1910b. Bacillus thermophilus vranjensis. Arch Hyg Sci 72, 201e210. Gilbert, 1904. Ueber Actinomyces thermophilue und andere Aktinomyceten. Z Hyg Infektionskrankh 47, 383e405. Godfrey, T., West, S., 1996. Industrial Enzymology. Macmillan Press Ltd, London. Goh, K.M., Kahar, U.M., Chai, Y.Y., Chong, C.S., Chai, K.P., Ranjani, V., et al., 2013. Recent discoveries and applications of Anoxybacillus. Appl. Microbiol. Biotechnol. 97, 1475e1488. Golikowa, S.M., 1926. Zur Frage der Thermobiose Zentr Bakt Parasitenk Infek II. 69, 178e185. Gurung, N., Ray, S., Bose, S., Rai, V., 2013. A broader view: microbial enzymes and their relevance in industries, medicine, and beyond. BioMed Res. Int. 2013, 329121, 10.1155/2013/329121. Haki, G.D., Rakshit, S.K., 2003. Developments in industrially important thermostable enzymes. Bioresour. Technol. 89, 17e34. Han, Y.L., Tainam, T.W., Guo, T.R., Zhubei, T.W., Chang, J.S., Taichung, T.W., Chou, I.J., 2014. Thermophilic Bacterium and Uses of Extracellular Proteins There from. US 8828238 B2. Henne, A., Bruggemann, H., Raasch, C., Wiezer, A., Hartsch, T., Liesegang, H., Johann, A., Lienard, T., Gohl, O., Martinez-Arias, R., Jacobi, C., 2004. The genome sequence of the extreme thermophile Thermus thermophiles. Nat. Biotechnol. 22, 547e553. Hicks, P.M., Adams, M.W.W., Kelly, R.M., 1999. Enzymes, extremely thermostable. In: Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis, and Bioseparation. John Wiley & Sons, Inc., New York, N.Y, pp. 987e1004. Hicks, P.M., Adams, M.W.W., Kelly, R.M., 1999. Enzymes, extremely thermostable. In: Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis, and Bioseparation. John Wiley & Sons, Inc., New York, N.Y, pp. 2536e2552. Ilyas, S., Pendyala, R., Marneni, N., 2014. Preparation, sedimentation, and agglomeration of nanofluids. Chem. Eng. Tech. 37, 2011e2021. Kirk, O., Borchert, T.V., Fuglsang, C.C., 2002. Industrial enzyme applications. Curr. Opin. Biotechnol. 13, 345. Koga, Y., 2012. Thermal adaptation of the archaeal and bacterial lipid membranes. Archaea 2012, 6. Krahe, M., Antranikian, G., Markl, H., 1996. Fermentation of extremophilic microorganisms. FEMS Microbiol. Rev. 18, 271e285. Kranenburg, R.V., Wageningen, N.L., Hartskamp, M.V., Gorinchem, N.L., 2014. Fermentation of Moderately Thermophilic Bacilli on Sucrose. US 8663954 B2. Kristjansson, J.K., Stetter, K.O., 1992. Thermophilic Bacteria. In: Kristjansson, J.K. (Ed.), Thermophilic Bacteria. CRC Press Inc,, London, pp. 1e18. € K€ uhne, W., 1877. Uber das Verhalten verschiedener organisirter und sog. ungeformter Fermente. Verhandlungen des naturhistorisch-medicinischen Vereins zu Heidelberg 1, 190e193. Lao, P.J., Forsdyke, D.R., 2000. Thermophilic bacteria strictly obey Szybalski’s transcription direction rule and politely purine-load RNAs with both adenine and guanine. Genome Res. 10 (2), 228e236. MacFadyen, A., Blaxall, F.R., 1896. Thermophilic bacteria. J. Pathol. Bacteriol. 3, 87e99. Brit Med J 2, 644. McClendon, S.D., Batth, T., Petzold, C.J., Adams, P.D., Simmons, B.A., Singer, S.W., 2012. Thermoascus aurantiacus is a promising source of enzymes for biomass deconstruction under thermophilic conditions. Biotechnol. Biofuels 5, 1e10. Maheshwari, R., Bharadwaj, G., Bhat, M.K., 2000. Thermophilic fungi: their physiology and enzymes. MMBR (Microbiol. Mol. Biol. Rev.) 64, 461e488. Mehta, R., Singhal, P., Singh, H., Damle, D., Sharma, A.K., 2016. Insight into thermophiles and their widespectrum applications. 3 Biotech 6 (1), 81.

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Mehta, D., Satyanarayana, T., 2013. Diversity of hot environments and thermophilic microbes. In: Satyanarayana, T., Littlechild, J., Kawarabayasi, Y. (Eds.), Thermophilic Microbes in Environmental and Industrial Biotechnology. Springer, Dordrecht. Mojsov, K., Janevski1, A., Andronikov, D., Zezova, S., 2014. Hyperthermophilic enzymes with industrial applications. Int. J. Innov. Res. Sci. Eng. (IJIRSE) 2 (12), 806e810. Mozhaev, V.V., 1993. Mechanism-based strategies for protein thermostabilization. Trends Biotechnol. 11, 88e95. Negre, L., 1913. Bacteries thermophiles des sables du Sahara. C R Biol. 74, 814e816. Novozymes, 2011. Enzymes at Work. http://www.novozymes.com/en/aboutus/brochures/Documents. O’Driscoll, K., Princeton, N.J., Sambrotto, R., Blauvelt, N.Y., DiFilippo, R., West Chester, P.A., Piccillo, P., Chapel Hill, N.C., 2014. Bioremediation of Persistent Organic Pollutants Using Thermophilic Bacteria. US 2014/0042087 A1. OECD, 1998. Biotechnology for Clean Industrial Products and Processes. OECD, Paris, France. Oshima, T., Moriya, T., 2008. A preliminary analysis of microbial and biochemical properties of hightemperature compost. Ann. N. Y. Acad. Sci. 1125, 338e344. Panke, S., Wubbolts, M.G., 2002. Enzyme technology and bioprocess engineering. Curr. Opin. Biotechnol. 13, 111e116. Parmar, A., Singh, N.K., Pandey, A., Gnansounou, E., Madamwar, D., 2011. Cyanobacteria and 1152 microalgae: a positive prospect for biofuels. Bioresour. Technol. 102, 10163e10172. Payen, A., Persoz, J.F., 1833. Memoir on diastase, the principal products of its reactions, and their applications to the industrial arts. Ann. Chem. Phys. 53, 73e92. Paz, A., Mester, D., Baca, I., Nevo, E., Korol, A., 2004. Adaptive role of increased frequency of polypurine tracts in mRNA sequences of thermophilic prokaryotes. Proc. Natl. Acad. Sci. U.S.A. 101 (9), 2951e2956. Rees, D.C., Adams, M.W.W., 1995. Hyperthermophiles: taking the heat and loving it. Structure 3 (3), 251e254. Sames, T., 1900. Zur Kenntniss der bei hoherer Temperatur wachsenden Bakterienund Streptothrixarten. Z Hyg Infektionskrankh 33, 313e362. Santos, H., Da Costa, M.S., 2002. Compatible solutes of organisms that live in hot saline environments. Environ. Microbiol. 4, 501e509. Sarmiento, F., Peralta, R., Blamey, J.M., 2015. Cold and hot extremozymes: industrial relevance and current trends. Front. Bioeng. Biotechnol. 3, 148. Schafer, T., Kirk, O., Borchert, T.V., Fuglsang, C.C., Pedersen, S., Salmon, S., Olsen, H.S., Deinhammer, R., Lund, H., 2002. Enzymes for technical applications. In: Fahnestock, S.R., Steinb€ uchel, S.R. (Eds.), Biopolymers. Wiley-VCH, Weinheim, Germany, pp. 377e437. Schiraldi, C., De Rosa, M., 2002. The production of biocatalysts and biomolecules from extremophiles. Trends Biotechnol. 20, 515e521. Schmid, A., Dordick, J.S., Hauer, B., Kiener, A., Wubbolts, M., Witholt, B., 2001. Industrial biocatalysis today and tomorrow. Nature 409, 258e268. Setchell, W.A., 1903. The upper temperature limits of life. Science 17, 934e937. Sprott, G.D., Meloche, M., Richards, J.C., 1991. Proportions of diether, macrocyclic diether, and tetraether lipids in Methanococcus jannaschii grown at different temperatures. J. Bacteriol. 173 (12), 3907e3910. Stetter, K.O., 1996. Hyperthermophilic prokaryotes. FEMS (Fed. Eur. Microbiol. Soc.) Microbiol. Rev. 18, 149e158. Stetter, K.O., 2013. A brief history of the discovery of hyperthermophilic life. Biochem. Soc. Trans. 41, 416e420. Synowiecki, J., 2008. Thermostable Enzymes in Food Processing in Enzymes as Additives or Processing Aids, pp. 29e54. Thompson, M.J., Eisenberg, D., 1999. Transproteomic evidence of a loop-deletion mechanism for enhancing protein thermostability. J. Mol. Biol. 290 (2), 595e604. Tirelli, E., 1907. I termofili delle acque potabili Zentr Bakt Parasitenk Infek II 19, 328. Turner, P., Mamo, G., Karlsson, E.N., 2007. Potential and utilization of thermophiles and thermostable enzymes in biorefining. Microb. Cell Factories 6, 9. Urbieta, M.S., Donati, E.R., Chan, K.G., Shahar, S., Sin, L.L., Goh, K.M., 2015. Thermophiles in the genomic era: biodiversity, science, and applications. Biotechnol. Adv. 33 (6 Pt 1), 633e647. van Beilen, J.B., Li, Z., 2002. Enzyme technology: an overview. Curr. Opin. Biotechnol 13 (4), 338e344.

General background

Vieille, C., Burdette, D.S., Zeikus, J.G., 1996. Thermozymes. Biotechnol. Ann. Rev. 2, 1e83. Vieille, C., Zeikus, G.J., 2001. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65, 1e43. Wyss, M., Brugger, R., Kronenberger, A., Remy, R., Fimbel, R., Osterhelt, G., Lehmann, M., Van Loon, A.P.G.M., 1999. Biochemical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolase): catalytic properties. Appl. Environ. Microbiol. 65, 367e373.

Relevant websites https://www.expertmarketresearch.com/reports/enzymes-market. https://ecampusontario.pressbooks.pub/microbio/chapter/temperature-and-microbial-growth/. https://microbeonline.com/psychrophiles-mesophiles-thermophiles. https://ecampusontario.pressbooks.pub/microbio/chapter/temperature-and-microbial-growth/.

Further reading Niehaus, F., Bertoldo, C., K€ahler, M., Antranikian, G., 1999. Extremophiles as a source of novel enzymes for industrial application. Appl. Microbiol. Biotechnol. 51 (6), 711e729. Schmidt-Dannert, C., Arnold, F.H., 1999. Directed evolution of industrial enzymes. Trends Biotechnol. 17, 135e136.

15

CHAPTER 2

Physiological and morphological aspects of thermophiles and hyperthermophiles 2.1 Introduction “The existence of life at high temperatures is quite fascinating. At both ends of the temperature range compatible with life, only microorganisms are capable of growth and survival” (Mehta and Satyanarayana, 2013). Several microorganisms are found to survive and can grow at higher temperatures. Many thermophilic microorganisms have been isolated from manmade and natural thermal habitats all over the world (Mehta and Satyanarayana, 2013). - Manmade thermal habitats Biological wastes and waste treatment plants, acid mine effluents, compost piles. - Natural thermal habitats Terrestrial fumaroles, terrestrial hot springs, volcanic areas, geothermal areas, deep-sea hydrothermal vents, sun-heated soils/sediments, geothermally heated oil, and petroleum reserves. Enzymes isolated from these fascinating microorganisms show exceptional features. These are remarkably thermostable. These are generally resistant toward chemical denaturants such as organic solvents, detergents, and chaotropic agents. Therefore, these enzymes can be used as a model for creating and designing proteins with novel properties that can be used for industrial applications (Niehaus et al., 1999). Culture-independent and culture-dependent strategies have been used to understand the diversity of microorganisms in hot environments. These microorganisms are known as thermophiles/thermophilic microorganisms and are able to withstand high temperatures and also generally need these for their growth and continued existence. This group includes prokaryotic (bacteria and archaea) and also eukaryotic microorganisms. Interest in their physiology, biochemistry, diversity, and ecology has enhanced considerably during the last few decades. These microorganisms have evolved many structural and chemical adaptations that enable them to stay alive and nurture at higher temperatures (Mehta and Satyanarayana, 2013).

Developments and Applications of Enzymes From Thermophilic Microorganisms ISBN 978-0-443-19197-8, https://doi.org/10.1016/B978-0-443-19197-8.00022-0

© 2023 Elsevier Inc. All rights reserved.

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Developments and Applications of Enzymes From Thermophilic Microorganisms

2.2 Physiological and morphological properties Most of the bacterial thermophilic anaerobes are chemoorganotrophic. These bacteria get energy from the oxidation of reduced organic compounds. These bacteria are also known as “heterotrophic bacteria” and contain a wide variety of different bacteria with quite different nutrient requirements. These bacterial thermophilic anaerobes belong to almost the same variety of nutritional categories as mesophilic bacteria. Thermotoga, which is a hyperthermophilic bacteria, is found to ferment different carbohydrates example xylan, glucose, and starch forming acetate, L-lactate, carbon dioxide, and hydrogen as the final product (Stetter, 1996). Thermotoga species have been isolated from geothermally heated environments around the world, which include submarine hot springs, oil reservoirs, and continental solfataric springs (Table 2.1). The genus Thermotoga comprises some of the most thermophilic and hypothermophilic bacteria. These bacteria are able to grow optimally at 80 C (Belkin et al., 1986; Huber et al., 1986; Jannasch et al., 1988) and are able to degrade a broad range of simple and complex carbohydrates, produce hydrogen at a higher yield, and catalyze reactions at a higher temperature, which have been the basis for several biotechnological applications (Conners et al., 2006). These bacteria appear as a model system to investigate microbial evolution and adaptation to higher temperatures because they defy conventional classification (Nesbo et al., 2006; Mongodin et al., 2005). For Thermotoga species, several genomic sequences have become available in the last few years. These offer further insights into the biology of these fascinating microorganisms and suggest biotechnological applications (Zhaxybayeva et al., 2009). Members of the order Thermotogales are anaerobic, rod-shaped bacteria that are encapsulated by a distinctive “toga”-like outer membrane. Substrates on which Thermotoga grow include xylans, glucans, amorphous cellulose, pentoses, hexoses, glucomannan, galactomannan, pectin, and chitin (Huber et al., 1986; Conners et al., 2005). This multiplicity of substrates is found to correlate with the abnormally larger fraction of Thermotoga genes involved in the use and degradation of carbohydrates (Chhabra et al., 2002). The main fermentation products are acetate, carbon dioxide, and hydrogen, though alanine, ethanol, lactate, and a-aminobutyrate have also been found (Balk et al., 2002; Takahata et al., 2001; Huber et al., 1986). Some Thermotoga species can use thiosulfate, sulfur, and iron as electron acceptors (Vargas et al., 1998). The hyperthermophilic Aquifex is strictly chemolithoautotrophic. For metabolic processes, these microorganisms get the required carbon from carbon dioxide in their environment. They can also use inorganic compounds for the energy to power these processes. It uses molecular hydrogen, thiosulfate and nitrate and oxygen as electron acceptors and elemental sulfur as electron donors (Huber et al., 1992). Aquifex pyrophilus and A. aeolicus show the optimum temperature near 95 C. These are among the most thermophilic bacteria known. These species mostly relate to

Physiological and morphological aspects of thermophiles and hyperthermophiles

Table 2.1 Thermotoga species. Opt. temp. 8C

Genome size (bp)

Thermotoga thermarum LA3

70

N/A

Thermotoga neapolitana NS-E Thermotoga petrophila RKU-1 Thermotoga naphthophila RKU-10 Thermotoga maritima MSB8 80 Thermotoga sp. strain RQ2

77

1,884,562

80

Thermotoga species

Thermotoga lettingae TMO Thermotoga elfii SEBR 6459 Thermotoga hypogea SEBR 7054 Thermotoga subterranea SL1

Isolation site

Country

References

Djibouti

1,823,511

Continental solfataric springs Shallow submarine hot springs Oil reservoir

Japan

80

1,809,823

Oil reservoir

Japan

80

1,860,725

Italy

76e82

1,877,693

65

2,135,243

66

N/A

Geothermallyheated sea floors Geothermallyheated sea floors Sulfate-reducing bioreactor Oil field

Windberger et al. (1989) Jannasch et al. (1988) Takahata et al. (2001) Takahata et al. (2001) Huber et al. (1986)

70

N/A

70

N/A

Oil-producing well Continental oil reservoir

Italy

Azores

Huber et al. (1986)

Netherlands

Balk et al. (2002) Ravot et al. (1995) Fardeau et al. (1997) Jeanthon et al. (1995)

Sudan Cameroon France

Based on Frock, A.D., Notey, J.S., Kelly, R.M., 2010. The genus Thermotoga: recent developments. Environ. Technol. 31 (10), 1169e1181.

filamentous bacteria that were first seen at the turn of the century, growing at 89 C in the outflow of hot springs in Yellowstone National Park (Reysenbach et al., 1994; Setchell, 1903; Huber et al., 1992). “The observation of these macroscopic assemblages would later be instrumental in the drive to grow hyperthermophilic organisms” (Brock, 1995). According to phylogenetic analysis of 16S ribosomal RNA, the Aquificaceae represent the bacterial domain’s most intensely branching family, though analyses of individual protein sequences have different conclusions about where Aquifex fits in relation to other groups (Burggraf et al., 1992; Pitulle et al., 1994; Baldauf et al., 1996; Klenk et al., 1994; Bocchetta et al., 1995; Wetmur et al., 1994). The genera in this group, Hydrogenobacter

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Developments and Applications of Enzymes From Thermophilic Microorganisms

and Aquifex, are thermophilic microaerophilic and obligate chemolithoautotrophs that oxidize hydrogen (Kristjannson et al., 1985; Kryukov et al., 1983; Huber et al., 1992; Kawasumi et al., 1984). A. aeolicus (isolated by Dr. Stetter) was grown at 85 C under hydrogen/carbon dioxide/oxygen (79.5:19.5:1.0) atmosphere in a medium having only inorganic components. Many organic substrates (amino acids, sugars, meat extract, or yeast extract) do not support A. aeolicus growth. It has not been demonstrated that A. aeolicus can grow anaerobically in the lab using nitrate as an electron acceptor, unlike its close relative A. pyrophilus. The physiology of the organism can be studied to make a number of predictions. A. aeolicus must possess a full complement of biosynthetic genes in addition to those that code for proteins involved in one or more forms of carbon fixation. Due to the fact that autotrophy is a trait shared by all Archaea and Bacteria, it is expected that the majority of the associated genes are of very ancient origin and are undoubtedly related to those described elsewhere. Most of the associated genes should have a long history and be clearly related to other genes that have been identified. Obligate autotrophy suggests a biosynthetic character as opposed to a degradative one. The presence of corresponding utilization and tolerance genes is implied by oxygen respiration. The early divergence of the Aquificaceae inferred from ribosomal RNA sequences raises a number of questions. Are there similarities or differences between the mechanisms for oxygen consumption and tolerance found in mitochondria and in other, well-studied organisms like Escherichia coli? Is there evidence for the use of alternative oxidants if there was much less oxygen when the lineage first began? (Deckert et al., 1998a,b). Generally, the physiological process of adaptation to environmental stress in anaerobic bacteria seems to involve different factors than in aerobic bacteria. Firstly, anaerobic organisms have limited energy during chemoorganotrophic growth as they cannot couple dehydrogenation reactions to oxygen reduction and obtain more chemical-free energy. Secondly, the majority of chemoorganotrophic anaerobic organisms (aside from methanogens) produce noxious end products as they grow, necessitating the development of a dynamic adaptation mechanism or resistance to those end products in anaerobic organisms. Hyperthermophiles are the most attention-grabbing group of thermophilic microorganisms as the isolation of these microbes has caused a reevaluation of the possible habitation for microbes and has raised the temperature limits at which life can exist. Hyperthermophilic anaerobic archaea range in size from 0.5 to 2.0 mm. Even though some of them have unusual morphological characteristics, this size is almost identical to one typical procaryotic cell. In terms of metabolism, hyperthermophiles are rather varied because they include methanogens, nitrate-reducers, aerobic respirers, and sulfate reducers. Nevertheless, the vast majority of species currently recognized are solely anaerobic heterotrophic S0 reducers. There are three distinct groups of terrestrial Archaea. Extremely acidophilic thermophiles are only found in continental solfataric fields.

Physiological and morphological aspects of thermophiles and hyperthermophiles

The creatures are strict, facultative aerobes with a coccoid shape, and they need an acidic pH (optimum approximately pH 3.0) to grow (Stetter, 1996; Kengen et al., 1996). They are members of the archaeal generadDesulfurolobus, Acidianus, Metallosphaera, and Sulfolobus. Contrastingly, the mildly acidophilic and neutrophilic thermophiles are present in both subaerial hydrothermal systems and continental solfataric fields. They are all strictly anaerobes. Members of the genera, Desulfurococcus, Methanothermus, Pyrobaculum, Thermophilum, and Thermoproteus, can be found in solfataric fields. Pyrobaculum islandicum has both the ability to grow heterotrophically through sulfur respiration and autotrophically through the anaerobic reduction of S0 with hydrogen as an electron donor. Obligate heterotrophs include Thermophilum and Pyrobaculum organotrophum strains (Stetter, 1996; Nissen et al., 1992). They utilize various organic substrates to grow through sulfur respiration. A lipid fraction of Thermoproteux tenax is interestingly required by Thermophilum pendens. The crenarchaeal genera, Hyperthermus, Methanopyrus, Pyrococcus, Thermococcus, Archaeoglobus, Pyrodictium, Thermodiscus, and Staphylothermus, and some members of Methanococcus represent the diversity of hyperthermophilic archaea adapted to the marine environments. The ideal temperatures for the growth of these organisms range from 75 to 105 C, and the highest temperature at which they can grow is 113 C (Pyrobolus) or even 110 C (Pyrodictium occultum). They cannot grow below 80 C because they are very well adapted to high temperatures (Stetter, 1996). Crenarchaeota like all Archaea are prokaryotic and are surrounded by isoprenoid side chains rather than fatty acids in their ether-linked lipid membranes. Cocci with a diameter of less than 1 m and filaments longer than 100 m make up the majority of cells. There are many different cell shapes found in different species, including regular cocci grouped in grape-like aggregates (Staphylothermus), discs (Thermodiscus), lobed cells (Sulfolobus), irregular, extremely thin filaments (0.5 m diameter; Thermofilum), and nearly rectangular rods (Pyrobaculum; Thermoproteus). Most species are motile and have flagella. Several Crenarchaeota species have peculiar morphologies: Pyrodictium occultum and Pyrodictium brockii are unusual organisms that grow as a mold-like layer on sulfur. These organisms have unusual cells that are irregularly shaped into discs and dishes, and their production may give the organism an adaptive advantage by trapping nutrients. A network of incredibly thin, hollow tubules links the cells together (Stetter, 1996). Most Pyrodictium strains are chemolitoautotrophs that produce energy by reducing S0 with hydrogen. Although yeast extract promotes growth, both species of Pyrodictium are solely reliant on hydrogen. As a heterotroph that grows by fermenting peptides, Pyrodictium abyssi cannot grow chemolithotrophically on carbon dioxide/hydrogen, either in the absence of S0 or S2O3-2. The cells of Pyrodictium abyssi are highly polymorphous, frequently diskshaped, and exhibit ultra-flat areas, just like the other members of the genus. The

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cytoplasmic membrane, the periplasmic space, and the surface layer protein make up the cell envelope. The zigzag structure of the S-layer is also visible in the ultra-thin sections. Typically, S-layer proteins are extremely stable, keep bacterial cells’ structural integrity in the face of harsh environmental conditions, and do not easily dissociate by mechanical disruption, chemical treatment, and high temperature (Andrade et al., 1999; Rieger et al., 1995; Messner et al., 1986). Many hyperthermophiles are found to grow at temperatures in the range of 80e108 C. Extreme thermophilic microorganisms, which are able to grow between 60 and 80 C, are extensively distributed among the genera, Fervidobacterium, Thermus, Thermotoga, Thermoanaerobacter, Bacillus, Aquifex, and Clostridium. Microbes that can grow in the temperature range of 50e60 C are called moderately thermophilic. The majority of these microbes belong to several dissimilar taxonomic groups of eukaryotic and prokaryotic microbes like protozoa, streptomycetes, cyanobacteria, fungi, and algae, which contain mostly mesophiles. The comparative abundance of archaea and bacteria in hot environments was mostly studied by cultivation-based procedures. Archaea are frequently isolated from these environments, suggesting that they dominate the hotter biotopes. Recently, the application of molecular biological techniques has shown that bacterial communities are also abundant in these environments. This indicates that archaea in temperate environments are generally found in lower abundance than expected from cultivation-based experiments. But the factors that allow bacteria to thrive in warmer habitats once thought to be the archaeal kingdoms are still unknown (Bertoldo and Antranikian, 2009). The most significant biotopes for microorganisms, which prefer to live in both acidic and thermophilic environments, are solfatar fields. The two layers of the solfatalic floor are easily distinguishable by their distinct colors: upper and lower layers. The upper layer is an aerobic layer. It has an ochre color due to the presence of ferric iron. The lower layer is an anaerobic layer that looks rather blackish-blue because of the presence of ferrous iron. Different kinds of microbes can be obtained from these habitats in accordance with the chemical parameters of the two layers. Thermophilic acidophiles, which show optimum growth in the range of 60 and 90 C and pH 0.7e5.0 are normally found in the aerobic upper layer. These belong to the genera Acidianus, Picrophilus, Sulfolobus, and Thermoplasma. From the lower layer, slightly acidophilic or neutrophilc anaerobes (Methanothermus fervidus or Thermoproteus tenax) can be obtained. Species of Thermoplasma showing optimum growth at a pH of 2.0 and a temperature of 60 C have been found in solfataras, hot springs, and coal refuse piles. The closest known phylogenetic relative, also found in Solfatarus, belongs to the genus Picrophilus. These are the most extreme acidophiles ever known. These can grow closer to pH 4.0. P. torridus and Picrophilus oshimae are both aerobic, heterotrophic archaea that show optimum growth at a temperature of 60 C and pH 0.7. These use a variety of polymers as substrates. Members of the genus Sulfolobus are found to be strict aerobes that grow either autotrophically,

Physiological and morphological aspects of thermophiles and hyperthermophiles

heterotrophically, or facultatively heterotrophically. S0, S2, and hydrogen are oxidized to sulfuric acid and hydrogen or water as end products during autotrophic growth. Sulfolobus metallicus and S. brierley are found to grow by oxidation of sulfidic ores. A thick biofilm of these microbes is responsible for the microbial ore leaching process, in which heavy metal ions such as iron, zinc, and copper are solubilized. Other thermoacidophilic microorganisms have been affiliated with the genera Metallosphaera, Acidianus, and Stygioglobus. Metallosphaera shows growth in the pH range of 1e4.5 and temperature range of 50e80 C, Acidianus shows growth in the pH range of 1.5e5 and temperature range of 60e95 C, and Stygioglobus shows growth in the pH range of pH 11e5.5 and temperature range of 57e90 C. Conversely, the alkaliphilic microorganisms that grow at higher pH values are extensively found all over the world. These were found in alkaline soils and carbonate-rich springs where the pH can be higher around 10.0 or higher, although the internal pH is maintained around 8.0. In these places, many species of Bacillus and cyanobacteria are usually found in abundance and make available organic matter for different groups of heterotrophs. Alkaliphilic organisms need sodium ions and alkaline environments to grow and also for sporulation and germination. In alkaliphilic microbes, sodium ion-dependent uptake of nutrients has been observed. Most of these microbes need several nutrients for growth. Few alkaliphilic Bacillus strains are able to grow in simple minimal media having glutamic acid, glycerol, and citric acid. The cultivation temperature generally ranges from 20 to 55 C. Moreover, halo-alkaline microorganisms isolated from alkaline hypersaline lakes can grow on alkaline media containing 20% sodium chloride. In Kenya’s Rift Valley, soda lakes and similar lakes found elsewhere on Earth are highly alkaline. The pH values range from 8.0 to 12.0 and represent a usual habitat from where alkaliphilic microbes can be isolated. An anaerobic spore-forming thermophilic alkalophilic bacterium, thermoalkalophilic Clostridium, was isolated from a sewage treatment plant. From the sewage plants, anaerobic spore-forming thermophilic alkaliphiles, thermoalkaliphilic Clostridia, were isolated. Thermoalkaliphilic bacteria, Anaerobranca gottschalkii, have been isolated from Lake Bogoria in Kenya and Anaerobranca horikoshii have been isolated from Yellowstone National Park. These new isolates represent a new line within the Clostridium/Bacillus subphylum. Thermococcus alcaliphilus and Thermococcus acidoaminivorans are the archaeal thermoalkaliphiles identified. These microorganisms grow at a pH of 9.0 and a temperature of 85 C. Alkaliphilic enzymes are mostly used in the detergent industry. These enzymes account for about 30% of global enzyme production. In the hide-dehairing process, alkaline enzymes have been also used where dehairing is performed at pH values ranging from 8.0 to 10.0 (Bertoldo and Antranikian, 2009). Figs. 2.1 and 2.2 show Bright orange thermophiles, microorganisms in hot springs thermal environments, Yellowstone National Park, USA, and Bright orange thermophiles, microorganisms in Black Warrior Lake, Yellowstone National Park, USA.

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Figure 2.1 Bright orange thermophiles, microorganisms in hot springs thermal environments, Yellowstone National Park, USA.

Figure 2.2 Bright orange thermophiles, microorganisms in Black Warrior Lake, Yellowstone National Park, USA.

Physiological and morphological aspects of thermophiles and hyperthermophiles

References Andrade, C.M., Pereira, N., Antranikian, G., 1999. Extremely thermophilic microorganisms and their polymer-hidrolytic enzymes. Rev. Microbiol. 30, 287e298. Baldauf, S.L., Palmer, J.D., Doolittle, W.F., 1996. The root of the universal tree and the origin of eukaryotes based on elongation factor phylogeny. Proc. Natl. Acad. Sci. U.S.A. 93, 7749e7754. Balk, M., Weijma, J., Stams, A.J., 2002. Thermotoga lettingae sp. nov., a novel thermophilic, methanol degrading bacterium isolated from a thermophilic anaerobic reactor. Int. J. Syst. Evol. Microbiol. 52, 1361e1368. Belkin, S., Wirsen, C.O., Jannasch, H.W., 1986. A new sulfur-reducing, extremely thermophilic eubacterium from a submarine thermal vent. Appl. Environ. Microbiol. 51, 1180e1185. Bertoldo, C., Antranikian, G., 2009. Thermoactive enzymes in biotechnological applications. Extremophiles I (3), 294. Bocchetta, M., Ceccarelli, E., Creti, R., Sanangelantoni, A.M., Tiboni, O., Cammarano, P., 1995. Arrangement and nucleotide sequence of the gene (fus) encoding elongation factor G (EF-G) from the hyperthermophilic bacterium Aquifex pyrophilus: phylogenetic depth of hyperthermophilic bacteria inferred from analysis of the EF-G/fus sequences. J. Mol. Evol. 41 (6), 803e812. Brock, T.D., 1995. The road to Yellowstonedand beyond. Annu. Rev. Microbiol. 49, 1e28. Burggraf, S., Olsen, G.J., Stetter, K.O., Woese, C.R., 1992. A phylogenetic analysis of Aquifex pyrophilus. Syst. Appl. Microbiol. 15, 353e356. Chhabra, S.R., Shockley, K.R., Ward, D.E., Kelly, R.M., 2002. Regulation of endo-acting glycosyl hydrolases in the hyperthermophilic bacterium Thermotoga maritima grown on glucan and mannan-based polysaccharides. Appl. Environ. Microbiol. 68, 545e554. Conners, S., Montero, C., Comfort, D., Shockley, K., Johnson, M., Chhabra, S., Kelly, R., 2005. Prediction of carbohydrate transport and utilization regulons in the hyperthermophilic bacterium Thermotoga maritima through the use of carbohydrate-specific transcriptional response. Abstr. Pap. Am. Chem. Soc. 229, U240. Conners, S.B., Mongodin, E.F., Johnson, M.R., Montero, C.I., Nelson, K.E., Kelly, R.M., 2006. Microbial biochemistry, physiology, and biotechnology of hyperthermophilic Thermotoga species. FEMS Microbiol. Rev. 30, 872e905. Deckert, G., Warren, P.V., Gaasterland, T., Young, W.G., Lenox, A.L., Graham, D.E., Overbeek, R., Snead, M.A., Keller, M., Aujay, M., Huber, R., Feldman, R.A., Short, J.M., Olsen, G.J., Swanson, R.V., 1998. The complete genome of the hyperthermophilic bacterium Aquifex aeolicus. Nature 392 (6674), 353e358. Deckert, G., Warren, P., Gaasterland, T., et al., 1998. The complete genome of the hyperthermophilic bacterium Aquifex aeolicus. Nature 392, 353e358. Fardeau, M.L., Ollivier, B., Patel, B.K., Magot, M., Thomas, P., Rimbault, A., Rocchiccioli, F., Garcia, J.L., 1997. Thermotoga hypogea sp. nov. a xylanolytic, thermophilic bacterium from an oil-producing well. Int. J. Syst. Bacteriol. 47, 1013e1019. Frock, A.D., Notey, J.S., Kelly, R.M., 2010. The genus Thermotoga: recent developments. Environ. Technol. 31 (10), 1169e1181. Huber, R., Langworthy, T.A., Koning, H., Thomm, M., Woese, C.R., Sleytr, U.B., Stetter, K.O., 1986. Thermotoga maritima sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 90 C. Arch. Microbiol. 144, 324e333. Huber, R., Wilharm, T., Huber, D., Trincone, A., Burggraf, S., König, H., Rachel, R., Rockinger, I., Fricke, H., Stetter, K.O., 1992. Aquifex pyrophilus gen. Nov. sp. nov., represents a novel group of marine hyperthermophilic hydrogen-oxidising bacteria. Syst. Appl. Microbiol. 15, 340e351. Jannasch, H.W., Huber, R.B.S., Stetter, K.O., 1988. Thermotoga neapolitana sp. nov. of the extremely thermophilic, eubacterial genus Thermotoga. Arch. Microbiol. 150, 103e104. Jeanthon, C., Reysenbach, A.L., L’Haridon, S., Gambacorta, A., Pace, N.R., Glenat, P., Prieur, D., 1995. Thermotoga subterranea sp. nov., a new thermophilic bacterium isolated from a continental oil reservoir. Arch. Microbiol. 164, 91e97.

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Kawasumi, T., Igarashi, Y., Kodama, T., Minoda, Y., 1984. Hydrogenobacter thermophilus gen. nov., sp. nov. an extremely thermophilic, aerobic, hydrogen-oxidizing bacterium. Int. J. Syst. Bacteriol. 34, 5e10. Kengen, S.W.M., Stams, A.J.M., de Vos, W.M., 1996. Sugar metabolism of hyperthermophiles. FEMS Microbiol. Rev. 18, 119e137. Klenk, H.P., Palm, P., Zillig, W., 1994. In: Pfeifer, F., Palm, P., Scleeifer, K.H. (Eds.), Molecular Biology of the Archaea, pp. 139e147. Kristjannson, J., Ingason, A., Alfredsson, G.A., 1985. Isolation of thermophilic obligately autotrophic hydrogen-oxidizing bacteria, similar to Hydrogenobacter thermophilus, from Icelandic hotsprings. Arch. Microbiol. 140, 321e325. Kryukov, V.R., Savel’eva, N.D., Pusheva, M.A., 1983. Calderobacterium hydrogenophilum gen. nov., sp. nov. an extreme thermophilic bacterium and its hydrogenase activity. Microbiology (Engl. Trans. Mikrobiologiya) 52, 611e618. Mehta, D., Satyanarayana, T., 2013. Diversity of hot environments and thermophilic microbes. In: Satyanarayana, T., Littlechild, J., Kawarabayasi, Y. (Eds.), Thermophilic Microbes in Environmental and Industrial Biotechnology. Springer, Dordrecht. Messner, P., Pum, D., Sara, M., Stetter, K.O., Sleytr, U.B., 1986. Ultrastructure of the cell envelope of the archaebacteria Thermoproteus tenax and Thermoproteus neutrophilus. J. Bacteriol. 166, 1046e1054. Mongodin, E.F., Hance, I.R., Deboy, R.T., Gill, S.R., Daugherty, S., Huber, R., Fraser, C.M., Stetter, K., Nelson, K.E., 2005. Gene transfer and genome plasticity in Thermotoga maritima a model hyperthermophilic species. J. Bacteriol. 187, 4935e4944. Nesbo, C.L., Dlutek, M., Doolittle, W.F., 2006. Recombination in Thermotoga: implications for species concepts and biogeography. Genetics 172, 759e769. Niehaus, F., Bertoldo, C., K€ahler, M., Antranikian, G., 1999. Extremophiles as a source of novel enzymes for industrial application. Appl Microbiol. Biotechnol. 51 (6), 711e729. Nissen, A.N., Anker, L., Munk, N., Lange, N.K., 1992. Xylanases for the pulp and paper industry. In: Visser, J., Beldman, G., Kusters -van Someren, M.A., Voragen, A.G.J. (Eds.), Xylan and Xylanases. Elsevier Science Publishers, Amsterdam, pp. 325e337. Pitulle, C., Yang, Y., Marchiani, M., Moore, E.R.B., Siefert, J.I., Aragno, M., Jurtshuk Jr., P., Fox, G., 1994. Phylogenetic position of the genus Hydrogenobacter. Int. J. Syst. Bacteriol. 44, 620e626. Ravot, G., Ollivier, B., Magot, M., et al., 1995. Thiosulfate reduction, an important physiological feature shared by members of the order thermotogales. Appl. Environ. Microbiol. 61 (5), 2053e2055. Reysenbach, L., Wickham, G.S., Pace, N.R., 1994. Phylogenetic analysis of the hyperthermophilic pink filament community in Octopus Spring, Yellowstone National Park. Appl. Environ. Microbiol. 60, 2113e2119. Rieger, G., Rachel, R., Hermann, R., Stetter, K.O., 1995. Ultrastructure of the hyperthermophilic archaeon Pyrodictium abyssi. J. Struct. Biol. 115, 78e87. Setchell, W.A., 1903. The upper temperature limits of life. Science 17, 934e937. Stetter, K.O., 1996. Hyperthermophilic procaryotes. FEMS Microbiol. Rev. 18, 149e158. Takahata, Y., Nishijima, M., Hoaki, T., Maruyama, T., 2001. Thermotoga petrophila sp. nov. and Thermotoga naphthophila sp. nov., two hyperthermophilic bacteria from the Kubiki oil reservoir in Niigata, Japan. Int. J. Syst. Evol. Microbiol. 51, 1901e1909. Vargas, M., Kashefi, K., Blunt-Harris, E.L., Lovley, D.R., 1998. Microbiological evidence for Fe(III) reduction on early Earth. Nature 395, 65e67. Wetmur, J.G., Wong, D.M., Ortiz, B., Tong, J., Reichert, F., Gelfand, D.H., 1994. Cloning, sequencing, and expression of RecA proteins from three distantly related thermophilic eubacteria. J. Biol. Chem. 269 (41), 25928e25935. Windberger, E., Huber, R., Trincone, A., Fricke, H., Stetter, K.O., 1989. Thermotoga thermarum sp. nov. and Thermotoga neapolitana occurring in African continental solfataric springs. Arch. Microbiol. 151, 506e512. Zhaxybayeva, O., Swithers, K., Lapierre, P., Fournier, G., Bickhart, D., Deboy, R., Nelson, K., Nesbo, C., Doolittle, W., Gogarten, J., Noll, K., 2009. On the chimeric nature, thermophilic origin, and phylogenetic placement of the Thermotogales. Proc. Natl. Acad. Sci. U. S. A. 106, 5865e5870.

Physiological and morphological aspects of thermophiles and hyperthermophiles

Relevant websites https://www.biologyonline.com/dictionary/thermophile. http://archive.bio.ed.ac.uk/jdeacon/microbes/thermo.htm. https://serc.carleton.edu/microbelife/extreme/extremeheat/index.html.

Further reading Bertoldo, C., Antranikian, G., 2003. Thermoactive enzymes in biotechnological applications, in extremophiles (life under extreme environmental condition), vol. I. In: Gerday, C., Glansdorff, N. (Eds.), Encyclopedia of Life Support Systems (EOLSS), Developed under the Auspices of the UNESCO, 3. Eolss Publishers, Paris, France, p. 294. http://www.eolss.net. Jorgensen, S., Vorgias, C.E., Antranikian, G., 1997. Cloning, sequencing and expression of an extracellular a-amylase from the hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli and Bacillus subtilis. J. Biol. Chem. 272, 16335e16342.

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CHAPTER 3

Biodiversity of thermotolerant microorganisms 3.1 Introduction During the last 2 decades, the scientific community has been more and more focused on the rich microbial diversity of natural hot spring sources to investigate the advantages of thermophilic/hyperthermophilic microorganisms in biotechnological and industrial fields. The interest shown by researchers in hyperthermophilic microorganisms has continuously increased during the last 5 decades. The increasing attentiveness is shown by the increasing number of hyperthermophiles that have been reported and by the most important position taken by hyperthermophilic species in global genomic sequencing projects. The study of environmental 16S rRNA sequences analysis has shown that known hyperthermophilic microorganisms represent only a fraction of hyperthermophiles diversity (Barns et al., 1996). A very good example of this is the bacterium Thermocrinis ruber (Vielle and Zeikus, 2001).

3.2 Thermophile/hyperthermophile diversity Thermophilic and hyperthermophilic microorganisms are mostly found in heated environments. The optimum growth temperature of thermophilic microorganisms is normally more than 55
C, while that for hyperthermophilic microorganisms is more than 80
C. Hyperthermophilic microorganisms have been obtained from hot natural as well as industrial environments (for instance, sewage sludge treatment plant, geothermal power plant wastewater) with temperatures ranging from 80 to 115
C (Satyanarayana et al., 2005; Eichler, 2001). Deep-sea hyperthermophilic microorganisms flourish in surroundings with hydrostatic pressures in the range from 200 to 360 atm. Hot springs are one of the primary places where hyperthermophilic and thermophilic microorganisms are isolated, but they can also flourish in artificial environments like composting facilities (Rastogi et al., 2010). Fig. 3.1 shows the pictures of numerous types of hot springs and biomats taken from around the world. Hot springs nearby to volcanic environments are generally acidic, while areas close to limestone have a neutral or slightly alkaline pH. Thermophiles can survive in severe conditions with extreme pH levels or high salinity. Most of the early biodiversity research took a culture-dependent approach. Results from these studies Developments and Applications of Enzymes From Thermophilic Microorganisms ISBN 978-0-443-19197-8, https://doi.org/10.1016/B978-0-443-19197-8.00023-2

© 2023 Elsevier Inc. All rights reserved.

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Figure 3.1 Photographs of hot springs in the form of a pool or basin (AeC), a stream (D), heated mud (EeF), and a pot (G). Various colors of biomats and sediments that can formin hot springs (HeL) (Reproduced with permission Urbieta, M.S., Donati, E.R., Chan, K.G., Shahar, S., Sin, L.L., Goh, K.M., 2015. Thermophiles in the genomic era: biodiversity, science, and applications. Biotechnol. Adv. 33 (6 Pt 1), 633e647.)

Biodiversity of thermotolerant microorganisms

show that normally only 1%e10% of the total biosphere population can be cultivated. Due to this limitation, scientists afterward selected a culture-independent approach that bypasses the culture and isolation steps to directly amplify the near-complete sequence of the 16S rRNA gene from the bulk genome (Urbieta et al., 2015, 2014a,b; Futterer et al., 2004; Giaveno et al., 2013; Ruepp et al., 2000). Hyperthermophilic communities are complex systems. These are the major producers and decomposers of organic matter. All hyperthermophiles are chemolithoautotrophs (methanogens and sulfur reducers and oxidants) (Lowe et al., 1993). Concerning the higher sulfur content of most high-temperature natural biotopes, most hyperthermophiles are either facultative or obligate chemolithotrophs, either reduce sulfur with hydrogen to form hydrogen sulfide (anaerobes), or oxidize sulfur with oxygen to form sulfuric acid (aerobic organisms). The hyperthermophiles highly acidophilic belong to the order Sulfolobales. These are facultative aerobes (such as Acidianus) or strictly aerobes (such as Sulfolobus) (Fujiwara, 2002). Hyperthermophiles are often within the domain Archaea. Thermotogales and Aquificales are the only bacteria. These enzymes have developed exceptional structurefunction characteristics of higher thermostability and optimum activity at temperatures more than 70
C. Some of these enzymes can perform at temperatures above 110
C (Vieille et al., 1996). Hyperthermophilic enzymes serve as model systems for scientists studying the evolution of enzymes, the molecular mechanisms underlying protein thermostability, and the upper-temperature limits of enzyme function. Thermophilic and hyperthermophilic enzymes are inherently stable and active at high temperatures, offering superior biotechnological benefits over mesophilic enzymes (Fakruddin, 2017; Bouzas et al., 2006). Thermophilic microbes include both archea and bacteria. “The most frequently isolated thermophilic strains have been reported to belong to the genus Bacillus (Abootalebi et al., 2020; Ladeira et al., 2015; Shen et al., 2021; Verma et al., 2018; Wang et al., 2006). The presence of thermophilic Bacilli has been investigated in hot springs and compost sources around the world (Yanmis et al., 2015), such as those in India (Verma et al., 2018), Iran (Abootalebi et al., 2020), Indonesia (Safitri et al., 2020), Jordan (Mohammad et al., 2017), Faisalabad (Saleh et al., 2020), Tunisia (Salem et al., 2020), Brazil (Bernardo et al., 2020), Egypt (Saeed et al., 2020), Malaysia (Msarah et al., 2020), Vietnam (Dang et al., 2018), Saudi Arabia (Al-Johani et al., 2016), South Africa (Tsotetsi et al., 2020) and Japan (Akita et al., 2017)” (Ulucay et al., 2022). Generally, geothermal hot springs are inhabited by microorganisms, and depending on the environment, they are active under extreme conditions and produce various enzymes. These microorganisms have attracted the attention of scientists as an important source of new thermostable enzymes such as xylanases, cellulases, chitinases, lipases, amylases, proteases, pectinases, phytases, and DNA polymerases. These enzymes of microbial origin are often preferred in the technical field as well. Enzymes are extensively used

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Developments and Applications of Enzymes From Thermophilic Microorganisms

in several biotechnology fields, like food processing, organic synthesis, biofuel production, renewable energy, molecular biology, medicine, vaccine development, and genetics. One of the most crucial habitats for thermophilic microorganisms is hot springs. On the other hand, several enzymes produced by microorganisms capable of surviving under severe conditions remain undetected (Ulucay et al., 2022; Thakur et al., 2021; Aanniz et al., 2015; Eriksen 2008; Falcicchio et al., 2021; Hogendoorn et al., 2021; Khan and Sathya 2018; Lee et al., 2010; Luo et al., 2017; Mehta et al., 2016; Msarah et al., 2020; Panosyan et al., 2020; Short 1998; Tango and Islam 2002; Zakzeski et al., 2010). Examples of hyperthermophilic microorganisms are sulfur-breathing heterotrophs/ autotrophs, sulfate reducers, nitrate reducers, glycolytic/proteolytic heterotrophs, iron oxidizers, microaerophiles, and methanogens. Hyperthermophilic microorganisms from a single geothermal site are extremely diverse from a physiological and phylogenetic perspective. A single hot spring in Yellowstone National Park was found to have an astounding diversity of hyperthermophilic organisms using 16S rRNA phylogeny (Stetter, 1996; Barns et al., 1994). The development of new isolation methods designated for hyperthermophilic microorganisms, as opposed to thermophilic microorganisms, is a major challenge for researchers to discover more hyperthermophilic enzymes in the near future, and the cultivation of newly isolated hyperthermophiles remains a challenge. For example, Brock (1985) described Thermocrinis ruber, a pink filament-forming bacterium in early 1967. However, it has taken about a quarter of a century to successfully cultivate it in pure culture (Huber et al., 1998a). Aquifex pyrophilus demonstrated the maximum growth temperatures of 95
C and optimal temperatures of 85
C among the hyperthermophilic bacteria. The highest growth temperatures for Thermotoga maritima were 90
C, and the best temperatures were 80
C. The highest optimum growth temperatures for archaea, however, were demonstrated by various species of Methanopyrus, Pyrolobus, Pyrococcus, and Pyrobaculum which could reach 106
C. The most prevalent hyperthermophilic bacterium, Pyrolobus fumarii, has an ideal growth temperature range of 90e113
C, but it is still unknown what the maximum growth temperature is? This illustrates the value of hyperthermophilic enzymes in the effective pretreatment of lignocelluloses at higher temperatures (Ebaid et al., 2019; Huber et al., 1986, 1992; Vieille and Zeikus, 2001). Hyperthermophiles are found in hot natural environments, which include deep sea hot sediments, continental solfataras, deep geothermally heated oil-containing stratifications, and hydrothermal vents located as far as 4000 m below the sea level (Table 3.1) (Ebaid et al., 2019). Deep-sea hyperthermophiles are barophilic or barotolerant (Marteinsson et al., 1999; Erauso et al., 1993; Reysenbach and Deming. 1991; Nelson et al., 1992). P. fumarii, the most thermophilic organism known, is found to grow in the temperature range of 90e113
C (Rothschild and Manicineli, 2001). Lifetime upper-temperature limit

Table 3.1 Hypothermophile diversity. Order

Genus

Species

Topt.(8C)

Isolation/habitat

References

Aquifer

A. pyrophilus

85

Huber et al. (1992)

A. aeolicus

85

T. tuber T. maritima

80 80

T. thermarum

77

T. neapolitana

77

A. nodosum F. islandicum T. africanus

65e70 65 75

Hot marine sediments at the Kolbeinsey Ridge, Iceland Hot marine sediments at the Kolbeinsey Ridge, Iceland Octopus spring, Yellowstone Heated sea floors, Vulcan, Italy, and Azores Water well, Lac Abbi, Djibouti, Africa Shallow marine hot spring, Naples, Italy New Zealand hot spring Icelandic hot spring Marine hydrothermal area at Obock, Djibouti, Africa

S. acidocal darius

70e75

S. metallicus S. shibatae

65e70 81

S. solfataricus

87

Bacteria

Aquificales

Thermotogales

Thermocrinis Thermotoga

Fervidobacterium

Huber et al. (1998a) Huber et al. (1986) Windberger et al. (1989) Belkin et al. (1986) and Jannasch et al. (1988) Patel et al. (1985) Huber et al. (1990) Huber et al. (1989a)

Archaea

Sulfolobales

Sulfolobus

Locomotive Spring in Yellowstone National Park, Italy Icelandic solfataric fields Acidic geothermal spring, Beppu, Kiushu Island, Japan Solfataric fields

Brock et al. (1972)

Huber and Stetter (1991) Grogan et al. (1990) Zillig et al. (1980) Continued

Biodiversity of thermotolerant microorganisms

Thermosipho

Huber et al. (1992)

33

34

Order

Genus

Species

Topt.(8C)

Isolation/habitat

References

Metallosphaera

M. prunae

75

Fuchs et al. (1995)

Stygiolobus

M. sedula S. azoricus

75 80

Acidianus

A. infernus

90

A. ambivalens

80

T. tenax

88

A smoldering slag heap of a uranium mine in Thuringen, Germany Solfataric field in Italy Solfataric fields, Sao Miguel Island, Azores Hot water, mud, and marine sediments at hot springs in Italy, the Azores, and the United States Solfataric source, Leirhnukur fissure, Iceland Thermal spring drainage in Yellowstone National Park, Italy Solfataric fields, Iceland

T. neuttrophilus

85

Hot spring, Iceland

T. uzoniensis

90

P. islandicum

100

P. organotrophum

102

P. aerophilum

100

T. pendens

85e90

Uzon caldera, Kamchatka peninsula Geothermal power plant, Iceland Solfataric fields, Iceland, Italy, and Azores Shallow marine boiling-water holes, Iischia, Italy Solfataric fields, Iceland

A. brierleyi

Thermoproteales

Thennoproteus

Pyrobaculum

Thermofilum

Huber et al. (1989b) Segerer et al. (1991) Segerer et al. (1986)

Fuchs et al. (1996) and Zillig et al. (1987) Brierley and Brierley (1973)

Zillig et al. (1981) and BonchOsmolovskaya et al. (1990) Fischer et al. (1983) and Stetter (1986) Bonch-Osmolovskaya et al. (1990) Huber et al. (1987) Huber et al. (1987) Volld et al. (1993) Zillig et al. (1983a)

Developments and Applications of Enzymes From Thermophilic Microorganisms

Table 3.1 Hypothermophile diversity.dcont’d

Desulfurococcales

Thermococcales

85

Solfataric fields, Iceland

D. amylolyticus

90e92

Staphylothermus Pyrolobus

D. mucosus S. marinus P. fumarli

85 92 106

Hyperthermus Thermosphaera Thermococcus

H. butylicus T. aggregans T. profundus

95e106 85 80

T. litoralis

85

T. celer

88

T. stetteri

75e95

T. chitonophagus

85

T. peptonophilus

85e90

T. fumicolans

85

T. hydrothermalis

85

Thermal springs, Kamchatka peninsula Acidic hot springs of Iceland Heated sea floor, Vulcan, Italy Deep-sea MHTV (3650 m), Mid-Atlantic Ridge Marine solfataric field, Azores Yellowstone, Obsidian pool MHTV (1400 m), MidOkinawa Trough, Japan Marine solfataras, Vulcan and Naples, Italy Shallow marine solfataric field, Vulcan, Italy Marine solfataric fields, Northern Kurils Deep-sea MHTV, Guaymas Mexico Deep-sea hydrothermal vent Ocean areas in the western Pacific Deep-sea hydrothermal vent in the North Fiji Basin Deep-sea MHTV, East Pacific Rise

Zillig et al. (1982) Zillig et al. (1983a) Bonch-Osmaolovskaya, (1989) Zillig et al. (1982) Fiala and Stetter (1986) Blöchl et al. (1997) Zillig et al. (1990) Huber et al. (1998a) Kobayashi et al. (1994) Neuner et al. (1990) Zillig et al. (1983b) Miroshnichenko et al. (1989) Huber et al. (1995) Gonzalez et al. (1995)

Godfroy et al. (1996) Godfroy et al. (1997) Continued

Biodiversity of thermotolerant microorganisms

D. mobilis

Desulfurococcus

35

36

Genus

Species

Topt.(8C)

Isolation/habitat

References

Pyrococcus

P. abyssi

96

Erauso et al. (1993)

P. woesei

100e103

P. furiosus

100

P. horikoshii

98

Archaeoglobus

A. fulgidus A. profundus

83 82

Fen oglobus

F. placidus

85

Methanobacteriales

Methanothermus

M. fervidus M. sociabilis

83 88

Methanopyrales

Methanopyrus

M. kandleri

98

Methanococcales

Methanococcus

M. thermolithotrophicus

65

M. jannaschii

85

M. infernus

85

M. igneus

88

Deep-sea MHTV, North Fiji Basin Marine Solfataras, Vulcan, Italy Marine Solfataric fields, Vulcan, Italy Okinawa Trough, western Pacific Heated sea floor, Vulcan, Italy Deep-sea MHTV, Guaymas, Mexico Shallow submarine hydrothermal system at Vulcan, Italy Icelandic hot spring Continental solfataras fields, Iceland Deep-sea MHTV, Guaymas Mexico Geothermally heated sea sediments close to Naples, Italy Deep-sea MHTV (2600 m), East Pacific Rise Deep-sea MHTV, MidAtlantic Ridge Shallow MHTV, Mid-Atlantic Ridge, north off Iceland

Order

Archaeoglobales

Zillig et al. (1987) Fiala and Stetter (1986) Gonzalez et al. (1998) Stetter (1988) Burggraf et al. (1990b) Hafenbradl et al. (1996)

Stetter et al. (1981) Lauerer et al. (1986) and Sako et al. (1996) Huber et al. (1989c); Kurr et al. (1991) Huber et al. (1982)

Jones et al. (1983) Jeanthon et al. (1998) Burggraf et al. (1990a)

Reproduced with permission Ebaid, R., Wang, H., Sha, C., Abomohra, A.E., Shao, W., 2019. Recent trends in hyperthermophilic enzymes production and future perspectives for biofuel industry: a critical review. J. Clean. Prod. 238, 117925.

Developments and Applications of Enzymes From Thermophilic Microorganisms

Table 3.1 Hypothermophile diversity.dcont’d

Biodiversity of thermotolerant microorganisms

extended to 121
C, 8
C higher than the earlier record holder. The hardy organism, named Strain 121, was found at a hydrothermal vent on the floor of the northeast Pacific Ocean. At a temperature higher than 110
C, amino acids and metabolites get highly unstable, and hydrophobic interactions grow weaker considerably (Jaenicke, 1998). ATP gets voluntarily hydrolyzed in an aqueous solution at temperatures lower than 140
C. Stetter (1996) described more than 29 genera, 70 species, and 10 orders of hyperthermophiles most of these are archaea. Aquificales and Thermotogales are the only bacteria; these are the deepest branches in the bacterial genealogy (the study of family origins and history), and so they represent noticeable importance in evolutionary studies (Achenbach-Richter et al., 1987). One of the most remarkable discoveries taken out from the Thermotoga maritima genome sequence is the abundant proof supporting lateral gene transfer between bacteria and archaea (Nelson et al., 1999). - 24% of the Thermotoga maritima open reading frames in comparison to 16% in Aquifex aeolicus encode proteins that are identical to archaeal than to bacterial proteins - These “archaea-like genes” are not evenly distributed among the biological groups - 81 of these genes are grouped in 15 4e20-kb regions, in which the gene order can be similar as in archaea - The G1C content is not homogenous in T. maritima genome sequence. Among the 50 regions having appreciably different G1C contents, 42 have “archaea-like” genes. The archaeal domain consists of two branches: Euryarchaeota and Crenarchaeota. A 16S rRNA obtained from a hyperthermophilic environment was sequenced which is unrelated to any other archaeal rRNA. This new rRNA species points to the presence of a third branch of the archaeal domain, the Korarcheota, which branches deeper into the archaeal tree than the Euryarchaeota and the Crenarchaeota (Barns et al., 1996). Hyperthermophilic microorganisms are represented by Euryarchaeota and Crenarchaeota, methodologically representing the deepest and shortest family of these two branches. Crenarchaeota include halophiles in addition to thermoacidophiles. In Euryarchaeota, methanogens have mesophilic relatives. Hyperthermophiles are the primary producers and decomposers of organic matter. All the hyperthermophilic primary producers are chemolithoautotrophs (sulfur reducers and oxidizers and methanogens) (Fischer et al., 1983; Lowe et al., 1993). Most hyperthermophilic microorganisms are either obligate or facultative chemolithotrophs, which means that they either oxidize sulfur with oxygen to produce sulfuric acid or reduce S0 with hydrogen to produce hydrogen sulfide (the anaerobes) (the aerobes). The order Sulfolobales contains acidophilic hyperthermophilic microorganisms. These are almost exclusively facultative or obligate aerobes that have been isolated from continental solfataras. Sulfolobus or Acidianus are two examples. All members of

37

38

Developments and Applications of Enzymes From Thermophilic Microorganisms

Table 3.2 Thermophilic and hyperthermophilic archaea isolated from thermal springs of Vulcano Island. Phylum

Class

Species

References

Crenarchaeota

Thermoprotei

Acidianus brierleyi

Simmons and Norris (2002) Segerer et al. (1986) Stetter et al. (1983) Fiala et al. (1986) Stetter (1986) Stetter (1988) Hafenbradl et al. (1996) Schmid et al. (1984)

Archaeoglobi Methanococci Euryarchaeota

Thermococci

Thermoplasmata

Acidianus infernus Pyrodictium occultum Staphylothermus marinus Thermodiscus maritimus Archeoglobus fulgidus Ferroglobus placidus Methanococcus aeolicus PL1/5H Palaeococcus helgesonii Pyrococcus furiosus Pyrococcus woesei Thermococcus acidaminovorans Thermococcus alcaliphilus Thermococcus celer Thermoplasma volcanium

Amend et al. (2003) Fiala and Stetter (1986) Zillig et al. (1987) Dirmeier et al. (1998) Keller et al. (1995) Zillig et al. (1983b) Segerer et al. (1988)

Gugliandolo, C., Maugeri, T.L., 2019. Phylogenetic diversity of archaea in shallow hydrothermal vents of Eolian Islands, Italy. Diversity 11 (9), 156. This chapter is distributed under the terms of the Creative Commons Attribution 4.0 International License.

the Thermotogales and the majority of the Thermococcales and Pyrococcales are able to grow independently of S0 because most heterotrophs are obligate sulfur reducers, and they do so by obtaining energy from fermentation (Table 3.1). Due to the very little organic matter content of the aquatic environment, carbon and energy are typically obtained by heterotrophic hyperthermophiles from a complex mixture of peptides that result from the breakdown of primary producers. Some species make use of polysaccharides like starch, glycogen, pectin, and chitin. The only species that has been found to use organic acids is Archeoglobus profundus. Table 3.2 shows thermophilic and hyperthermophilic archaea isolated from the thermal springs of Vulcano Island.

References Aanniz, T., Ouadghiri, M., Melloul, M., Swings, J., Elfahime, E., Ibijbijen, J., Ismaili, M., Amar, M., 2015. Thermophilic bacteria in Moroccan hot springs, salt marshes and desert soils. Braz. J. Microbiol. 46 (2), 443e453. Abootalebi, S.N., Saeed, A., Gholami, A., Mohkam, M., Kazemi, A., Nezafat, N., Mousavi, S.M., Hashemi, S.A., Shorafa, E., 2020. Screening, characterization and production of thermostable alphaamylase produced by a novel thermophilic Bacillus megaterium isolated from pediatric intensive care unit. J. Environ. Treat. Tech. 8 (3), 952e960.

Biodiversity of thermotolerant microorganisms

Achenbach-Richter, L., Gupta, R., Stetter, K.O., Woese, C.R., 1987. Were the original eubacteria thermophiles? Syst. Appl. Microbiol. 9, 34e39. Akita, H., Kimura, Z.-I., Matsushika, A., 2017. Complete genome sequence of Ureibacillus thermosphaericus A1, a thermophilic bacillus ısolated from compost. Genome Announc. 5 (38), e00910ee917. Al-Johani, N.B., Al-Seeni, M.N., Ahmed, Y.M., 2016. Optimizatıon of alkaline a-amylase production by thermophilic Bacillus subtilis. Afr. J. Tradit. Complement. Altern. Med. 14 (1), 288e301. Amend, J.P., Meyer-Dombard, D.R., Sheth, S.N., Zolotova, N., Amend, A.C., 2003. Palaeococcus helgesonii sp. nov., a facultatively anaerobic, hyperthermophilic archaeon from a geothermal well on Vulcano Island, Italy. Arch. Microbiol. 179, 394e401. Barns, S.M., Fundyga, R.E., Jeffries, M.W., Pace, N.R., 1994. Remarkable archaeal diversity detected in a Yellowstone National Park hot spring environment. Proc. Natl. Acad. Sci. USA 91 (5), 1609e1613. Barns, S.M., Delwiche, C.F., Palmer, J.D., Pace, N.R., 1996. Perspectives on archaeal diversity. thermophily and monophyly from environmental rRNA sequences. Proc. Natl. Acad. Sci. U.S.A. 93, 9188e9193. Belkin, S., Wirsen, C.O., Jannasch, H.W., 1986. A new sulfur-reducing, extremely thermophilic eubacterium from a submarine thermal vent. Appl. Environ. Microbiol. 51 (6), 1180e1185. Bernardo, S.P.C., Rosana, A.R.R., de Souza, A.N., Chiorean, S., Martins, M.L.L., Vederas, J.C., 2020. Draft genome sequence of the thermophilic bacterium Bacillus licheniformis SMIA-2, an antimicrobialand thermostable enzyme-producing ısolate from Brazilian soil. Microbiol. Resourc. Announc. 9 (17), e00106ee120. Blöchl, E., Rachel, R., Burggraf, S., Hafenbradl, D., Jannasch, H.W., Stetter, K.O., 1997. Pyrolobus fumarii, gen. and sp. nov, represents a novel group of archaea, extending the upper temperature limit for life to 113
C. Extremophiles 1 (1), 14e21. Bonch-Osmaolovskaya, E., 1989. Characteristics of Desulfurococcus amylolyticus n. sp.-a new extremely thermophilic archaebacterium isolated from thermal springs of Kamchatka and Kunashir island. Mikrobiologiya 57, 78e85. Bonch-Osmolovskaya, E., Miroshnichenko, M., Kostrikina, N., Chernych, N., Zavarzin, G., 1990. Thermoproteus uzoniensis sp. nov, a new extremely thermophilic archaebacterium from Kamchatka continental hot springs. Arch. Microbiol. 154 (6), 556e559. Bouzas, T.M., Barros-Velazquez, J., Villa, T.G., 2006. Industrial applications of hyperthermophilic enzymes: a review. Protein Pept. Lett. 13, 645e651. Brierley, C.L., Brierley, J.A., 1973. A chemoautotrophic and thermophilic microorganism isolated from an acid hot spring. Can. J. Microbiol. 19 (2), 183e188. Brock, T.D., 1985. Life at high temperatures. Science 230 (4722), 132e138. Brock, T.D., Brock, K.M., Belly, R.T., Weiss, R.L., 1972. Sulfolobus: a new genus of sulfur oxidizing bacteria living at low pH and high temperature. Arch. Mikrobiol. 84 (1), 54e68. Burggraf, S., Fricke, H., Neuner, A., Kristjansson, J., Rouvier, P., Mandelco, L., Woese, C.R., Stetter, K.O., 1990a. Methanococcus igneus sp. nov, a novel hyperthermophilic methanogen from a shallow submarine hydrothermal system. Syst. Appl. Microbiol. 13 (3), 263e269. Burggraf, S., Jannasch, H.W., Nicolaus, B., Stetter, K.O., 1990b. Archaeoglobus profundus sp. nov, represents a new species within the sulfate-reducing archaebacteria. Syst. Appl. Microbiol. 13 (1), 24e28. Dang, T.C.H., Nguyen, D.T., Thai, H., Nguyen, T.C., Tran, T.T.H., Le, V.H., Van Huynh, N., Tran, X.B., Pham, T.P.T., Nguyen, T.G., Nguyen, Q.T., 2018. Plastic degradation by thermophilic Bacillus sp. BCBT21 isolated from composting agricultural residual in Vietnam. Adv. Nat. Sci. Nanosci. Nanotechnol. 9 (1), 015014. Dirmeier, R., Keller, M., Hafenbradl, D., Braun, F.J., Rachel, R., Burggraf, S., Stetter, K.O., 1998. Thermococcus acidaminovorans sp. nov., a new hyperthermophilic alkalophilic archaeon growing on amino acids. Extremophiles 2, 109e114. Ebaid, R., Wang, H., Sha, C., Abomohra, A.E., Shao, W., 2019. Recent trends in hyperthermophilic enzymes production and future perspectives for biofuel industry: a critical review. J. Clean. Prod. 238, 117925. Eichler, J., 2001. Biotechnological uses of archaeal enzymes. Biotechnol. Adv. 19, 261e278.

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Erauso, G., Reysenbach, A.L., Godfroy, A., Meunier, J.R., Crump, B., Partensky, F., Baross, J.A., Marteinsson, V., Barbier, G., Pace, N.R., Prieur, D., 1993. Pyrococcus abyssi sp. nov., a new hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent. Arch. Microbiol. 160 (5), 338e349. Eriksen, N.T., 2008. Production of phycocyaninda pigment with applications in biology, biotechnology, foods and medicine. Appl. Microbiol. Biotechnol. 80 (1), 1e14. Fakruddin, M., 2017. Thermostable enzymes and their industrial application: a review. Discovery 53 (254), 147. Falcicchio, P., Levisson, M., Kengen, S.W., Koutsopoulos, S., van der Oost, J., 2021. (Hyper) thermophilic enzymes: production and purification. In: Labrou, N.E. (Ed.), Protein Downstream Processing. Springer, Berlin, pp. 469e478. Fiala, G., Stetter, K.O., Jannasch, H.W., Langworthy, T.A., Madon, J., 1986. Staphylothermus marinus sp. nov. represent a novel genus of extremely thermophilic submarine heterotrophic archaebacteria growing up to 98
C. Syst. Appl. Microbiol. 8, 106e113. Fiala, G., Stetter, K.O., 1986. Pyrococcus furiosus sp. nov. represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100
C. Arch. Microbiol. 145, 56e61. Fischer, F., Zillig, W., Stetter, K.O., Schreiber, W.G., 1983. Chemolithoautotrophic metabolism of anaerobic extremely thermophilic archaebacteria. Nature 301, 511e513. Fuchs, T., Huber, H., Burggraf, S., Stetter, K.O., 1996. 16S rDNA-based phylogeny of the archaeal order Sulfolobales and reclassification of Desulfurolobus ambivalens as Acidianus ambivalens comb. nov. Syst. Appl. Microbiol. 19 (1), 56e60. Fuchs, T., Huber, H., Teiner, K., Burggraf, S., Stetter, K.O., 1995. Metallosphaera prunae, sp. nov, a novel metal-mobilizing, thermoacidophilic archaeum, isolated from a uranium mine in Germany. Syst. Appl. Microbiol. 18 (4), 560e566. Fujiwara, S., 2002. Extremophiles: developments of their special functions and potential resources. J. Biosci. Bioeng. 94, 518e525. F€ utterer, O., Angelov, A., Liesegang, H., Gottschalk, G., Schleper, C., Schepers, B., Dock, C., Antranikian, G., Liebl, W., 2004. Genome sequence of Picrophilus torridus and its implications for life around pH 0. Proc. Natl. Acad. Sci. U. S. A. 101 (24), 9091e9096. Giaveno, M.A., Urbieta, M.S., Ulloa, J.R., Toril, EG.l, Donati, E.R., 2013. Physiologic versatility and growth flexibility as the main characteristics of a novel thermoacidophilic Acidianus strain isolated from Copahue geothermal area in Argentina. Microb. Ecol. 65, 336e346. Godfroy, A., Lesongeur, F., Raguenes, G., Querellou, J., Antoine, E., Meunier, J.R., Guezennec, J., Barbier, G., 1997. Thermococcus hydrothermalis sp. nov, a new hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent. Int. J. Syst. Evol. Microbiol. 47 (3), 622e626. Godfroy, A., Meunier, J.R., Guezennec, J., Lesongeur, F., Raguenes, G., Rimbault, A., Barbier, G., 1996. Thermococcus fumicolans sp. nov, a new hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent in the North Fiji Basin. Int. J. Syst. Evol. Microbiol. 46 (4), 1113e1119. Gonzalez, J.M., Kato, C., Horikoshi, K., 1995. Thermococcus peptonophilus sp. nov, a fast growing, extremely thermophilic archaebacterium isolated from deep-sea hydrothermal vents. Arch. Microbiol. 164 (3), 159e164. Gonzalez, J.M., Masuchi, Y., Robb, F.T., Ammerman, J.W., Maeder, D.L., Yanagibayashi, M., Tamaoka, J., Kato, C., 1998. Pyrococcus horikoshii sp. nov, a hyperthermophilic archaeon isolated from a hydrothermal vent at the Okinawa Trough. Extremophiles 2 (2), 123e130. Grogan, D., Palm, P., Zillig, W., 1990. Isolate B12, which harbours a virus-like element, represents a new species of the archaebacterial genus Sulfolobus, Sulfolobus shibatae, sp. nov. Arch. Microbiol. 154 (6), 594e599. Gugliandolo, C., Maugeri, T.L., 2019. Phylogenetic diversity of archaea in shallow hydrothermal vents of Eolian Islands, Italy. Diversity 11 (9), 156. Hafenbradl, D., Keller, M., Dirmeier, R., Rachel, R., Roßnagel, P., Burggraf, S., Huber, H., Stetter, K.O., 1996. Ferroglobus placidus gen. nov, sp. nov, a novel hyperthermophilic archaeum that oxidizes Fe2þ at neutral pH under anoxic conditions. Arch. Microbiol. 166 (5), 308e314.

Biodiversity of thermotolerant microorganisms

Hogendoorn, C., Picone, N., van Hout, F., Vijverberg, S., Poghosyan, L., van Alen, T.A., Frank, J., Pol, A., Gagliano, A.L., Jetten, M.S.M., 2021. Draft genome of a novel methanotrophic Methylobacter sp. from the volcanic soils of Pantelleria Island. Antonie van Leeuwenhoek 114 (3), 313e324. Huber, H., Thomm, M., König, H., Thies, G., Stetter, K.O., 1982. Methanococcus thermolithotrophicus, a novel thermophilic lithotrophic methanogen. Arch. Microbiol. 132 (1), 47e50. Huber, R., Langworthy, T.A., König, H., Thomm, M., Woese, C.R., Sleytr, U.B., Stetter, K.O., 1986. Thermotoga maritima sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 90
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C. Arch. Microbiol. 149 (2), 95e101. Huber, R., Woese, C., Langworthy, T.A., Fricke, H., Stetter, K.O., 1989a. Thermosipho africanus gen. nov, represents a new genus of thermophilic eubacteria within the “Thermotogales”. Syst. Appl. Microbiol. 12 (1), 32e37. Huber, G., Spinnler, C., Gambacorta, A., Stetter, K.O., 1989b. Metallosphaera sedula gen. and sp. nov. represents a new genus of aerobic, metal-mobilizing, thermoacidophilic archaebacteria. Syst. Appl. Microbiol. 12, 38e47. Huber, R., Kurr, M., Jannasch, H., Stetter, K., 1989c. A novel group of abyssal methanogenic archaebacteria (Methanopyrus) growing at 110
C. Nature 342 (6251), 833. Huber, R., Woese, C.R., Langworthy, T.A., Kristjansson, J.K., Stetter, K.O., 1990. Fervidobacterium islandicum sp. nov, a new extremely thermophilic eubacterium belonging to the “Thermotogales”. Arch. Microbiol. 154 (2), 105e111. Huber, G., Stetter, K.O., 1991. Sulfolobus metallicus, sp. nov, a novel strictly chemolithoautotrophic thermophilic archaeal species of metal-mobilizers. Syst. Appl. Microbiol. 14 (4), 372e378. Huber, R., Wilharm, T., Huber, D., Trincone, A., Burggraf, S., König, H., Reinhard, R., Rockinger, I., Fricke, H., Stetter, K.O., 1992. Aquifex pyrophilus gen. nov. sp. nov, represents a novel group of marine hyperthermophilic hydrogen-oxidizing bacteria. Syst. Appl. Microbiol. 15 (3), 340e351. Huber, R., Stöhr, J., Hohenhaus, S., Rachel, R., Burggraf, S., Jannasch, H.W., Stetter, K.O., 1995. Thermococcus chitonophagus sp. nov, a novel, chitin-degrading, hyperthermophilic archaeum from a deep-sea hydrothermal vent environment. Arch. Microbiol. 164 (4), 255e264. Huber, R., Dyba, D., Huber, H., Burggraf, S., Rachel, R., 1998a. Sulfur-inhibited Thermosphaera aggregans sp. nov, a new genus of hyperthermophilic archaea isolated after its prediction from environmentally derived 16S rRNA sequences. Int. J. Syst. Evol. Microbiol. 48 (1), 31e38. Jaenicke, R., 1998. What ultrastable globular proteins teach us about protein stabilization. Biochemistry 63, 312e321. Jannasch, H.W., Huber, R., Belkin, S., Stetter, K.O., 1988. Thermotoga neapolitana sp. nov. of the extremely thermophilic, eubacterial genus Thermotoga. Arch. Microbiol. 150 (1), 103e104. Jeanthon, C., L’haridon, S., Reysenbach, A., Vernet, M., Messner, P., Sleytr, U., Prieur, D., 1998. Methanococcus infernus sp. nov, a novel hyperthermophilic lithotrophic methanogen isolated from a deep-sea hydrothermal vent. Int. J. Syst. Evol. Microbiol. 48 (3), 913e919. Jones, W., Leigh, J., Mayer, F., Woese, C., Wolfe, R., 1983. Methanococcus jannaschii sp. nov, an extremely thermophilic methanogen from a submarine hydrothermal vent. Arch. Microbiol. 136 (4), 254e261. Keller, M., Braun, F.J., Dirmeier, R., Hafenbradl, D., Burggraf, S., Rachel, R., 1995. Thermococcus alcaliphilus sp. nov., a new hyperthermophilic archaeum growing on polysulfide at alkaline pH. Arch. Microbiol. 164, 390e395. Khan, M., Sathya, T.A., 2018. Extremozymes from metagenome: potential applications in food processing. Crit. Rev. Food Sci. Nutr. 58 (12), 2017e2025. Kobayashi, T., Kwak, Y.S., Akiba, T., Kudo, T., Horikoshi, K., 1994. Thermococcus profundus sp. nov, a new hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent. Syst. Appl. Microbiol. 17 (2), 232e236. Kurr, M., Huber, R., König, H., Jannasch, H.W., Fricke, H., Trincone, A., Kristjansson, J.K., Stetter, K.O., 1991. Methanopyrus kandleri, gen. and sp. nov. represents a novel group of hyperthermophilic methanogens, growing at 110
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Ruepp, A., Graml, W., Santos-Martinez, M.-L., Koretke, K.K., Volker, C., Mewes, H.W., Frishman, D., Stocker, S., Lupas, A.N., Baumeister, W., 2000. The genome sequence of the thermoacidophilic scavenger Thermoplasma acidophilum. 1184. Nature 407, 508e513. Saeed, A.M., Sayed, H.A.E., El-Shatoury, E.H., 2020. Optimizing the reduction of molybdate by two novel thermophilic bacilli isolated from Sinai, Egypt. Curr. Microbiol. 77 (5), 786e794. Safitri, R., Kusumawardhani, D.P., Annisa, A., Partasasmita, R., Asharina, S., Maskoen, A.M., 2020. Characterization and identification of three thermophilic Bacillus strain isolated from Domas Crater, Mt. Tangkuban Perahu. Indonesia. Biodiver. J. Biol. Divers. Sako, Y., Takai, K., Uchida, A., Ishida, Y., 1996. Purification and characterization of phosphoenolpyruvate carboxylase from the hyperthermophilic archaeon Methanothermus sociabilis. FEBS Lett. 392 (2), 148e152. Saleh, F., Hussain, A., Younis, T., Ali, S., Rashid, M., Ali, A., Mustafa, G., Jabeen, F., Al-Surhanee, A.A., Alnoman, M.M., Qamer, S., 2020. Comparative growth potential of thermophilic amylolytic Bacillus sp. on unconventional media food wastes and its industrial application. Saudi J. Biol. Sci. 27 (12), 3499e3504. Salem, R.B., Abbassi, M.S., Cayol, J.-L., Bourouis, A., Mahrouki, S., Fardeau, M.-L., Belhadj, O., 2020. Thermophilic Bacillus licheniformis RBS 5 isolated from hot Tunisian spring coproducing alkaline and thermostable a-amylase and protease enzymes. J. Microbiol. Biotechnol. Food Sci. 5 (6), 557e562. Satyanarayana, T., Raghukumar, C., Shivaji, S., 2005. Extremophilic microbes: diversity and perspectives. Curr. Sci. 89, 78e90. Schmid, K., Thomm, M., Laminet, A., Laue, F.G., Kessler, C., Stetter, K.O., Schmitt, R., 1984. Three new restriction endonucleases MaeI, MaeII and MaeIII from Methanococcus aeolicus. Nucleic Acids Res. 12, 2619e2628. Segerer, A., Langworthy, T.A., Stetter, K.O., 1988. Thermoplasma acidophilum and Thermoplasma volcanium sp. nov. from solfatara fields. Syst. Appl. Microbiol. 10, 161e171. Segerer, A., Neuner, A., Kristjansson, J.K., Stetter, K.O., 1986. Acidianus infernus gen. nov, sp. nov, and Acidianus brierleyi comb. nov.: facultatively aerobic, extremely acidophilic thermophilic sulfur-metabolizing archaebacteria. Int. J. Syst. Evol. Microbiol. 36 (4), 559e564. Segerer, A.H., Trincone, A., Gahrtz, M., Stetter, K.O., 1991. Stygiolobus azoricus gen. nov, sp. nov. represents a novel genus of anaerobic, extremely thermoacidophilic archaebacteria of the order Sulfolobales. Int. J. Syst. Evol. Microbiol. 41 (4), 495e501. Shen, P., Niu, D., Liu, X., Tian, K., Kugenthiren, P., Singh, S., Wang, Z., 2021. Overexpression of thermophilic a-amylase in Bacillus licheniformis using a high efficiency chromosomal integration and amplification strategy. Microb. Cell Factories. Short, J.M., 1998. Directed Evolution of Thermophilic Enzymes. US5830696A. Simmons, S., Norris, R., 2002. Acidophiles of saline water at thermal vents of Vulcano, Italy. Extremophiles 6, 201e207. Stetter, K.O., 1996. Hyperthermophilic procaryotes. FEMS Microbiol. Rev. 18, 149e158. Stetter, K.O., 1986. Diversity of extremely thermophilic archaebacteria. In: Brock, T. (Ed.), The Thermophiles: General, Molecular and Applied Microbiology. Weiley, New York, pp. 39e74. Stetter, K.O., 1988. Archaeoglobus fulgidus gen. nov., sp. nov.: a new taxon of extremely thermophilic archaebacteria. Syst. Appl. Microbiol. 10, 172e173. Stetter, K.O., Konig, H., Stackebrandt, E., 1983. Pyrodictium gen. nov., a new genus of submarine disc shaped sulphur reducing archaebacteria growing optimally at 105
C. Syst. Appl. Microbiol. 4, 535e551. Stetter, K.O., Thomm, M., Winter, J., Wildgruber, G., Huber, H., Zillig, W., Jane-Covic, D., König, H., Palm, P., Wunderl, S., 1981. Methanothermus fervidus, sp. nov, a novel extremely thermophilic methanogen isolated from an Icelandic hot spring. Zentralblatt f€ ur Bakteriologie Mikrobiologie und Hygiene: I. € Abt. Originale C: Allgemeine, Angewandte und Okologische Mikrobiologie 2 (2), 166e178. Tango, M.S.A., Islam, M.R., 2002. Potential of extremophiles for biotechnological and petroleum applications. Energy Sources 24 (6), 543e559.

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Thakur, A., Sharma, K., Mutreja, R., Goyal, A., 2021. Thermostable enzymes from Clostridium thermocellum. In: Thatoi, H., Mohapatra, S., Das, S.K. (Eds.), Bioprospecting of Enzymes in Industry, Healthcare and Sustainable. Springer, Berlin, pp. 251e267. Tsotetsi, L., Prenaven, R., Modise, S., Monapathi, M., 2020. Isolation and identification of xylanase producing thermophilic bacteria from compost piles and optimization of xylanase production. J. Biotech Res. 11, 122e132. Ulucay, O., Gormez, A., Ozic, C., 2022. Identification, characterization and hydrolase producing performance of thermophilic bacteria: geothermal hot springs in the Eastern and Southeastern Anatolia Regions of Turkey. Antonie Leeuwenhoek 115 (2), 253e270. Urbieta, M.S., Rascovan, N., Castro, C., Revale, S., Giaveno, M.A., Vazquez, M., Donati, E.R., 2014a. Draft genome sequence of the novel thermoacidophilic archaeon Acidianus copahuensis strain ALE1, isolated from the Copahue volcanic area in Neuquen, Argentina. Genome Announc. 2, e00259-4. Urbieta, M.S., Toril, E.G., Giaveno, M.A., Bazan, A.A., Donati, E.R., 2014b. Archaeal and bacterial diversity in five different hydrothermal ponds in the Copahue region in Argentina. Syst. Appl. Microbiol. 37, 429e441. Urbieta, M.S., Donati, E.R., Chan, K.G., Shahar, S., Sin, L.L., Goh, K.M., 2015. Thermophiles in the genomic era: biodiversity, science, and applications. Biotechnol. Adv. Nov 1 33 (6 Pt 1), 633e647. Verma, J.P., Jaiswal, D.K., Krishna, R., Prakash, S., Yadav, J., Singh, V., 2018. Characterization and screening of thermophilic bacillus strains for developing plant growth promoting consortium from hot spring of Leh and Ladakh Region of India. Front. Microbiol. 9, 1293. Vieille, C., Zeikus, G.J., 2001. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65 (1), 1e43. Vieille, C., Burdette, D.S., Zeikus, J.G., 1996. Thermozymes. Biotechnol. Annu. Rev. 2, le8. Völkl, P., Huber, R., Drobner, E., Rachel, R., Burggraf, S., Trincone, A., Stetter, K.O., 1993. Pyrobaculum aerophilum sp. nov, a novel nitrate-reducing hyperthermophilic archaeum. Appl. Environ. Microbiol. 59 (9), 2918e2926. Wang, L., Tang, Y., Wang, S., Liu, R.L., Liu, M.Z., Zhang, Y., Liang, F.L., Feng, L., 2006. Isolation and characterization of a novel thermophilic Bacillus strain degrading long-chain n-alkanes. Extremophiles 10 (4), 347e356. Windberger, E., Huber, R., Trincone, A., Fricke, H., Stetter, K.O., 1989. Thermotoga thermarum sp. nov. and Thermotoga neapolitana occurring in African continental solfataric springs. Arch. Microbiol. 151 (6), 506e512. Yanmis, D., Baltaci, M.O., Gulluce, M., Adiguzel, A., 2015. Identification of thermophilic strains from geothermal areas in Turkey by using conventional and molecular techniques. Res. J. Biotechnol. 10 (1), 39e45. Zakzeski, J., Bruijnincx, P.C., Jongerius, A.L., Weckhuysen, B.M., 2010. The catalytic valorization of lignin for the production of renewable chemicals. Chem. Rev. 110 (6), 3552e3599. Zillig, W., Stetter, K.O., Wunderl, S., Schulz, W., Priess, H., Scholz, I., 1980. The Sulfolobus-“Caldariella” group: taxonomy on the basis of the structure of DNA-dependent RNA polymerases. Arch. Microbiol. 125 (3), 259e269. Zillig, W., Stetter, K., Sch€afer, W., Janekovic, D., Wunderl, S., Holz, I., Palm, P., 1981. Thermoproteales: a novel type of extremely thermoacidophilic anaerobic archaebacteria isolated from Icelandic solfataras. Zentralblatt f€ ur Bakteriologie Mikrobiologie und Hygiene: I. Abt. Originale C: Allgemeine, angewandte und ökologische Mikrobiologie 2 (3), 205e227. Zillig, W., Stetter, K., Prangishvilli, D., Sch€afer, W., Wunderl, S., Janekovic, D., Holz, I., Palm, P., 1982. Desulfurococcaceae, the second family of the extremely thermophilic, anaerobic, sulfur-respiring Thermoproteales. Zentralblatt f€ ur Bakteriologie Mikrobiologie und Hygiene: I. Abt. Originale C: All€ gemeine, Angewandte Und Okologische Mikrobiologie 3 (2), 304e317. Zillig, W., Gierl, A., Schreiber, G., Wunderl, S., Janekovic, D., Stetter, K., Klenk, H., 1983a. The archaebacterium Thermofilum pendens represents, a novel genus of the thermophilic, anaerobic sulfur respiring Thermoproteales. Syst. Appl. Microbiol. 4 (1), 79e87.

Biodiversity of thermotolerant microorganisms

Zillig, W., Holz, I., Janekovic, D., Sch€afer, W., Reiter, W., 1983b. The archaebacterium Thermococcus celer represents, a novel genus within the thermophilic branch of the archaebacteria. Syst. Appl. Microbiol. 4 (1), 88e94. Zillig, W., Holz, I., Klenk, H.P., Trent, J., Wunderl, S., Janekovic, D., Imsel, E., Haas, B., 1987. Pyrococcus woesei, sp. nov, an ultra-thermophilic marine archaebacterium, representing a novel order, Thermococcales. Syst. Appl. Microbiol. 9 (1e2), 62e70. Zillig, W., Holz, I., Janekovic, D., Klenk, H., Imsel, E., Trent, J., Wunderl, S., Forjaz, V., Coutinho, R., Ferreira, T., 1990. Hyperthermus butylicus, a hyperthermophilic sulfur reducing archaebacterium that ferments peptides. J. Bacteriol. 172 (7), 3959e3965.

Relevant websites https://worldwidescience.org/. https://bio.libretexts.org/. https://microbewiki.kenyon.edu/.

Further reading Huber, R., Eder, W., Heldwein, S., Wanner, G., Huber, H., Rachel, R., Stetter, K.O., 1998b. Thermocrinis ruber gen. nov, sp. nov, a pink-filament-forming hyperthermophilic bacterium isolated from Yellowstone National Park. Appl. Environ. Microbiol. 64 (10), 3576e3583. Ravot, G., Magot, M., Fardeau, M.L., Patel, B., Prensier, G., Egan, A., Garcia, J.L., Ollivier, B., 1995. Thermotoga elfii sp. nov, a novel thermophilic bacterium from an African oil producing well. Int. J. Syst. Evol. Microbiol. 45 (2), 308e314.

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CHAPTER 4

Properties of thermophilic/ hyperthermophilic enzymes 4.1 Enzymes as biocatalyst Enzymes are highly efficient catalysts. These biocatalysts carry out chemical reactions at a very high rate under mild reaction conditions with regard to pressure, pH, and temperature. The use of enzymes in laboratory and industry is the result of higher substrate specificity, higher activity in aqueous media, and product selectivity (Woodley, 2008; Straathof et al., 2002; Pollard and Woodley, 2007; Schmid et al., 2001). These advantages have been shown to translate into reduced operating costs when they are effectively used as biocatalysts in chemical processes. But, few enzymes have been chosen through normal evolution for carrying out physiological tasks under very severe conditions and perform optimally at pH, temperature, salinity, and in a solvent environment that may be relatively far from their physiological environments (Cowan et al., 2010; Cowan and Fernandez-Lafuente, 2011; Egorova and Antranikian, 2005). For doing well in the industry, biochemical catalysts should be firm and functional under conditions that fall well outside the typical working ranges (Durand et al., 2007; Sheldon 2005; Cantone et al., 2007). In general, normal enzymes do not meet all the criteria of an industrial biochemical catalyst and need enhancement of one or more properties before application (Schoemaker et al., 2003; Meyer, 2006; Polizzi et al., 2007). This type of improvement can be achieved using diverse tools. For instance, the metagenomic genetic discovery has made thermophilic enzymes promptly reachable, even when the parent microbes could not be grown (Meiring et al., 2011). The fast cloning and overexpression of target enzyme genes in appropriate hosts have now become a regular and quick technique (Samuelson 2011; Smith 1996). It has been possible to maneuver almost any enzyme by directed evolution techniques and site-directed mutagenesis (Jackel et al., 2008; Reetz, 2007; Schmidt et al., 2009; Cowan and Fernandez-Lafuente 2011; Kurtovic and Mannervik, 2009). Cowan and Fernandez-Lafuente (2011) comprehensively reviewed the literature concerning chemical modification and immobilization of dissimilar types of the enzyme from thermophilic microorganisms, with stress on the chemistries involved and their implication for the alteration of the functional properties of the enzyme. New immobilization supports have been designed, to develop more efficient immobilization techniques and improved bioreactors.

Developments and Applications of Enzymes From Thermophilic Microorganisms ISBN 978-0-443-19197-8, https://doi.org/10.1016/B978-0-443-19197-8.00003-7

© 2023 Elsevier Inc. All rights reserved.

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Developments and Applications of Enzymes From Thermophilic Microorganisms

The various methods of enhancing enzyme performancedchemical versus geneticd have traditionally been applied as nearly independent tactics. However, combining various approaches has demonstrated a synergistic effect in which, for instance, the properties of genetically altered enzymes may be enhanced further through immobilization or chemical modification, whereas chemically altered enzymes may be further stabilized through immobilization. There are numerous other instances of the coordinated use of various methods for enhancing the performance of the enzyme. The issue of enzyme stability has frequently been addressed by the suggestion of using enzymes from thermophilic microorganisms. Whereas the intracellular enzymes of other extremophiles, such as acidophilic and alkalophilic bacteria, may not be exposed to the harsh conditions of the external environment, thermophiles are in perfect thermal equilibrium with their microenvironment. It is essential from an evolutionary point of view that these enzymes are adequately stable at room temperature for supporting biological function. Even these stable enzymes still have some room for improvement in their properties (Baker-Austin and Dopson, 2007; van de Vossenberg et al., 1998; Olsson et al., 2003; Cowan et al., 2010; Demirjian et al., 2001; Hough and Danson, 1999; Cowan and Fernandez-Lafuente, 2011).

4.2 Properties of thermophilic/hyperthermophilic enzymes Generally, enzymes from thermophiles/hyperthermophiles perform perfectly at or closer to the optimum growth temperature of the microorganism. Moderate thermophiles are optimally active at 50e60 C, extreme thermophiles show optimal activity at 60e80 C while hyperthermophiles show optimal activity at 80e120 C. Generally, intracellular enzymes follow this “rule” more accurately, whereas extracellular enzymes are found to be stable at temperatures very much higher than the optimal growth temperature (Segerer et al., 1993). All enzymes can be classified according to their Tm (melting temperature), which is the temperature at which a protein undergoes reversible (or irreversible) unfolding. Circular dichroism or intrinsic fluorescence monitoring are spectral techniques that can be used to visualize such significant conformational changes. In practice, however, the most common determinant of protein stability is loss of function, which for enzymes can be easily measured by loss of catalytic activity, leading to half-life and temperature inactivation (Tinact) (Tinact is the rate constant of enzyme inactivation under certain circumstances). As the conformational changes that lead to the catalytic function being inactivated may be much smaller as compared to those involved in full protein unfolding, the correlation between Tm and Tinact is not exact. Understanding the molecular mechanisms of protein stability is one of the most active areas of thermophilic research. Several researchers have examined this field. Many studies, including site-directed and random

Properties of thermophilic/hyperthermophilic enzymes

mutagenesis, comparative analysis of structural analogs from different thermal groups, and other studies, show that the required stability of proteins is established by a variety of subtle molecular mechanisms. The majority of these processes take place intramolecularly and are not always obvious or predictable. However, the majority of these subtle molecular mechanisms work by reducing conformational mobility and boosting the complement of noncovalent intracellular cross-linking. Although protein engineering studies have shown that these two processes can be decoupled, it is generally accepted that decreased conformational mobility is associated with decreased enzymatic activity (Cowan, 1997, 1999; Razvi and Scholtz, 2006; Van Mierlo and Steensma 2000; Scandurra et al., 1998; Daniel and Cowan, 2000; Ladenstein and Antranikian, 1998; Daniel et al., 2008; Lebbink et al., 1999; Cowan and Fernandez-Lafuente, 2011; Walters et al., 2011; Tian et al., 2010). Thermostability and optimal performance at higher temperatures are the essential properties of the enzymes obtained from hyperthermophiles. As most of the proteins are not active at higher temperatures, the mechanism of the thermal stability of hyperthermophilic enzymes is attracting the interest of researchers from the time when they were first discovered. The analysis of their amino acid sequence showed nothing remarkable. The sequences from homologous mesophilic and hyperthermophilic enzymes were found to be highly similar. They show the same catalytic mechanisms and their threedimensional structures are superimposable (Vieille and Zeikus, 2001). However, the information that these enzymes stay thermostable once expressed in mesophilic organisms shows that thermoresistance exists in the genetic sequence. It has been observed that thermophilic enzymes have a tendency to have hydrophobic cores because of a different folding with respect to mesophilic host, which most likely imparts them greater stability (Kirino et al., 1994). So, the simple fact of substituting one amino acid with another one influencing the folding is sufficient to make protein thermostable. Although no specific rules for thermostability or generalizations can be obtained from a methodical analysis of the sequences of homologous thermophilic and mesophilic proteins, some hydrophobic substitutions in the protein core studied by site-specific or directed mutagenesis showed improved thermostability (Hough and Danson, 1999). A broad array of genes from hyperthermophilic microorganisms have been effectively cloned and expressed in mesophilic hosts. Therefore, most archaeal genes cloned in Escherichia coli have been effectively expressed under the control of strong promoters like pTac, pLac, or the T7 RNA polymerase promoter, because of the significant differentiation between bacterial and archaeal transcription systems (Vieille and Zeikus, 2001). From hyperthermophilic archaea, a small number of genes have been also effectively expressed in yeasts which harbor transcription systems more strongly related to theirs (D’Auria et al., 1996). Of all the hyperthermophilic proteins expressed till now in E. coli, less than 10% show stabilities and properties nonidentical from those of the native

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Developments and Applications of Enzymes From Thermophilic Microorganisms

enzymes (Vieille and Zeikus, 2001). Such enzymes might need posttranslational modifications or molecular chaperones for obtaining their stable folded state. Thermophilic and hyperthermophilic microorganisms have a special type of proteins, called “chaperonins.” These proteins are thermoresistant and resist denaturation. These types of proteins help other proteins to revert to their original structure and restore function after denaturation (Everly and Alberto, 2000). Aromatic interactions, hydrogen bonds, disulfide bonds, etc. contribute to protein stability at higher temperatures (Vieille and Zeikus, 2001). But, there is a significant dissimilarity between the inference reached with different types of proteins, and so enough proof for a general rule for the structural basis of stability is not available. As several regular enzymatic reactions are conducted at higher temperatures, thermostable enzymes are attracting a lot of attention in industry and biotechnology in recent times. Their enhanced stability with respect to mesophilic enzymes makes them more appropriate for severe industrial processes. Furthermore, their thermostability is generally linked with a greater resistance to chemical denaturants normally utilized in several industrial processes. When the enzyme reactions are conducted at higher temperatures enhanced reaction rates and process yields are achieved. This can be attributed to the following (Haki and Raksht, 2003): - Reduction in viscosity - Enhancement in the diffusion coefficient of substrates - Enhancement in the solubility of substrates and products - Suitable equilibrium displacement in endothermal reactions Furthermore, contamination by common mesophilic species reduces with an increase in reaction temperature. Lastly, a major advantage from the perspective of enzyme production and purification is that thermophilic and hyperthermophilic enzymes, once expressed in mesophilic organisms, can be easily purified by heat treatment (Vieille and Zeikus, 2001). Several enzymes from hyperthermophiles have been characterized. The characterization of a variety of enzymes and proteins from these microorganisms shows that most of these are undeniably exceptionally thermostable (Adams and Kelly, 1998). They function optimally above 100 C and most of them are stable at these temperatures for long periods, in a few cases for several days (Adams and Kletzin, 1996; Daniel et al., 1996). The query is, how these proteins are different from their mesophilic counterparts? The reply is, not by very much! Direct sequence comparisons show that mesophilic and hyperthermophilic versions of the same enzyme typically share about 30%e50% identity, whereas methodological analysis suggests that these are small proteins and provide no evidence as to why one is more stable than the other (Adams and Kelly, 1998).

Properties of thermophilic/hyperthermophilic enzymes

A hyperthermophilic protein’s first crystal structure, which revealed that it had only 53 amino acids, also revealed that it could almost be superimposed on its mesophilic counterpart. It is clear that protein hyperthermal stability is caused by relatively small changes in protein structure but there is no consensus so far. It seems that additional salt bridges and hydrogen bonds, extended secondary structures, and abbreviated loops play a role in some (but not all) cases. Crystal structures of three enzymes and one DNA-binding protein from organisms that can grow at 100 C have been reported. It is truly amazing how cleverly nature has concealed the secret to extreme thermal stability; the underlying mechanisms seem to be particular to each type of enzyme. However, it is clear that this stability is an inherent quality of the protein and that there are not any “new rules” at all because mesophilic and hyperthermophilic proteins are both stabilized by similar kinds of intramolecular forces, though the specifics of how are still unknown. Unfortunately, research to date has revealed two important facts: first, structural data are a requirement because amino acid sequence analyses do not reveal stabilizing mechanisms, which is bad news for choosing helpful enzymes from the massive amount of sequence data provided by “genomics”; and second, there does not seem to be a general method for conversion of a mesophilic enzyme using site-directed mutagenesis methods into a hyperthermophilic enzyme. As a result, organisms or genes derived from them will be the only sources of hyperthermophilic enzymes for the foreseeable future (Day et al., 1992; Chan et al., 1995; Yip et al., 1995; Russell et al., 1997; Dedecker et al., 1996; Cowan and Fernandez-Lafuente, 2011). The hyperthermophilic enzymes are thermostable and show optimal activity at higher temperatures. Thermostability includes thermodynamic and kinetic stabilities. For the enzymes which unfold permanently, only Tm can be determined. Kinetic stability relies upon the energy barrier to unfolding. The kinetic stability of the enzyme is usually expressed as its half-life at described temperatures. Most of the enzymes obtained from hyperthermophilic microorganisms show optimum activity at temperatures closer to the host organism normally 70e125 C (Hicks et al., 1999; Vieille et al., 1996). Cellbound and extracellular hyperthermophilic enzymes (for instance proteases and saccharidases) show optimal activity at temperatures above in a few cases far above the optimum growth temperature of the host organism. For instance, Thermococcus litoralis amylopullulanase shows optimal activity at 117 C, whereas the optimal growth temperature of an organism is 88 C, which is 29 C higher (Brown and Kelly, 1993). Whereas these are generally less thermophilic as compared to extracellular enzymes obtained from a similar host, intracellular enzymes (like xylose isomerase) function optimally at the optimal

51

52

Developments and Applications of Enzymes From Thermophilic Microorganisms

growth temperature of the organism. Not many enzymes that perform optimally at 10e20 C below the optimum growth temperature of the organism (Fujiwara et al., 1996; Kunow et al., 1994; Purcarea et al., 1994). Whereas many hyperthermophilic enzymes are inherently more stable, and few intracellular enzymes derive greater thermostability from intracellular factors like coenzymes, substrates, activators, higher protein concentrations, salts, or general stabilizers like thermamine. Arrhenius plots for mesophilic and hyperthermophilic enzymes are generally linear (Bauer et al., 1999; Chen and Roberts, 1999). Not all mesophilic and hyperthermophilic enzymes change in their respective temperature ranges. If the structure of an enzyme changes in a catalytically significant way with an increase in temperature, we observe (1) nonlinear Arrhenius plots for most enzymes and (2) different types of plots for different classes of enzymes. Biphasic Arrhenius plots have been reported for several enzymes from hyperthermophiles. This represents an important exception to the typical Arrhenius-like behavior (Cannio et al., 1996; Fabry and Hensel, 1987; Faraone-Mennella et al., 1998; Hensel et al., 1987; Wrba et al., 1990). Biphasic Arrhenius plots are frequently correlated with functionally important conformational changes, found by spectroscopic techniques (Faraone-Mennella et al., 1998; Hensel et al., 1987; Londesborough, 1980). Not sufficient data is generally available which shows the effect of temperature on the activity of mesophilic enzymes. Bent Arrhenius plots are observed in the case of a few mesophilic enzymes (Fusek et al., 1990). This proposes that such breaks are not a specific attribute of enzymes from hyperthermophiles. Hyperthermophilic enzymes are very similar to mesophilic enzymes, except for phylogenetic variation. The difference between mesophilic and hyperthermophilic enzymes is the temperature range in which they are active and stable. (1) The sequences of homologous mesophilic and hyperthermophilic enzymes are normally 40%e85% alike (Davies et al., 1993; Vieille et al., 1995). (2) The three-dimensional structures are superposable (Maes et al., 1999; Russell et al., 1997; Tahirov et al., 1998; Auerbach et al., 1998; Chi et al., 1999; Hopfner et al., 1999; Isupov et al., 1999). (3) The catalytic mechanisms are similar (Bauer and Kelly, 1998; Zwickl et al., 1990; Vieille et al., 1995). In comparison to mesophilic enzymes, hyperthermophilic enzymes are more rigid at mesophilic temperatures. Actually, this rigidity is a requirement for higher thermostability. Experimental data, such as frequency-domain fluorescence analysis, anisotropic decay, tryptophan phosphorescence experiments, and hydrogen-deuterium exchange, support this hypothesis (Jaenicke and Bohm. 1998; Zavodszky et al., 1998; Gershenson et al., 2000; Bonisch et al., 1996). Table 4.1 presents some hyperthermostable proteins showing optimal operation temperatures at or above 100 C.

Table 4.1 Hyperthermostable enzymes with commercial interest and optimal activity over 100 C in aqueous media. Molecular mass (kDa)

References

106 100 100 100

6.5e7.5 4.5 5.5 5.0

129 (a2) 54 68 e

Brown et al. (1990) Jørgensen et al. (1997) Koch et al. (1991) Bragger et al. (1989)

85

120

5.0e8.0

e

Kim et al. (2001)

100 100 98 85

100 102 105 100

6.0 6.0 9.0 6.5

90 89 e 83

Rudiger et al. (1995) Brown et al. (1990) Andrade et al. (2001) Niehaus et al. (2000)

100

102

7.4

65 (a2)

Hansen et al. (2004)

80 100 80

105 105 130

6.5e7.5 7.4 e

180 (a4) 220 (a4) 63

Brown et al. (1993) Bauer et al. (1997) Piller et al. (1996)

80

120

5.5

57

100 88 100 100 95 80

100 >120 115 105 >100 103

5.0e5.5 4.5 5.0e6.0 e 6.0 7.0e7.5

90 80 135 232 (a4) 35 61

Galichet and Belarbi (1999) L
eveque et al. (2000) Rolfsmeier et al. (1998) Brown and Kelly (1993) Kengen et al. (1993) Matsui et al. (2000) Duffaud et al. (1997)

Microorganism

a-Amylase (a-glucosidic bonds)

Pyrococcus furiosus Pyrococcus furiosus Pyrococcus woesei Staphylothermus marinus Methanococcus jannaschii Pyrococcus woesei Pyrococcus furiosus Pyrodictium abyssi Thermococcus aggregans

100 100 100 90

Pyrobaculum aerophilum Thermotoga maritima Pyrococcus furiosus Thermococcus strain AN1 Thermococcus hydrothermalis Pyrococcus woesei Sulfolobus solfataricus Pyrococcus furiosus Pyrococcus furiosus Pyrococcus horikoshii Thermotoga neapolitana

Pullulanase type II (a-1,6 glycosidic bonds) Pullulan hydrolase III (a-1,6 and a-1,4 glycosidic bonds) Phospho-glucose/ mannose isomerase Glucose isomerase b-Mannosidase a-Glucosidase

b-Glucosidase a-Galactosidase

Protein Topt. (8C)

Continued

Properties of thermophilic/hyperthermophilic enzymes

Optimal pH

Topt. (8C)

Enzyme

53

54

Optimal pH

Molecular mass (kDa)

100

10.0

155

100 100 95

100 100 100

6.1e8.8 6.2e6.6 7.0e7.5

32 59 330 (a8)

80

>100

6.0e7.0

40

Machielsen and van der Oost (2006) Machielsen et al. (2006) Cheng et al. (1999) Mori and Ishikawa (2005) Lee et al. (2006)

100 100 100 88 95

>100 105 110 105 >100

8.0 e e e 7.0

38 93 114 52 45

Story et al. (2005) Badr et al. (1994) Kengen et al. (1995) Cowan et al. (1987) Morikawa et al. (1994)

100 100 100

100 104 >100

6.3 6.0e7.0 6.0

124 (a6) 30 205 (a4)

Halio et al. (1997) Gueguen et al. (1997) Johnsen et al. (2003)

93 80 100 87 95

>100 >100 125 120 >100

6.1 5.9 7.4 7.4 5.0

207 190 180 160 312

87

100

e

133 (a4)

Buchanan et al. (1999)

93 100 85

>100 >100 >100

6.2 7.5 7.5

36 98 (a2) 52

Hansen et al. (2002) Koga et al. (2000) Koga et al. (2000)

Enzyme

Microorganism

Topt. (8C)

Threonine (alcohol) dehydrogenase Alcohol dehydrogenase Carboxypeptidase Aminopeptidase

Pyrococcus furiosus

100

Pyrococcus furiosus Pyrococcus furiosus Pyrococcus horikoshii

Glukokinase Sucrose a-glucohydrolase Serine protease Thiol protease Metalloprotease b-1,4-endoglucanase Pyruvate kinase

Methylthioadenosine phosphorylase Fructose 1,6-biphosphate aldolase 2-keto-3-deoxygluconate aldolase Glucokinase ADP-dependent glucokinase

Thermococcus strain NA1 Pyrococcus furiosus Pyrococcus furiosus Pyrococcus furiosus Desulfurococcus mucosus Thermoc. kodakaraensis KOD1 Pyrococcus furiosus Pyrococcus furiosus Pyrobaculum aerophilum Aeropyrum pernix Thermotoga maritima Pyrococcus furiosus Sulfolobus solfataricus Thermoc. kodakaraensis KOD1 Sulfolobus-solfataricus Aeropyrum pernix Pyrococcus furiosus Thermococcus litoralis

Protein Topt. (8C)

(a4) (a4) (a4) (a6) (a10)

References

Johnsen et al. (2003) Johnsen et al. (2003) Cacciapuoti et al. (2003) Cacciapuoti et al. (1994) Imanaka et al. (2002)

Developments and Applications of Enzymes From Thermophilic Microorganisms

Table 4.1 Hyperthermostable enzymes with commercial interest and optimal activity over 100 C in aqueous media.dcont’d

Glucanotransferase 4-a-glucanotransferase

Esterase Metalloproteinase Aminoacylase

Thermococcus strain B1001 Pyrococcus furiosus KO D1 Pyrococcus furiosus Aeropyrum pernix K1 Pyrococcus furiosus

85

110

5.0e5.5

83

Tachibana et al. (1999)

100

100

6.0e8.0

77

Tachibana et al. (1997)

100 90 100

100 100 100

7.6 5.0e9.0 6.5

e 52 170 (a4)

Ikeda and Clark (1998) Sako et al. (1997) Story et al. (2001)

Reproduced with permission Unsworth, L.D., van der Oost, J., Koutsopoulos, S., 2007. Hyperthermophilic enzymesdstability, activity and implementation strategies for high temperature applications. FEBS J. 274, 4044e4405.

Properties of thermophilic/hyperthermophilic enzymes

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Developments and Applications of Enzymes From Thermophilic Microorganisms

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Properties of thermophilic/hyperthermophilic enzymes

Chan, M.K., Mukund, S., Kletzin, A., Adams, M.W., Rees, D.C., 1995. Structure of a hyperthermophilic tungstopterin enzyme, aldehyde ferredoxin oxidoreductase. Science 267 (5203), 1463e1469. Chen, L., Roberts, M.F., 1999. Characterization of a tetrameric inositol monophosphatase from the hyperthermophilic bacterium Thermotoga maritima. Appl. Environ. Microbiol. 65, 4559e4567. Cheng, T.C., Ramakrishnan, V., Chan, S.I., 1999. Purification and characterization of cobalt-activated carboxypeptidase from the hyperthermophilic archaeon Pyrococcus furiosus. Protein Sci. 8, 2474e2486. Chi, Y.I., Martinez-Cruz, L.A., Jancarik, J., Swanson, R.V., Robertson, D.E., Kim, S.H., 1999. Crystal structure of the beta-glycosidase from the hyperthermophile Thermosphaera aggregans: insights into its activity and thermostability. FEBS Lett. 445, 375e383. Cowan, D.A., 1997. Thermophilic proteins: stability and function in aqueous and organic solvents. Comp. Biochem. Physiol. A Physiol. 118, 429e438. Cowan, D.A., 1999. Life in extreme thermal environments: implications for exobiology. In: Life on Mars. British Interplanetary Society Special Publication, pp. 37e48. Cowan, D.A., Fernandez-Lafuente, R., 2011. Enhancing the functional properties of thermophilic enzymes by chemical modification and immobilization. Enzym. Microb. Technol. 49 (4), 326e346. Cowan, D.A., Smolenski, K.A., Daniel, R.M., Morgan, H.W., 1987. An extremely thermostable extracellular proteinase from a strain of the archaebacterium Desulfurococcus growing at 88 C. Biochem. J. 247, 121e133. Cowan, D.A., Kirby, B.M., Meiring, T.L., Ferrer, M., Guazzaroni, M.-E., Golyshina, O.V., Golyshin, P.N., 2010. Enzymes from extreme environments Ch. 4. In: Baltz, R.H., Davies, J.E., Demain, A.L. (Eds.), Manual of Industrial Microbiology and Biotechnology, third ed. ASM Press. ISBN: 978-1-55581512-7. Daniel, R.M., Cowan, D.A., 2000. Biomolecular stability and life at high temperatures. Cell. Mol. Life Sci. 57, 250e264. Daniel, R.M., Danson, M.J., Hough, D.W., Lee, C.K., Peterson, M.E., Cowan, D.A., 2008. In: Thomas, T. (Ed.), Enzyme Stability and Activity at High Temperatures in Anatomy and Physiology of Extremophilic Proteins. Nova Science, Sidiqui, pp. S1eS34. Daniel, R.M., Dines, M., Petach, H.H., 1996. The denaturation and degradation of stable enzymes at high temperatures. Biochem. J. 317, 1e11. D’Auria, S., Morana, A., Febbraio, F., Vaccaro, C., De Rosa, M., Nucci, R., 1996. Functional and structural properties of the homogeneous beta-glycosidase from the extreme thermoacidophilic archaeon Sulfolobus solfataricus expressed in Saccharomyces cerevisiae. Protein Expr. Purif. May 7 (3), 299e308. Davies, G.J., Gamblin, S.J., Littlechild, J.A., Watson, H.C., 1993. The structure of a thermally stable 3phosphoglycerate kinase and a comparison with its mesophilic equivalent. Proteins Struct. Funct. Genet. 15, 283e289. Day, M.W., Hsu, B.T., Joshua-Tor, L., Park, J.B., Zhou, Z.H., Adams, M.W.W., Rees, D.C., 1992. X-ray crystal structures of the oxidized and reduced forms of the rubredoxin from the marine hyperthermophilic archaebacterium Pyrococcus furiosus. Protein Sci. 1, 1494e1507. DeDecker, B.S., O’Brien, R., Fleming, P.J., Geiger, J.H., Jackson, S.P., Sigler, P.B., 1996. The crystal structure of a hyperthermophilic archaeal TATA-box binding protein. J. Mol. Biol. 264 (5), 1072e1084. Demirjian, D.C., Moris-Varas, F., Cassidy, C.S., 2001. Enzymes from extremophiles. Curr. Opin. Chem. Biol. 5, 144e151. Duffaud, G.D., McCutchen, C.M., Leduc, P., Parker, K.N., Kelly, R.M., 1997. Purification and characterization of extremely thermostable beta-mannanase, beta-mannosidase, and alpha-galactosidase from the hyperthermophilic eubacterium Thermotoga neapolitana 5068. Appl. Environ. Microbiol. 63, 169e177. Durand, J., Teuma, E., Gomez, M., 2007. Ionic liquids as a medium for enantioselective catalysis. C R Chim 10, 152e177. Egorova, K., Antranikian, G., 2005. Industrial relevance of thermophilic Archaea. Curr. Opin. Microbiol. 8, 649e655. Everly, C., Alberto, J., 2000. Stressors, stress and thermostable extracellular protease of bacillus survival: overview front. Bioscience 5, 780e786.

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Fabry, S., Hensel, R., 1987. Purification and characterization of D-glyceraldehyde-3-phosphate dehydrogenase from the thermophilic archaebacterium Methanothermus fervidus. Eur. J. Biochem. 165, 147e155. Faraone-Mennella, M.R., Gambacorta, A., Nicolaus, B., Farina, B., 1998. Purification and biochemical characterization of a poly(ADP-ribose) polymerase-like enzyme from the thermophilic archaeon Sulfolobus solfataricus. Biochem. J. 335, 441e447. Fujiwara, S., Lee, S., Haruki, M., Kanaya, S., Takagi, M., Imanaka, T., 1996. Unusual enzyme characteristics of aspartyl-tRNA synthetase from hyperthermophilic archaeon Pyrococcus sp. KOD1. FEBS Lett. 394, 66e70. Fusek, M., Lin, X., Tang, J., 1990. Enzymic properties of thermopsin. J. Biol. Chem. 265, 1496e1501. Galichet, A., Belarbi, A., 1999. Cloning of an alpha-glucosidase gene from Thermococcus hydrothermalis by functional complementation of a Saccharomyces cerevisiae mal11 mutant strain. FEBS Lett. 458, 188e192. Gershenson, A., Schauerte, J.A., Giver, L., Arnold, F.H., 2000. Tryptophan fluorescence study of enzyme flexibility and unfolding in laboratory evolved thermostable esterases. Biochemistry 39, 4658e4665. Gueguen, Y., Voorhorst, W.G.B., van der Oost, J., de Vos, W.M., 1997. Molecular and biochemical characterization of an endo-b-1,3-glucanase of the hyperthermophilic archaeon Pyrococcus furiosus. J. Biol. Chem. 272, 31258e31264. Haki, G.D., Rakshit, S.K., 2003. Developments in industrially important thermostable enzymes. Bioresour. Technol. 89, 17e34. Halio, S.B., Bauer, M.W., Mukund, S., Adams, M.W.W., Kelly, R.M., 1997. Purification and characterization of two functional forms of intracellular protease PfpI from the hyperthermophilic archaeon Pyrococcus furiosus. Appl. Environ. Microbiol. 63, 289e295. Hansen, T., Reichstein, B., Schmid, R., Schonheit, P., 2002. The first archaeal ATP-dependent glucokinase, from the hyperthermophilic crenarchaeon Aeropyrum pernix, represents a monomeric, extremely thermophilic ROK glucokinase with broad hexose specificity. J. Bacteriol. 184, 5955e5965. Hansen, T., Urbanke, C., Schonheit, P., 2004. Bifunctional phopshoglucose/phosphomannose isomerase from the hyperthermophilic archaeon Pyrobaculum aerophilum. Extremophiles 8, 507e512. Hensel, R., Laumann, S., Lang, J., Heumann, H., Lottspeich, F., 1987. Characterization of two D-glyceraldehyde-3-phosphate dehydrogenases from the extremely thermophilic archaebacterium Thermoproteus tenax. Eur. J. Biochem. 170 (1e2), 325e333. Hicks, P.M., Adams, M.W.W., Kelly, R.M., 1999. Enzymes, extremely thermostable. In: Flickinger, M.C., Drew, S.W. (Eds.), Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis, and Bioseparation. John Wiley & Sons, Inc., New York, NY, pp. 987e1004. Hopfner, K.P., Eichinger, A., Engh, R.A., Laue, F., Ankenbauer, W., Huber, R., Angerer, B., 1999. Crystal structure of a thermostable type B DNA polymerase from Thermococcus gorgonarius. Proc. Natl. Acad. Sci. U. S. A. 96 (7), 3600e3605. Hough, D.W., Danson, M.J., 1999. Extremozymes. Curr. Opin. Chem. Biol. 3, 39e46. Ikeda, M., Clark, D.S., 1998. Molecular cloning of extremely thermostable esterase gene from hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli. Biotechnol. Bioeng. 57, 624e629. Imanaka, H., Fukui, T., Atomi, H., Imanaka, T., 2002. Gene cloning and characterization of fructose-1,6bisphosphate aldolase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. J. Biosci. Bioeng. 94, 237e243. Isupov, M.N., Fleming, T.M., Dalby, A.R., Crowhurst, G.S., Bourne, P.C., Littlechild, J.A., 1999. Crystal structure of the glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Sulfolobus solfataricus. J. Mol. Biol. 291, 651e660. Jackel, C., Kast, P., Hilvert, D., 2008. Protein design by directed evolution. Annu. Rev. Biophys. 37, 153e173. Jaenicke, R., Bohm, G., 1998. The stability of proteins in extreme environments. Curr. Opin. Struct. Biol. 8, 738e748. Johnsen, U., Hansen, T., Schonheit, P., 2003. Comparative analysis of pyruvate kinases from the hyperthermophilic archaea Archaeoglobus fulgidus, Aeropyrum pernix, and Pyrobaculum aerophilum and the hyperthermophilic bacterium Thermotoga maritima: unusual regulatory properties in hyperthermophilic archaea. J. Biol. Chem. 278, 25417e25427.

Properties of thermophilic/hyperthermophilic enzymes

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eveque, E., Janecek, S., Belarbi, H.B., 2000. Thermophilic archaeal amylolytic enzymes. Enzym. Microb. Technol. 26, 3e14. Lebbink, J.H., Knapp, S., van der Oost, J., Rice, D., Ladenstein, R., de Vos, W.M., 1999. Engineering activity and stability of Thermotoga maritima glutamate dehydrogenase. II: construction of a 16-residue ionpair network at the subunit interface. J. Mol. Biol. 289, 357e369. Lee, H.S., Kim, Y.J., Bae, S.S., Jeon, J.H., Lim, J.K., Jeong, B.C., Kang, S.G., Lee, J.H., 2006. Cloning, expression, and characterization of aminopeptidase P from the hyperthermophilic archaeon Thermococcus sp. strain NA1. Appl. Environ. Microbiol. 72, 1886e1890. Londesborough, J., 1980. The causes of sharply bent or discontinuous Arrhenius plots for enzyme-catalyzed reactions. Eur. J. Biochem. 105, 211e215. Machielsen, R., Uria, A.R., Kengen, S.W.M., van der Oost, J., 2006. Production and characterization of a thermostable alcohol dehydrogenase that belongs to the aldoketo reductase superfamily. Appl. Environ. Microbiol. 72, 233e238. Machielsen, R., van der Oost, J., 2006. Production and characterization of a thermostable l-threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus. FEBS J. 273, 2722e2729. Maes, D., Zeelen, J.P., Thanki, N., Beaucamp, N., Alvarez, M., Thi, M.H., Backmann, J., Martial, J.A., Wyns, L., Jaenicke, R., Wierenga, R.K., 1999. The crystal structure of triosephosphate isomerase (TIM) from Thermotoga maritima: a comparative thermostability structural analysis of ten different TIM structures. Proteins 37, 441e453. Matsui, I., Sakai, Y., Matsui, E., Kikuchi, H., Kawarabayasi, Y., Honda, K., 2000. Novel substrate specificity of a membrane-bound beta-glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii. FEBS Lett. 467, 195e200. Meiring, T.L., Bauer, R., Scheepers, I., Ohloff, C., Tuffin, I.M., Cowan, D.A., 2011. Metagenomics and beyond: current approaches and integration with complementary technologies. In: Marco, D. (Ed.), Metagenomics: Current Innovations and Future Trends. Horizon Press, pp. 1e19.

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Meyer, H.-P., 2006. Chemocatalysis biocatalysis (biotransformation): some thoughts of a chemist and of a biotechnologist. Org. Process Res. Dev. 10, 572e580. Mori, K., Ishikawa, K., 2005. New deblocking aminopeptidases from Pyrococcus horikoshii. Biosci. Biotechnol. Biochem. 69, 1854e1860. Morikawa, M., Izawa, Y., Rashid, N., Hoaki, T., Imanaka, T., 1994. Purification and characterization of a thermostable thiol protease from a newly isolated hyperthermophilic Pyrococcus sp. Appl. Environ. Microbiol. 60, 4559e4566. Niehaus, F., Peters, A., Groudieva, T., Antranikian, G., 2000. Cloning, expression and biochemical characterization of a unique thermostable pullulan-hydrolysing enzyme from the hyperthermophilic archaeon Thermococcus aggregans. FEMS Microbiol. Lett. 190, 223e239. Olsson, K., Keis, S., Morgan, H.W., Dimroth, P., Cook, G.M., 2003. Bioenergetic properties of the thermoalkaliphilic Bacillus sp. strain TA2.A1. J. Bacteriol. 1852, 461e465. Piller, K., Daniel, P.M., Petach, H.H., 1996. Properties and stabilization of an extracellular alpha-glucosidase from the extremely thermophilic archaebacteria Thermococcus strain AN1: enzyme activity at 130 C. Biochim. Biophys. Acta Protein Struct. Mol. Enzymol. 1292, 197e205. Polizzi, K.M., Bommarius, A.S., Broering, J.M., Chaparro-Riggers, J.F., 2007. Stability of biocatalysts. Curr. Opin. Chem. Biol. 11, 220e225. Pollard, D.J., Woodley, J.M., 2007. Biocatalysis for pharmaceutical intermediates: the future is now. Trends Biotechnol. 25, 66e73. Purcarea, C., Erauso, G., Prieur, D., Herv
e, G., 1994. The catalytic and regulatory properties of aspartate transcarbamoylase from Pyrococcus abyssi, a new deep-sea hyperthermophilic archaebacterium. Microbiology 140, 1967e1975. Razvi, A., Scholtz, J.M., 2006. Lessons in stability from thermophilic proteins. Protein Sci. 15, 1569e1578. Reetz, M.T., 2007. Controlling the selectivity and stability of proteins by new strategies in directed evolution: the case of organocatalytic enzymes. Ernst Schering Found. Symp. Proc. 321e340. Rolfsmeier, M., Haseltine, C., Bini, E., Clark, A., Blum, P., 1998. Molecular characterization of the a-glucosidase gene (malA) from the hyperthermophilic archaeon Sulfolobus solfataricus. J. Bacteriol. 180, 1287e1295. Rudiger, A., Jørgensen, P.L., Antranikian, G., 1995. Isolation and characterization of a heat stable pullulanase from the hyperthermophilic archeon Pyrococcus woesei after cloning and expression of its gene in Escherichia coli. Appl. Environ. Microbiol. 61, 567e575. Russell, R.J., Ferguson, J.M., Hough, D.W., Danson, M.J., Taylor, G.L., 1997. The crystal structure of citrate synthase from the hyperthermophilic archaeon Pyrococcus furiosus at 1.9  A resolution. Biochemistry 36, 9983e9994. Sako, Y., Croocker, P.C., Ishida, Y., 1997. An extremely heat-stable extracellular proteinase (aeropyrolysin) from the hyperthermophilic archaeon Aeropyrum pernix K1. FEBS Lett. 415, 329e334. Samuelson, J.C., 2011. Recent developments in difficult protein expression: a guide to E. coli strains, promoters, and relevant host mutations. Methods Mol. Biol. 705, 195e209. Scandurra, R., Consalvi, V., Chiaraluce, R., Politi, L., Engel, P.C., 1998. Protein thermostability in extremophiles. Biochimie 80, 933e941. Schmid, A., Dordick, J.S., Hauer, B., Kiener, A., Wubbolts, M., Witholt, B., 2001. Industrial biocatalysis today and tomorrow. Nature 409, 258e268. Schmidt, M., Bottcher, D., Bornscheuer, U.T., 2009. Protein engineering of carboxyl esterases by rational design and directed evolution. Protein Pept. Lett. 16, 1162e1171. Schoemaker, H.E., Mink, D., WubboLts, M.G., 2003. Dispelling the mythsdbiocatalysis in industrial synthesis. Science 299, 1694e1697. Segerer, A.H., Burggraf, S., Fiala, G., Huber, G., Huber, R., Pley, U., Stetter, K.O., 1993. Life in hot springs and hydrothermal vents. Orig. Life Evol. Biosph. 23, 77e90. Sheldon, R.A., 2005. Green solvents for sustainable organic synthesis: state of the art. Green Chem. 7, 267e278. Smith, D.W., 1996. Problems of translating heterologous genes in expression systems: the role of tRNA. Biotechnol. Prog. 12, 417e422.

Properties of thermophilic/hyperthermophilic enzymes

Story, S.V., Grunden, A.M., Adams, M.W.W., 2001. Characterization of an aminoacylase from the hyperthermophilic archaeon Pyrococcus furiosus. J. Bacteriol. 183, 4259e4268. Story, S.V., Shah, C., Jenney, F.E., Adams, M.W.W., 2005. Characterization of a novel zinc-containing, lysine-specific aminopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus. J. Bacteriol. 187, 2077e2083. Straathof, A.J.J., Panke, S., Schmid, A., 2002. The production of fine chemicals by biotransformations. Curr. Opin. Biotechnol. 13, 548e556. Tachibana, Y., Fujiwara, S., Takagi, M., Imanaka, T., 1997. Cloning and expression of the 4-alpha-glucanotransferase gene from the hyperthermophilic archaeon Pyrococcus sp. KOD1, and characterization of the enzyme. J. Ferment. Bioeng. 83, 540e548. Tachibana, Y., Kuramura, A., Shirasaka, N., Suzuki, Y., Yamamoto, T., Fujiwara, S., Takagi, M., Imanaka, T., 1999. Purification and characterization of an extremely thermostable cyclomaltodextrin glucanotransferase from a newly isolated hyperthermophilic archaeon, a Thermococcus sp. Appl. Environ. Microbiol. 65, 1991e1997. Tahirov, T.H., Oki, H., Tsukihara, T., Ogasahara, K., Yutani, K., Ogata, K., Izu, Y., Tsunasawa, S., Kato, I., 1998. Crystal structure of methionine aminopeptidase from hyperthermophile, Pyrococcus furiosus. J. Mol. Biol. 284, 101e124. Tian, J., Wang, P., Gao, S., Chu, X., Wu, N., Fan, Y., 2010. Enhanced thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 by rational engineering of a glycine to proline mutation. FEBS J. 277, 4901e4908. Unsworth, L.D., van der Oost, J., Koutsopoulos, S., 2007. Hyperthermophilic enzymesdstability, activity and implementation strategies for high temperature applications. FEBS J. 274, 4044e4405. van de Vossenberg, J.L.C.M., Driessen, A.J., Zillig, W., Konings, W.N., 1998. Bioenergetics and cytoplasmic membrane stability of the extremely acidophilic, thermophilic archaeon Picrophilus oshimae. Extremophiles 2, 67e74. Van Mierlo, C.P.M., Steensma, E., 2000. Protein folding and stability investigated by fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy: the flavodoxin story. J. Biotechnol. 79, 281e298. Vieille, C., Zeikus, G.J., 2001. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65, 1e43. Vieille, C., Burdette, D.S., Zeikus, J.G., 1996. Thermozymes. Biotechnol. Annu. Rev. 2, 1e83. Vieille, C., Hess, J.M., Kelly, R.M., Zeikus, J.G., 1995. xylA cloning and sequencing and biochemical characterization of xylose isomerase from Thermotoga neapolitana. Appl. Environ. Microbiol. 61, 1867e1875. Walters, J., Swartz, P., Mattos, C., Clark, A.C., 2011. Thermodynamic, enzymatic and structural effects of removing a salt bridge at the base of loop 4 in (pro)caspase-3. Arch. Biochem. Biophys. 508, 31e38. Woodley, J.M., 2008. New opportunities for biocatalysis: making pharmaceutical processes greener. Trends Biotechnol. 26, 321e327. Wrba, A., Schweiger, A., Schultes, V., Jaenicke, R., Zavodsky, P., 1990. Extremely thermostable D-glyceraldehyde-3-phosphate dehydrogenase from the eubacterium Thermotoga maritima. Biochemistry 29, 7584e7592. Yip, K.S., Stillman, T.J., Britton, K.L., Artymiuk, P.J., Baker, P.J., Sedelnikova, S.E., Engel, P.C., Pasquo, A., Chiaraluce, R., Consalvi, V., Scandurrra, R., Rice, D.W., 1995. The structure of Pyrococcus furiosus glutamate dehydrogenase reveals a key role for ion-pair networks in maintaining enzyme stability at extreme temperatures. Structure 3 (11), 1147e1158. Zavodszky, P., Kardos, J., Svingor, A., Petsko, G.A., 1998. Adjustment of conformational flexibility is a key event in the thermal adaptation of proteins. Proc. Natl. Acad. Sci. U. S. A. 95, 7406e7411. Zwickl, P., Fabry, S., Bogedain, C., Haas, A., Hensel, R., 1990. Glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei: characterization of the enzyme, cloning and sequencing of the gene, and expression in Escherichia coli. J. Bacteriol. 172, 4329e4338.

Further reading Adams, M.W., 1993. Enzymes and proteins from organisms that grow near and above 100 C. Annu. Rev. Microbiol. 47, 627e658.

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Bauer, R., Mutondo, M., Huddy, R.J., Tuffin, I.M., Cowan, D.A., 2011. Metagenomic gene discovery. In: Li, R.W. (Ed.), Ch. 15 in Metagenomics and its Applications in Agriculture, Biomedicine and Environmental Studies. Nova Science Publisher’s, pp. 287e320. Beaucamp, N., Hofmann, A., Kellerer, B., Jaenicke, R., 1997. Dissection of the gene of the bifunctional PGK-TIM fusion protein from the hyperthermophilic bacterium Thermotoga maritima: design and characterization of the separate triosephosphate isomerase. Protein Sci. 6, 2159e2165. Bischof, J.C., He, X., 2005. Thermal stability of proteins. Ann. N. Y. Acad. Sci. 1066, 12e33. D’Auria, S., Nucci, R., Rossi, M., Bertoli, E., Tanfani, F., Gryczynski, I., Malak, H., Lakowicz, J.R., 1999. b-Glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: structure and activity in the presence of alcohols. J. Biochem. 126, 545e552. De Montigny, C., Sygusch, J., 1996. Functional characterization of an extreme thermophilic class II fructose1,6-bisphosphate aldolase. Eur. J. Biochem. 241, 243e248. Giver, L., Gershenson, A., Freskgard, P.O., Arnold, F.H., 1998. Directed evolution of a thermostable esterase. Proc. Natl. Acad. Sci. USA 95, 12809e12813. Glasemacher, J., Bock, A.-K., Schmidt, R., Schönheit, P., 1999. Purification and characterization of two extremely thermostable enzymes, phosphate acetyltransferase and acetate kinase, from the hyperthermophilic eubacterium Thermotoga maritima. J. Bacteriol. 181, 1861e1867. Hernandez, G., Jenney Jr., F.E., Adams, M.W., LeMaster, D.M., 2000. Millisecond time scale conformational flexibility in a hyperthermophile protein at ambient temperature. Proc. Natl. Acad. Sci. U. S. A. 97 (7), 3166e3170. Ichikawa, J.K., Clarke, S., 1998. A highly active protein repair enzyme from an extreme thermophile: the Lisoaspartyl methyltransferase from Thermotoga maritima. Arch. Biochem. Biophys. 358, 222e231. Kohen, A., Cannio, R., Bartolucci, S., Klinman, J.P., 1999. Enzyme dynamics and hydrogen tunnelling in a thermophilic alcohol dehydrogenase. Nature 399, 496e499. Kristjannson, J.K., Stetter, K.O., 1991. Thermophilic Bacteria, in Thermophilic Bacteria. CRC Press, Boca Raton, pp. 2e13. Kujo, C., Oshima, T., 1998. Enzymological characteristics of the hyperthermostable NAD-dependent glutamate dehydrogenase from the archaeon Pyrobaculum islandicum and effects of denaturants and organic solvents. Appl. Environ. Microbiol. 64, 2152e2157. Lazaridis, T., Lee, I., Karplus, M., 1997. Dynamics and unfolding pathways of a hyperthermophilic and a mesophilic rubredoxin. Protein Sci. 6, 2589e2605. Liang, X., Arunima, A., Zhao, Y., Bhaskaran, R., Shende, A., Byrne, T.S., Fleeks, J., Palmier, M.O., Van Doren, S.R., 2010. Apparent tradeoff of higher activity in MMP-12 for enhanced stability and flexibility in MMP-3. Biophys. J. 99, 273e283. Manco, G., Giosue, E., D’Auria, S., Herman, P., Carrea, G., Rossi, M., 2000. Cloning, overexpression, and properties of a new thermophilic and thermostable esterase with sequence similarity to hormonesensitive lipase subfamily from the archaeon Archaeoglobus fulgidus. Arch. Biochem. Biophys. 373, 182e192. Merz, A., Knöchel, T., Jansonius, J.N., Kirschner, K., 1999. The hyperthermostable indoleglycerol phosphate synthase from Thermotoga maritima is destabilized by mutational disruption of two solventexposed salt bridges. J. Mol. Biol. 288 (4), 753e763. Sterner, R., Kleemann, G.R., Szadkowski, H., Lustig, A., Hennig, M., Kirschner, K., 1996. Phosphoribosyl anthranilate isomerase from Thermotoga maritima is an extremely stable and active homodimer. Protein Sci. 5, 2000e2008. Vihinen, M., 1987. Relationship of protein flexibility to thermostability. Protein Eng. 1, 477e480. Wilquet, V., Gaspar, J.A., van de Lande, M., Van de Casteele, M., Legrain, C., Meiering, E.M., Glansdorff, N., 1998. Purification and characterization of recombinant Thermotoga maritima dihydrofolate reductase. Eur. J. Biochem. 255 (3), 628e637. Zhao, H., Arnold, F.H., 1999. Directed evolution converts subtilisin E into a functional equivalent of thermitase. Protein Eng. 12, 47e53.

CHAPTER 5

Enzyme production by thermophiles 5.1 Introduction Thermophiles are able to survive at temperatures up to 122 C. Based on their optimum growth temperature, these are classified into moderate thermophiles, extreme thermophiles, and hyperthermophiles (Zuliani et al., 2021). The optimum growth temperature of moderate thermophiles, extreme thermophiles, and hyperthermophiles is in the range of 50e60 C; 60e80 C, and 80e110 C, respectively (Zuliani et al., 2021). These microbes are a source of important industrial enzymes and are attracting substantial attention in the last few years (Singh et al., 2011; Rigoldi et al., 2018). Thermophilic enzymes show a high unfolding temperature and a long half-life (t1/2) at a selected higher temperature and in the presence of organic solvents also. This is quite the opposite of mesophilic enzymes (Böhme et al., 2020). So, thermostable enzymes can survive severe conditions in the industry (i.e., very high pH and temperature, presence of organic solvents, longer reaction time, etc.), while making sure the requisite standards of repeatability.

5.2 Enzyme production by thermophilic microorganisms Growing the thermophilic microorganisms at higher temperatures is economically interesting because the threat of contamination is reduced, and viscosity is also reduced. Because of this, the mixing becomes easier, which results in a higher amount of substrate solubility. But, in comparison to mesophilic organisms, the biomass production by these microorganisms is generally very low. The reduced biomass yield creates difficulty for small as well as commercial production. This makes comprehensive studies of their enzymes not very easy. This has prompted substantial R&D for improving biomass yield. Numerous reports are available on the optimization of culture and media compositions of thermophiles (Krahe et al., 1996). Specific processes and special equipment have been developed for improving the fermentation processes of thermophilic and hyperthermophilic microorganisms (Schiraldi and De Rosa, 2002). But, growing thermophiles on an industrial scale for enzyme production remains challenging owing to the factors like the requirement of complex and costly media, reduced growth rate and reduced solubility of gas at higher temperatures, and product inhibition (Krahe et al., 1996; Schiraldi and De Rosa, 2002). The exorbitant cost of commercial fermentation

Developments and Applications of Enzymes From Thermophilic Microorganisms ISBN 978-0-443-19197-8, https://doi.org/10.1016/B978-0-443-19197-8.00009-8

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processes for producing enzymes by thermophilic and hyperthermophilic microorganisms is justified only for very few precise applications. There is a need for reducing the cost of production of thermophilic enzymes for obtaining advancement on a commercial scale. Genetic engineering methods can be used to reduce the cost of production and enhance the yield. Several thermostable enzymes have been cloned and successfully expressed in mesophilic hosts, for example, Kluyveromyces lactis, Trichoderma reesei, Aspergillus oryzae, Saccharomyces cervisae, Escherichia coli, Bacillus subtilis, Pichia pastoris, etc. (Bergquist et al., 1995, 2002; Moracci et al., 1992; Ramchuran et al., 2002; Shinohara et al., 2001; Soutschek-Bauer and Staudenbauer, 1987; Walsh et al., 1998). But, dissimilarity in the usage of codon or inappropriate folding of the proteins may result in low enzyme activity or reduced level of expression (Duffner et al., 2000; Ciaramella et al., 1995). Furthermore, several complex enzymes, such as heterooligomers or those that need covalently bound cofactors are not very easy to produce in mesophilic organisms. This started the investigation of genetic tools for overexpressing these enzymes in thermophilic hosts. Up to now, several vectors have been developed for expressing the proteins in several thermophilic organisms (Table 5.1). However, the use of the novel thermophilic expression systems is at the R&D stage only and further work needs to be done before use on a commercial scale can be considered.

Table 5.1 Vectors constructed for thermophilic expression system.

Thermus thermophilus Thermus thermophilus Sulfolobus solfataricus Talaromyces sp. CL240

pMKMOO1 pMKE1 pEXSs pUT737

Rhodothermus marinus

pRM100

Pyrococcus abyssi Thermoanaerobacterium saccharolyticum Thermoanaerobacterium saccharolyticum Sulfolobus acidocaldarius

pYS2 pRKM1, pRUKM pUXK, pUXKC I pAG1/pAG2

Pyrococcus furiosus

pAG1/pAG2

Shuttle Shuttle Shuttle Shuttle, Integration Shuttle Shuttle Shuttle Integration Shuttle Shuttle

Mather and Fee (2007) Moreno et al. (2003) Contursi et al. (2003) Jain et al. (1992) Bjornsdottir et al. (2005) Lucas et al. (2002) Mai and Wiegel (2000) Mai and Wiegel (2000) Aravalli and Garrett (1997) Aravalli and Garrett (1997)

Turner, P., Mamo, G., Karlsson, E.N., 2007. Potential and utilization of thermophiles and thermostable enzymes in biorefining. Microb. Cell Factories 6, 9. Distributed under the Creative Commons CC BY license.

Enzyme production by thermophiles

5.2.1 Use of whole cells or isolated enzymes Thermophilic enzymes are used in several industrial processes mostly because of their remarkable performance at elevated temperatures and their capability to tolerate denaturants. These enzymes are used in paper, textile, food, chemical, pharmaceuticals, and other industries (Zaks, 2001; Gomes and Steiner, 2004; van Beilen and Li, 2002; Demirjian et al., 2001). The majority of these applications use genetically modified thermostable enzymes that have been expressed in mesophilic organisms depending upon the nature of the application, the type of reaction, and the purity of the product. The enzyme preparation can be cell-associated or cell-free (the solution is obtained by breaking the cells and removing all particulate matter). For instance, the use of cell-free dehydrogenase is hindered by the requirement of costly cofactors (Stewart, 2001) whereas the transaminases suffer from unsuitable reaction equilibria (Taylor et al., 1998). On this point, whole cells appear to be more striking. In food processing, whole cells have been used. Recombinant thermostable b-glucosidase enzymes expressed in Lactococcus lactis have been used (Giuliano et al., 2004). The use of whole cells is of special interest for the conversion of lignocellulosic raw materials. The steps involved in the conversion of lignocellulosics are saccharification and fermentation. In the saccharification step, the carbohydrates (celluloses and hemicelluloses) are hydrolyzed into sugars. These sugars are then used as raw material in the fermentation step by microbes that convert it into ethanol. Whole-cell biocatalytic processes offer a striking option of a single-step conversion, in which the microbes produce enzymes capable of splitting sugars that degrade the lignocelluloses and ferment the released sugars, which might lead to enhance efficacy in comparison to the common multistep conversion of lignocelluloses (Lynd et al., 2002, 2005). In comparison to starch or sugar-containing crops, for example, maize or sugar cane, lignocelluloses are not easy to break down into monomers at high yields because hemicelluloses and cellulose are closely associated with lignin in the plant cell wall. Pretreatment with the use of steam, acid, or alkali is therefore required for making the carbohydrates available for enzymatic hydrolysis and fermentation (Klinke et al., 2004; Galbe and Zacchi 2002). Among the several pretreatment methods reported, pretreatment with hot water is found to make the biomass (particularly the cellulosic part) more available for enzymatic attack. The use of thermophilic microorganisms is here appealing because energy can be saved by the reduction of the cost of cooling after pretreatment with steam, reducing the danger of contamination, and enhancing the rates of saccharification and fermentation. Furthermore, the use of thermophilic conditions results in continuous evaporation of ethanol that allows harvest during fermentation. Concurrent fermentation and product recovery can reduce product inhibition by ethanol and also decrease the volume of water used for distillery cooling and the time needed for distillation. All this would lead to a more potent process. However, a difficulty associated with the pretreatment of lignocellulose is that the degradation products are liberated, which may inhibit the growth of microbes (Klinke et al., 2004). However, very good results have been obtained using thermophilic bacteria in the fermentation of

65

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Developments and Applications of Enzymes From Thermophilic Microorganisms

lignocellulosic hydrolysates to ethanol. Thermoanaerobacter mathranii, which is an anaerobic thermophilic bacterium, is found to ferment the xylose in the hemicellulosic fraction from alkaline wet oxidation of wheat straw to ethanol with no preceding detoxification (Klinke et al., 2004). The growth of pretreated lignocellulosic material is found to vary dependent upon both the microorganism and the substrate origin (Lynd et al., 2002). Furthermore, the insolubility of lignocellulose poses problems in maintaining homogeneity within the reactor, making it difficult to monitor and control process parameters. Therefore, similar to mesophiles, the efficient utilization of thermophiles in integrated bioprocesses should be thoroughly investigated. Solid culture of thermophiles on lignocellulose has recently been reported (Hatzinikolaou et al., 2001; Patel et al., 2006). In some cases, better conversions were achieved in solid cultures in comparison to more traditional submerged liquid fermentations (Chinn et al., 2006). However, using natural microorganisms is generally not proficient enough to convert substrates into more valuable products. Therefore, it is imperative to improve microbial robustness for increasing substrate hydrolysis and product yield by the use of metabolic engineering. This was performed in a mesophilic host that resulted in strains of biorefinery interest. These strains produced higher yields of ethanol, acetate, adipic acid, propanediol, lactic acid, and succinic acid. But, not many reports of such metabolic engineering for thermophiles are available (Altaras and Cameron, 1999; Nakamura et al., 2000; Causey et al., 2003; Nie et al., 2002; Vemuri et al., 2002; Zhou et al., 2003; Tao et al., 2001; Desai et al., 2004; Ingram et al., 1999). However, these may increase due to the availability or development of genetic tools. Some thermophiles like Clostridium thermohydrosulfuricum, Moorella sp. HUC22-1, and Thermoanaerobium brockii have been studied for producing bioethanol (Sakai et al., 2004; Ben-Bassat et al., 1981; Lovitt et al., 1984). Such metabolic engineering of thermophiles for improving ethanol productivity and efficiency using different substrates such as hemicelluloses, cellulose, and pectin is of great interest (Turner et al., 2007).

5.3 Thermophiles in biorefineries The application of thermophiles in lignocellulosic ethanol biorefining is gaining a lot of interest (Espliego et al., 2018; Margaryan et al., 2018). Thermophilic microorganisms are a source of efficient carbohydrate-degrading enzymes. In second-generation biorefineries, the use of thermophilic enzymes may allow the process to take place at high temperatures (Zuliani et al., 2021). The benefits are presented in Table 5.2 (Taylor et al., 2009; Viikari et al., 2007). It is vital that the production process for second-generation bioethanol must be able to utilize all types of sugars obtained from lignocellulose, including cellobiose, xylose, and arabinose. The economic feasibility can be improved if second-generation ethanol production makes use of all sugars obtained from lignocelluloses. Furthermore, the

Enzyme production by thermophiles

Table 5.2 Benefits of using thermophilic microorganisms in second-generation biorefineries.

Enhanced solubility of substrate and product Higher rate of reaction Reduced amount of required enzyme Better mixing because of reduction in viscosity of the medium Contamination risk is reduced Recovery of product becomes easier Costs for cooling the reaction vessel are reduced Based on Viikari, L., Alapuranen, M., Puranen, T., Vehmaanper€a, J., Siika-aho, M. (2007). Thermostable enzymes in lignocellulose hydrolysis. In: Biofuels. Olsson, L. (Ed.). Springer, Berlin/Heidelberg, Germany, pp. 121e145. ISBN 9783-540-73651-6; Taylor, M.P., Eley, K.L., Martin, S., Tuffin, M.I., Burton, S.G., Cowan, D.A. (2009). Thermophilic ethanologenesis: future prospects for second-generation bioethanol production. Trends Biotechnol. 27, 398e405.

likelihood of obtaining simultaneous saccharification and fermentation (SSF) process configuration is gathering considerable interest as it shows several benefits. These are listed below (Fonseca et al., 2008): (i) Saving in energy because of the reduced cooling cost (ii) High rate of saccharification and fermentation (iii) Continuous removal of ethanol under reduced working pressure (iv) Reduced risk of contamination Commercial ethanol biorefineries use S. cerevisiae, which is not able to ferment all the sugars obtained from the hydrolysis of lignocelluloses. Attempts were made to engineer S. cerevisiae for use of pentose sugars. It is a mesophile and therefore, its optimum temperature is 30 to 5 C. This limits the attainment of a proper one-pot/one-phase SSF (Jansen et al., 2017). K. marxianus is a thermotolerant ascomycetous yeast. This is able to grow up to temperatures of 52 C and is able to metabolize different types of substrates (including less conventional sugars like lactose, xylose, arabinose, and inulin to ethanol (Fonseca et al., 2008; Kuloyo et al., 2014). K. marxianus shows multiple biotechnological potentials for industrial applications. Interest in this yeast has significantly increased during the last 10 years for producing ethanol and other chemicals (Fonseca et al., 2008; Leonel et al., 2021; Nurcholis et al., 2020; Karim et al., 2020). Table 5.3 presents some examples of fermentations conducted with K. marxianus at a temperature of 40 C or higher in SSF. K. marxianus is a temperature tolerant (i.e., 48 C) yeast and can substitute S. cerevisiae (Madeira and Gombert 2018). da Silva et al. (2018) made a comparison of ethanol production using the SSF process from pretreated carnauba straw residues. They used S. cerevisiae or K. marxianus at 35, 40, and 45 C. Fermentation conducted at 45 C by K. marxianus showed higher ethanol production in comparison to S. cerevisiae at its optimum temperature (i.e., 35 C). de Araujo Guilherme et al. (2019) studied the production of ethanol from bagasse in the SSF process using S. cerevisiae and K. marxianus. Bagasse was pretreated with different

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68

Temp (8C)

Fermentation timea

Process scheme

Maximum ethanol titerb

Maximum ethanol yieldc

Prosopis juliflora woody stems

41

72 h (72 h)

SSF

0.67 g/g

0.67 g/g

Cashew apple bagasse

40

72 h (w28 h)

SSF

68 g/L

80.70%

Sugarcane leaves

40

132 h (w48 h)

SSF

5.59 g/L

0.10 g/g dry weight

Water hyacinth

42

24 h (24 h)

SSF

7.34 g/L

0.16 g/g biomass

Empty palm fruit bunches Carnauba straw residue

42

w28 h

SSF

45

48 h (12 h)

SSF

Sugarcane bagasse

43

24 h

SSF

Bamboo

42.5

108 h (108 h)

SSF

26.04 g/L

Rice husk

60

120 h

CBP

1 g/

Sugarcane bagasse

60

120 h

CBP

1.21 g/L

Corn straw

55

168 h (120 h)

CBP

0.45 g/L

Strain [reference]

Feedstock

K. marxianus MTCC 1389 (Sivarathnakumar et al., 2019) K. marxianus ATCC 36,907 (de Barros et al., 2017) K. marxianus S1.17 (Jutakanoke et al., 2017) K. marxianus K213 (Yan et al., 2015) K. marxianus KCTC7001 (Jung et al., 2015) K. marxianus ATCC 36,907 (da Silva et al., 2018) K. marxianus ATCC 36,907 (de Araujo et al., 2019) K. marxianus TY16 (Ganesan, 2019) C. thermocellum DSM 1313 (Nisha et al., 2017) C. thermocellum (Nisha et al., 2017) C. thermocellum ATCC 27,405 and T. thermosaccharolyticum DSM 571 (Pang et al., 2018)

7.80% 7.52 g/L

75.29%

4.18 g/100 g biomass

11.20%

Developments and Applications of Enzymes From Thermophilic Microorganisms

Table 5.3 Use of thermophiles for production of bioethanol using industrially relevant feedstocks.

a

Salix

55

168 h

CBP

0.2 g/L

11.10%

Banana Agro-waste

60

120 h

CBP

Sugarcane bagasse

55

168 h

CBP

10.60 mM

Rice straw slurry

60

144 h (144 h)

CBP

142 mM

48%

Sugarcane bagasse

60

60 h (w28 h)

CBP

0.86 g/L

83.30%

Switchgrass

75

50 h (15 h)

CBP

w2 mM

Poplar (transgenic)

65

168 h

CBP

18.3 mM

Food waste

60

120 h (120 h)

CBP

18.4 g/L

0.41 g/g substrate

0.24 g/g sugar

If available in the source paper, the time at which the ethanol titer reached its maximum is reported in brackets. Ethanol titers. c Yields are reported as specified in the source paper. Zuliani, L., Serpico, A., De Simone, M., Frison, N., Fusco, S., 2021. Biorefinery gets hot: thermophilic enzymes and microorganisms for second-generation bioethanol production. Processes 9 (9), 1583. https://doi.org/10.3390/pr9091583. Distributed under the Creative Commons CC BY 4.0 license. b

Enzyme production by thermophiles

C. thermocellum ATCC 27,405 and T. thermosaccharolyticum DSM 571 (Pang et al., 2019) C. thermocellum CT2 and Clostridium thermosaccharolyticum HG8 (Harish Kumar Reddy et al., 2010) C. thermocellum (Beri et al., 2021) Clostridium sp. DBT eIOCeC19 and Thermoanaerobacter sp. DBTeIOCeX2 (Singh et al., 2021) C. thermocellum DSM 1237 (Liu et al., 2020) Engineered C. bescii (Chung et al., 2015) C. bescii MACB 1058 (Straub et al., 2019) G. thermoglucosidasius and T. ethanolicus (Bibra et al., 2020)

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Developments and Applications of Enzymes From Thermophilic Microorganisms

methods. They found that K. marxianus showed a higher ethanol yield as compared to S. cerevisiae. With K. marxianus K213, the ethanol yield was higher by 1.78-fold when comparison was made with S. cerevisiae used in one-pot/one-phase SSF fermentation of pretreated water hyacinth (Yan et al., 2015). The performance of K. marxianus is comparable to or better than S. cerevisiae. So, more efforts must be made for implementing the use of K. marxianus (or other thermophilic microorganisms) instead of S. cerevisiae for establishing a one-pot/one-phase SSF process on a commercial scale for converting lignocelluloses into bioethanol. This will finally allow second-generation biorefineries to achieve the full advantages of an SSF technique, which is presently undervalorized given the present temperature compromise imposed using yeasts growing at mesophilic temperatures. But, the optimal temperature of 50 C remains unfulfilled by both presently used mesophilic and thermotolerant yeasts. Among ethanologenic thermophiles, many bacteria show optimum growth temperatures at or beyond the optimum activity (i.e., 45e55 C) of commercial enzyme cocktails presently used for saccharification. Furthermore, some of these are able to hydrolyze hemicelluloses and cellulose and ferment to ethanol, thereby representing an option to the presently used mesophiles. These features make these thermophilic microorganisms very interesting for achieving an inflexible one-pot/one-phase SSF process. Among the most characterized cellulolytic and ethanol-producing thermophiles is Clostridium thermocellum. It is able to solubilize lignocelluloses with a higher rate of conversion (Demain et al., 2005). So, it is a suitable organism for establishing consolidated bioprocessing (CBP) for the conversion of lignocelluloses to biofuels (Olson et al., 2012). C. thermocellum is a rod-shaped, gram-positive, obligate anaerobic bacterium. Its cellulolytic activity is due to individual free enzymes as well as secreted cell wallbound multihydrolytic complexes (i.e., cellulosomes). In particular, the comparison of its hydrolytic activity is often made with commercial enzyme mixtures. Furthermore, members of the genus Clostridium have been used in many biorefinery applications other than bioethanol production. Thermocellum cannot use C5 sugars and may also have inhibitory effects. C. thermocellum was engineered with the xylA and xylB genes from Thermoanaerobacter ethanolicus to facilitate xylose co-utilization with C6 sugars derived from cellulose. But, the practical application of genetically modified strains requires several studies to achieve effective degradation and cofermentation of cellulose as well as hemicellulose. Furthermore, despite numerous engineering endeavors on this organism, the ethanol produced by C. thermocellum remains inadequate to reach that of conventional fermentative microorganisms (e.g., S. cerevisiae and Z. mobilis). Though genetic advances have been made, C. thermocellum is often used as a lignocellulose solubilizer in cocultivation with ethanologenic partners that can utilize hemicellulosic sugars. Thermoanaerobacterium and Thermoanaerobacter are perfect fermentation partners as they are anaerobic xylanolytic thermophiles. These are able to tolerate higher incubation temperatures (55e65 C and 65e75 C, respectively) (Demain et al., 2005; Nisha et al., 2017; Lynd et al., 2016; Beri et al., 2020; Du et al., 2020; Liberato et al., 2019; Demain et al.,

Enzyme production by thermophiles

2005; Froese and Sparling, 2021; Beri et al., 2020; Xiong et al., 2018; Hon et al., 2017; Zuliani et al., 2021; Jiang et al., 2017; Bing et al., 2021; Scully and Orlygsson, 2015). Thermoanaerobacterium saccharolyticum can ferment hemicellulose but it is not able to ferment cellulose (Lynd et al., 2016; Herring et al., 2016). Genetic engineering has been used to remove fermentation pathways that produce by-products, thus allowing the development of a strain that can produce up to 90% ethanol (based on theoretical yield). But, with industrial raw materials, the inhibitory effect of the compounds generated, reduced the yield by about 76%. The final concentration of ethanol was around 30 g/L (Herring et al., 2016). Despite the development in genomic progress based on this bacterium, the finding that commercial enzyme mixtures become inactivated at lower redox conditions has prompted research on these microbes to concentrate on an expression and secretion strategy of heterologous cellulase enzymes (Herring et al., 2016; Currie et al., 2013). But, the expression levels of these enzymes are not found to be sufficient (Herring et al., 2016). Therefore, coculturing T. saccharolyticum with other thermophilic anaerobes (e.g., C. thermocellum) remains a pertinent strategy (He et al., 2011). Fermentation of corn straw (no pretreatment) and salix to bioethanol using a CBP coculture of Thermoanaerobacterium thermosaccharolyticum and C. thermocellum and at 55 C was studied by Pang et al. (2018, 2019). Ethanol yield of 11% was obtained. An ethanol concentration of 0.5 g/L from cofermentation of bagasse (sugarcane) by Thermoanaerobacterium aotearoense and C. thermocellum in a calcium carbonate buffered medium was obtained by Bu et al. (2017). The use of different thermophilic microorganisms for the production of bioethanol using industrially relevant raw materials is presented in Table 5.3.

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Bjornsdottir, S.H., Thorbjarnardottir, S.H., Eggertsson, G., 2005. Establishment of a gene transfer system for Rhodothermus marinus. Appl. Microbiol. Biotechnol. 66, 675e682. Böhme, B., Moritz, B., Wendler, J., Hertel, T.C., Ihling, C., Brandt, W., Pietzsch, M., 2020. Enzymatic activity and thermoresistance of improved microbial transglutaminase variants. Amino Acids 52, 313e326. Bu, J., Tian, Q.Q., Zhu, M.J., 2017. Enhanced biodegradation of sugar cane bagasse by coculture of Clostridium thermocellum and Thermoanaerobacterium aotearoense supplemented with CaCO3. Energy Fuels 31, 9477e9483. Causey, T.B., Zhou, S., Shanmugam, K.T., Ingram, L.O., 2003. Engineering the metabolism of Escherichia coli W3110 for the conversion of sugar to redox-neutral and oxidized products: homoacetate production. Proc. Natl. Acad. Sci. U. S. A 100, 825e832. Chinn, M.S., Nokes, S.E., Strobel, H.J., 2006. Screening of thermophilic anaerobic bacteria for solid substrate cultivation on lignocellulosic substrates. Biotechnol. Prog. 22, 53e59. Chung, D., Cha, M., Snyder, E.N., Elkins, J.G., Guss, A.M., Westpheling, J., 2015. Cellulosic ethanol production via consolidated bioprocessing at 750C by engineered Caldicellulosiruptor bescii. Biotechnol. Biofuels 8, 163. Ciaramella, M., Cannio, R., Moracci, M., Pisani, F.M., Rossi, M., 1995. Molecular biology of extremophiles. World J. Microbiol. Biotechnol. 11, 71e84. Contursi, P., Cannio, R., Prato, S., Fiorentino, G., Rossi, M., Bartolucci, S., 2003. Development of a genetic system for hyperthermophilic Archaea: expression of a moderate thermophilic bacterial alcohol dehydrogenase gene in Sulfolobus solfataricus. FEMS Microbiol. Lett. 218, 115e120. Currie, D.H., Herring, C.D., Guss, A.M., Olson, D.G., Hogsett, D.A., Lynd, L.R., 2013. Functional heterologous expression of an engineered full length CipA from Clostridium thermocellum in Thermoanaerobacterium saccharolyticum. Biotechnol. Biofuels 6, 32. da Silva, F.L., de Oliveira Campos, A., dos Santos, D.A., Batista Magalh~aes, E.R., de Macedo, G.R., dos Santos, E.S., 2018. Valorization of an agroextractive residuedcarnauba strawdfor the production of bioethanol by simultaneous saccharification and fermentation (SSF). Renew. Energy 127, 661e669. de Araujo Guilherme, A., Dantas, P.V.F., Padilha, C.E.A., dos Santos, E.S., de Macedo, G.R., 2019. Ethanol production from sugarcane bagasse: use of different fermentation strategies to enhance an environmental-friendly process. J. Environ. Manag. 234, 44e51. de Barros, E.M., Carvalho, V.M., Rodrigues, T.H.S., Rocha, M.V.P., Gonçalves, L.R.B., 2017. Comparison of strategies for the simultaneous saccharification and fermentation of cashew apple bagasse using a thermotolerant Kluyveromyces marxianus to enhance cellulosic ethanol production. Chem. Eng. J. 307, 939e947. Demain, A.L., Newcomb, M., Wu, J.H.D., 2005. Cellulase, clostridia, and ethanol. Microbiol. Mol. Biol. Rev. 69, 124e154. Demirjian, D.C., Moris-Varas, F., Cassidy, C.S., 2001. Enzymes from extremophiles. Curr. Opin. Chem. Biol. 5, 144e151. Desai, S.G., Guerinot, M.L., Lynd, L.R., 2004. Cloning of L-lactate dehydrogenase and elimination of lactic acid production via gene knockout in Thermoanaerobacterium saccharolyticum JW/SLYS485. Appl. Microbiol. Biotechnol. 65, 600e605. Du, Y., Zou, W., Zhang, K., Ye, G., Yang, J., 2020. Advances and applications of Clostridium co-culture systems in biotechnology. Front. Microbiol. 11, 1937. Duffner, F., Bertoldo, C., Andersen, J.T., Wagner, K., Antranikian, G., 2000. A new thermoactive pullulanase from Desulfurococcus mucosus: cloning, sequencing, purification and characterization of the recombinant enzyme after expression in Bacillus subtilis. J. Bacteriol. 182, 6331e6338. Espliego, J.M.E., Saiz, V.B., Torregrosa-Crespo, J., Luque, A.V., Camacho Carrasco, M.L., Pire, C., Bonete, M.J., Martínez-Espinosa, R.M., 2018. Extremophile enzymes and biotechnology. In: Extremophiles. CRC Press; Taylor & Francis, Boca Raton, FL, USA: Abingdon, UK, pp. 227e248. Fonseca, G.G., Heinzle, E., Wittmann, C., Gombert, A., 2008. The yeast Kluyveromyces marxianus and its biotechnological potential. Appl. Microbiol. Biotechnol. 79, 339e354. Froese, A.G., Sparling, R., 2021. Cross-feeding and wheat straw extractives enhance growth of Clostridium thermocellum-containing co-cultures for consolidated bioprocessing. Bioproc. Biosyst. Eng. 44, 819e830.

Enzyme production by thermophiles

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Leonel, L.V., Arruda, P.V., Chandel, A.K., Felipe, M.G.A., Sene, L., 2021. Kluyveromyces marxianus: a potential biocatalyst of renewable chemicals and lignocellulosic ethanol production. Crit. Rev. Biotechnol. 1e22, 1e39. Liberato, V., Benevenuti, C., Coelho, F., Botelho, A., Amaral, P., Pereira, N., Ferreira, T., 2019. Clostridium sp. as bio-catalyst for fuels and chemicals production in a biorefinery context. Catalysts 9, 962. Liu, Y., Xie, X., LiuW, X.H., Cao, Y., 2020. Consolidated bioprocess for bioethanol production from lignocellulosic biomass using Clostridium thermocellum DSM 1237. Bioresources 15, 8355e8368. Lovitt, R.W., Longin, R., Zeikus, J.G., 1984. Ethanol production by thermophilic bacteria: physiological comparison of solvent effects on parent and alcohol-tolerant strains of Clostridium thermohydrosulfuricum. Appl. Environ. Microbiol. 48, 171e177. Lucas, S., Toffin, L., Zivanovic, Y., Charlier, D., Moussard, H., Forterre, P., Prieur, D., Erauso, G., 2002. Construction of a shuttle vector for, and spheroplast transformation of, the hyperthermophilic archaeon Pyrococcus abyssi. Appl. Environ. Microbiol. 68, 5528e5536. Lynd, L.R., Van Zyl, W.H., McBride, J.E., Laser, M., 2005. Consolidated bioprocessing of cellulosic biomass: an update. Curr. Opin. Biotechnol. 16, 577e583. Lynd, L.R., Weimer, P.J., van Zyl, W.H., Pretorius, I.S., 2002. Microbial cellulose utilization: fundamentals and biotechnology. Microbiol. Mol. Biol. Rev. 66, 506e577. Lynd, L.R., Guss, A.M., Himmel, M.E., Beri, D., Herring, C., Holwerda, E.K., Murphy, S.J.L., Olson, D.G., Paye, J., Rydzak, T., Shao, X., 2016. Advances in consolidated bioprocessing using Clostridium thermocellum and Thermoanaerobacter saccharolyticum. In: Industrial Biotechnology. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, pp. 365e394. Madeira-Jr, J.V., Gombert, A.K., 2018. Towards high-temperature fuel ethanol production using Kluyveromyces marxianus: On the search for plug-in strains for the Brazilian sugarcane-based biorefinery. Biomass and Bioenergy 119, 217e228. Mai, V., Wiegel, J., 2000. Advances in development of a genetic system for Thermoanaerobacterium spp.: expression of genes encoding hydrolytic enzymes, development of a second shuttle vector, and integration of genes into the chromosome. Appl. Environ. Microbiol. 66, 4817e4821. Margaryan, A., Shahinyan, G., Hovhannisyan, P., Panosyan, H., Birkeland, N.K., Trchounian, A., 2018. Geobacillus and Anoxybacillus spp. from terrestrial geothermal springs worldwide: diversity and biotechnological applications. In: Egamberdieva, D., Birkeland, N.-K., Panosyan, H., Li, W.-J. (Eds.), Extremophiles in Eurasian Ecosystems: Ecology, Diversity, and Applications, vol 119e166. Springer, Singapore, ISBN 978-981-13-0329-6. Mather, M.W., Fee, J.A., 2007. Development of plasmid cloning vectors for Thermus thermophilus HB8: expression of a heterologous. Microb. Cell Factories 6, 9. Moracci, M., La Volpe, A., Pulitzer, J.F., Rossi, M., Ciaramella, M., 1992. Expression of the thermostable b-galactosidase gene from the archaebacterium Sulfolobus solfataricus in Saccharomyces cerevisiae and characterization of a new inducible promoter for heterologous expression. J. Bacteriol. 174, 873e882. Moreno, R., Zafra, O., Cava, F., Berenguer, J., 2003. Development of a gene expression vector for Thermus thermophilus based on the promoter of the respiratory nitrate reductase. Plasmid 49, 2e8. Nakamura, C.E., Gatenby, A.A., Hsu, A.K.-H., La Reau, R.D., Haynie, S.L., Diaz-Torres, M., Trimbur, D.E., Whited, G.M., Nagarajan, V., Payne, M.S., Picataggio, S.K., Nair, R.V., 2000. Method for the production of 1,3-propanediol by recombinant microorganisms. Patent. US 6013494. Nie, W., Draths, K.M., Frost, J.W., 2002. Benzene-free synthesis of adipic acid. Biotechnol. Prog. 18, 201e211. Nisha, M., Saranyah, K., Shankar, M., Saleena, L.M., 2017. Enhanced saccharification of lignocellulosic agricultural biomass and increased bioethanol titre using acclimated Clostridium thermocellum DSM1313. 3 Biotech 7, 35. Nurcholis, M., Lertwattanasakul, N., Rodrussamee, N., Kosaka, T., Murata, M., Yamada, M., 2020. Integration of comprehensive data and biotechnological tools for industrial applications of Kluyveromyces marxianus. Appl. Microbiol. Biotechnol. 104, 475e488. Olson, D.G., McBride, J.E., Joe Shaw, A., Lynd, L.R., 2012. Recent progress in consolidated bioprocessing. Curr. Opin. Biotechnol. 23, 396e405. Pang, J., Hao, M., Li, Y., Liu, J., Lan, H., Zhang, Y., Zhang, Q., Liu, Z., 2018. Consolidated bioprocessing using Clostridium thermocellum and Thermoanaerobacterium thermosaccharolyticum co-culture for enhancing ethanol production from corn straw. Bioresources 13, 8209e8221.

Enzyme production by thermophiles

Pang, J., Hao, M., Shi, Y., Li, Y., Zhu, M., Hu, J., Liu, J., Zhang, Q., Liu, Z., 2019. Enhancing the ethanol yield from salix using a Clostridium thermocellum and Thermoanaerobacterium thermosaccharolyticum co-culture system. Bioresources 13, 5377e5393. Patel, M.A., Ou, M.S., Harbrucker, R., Aldrich, H.C., Buszko, M.L., Ingram, L.O., Shanmugam, K.T., 2006. Isolation and characterization of acid-tolerant, thermophilic bacteria for effective fermentation of biomass- derived sugars to lactic acid. Appl. Environ. Microbiol. 72, 3228e3235. Ramchuran, S.O., Nordberg Karlsson, E., Velut, S., Mare de, L., Hagander, P., Holst, O., 2002. Production of heterologous thermostable glycoside hydrolases and the presence of host-cell proteases in substrate limited fed-batch cultures of Escherichia coli BL21(DE3). Appl. Microbiol. Biotechnol. 60, 408e416. Rigoldi, F., Donini, S., Redaelli, A., Parisini, E., Gautieri, A., 2018. Review: engineering of thermostable enzymes for industrial applications. APL Bioeng. 2, 011501. Sakai, S., Nakashimada, Y., Yoshimoto, H., Watanabe, S., Okada, H., Nishio, N., 2004. Ethanol production from H2 and CO2 by a newly isolated thermophilic bacterium, Moorella sp. HUC22-1. Biotechnol. Lett. 26, 1607e1612. Schiraldi, C., De Rosa, M., 2002. The production of biocatalysts and biomolecules from extremophiles. Trends Biotechnol. 20, 515e521. Scully, S.M., Orlygsson, J., 2015. Recent advances in second generation ethanol production by thermophilic bacteria. Energies 8, 1e30. Shinohara, M.L., Ihara, M., Abo, M., Hashida, M., Takagi, S., Beck, T.C., 2001. A novel thermostable branching enzyme from an extremely thermophilic bacterial species. Rhodothermus obamensis. Appl. Microbiol. Biotechnol. 57, 653e659. Singh, N., Gupta, R.P., Puri, S.K., Mathur, A.S., 2021. Bioethanol production from pretreated whole slurry rice straw by thermophilic co-culture. Fuel 301, 121074. Singh, S., Shukla, L., Khare, S., Nain, L., 2011. Detection and characterization of new thermostable endoglucanase from Aspergillus awamori strain F 18. J. Mycol. Plant Pathol. 41, 97e103. Sivarathnakumar, S., Jayamuthunagai, J., Baskar, G., Praveenkumar, R., Selvakumari, I.A.E., Bharathiraja, B., 2019. Bioethanol production from woody stem Prosopis juliflora using thermo tolerant yeast Kluyveromyces marxianus and its kinetics studies. Bioresour. Technol. 293, 122060. Soutschek-Bauer, E., Staudenbauer, W.L., 1987. Synthesis and secretion of a heat-stable carboxymethylcellulase from Clostridium thermocellum in Bacillus subtilis and Bacillus stearothermophilus. Mol. Gen. Genet. 208, 537e541. Stewart, J.D., 2001. Dehydrogenases and transaminases in asymmetric synthesis. Curr. Opin. Chem. Biol. 5, 120e129. Straub, C.T., Khatibi, P.A., Wang, J.P., Conway, J.M., Williams-Rhaesa, A.M., Peszlen, I.M., Chiang, V.L., Adams, M.W.W., Kelly, R.M., 2019. Quantitative fermentation of unpretreated transgenic poplar by Caldicellulosiruptor bescii. Nat. Commun. 10, 3548. Tao, H., Gonzalez, R., Martinez, A., Rodriguez, M., Ingram, L.O., Preston, J.F., Shanmugam, K.T., 2001. Engineering a homo-ethanol pathway in Escherichia coli: increased glycolytic flux and levels of expression of glycolytic genes during xylose fermentation. J. Bacteriol. 183, 2979e2988. Taylor, P.P., Pantaleone, D.P., Senkpeil, R.F., Fotheringham, I.G., 1998. Novel biosynthetic approaches to the production of unnatural amino acids using transaminases. Trends Biotechnol. 16, 412e418. Taylor, M.P., Eley, K.L., Martin, S., Tuffin, M.I., Burton, S.G., Cowan, D.A., 2009. Thermophilic ethanologenesis: future prospects for second-generation bioethanol production. Trends Biotechnol. 27, 398e405. Turner, P., Mamo, G., Karlsson, E.N., 2007. Potential and utilization of thermophiles and thermostable enzymes in biorefining. Microb. Cell Factories 6, 9. van Beilen, J.B., Li, Z., 2002. Enzyme technology: an overview. Curr. Opin. Biotechnol. 3, 338e344. Vemuri, G.N., Eiteman, M.A., Altman, E., 2002. Effects of growth mode and pyruvate decarboxylase on succinic acid production by metabolically engineered strains of Escherichia coli. Appl. Environ. Microbiol. 68, 1715e1727. Viikari, L., Alapuranen, M., Puranen, T., Vehmaanper€a, J., Siika-aho, M., 2007. Thermostable Enzymes in Lignocellulose Hydrolysis. In: Olsson, L. (Ed.), Biofuels. Springer, Berlin/Heidelberg, Germany, ISBN 978-3-540-73651-6, pp. 121e145.

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Walsh, D.J., Gibbs, M.D., Bergquist, P.L., 1998. Expression and secretion of a xylanase from the extreme thermophile Thermotoga strain FjSS3-B1 in Kluyveromyces lactis. Extremophiles 2, 2e16. Xiong, W., Reyes, L.H., Michener, W.E., Maness, P.C.C.K.J., 2018. Engineering cellulolytic bacterium Clostridium thermocellum to co-ferment cellulose- and hemicellulose-derived sugars simultaneously. Biotechnol. Bioeng. 115, 1755e1763. Yan, J., Wei, Z., Wang, Q., He, M., Li, S., Irbis, C., 2015. Bioethanol production from sodium hydroxide/ hydrogen peroxide-pretreated water hyacinth via simultaneous saccharification and fermentation with a newly isolated thermotolerant Kluyveromyces marxianu strain. Bioresour. Technol. 193, 103e109. Zaks, A., 2001. Industrial biocatalysis. Curr. Opin. Chem. Biol. 5, 130e136. Zhou, S.D., Causey, T.B., Hasona, A., Shanmugam, K.T., Ingram, L.O., 2003. Production of optically pure D-lactic acid in mineral salts medium by metabolically engineered Escherichia coli W3110. Appl. Environ. Microbiol. 69, 399e407. Zuliani, L., Serpico, A., De Simone, M., Frison, N., Fusco, S., 2021. Biorefinery gets hot: thermophilic enzymes and microorganisms for second-generation bioethanol production. Processes 9 (9), 1583. https:// doi.org/10.3390/pr9091583.

Further reading Bergquist, P.L., Gibbs, M.D., Morris, D., 1997. Xylanase from Dictyoglomus thermophilum and its use in bleaching of cellulose products. Patent. WO 9736995. Bhalla, A., Bansal, N., Kumar, S., Bischoff, K.M., Sani, R.K., 2013. Improved lignocellulose conversion to biofuels with thermophilic bacteria and thermostable enzymes. Bioresour. Technol. 128, 751e759. Bhalla, A., Bischoff, K.M., Sani, R.K., 2015. Highly thermostable xylanase production from a thermophilic Geobacillus sp. strain WSUCF1 utilizing lignocellulosic biomass. Front. Bioeng. Biotechnol. 3, 84. Blumer-Schuette, S.E., Brown, S.D., Sander, K.B., Bayer, E.A., Kataeva, I., Zurawski, J.V., Conway, J.M., Adams, M.W.W., Kelly, R.M., 2014. Thermophilic lignocellulose deconstruction. FEMS Microbiol. Rev. 38, 393e448. Kim, S.K., Jin, Y.S., Choi, I.G., Park, Y.C., Seo, J.H., 2015. Enhanced tolerance of Saccharomyces cerevisiae to multiple lignocelluloses derived inhibitors through modulation of spermidine contents. Metab. Eng. 29, 46e55. Kim, S.K., Westpheling, J., 2018. Engineering a spermidine biosynthetic pathway in Clostridium thermocellum results in increased resistance to furans and increased ethanol production. Metab. Eng. 49, 267e274. Limayem, A., Ricke, S.C., 2012. Lignocellulosic biomass for bioethanol production: current perspectives, potential issues and future prospects. Prog. Energy Combust. Sci. 38, 449e467. Patel, A., Shah, A.R., 2021. Integrated lignocellulosic biorefinery: gateway for production of second generation ethanol and value added products. J. Bioresour. Bioprod. 6, 108e128. Papanek, B., Biswas, R., Rydzak, T., Guss, A.M., 2015. Elimination of metabolic pathways to all traditional fermentation products increases ethanol yields in Clostridium thermocellum. Metab. Eng. 32, 49e54. Straub, C.T., Bing, R.G., Wang, J.P., Chiang, V.L., Adams, M.W.W., Kelly, R.M., 2020. Use of the lignocellulose-degrading bacterium Caldicellulosiruptor bescii to assess recalcitrance and conversion of wild-type and transgenic poplar. Biotechnol. Biofuels 13, 43. Straub, C.T., Khatibi, P.A., Otten, J.K., Adams, M.W.W., Kelly, R.M., 2019a. Lignocellulose solubilization and conversion by extremely thermophilic Caldicellulosiruptor bescii improves by maintaining metabolic activity. Biotechnol. Bioeng. 116, 1901e1908. Tian, L., Perot, S.J., Stevenson, D., Jacobson, T., Lanahan, A.A., Amador-Noguez, D., Olson, D.G., Lynd, L.R., 2017. Metabolome analysis reveals a role for glyceraldehyde 3-phosphate dehydrogenase in the inhibition of C. thermocellum by ethanol. Biotechnol. Biofuels 10, 276. Zhu, D., Adebisi, W.A., Ahmad, F., Sethupathy, S., Danso, B., Sun, J., 2020. Recent development of extremophilic bacteria and their application in biorefinery. Front. Bioeng. Biotechnol. 8, 483.

CHAPTER 6

Current status of hyperthermophilic enzyme production 6.1 Introduction Most of the enzymes are produced on an industrial scale by microorganisms by using submerged fermentation (SmF) or solid-state fermentation (SSF) (Chisti, 2010). Submerged fermentation uses a liquid medium for growing the microorganisms whereas in solid-state fermentation solid substrate is used for growing microorganisms. There are several benefits of producing enzymes by fermentation. It ensures a higher production yield and the product quality is also consistent. Also, enzymes specifically targeted to carry out explicit jobs under requisite conditions can be obtained. After the completion of the fermentation, the culture is made inactive and removed by filtration or centrifugation and the resulting enzyme is separated from the fermentation broth. Afterward, the enzyme concentrate is purified, standardized, and stabilized with diluents to deliver liquid or granulated enzyme products, depending upon the application it will be used in (https:// amfep.org/about-enzymes/production/). Table 6.1 shows various steps involved in enzyme production.

Table 6.1 Steps involved in enzyme production.

Step 1 Selection of suitable enzyme Step 2 Selection of suitable production strain Step 3 Production methodology Submerged fermentation Solid-state fermentation Step 4 Methods for enhancing production Step 5 Downstream processing Step 6 Formulation of a stable product Based on Singhania, R.R., Patel, A.K., Pandy, A., 2010. Industrial Biotechnology: Sustainable Growth and Economic Success. In: Soetaert, W., Vandamme, E.J., (Eds.), Wiley VCH Verlag GmbH: Weinheim, Germany, pp. 207e226.

Developments and Applications of Enzymes From Thermophilic Microorganisms ISBN 978-0-443-19197-8, https://doi.org/10.1016/B978-0-443-19197-8.00025-6

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Developments and Applications of Enzymes From Thermophilic Microorganisms

The main commercial enzymes are produced by generally recognized as safe (GRAS)dstatus microorganisms in the fermenters. But, such genetically modified microorganisms (GMMs) are not part of the final enzyme product. After fermentation, the microorganisms are inactivated and then removed from the fermentation broth (containing the enzyme). But, few enzymes are still obtained from plant or animal tissues. Enzymes of plant origin include proteasesdbromelain, papain, ficin, and other specialized enzymes like soybean lipoxygenase. Enzymes of animal origin include proteases such as renin and pepsin. As a rule, the producing organisms and often individual enzymes are genetically modified to achieve the highest production yields and ideal enzymatic properties. Subsequent processing depends on the required purity. This actually depends upon the type of application. The industrial enzymes produced on a large scale are not generally purified. These are sold as dry products in the granular form or concentrated liquids. For special applications like diagnostics and DNA technology, highly purified enzymes are required (Singhania et al., 2010).

6.2 Current status of hyperthermophilic enzyme production Microbes have played a very important role in the discovery and development of new industrial enzymes from mesophiles. Novozymes began commercial production and marketing of proteolytic enzymes from Bacillus sp. for application in detergents since 1960s. Haron et al. (2018) reported that roughly 80% of the laboratory studies were performed by the use of microbes growing at moderate temperatures. But, many of the existing industrial processes are performed under severe conditions of temperature, pH as well as salinity. The enzymes produced from mesophilic organisms, require a narrow range of optimal conditions for functioning, but are not able to withstand harsh conditions that lead to denaturation of the enzyme. So, the requirement of new enzymes that can tolerate harsh industrial conditions has drawn the attention of extremophiles as a sustainable and environmentally friendly option. Extremophilic microbes are a very good source of natural enzymes. These are much better in comparison to their mesophilic counterparts for use at severe conditions. Hyperthermophilic enzymes have been reported as a catalyst for several significant commercial processes due to their better stability at higher temperatures. But, the yields of these enzymes are generally lower, making it difficult to commercially produce the desired enzymes from the starting microorganism in an uncontaminated state. Therefore, an alternative is to express hyperthermophilic enzymes in their mesophilic counterparts as purification is facilitated and enzymes of fairly higher purity appropriate for commercial use can be obtained (Sarmiento et al., 2015). In the laboratory, hyperthermophiles produce large amounts of enzymes but on an industrial scale the quantity produced is not sufficient (Ebaid et al., 2019). These days, the development and characterization of enzymes from novel hyperthermophilic microorganisms is important for several industries which need severe

Current status of hyperthermophilic enzyme production

conditions. For instance, the optimal conditions of these enzymes correlate with the severe conditions needed for the liquefaction of starch, which is pH 4.0e5.0, and temperature, 100
C (Niehaus et al., 1999). Table 6.2 presents the examples of few most thermostable enzymes (Atalah et al., 2019). Table 6.3 presents some of the commercial hyperthermophilic enzymes for diverse applications. Thermococcus hydrothermalis, T. profundus, Pyrococcus furiosus, and P. woesei have produced the most prevalent hyperthermophilic amylases, with an optimal temperature of about 100
C. Both Escherichia coli and Bacillus subtilis were able to clone and express the extracellular amylase from P. furiosus with success (Jrgensen et al., 1997; Dong et al., 1997). To achieve a superior reduction in mash viscosity, Verenium Corporation is currently selling the Fuelzyme, which is a hyperthermophilic a-amylase for the liquefaction of starch under challenging conditions. This enzyme is used to improve starch conversion for higher ethanol yields and operates best at a temperature of 88e91
C. Another thermostable amylase made from B. subtilis is called Novamyl. Novozymes produces stearothermophilus for use in commercial baking. Additionally, B. subtilis produces the a-amylase, which is employed in brewing processes. This is sold under the trade name AlphaStar PLUS. Numerous xylose isomerases have been found in various organisms, such as Thermotoga maritima, T. thermophilus, T. neapolitana, and T. aquaticus, although they are not available commercially (Ebaid et al., 2019; Sarmiento et al., 2015). Xylanases have stimulated great interest because of their potential application in several industries. Use of xylanases in pulp and paper industry has attracted significant attention in recent years (Sarmiento et al., 2015). The most important application of xylanases is in the biobleaching of kraft pulp. Xylanases are also being used for reducing beating time in virgin pulps and the water retention and fibrillation of pulp. Xylanases have also been used for deinking of recycled fibers and increase freeness and restore bonding. A few other applications include the retting of flax fibers, selective removal of xylan from dissolving pulp, and reduced vessel picking in hardwood pulps. Xylanases also remove bark, shives, pitch, and slime (https://www.creative-enzymes.com/resource/application-ofenzymes-in-pulp-and-paper-industry_64.html). Xylanases degrade hemicelluloses found in plant cell walls by breaking down the b-1,4-xylan into xylose. This releases lignin (Shi et al., 2013; Jiang et al., 2006). Many microorganisms, including Streptomyces sp., Thermoascus aurantiacus, T. maritima, and T. thermarum, have been found to produce hyperthermophilic xylanases. Currently, some hyperthermophilic xylanases are available for use in pulp and paper applications. For instance, Verenium Company is marketing Luminase PB-200, which is a highly thermostable xylanase that can be used up to 90
C. Additionally, Megazyme has developed Xyn 10A, which is a recombinant T. maritima endo-1,4-b-d-xylanase. It exhibits thermal stability up to 90
C. Many highly thermostable laccases have been reported for bleaching applications. A patent for laccase,

79

80

Enzyme properties Type of enzyme

Organism

Enzyme expression

Optimal temperature

Optimal pH

Thermostability half-life

Lipase EC 3.1.1.3

Pyrococcus furiosus

Recombinant

80
C

7.0

6 h at 75
C

Bacillus sp. 1-1T19 Ureibocillus thermosphoericus Stenotrophomonas maltophilia Psi-1 Archaeoglobus fulgidus Dictyoglomus thermophilum Nonomuraea flexuosa

Recombinant Recombinant

70
C 80
C

9.0 8.0

2 h at 70
C >6 h at 70
C

Recombinant

70
C

8.0

N/A

Recombinant Recombinant

75
C 75
C

10.0 6.5

N/A >10 h at 80
C

Recombinant

80
C

8.0

4.5 h at 80
C

Caldicellulosiruptor owensensis Thermotoga neapolitana Pyrodictium abyssi

Recombinant

90
C

7.0

1 h at 80
C

Recombinant Native

102
C 110
C

5.5e6.0 5.5

2 h at 100
C 1.2 h at 105
C

Bacillus pollidus

Native

65
C

7.6

2.5 h a 60
C

Thermotoga maritima MS88. Geobacillus pallidus

Recombinant

45
C

7.5

1 h at 75
C

Recombinant

50
C

7.0

5 h at 60
C

Pyrococcus obyssi Pyrococcus sp. M24D13

Recombinant Native

80
C 90
C

7.4 7.0

6 h at 90
C 8 h at 90
C

Xylanase EC 3.2.1.8

Nitrilase EC 3.5.5.1

Reference

Alqueres et al. (2011) Li and Liu (2017) Samoylova et al. (2018) Parapouli et al. (2018) Chen et al. (2009) McCarthy et al. (2000) Hakulinen et al. (2003) Liu et al. (2018) Zverlov et al. (1996) Andrade et al. (2001) Almatawah et al. (1999) Chen et al. (2015) Makhongela et al. (2007) Mueller et al. (2006) Dennett and Blarney (2016)

Developments and Applications of Enzymes From Thermophilic Microorganisms

Table 6.2 Example of thermophilic enzymes.

Transaminase EC 2.6.1.X

GDH EC 1.4.1.2 GDH EC 1.4.1.3

GDH EC 1.4.1.4

Recombinant

80
C

7.5

5 h at 70
C

Recombinant

65
C

9.0

N/A

Recombinant

65
C

9.5

22 days at 50
C

Vulcanisaeta moutnovskia Cloned from a Metagenome Bacillus sp. Pyrococcus furiosus

Recombinant

90
C

8.0

>5 h at 70
C

Recombinant

80
C

9.0

>7 days at 80
C

Native Native

65
C 85
C

8.0 N/A

>8 h at 65
C 10.5 h at 100
C

Thermotoga maritima Thermococcus litoralis Aeropyrum pernix K1 Thermus thermophilus Aquifex aeolicus

Recombinant Native Native Recombinant Recombinant

75
C 95
C 95
C 92
C 75
C

N/A 8.0 7.0 5.5 7.0

1.8 h at 85
C 2 h at 100
C >5 h at 100
C 14 h at 85
C 1 h at 80
C

Thermobacullum terrenum Bacillus sp. PC-3

Recombinant

60
C

7.0

8 h at 80
C

M
arquez and Blamey (2019) Stekhanova et al. (2017) Ferrandi et al. (2017) Flores et al. (2016) Diruggiero and Robb (1995) Knapp et al. (1997) Ma et al. (1994) Helianti et al. (2001) Miyazaki (2005) Fernandes et al. (2007) Brander et al. (2015)

Native

60
C

7.0

3.75 h at 60
C

Sharma et al. (2019)

Mathew et al. (2016) Chen et al. (2016)

Atalah, J., C
aceres-Moreno, P., Espina, G., Blamey, J.M., 2019. Thermophiles and the applications of their enzymes as new biocatalysts. Bioresour. Technol. 280, 478e488. Reproduced with permission.

Current status of hyperthermophilic enzyme production

Laccase EC 1.3.10.2

Thermomicrobium roseum Geobacillus thermodenitrificans Alvidobulum sp. SLM16

81

82

Enzyme

Commercial product

Producer

Origin

Tmax(o C)

Applications

Amylases

AlphaStar PLUS TermamylÒ Novozymes LiquozymeÒ Novozymes FungamylÒ Novozymes NovamylÒ Novozymes Dexamyl-HTP

Dyadic Novozymes

Bacillus subtilis Bacillus licheniformis Bacillus licheniformis Aspergillus oryzae 85 Bacillus subtilis 80 Bacillus licheniformis Streptomyces murinus

90 105e105

Starch hydrolysis to form syrups. Modification of starch of coated paper. Industrial processes such as brewing, baking, digestive aids preparation, cakes, and fruit juices

Novozymes Novozymes Novozymes Varuna Biocell Novozymes

105e110 85 80 80e90

Glucose (xylose) isomerases

SweetzymeÒ

90

Glutamate Dehydrogenase

Glutamate dehydrogenase

Swissaustral

Escherichia coli

80

Amyloglucosidases protease PLUS xylanases, pectinases, mannanases, cellulases, b-xylosidases, a-larabinofuran osidases, amylases, protease, other

Protease AlphaStar CONC Xylanase PLUS Beta Glucanase BP CONC Panzea 10X BG

Dyadic Dyadic Dyadic Dyadic Dyadic Novozymes

Aspergillus niger Bacillus subtilis Trichoderma longibrachiatum Trichoderma longibrachiatum Bacillus Licheniformis

90 90 80 80

Isomerization equilibrium of glucose into fructose Research and diagnostics, aroma and flavor development in cheese, analysis in wine production Used in processing aids Hydrolysis of hemicellulose and cellulose to lower the molecular weight of polymers in brewing

Developments and Applications of Enzymes From Thermophilic Microorganisms

Table 6.3 Examples of some commercially available hyperthermophilic enzymes.

NovoCorÒ AD L LipozymeÒ TL IM ResinaseÒ HT

Novozymes Novozymes Novozymes

Candida antarctica Thermomyces lanuginosus Aspergillus oryzae

90 85 90

Glucose oxidase Xylanases

GluzymÒ LuminaseÒ PB-200 Xyn 10A

Novozymes VereniumMegazyme

Aspergillus niger Thermotoga maritima

80 90 90

Laccases

Laccases DeniliteÒ IIS

ASA Spezialenzyme Novozymes

Trametes sp. Myceliophthora thermophila

80 70

Cellulases/hemicellulases

FibreZymeÒ G5000 and FibreZymeÒ LBL CONC

Dyadic

Aspergillus oryzae

75

Stereoselective hydrolysis of esters and transesterification Pitch control Obtaining of stronger dough in bakery Kraft pulp treatment Used to obtain stronger glutenin bakery Bio-bleaching Research and diagnostic analysis Research and biobleaching Precipitation of phenolic substances, Enzymatic browning of food, and gluing of flake boards, Modification of elasticity and consistency of pastes, gums. Modify cellulose and hemicellulose components of virgin and recycled pulps

Ebaid, R., Wang, H., Sha, C., El-Fatah Abomohra, A., Shao, W., 2019. Recent trends in hyperthermophilic enzymes production and future perspectives for biofuel industry: a critical review. J. Clean. Prod. 238, 117925. Reproduced with permission.

Current status of hyperthermophilic enzyme production

Lipases and esterases

83

84

Developments and Applications of Enzymes From Thermophilic Microorganisms

which is thermally stable at 80
C, was granted by AB Vista in 2006 (Paloheimo et al., 2006). Swissaustral presently produces thermostable laccase up to 90
C for research and biobleaching. Hyperthermophilic lipases have been developed for reducing the deposition of the pitch for controlling pitch in the paper industry. Novozymes have developed Resinase HT. This lipase is produced from Aspergillus oryzae. It is stable up to a temperature of 90
C. One of the most significant hyperthermophilic enzymes with numerous industrial uses is cellulase. Currently, these are used in the pulp and paper industry for increasing refining efficiency and to enhance the brightness and strength properties of paper sheets (Atalah et al., 2019; Kuhad et al., 2011). Graham et al. (2011) reported a novel recombinant archaeal cellulase that exhibited optimal activity at extreme temperatures of 109
C. This enzyme showed higher resistance to ionic liquids, higher salinity, and strong detergents. Cellulase and Hemicellulase enzymes are now commercially produced by many companies. Dyadic International is marketing FibreZyme LDI, Fiberzyme G5000, FibreZyme LBL CONC, and FibreZyme G4. Despite the recent advances in developing new industrial hyperthermophilic enzymes spur further efforts. Research and development is needed for developing and discovering enzymes showing better performance on an industrial scale (Ebaid et al., 2019). For improving microbial biomass and product formation substantially in extremophiles, high cell density cultivation is important. But, only lower cell yields have been obtained. This makes the application not very easy. The important reason may be attributed to the problems mostly related to produce and purify large amounts of enzymes and cellular components. Furthermore, extremophiles need special equipment for reaching and maintaining the optimal growth temperature and very high pH. There are two dissimilar strategies to solve this issue: e Recombinant DNA method to increase enzyme production in mesophiles e Innovative design of bioreactor for improving the biomass yield. The buildup of toxic compounds is mainly accountable for reduced biomass yield. Therefore, dialysis fermentation with several extremophiles has been conducted for efficiently removing low molecular-weight components from the culture broth. This results in a substantial increase in biomass yield. Biomass yield of 2.6 g/L was obtained when Pyrococcus furiosus, which is a hyperthermophilic archaeon was grown at 90
C. With thermoacidophile Sulfolobus shibatae (grown at 75
C, pH 3.5), a biomass yield of 114 g/L was obtained. In the case of S. shibatae, which grows at lower pH, the selection of a proper membrane was important. Cuprophan membranes, consisting of regenerated cellulose and polyamide membrane, were damaged after 2 days of use, most likely because of enzyme action. Polyethersulphonic membrane (porous, nontransparent) has been found to be stable. The fermentation process could be scaled up from 3 L to 30 L and up to 300 L. The pilot plant offers the chance to transfer the fermentation performance to

Current status of hyperthermophilic enzyme production

industry standards. By using external dialysis modules, even the results of a 1 L dialysis reactor could be reproduced in a 30 L reactor. In addition to the dialysis method, a novel microfiltration reactor (based on a microfiltration hollow-fiber module placed inside the fermenter) was designed to improve the biomass yield and also enzyme productivity. When Sulfolobus solfataricus, which is a thermoacidophilic archaeon was grown, a biomass yield of 35 g/L dry weight was achieved. It was approximately 20 times higher as compared to results attained in traditional batch fermenters (Bertoldo and Antranikian, 2011). At present, the industrial requirement for cellulase enzymes is being met by using SmF process usually genetically engineered strains of Hypocrea jecorina (anamorph Trichoderma reesei). There is a move toward the SSF systems because of higher production costs in SmF systems because of the longer fermentation time with reduced productivity. However, SmF systems show the benefits of better monitoring and handling (Sukumaran et al., 2005). Although there are many studies on the production of cellulase enzymes by SSF, the industry is still using the established technology of SmF as the SSF is still uncompetitive. The suitable technology, operational controls, and better design of bioreactors may make it feasible. For instance, the enzyme obtained in the SSF process can be used directly for agro-biotechnological applications like silage or feed additive, hydrolysis of lignocellulose, and processing of natural fibers. SSF may become a competitive technique for producing cellulase enzymes as it offers several benefits like better productivity, fairly higher concentration of the products, and reduced generation of effluents. Tengerdy (1996) made a comparison of the production of cellulases in SmF and SSF systems. The production cost in SSF was 10 times lesser as compared to SmF. Pandey et al. (1999) reported the remarkable potential of SSF for producing a variety of industrial enzymes and their direct use as a feed additive, silage, lignocellulose hydrolysis and processing of natural fibers. Da-Silva et al. (2005) used the SSF technique for producing xylanase and CMCase with Theroascus aurantiacus. They used different agricultural residues as substrates. The enzymes were found to be stable at room temperature over a wide range of pH from 3.0 to 9.0 and were also stable at a temperature of 60
C for 1 hour. The microorganisms were able to grow rapidly in the simple and economical medium. The properties of extracellular enzymes were found to match with those often needed in an industrial environment. Attempts have been made to produce cellulase enzymes by a fed-batch process instead of the batch process that facilitates to supersede the repression caused by a buildup of reducing sugars (Acharya and Chaudhary, 2012).

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Current status of hyperthermophilic enzyme production

Jiang, Z.Q., Li, X.T., Yang, S.Q., Li, L.T., Li, Y., Feng, W.Y., 2006. Biobleach boosting effect of recombinant xylanase B from the hyperthermophilic Thermotoga maritima on wheat straw pulp. Appl. Microbiol. Biotechnol. 70 (1), 65e71. Jørgensen, S., Vorgias, C.E., Antranikian, G., 1997. Cloning, sequencing, characterization, and expression of an extracellular a-amylase from the hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli and Bacillus subtilis. J. Biol. Chem. 272 (26), 16335e16342. Knapp, S., de Vos, W.M., Rice, D., Ladenstein, R., 1997. Crystal structure of glutamate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima at 3.0 Å resolution. J. Mol. Biol. 267, 916e932. Kuhad, R.C., Gupta, R., Khasa, Y.P., Singh, A., Zhang, Y.H.P., 2011. Bioethanol production from pentose sugars: current status and future prospects. Renewable Sustainable Energy Rev 15 (9), 4950e4962. Li, J., Liu, X., 2017. Identification and characterization of a novel thermophilic, organic solvent stable lipase of Bacillus from a hot spring. Lipids 52, 619e627. Liu, X., Liu, T., Zhang, Y., Xin, F., Mi, S., Wen, B., Gu, T., Shi, X., Wang, F., Sun, L., 2018. Structural insights into the thermophilic adaption mechanism of endo-1,4-b-xylanase from Caldicellulosiruptor owensensis. J. Agric. Food Chem. 66, 187e193. Ma, K., Robb, F.T., Adams, M.W., 1994. Purification and characterization of NADP-specific alcohol dehydrogenase and glutamate dehydrogenase from the hyperthermophilic archaeon Thermococcus litoralis. Appl. Environ. Microbiol. 60, 562e568. Makhongela, H.S., Glowacka, A.E., Agarkar, V.B., Sewell, B.T., Weber, B., Cameron, R.A., Cowan, D.A., Burton, S.G., 2007. A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity. Appl. Microbiol. Biotechnol. 75, 801e811. M
arquez, S.L., Blamey, J.M. 2019. Isolation and partial characterization of a new moderate thermophilic Albidovulum sp. SLM16 with transaminase activity from Deception Island, Antarctica. Biol. Res. 52, 5. Mathew, S., Deepankumar, K., Shin, G., Hong, E.Y., Kim, B.G., Chung, T., Yun, H., 2016. Identification of novel thermostable u-transaminase and its application for enzymatic synthesis of chiral amines at high temperature. RSC Adv. 6, 69257e69260. McCarthy, A.A., Morris, D.D., Bergquist, P.L., Baker, E.N., 2000. Structure of XynB, a highly thermostable beta-1,4-xylanase from Dictyoglomus thermophilum Rt46B.1, at 1.8 A resolution. Acta Crystallogr. D. Biol. Crystallogr. 56, 1367e1375. Miyazaki, K., 2005. A hyperthermophilic laccase from Thermus thermophilus HB27. Extremophiles 9, 415e425. Mueller, P., Egorova, K., Vorgias, C.E., Boutou, E., Trauthwein, H., Verseck, S., Antranikian, G., 2006. Cloning, overexpression, and characterization of a thermoactive nitrilase from the hyperthermophilic archaeon Pyrococcus abyssi. Protein Expr. Purif. 47, 672e681. Niehaus, F., Bertoldo, C., Kahler, M., Antranikian, G., 1999. Extremophiles as a source of novel enzymes for industrial application. Appl. Microbiol. Biotechnol. 51, 711e729. Paloheimo, M., Puranen, T., Valtakari, L., Kruus, K., Kallio, J., M€antyl€a, A., Fagerström, R., Ojapalo, P., Vehmaanper€a, J., 2006. Novel laccase enzymes and their uses. US Patent 77321784. Pandey, A., Selvakumar, P., Soccol, R.C., Nigam, P., 1999. Solid state fermentation for the production of industrial enzymes. Current Sci. 77 (1), 149e162. Parapouli, M., Foukis, A., Stergiou, P.Y., Koukouritaki, M., Magklaras, P., Gkini, O.A., Papamichael, E.M., Afendra, A.S., Hatziloukas, E., 2018. Molecular, biochemical and kinetic analysis of a novel, thermostable lipase (LipSm) from Stenotrophomonas maltophilia Psi-1, the first member of a new bacterial lipase family (XVIII). J. Biol. Res. 25, 4. Samoylova, Y.V., Sorokina, K.N., Romanenko, M.V., Parmon, V.N., 2018. Cloning, expression and characterization of the esterase estUT1 from Ureibacillus thermosphaericus which belongs to a new lipase family XVIII. Extremophiles 22, 271e285. Sarmiento, F., Peralta, R., Blamey, J.M., 2015. Cold and hot extremozymes: industrial relevance and current trends. Front. Bioeng. Biotechnol. 3, 148. Sharma, V., Ayothiraman, S., Dhakshinamoorthy, V., 2019. Production of highly thermo-tolerant laccase from novel thermophilic bacterium Bacillus sp. PC-3 and its application in functionalization of chitosan film. J. Biosci. Bioeng. 127 (6), 672e678.

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Shi, H., Zhang, Y., Li, X., Huang, Y., Wang, L., Wang, Y., Ding, H., Wang, F., 2013. A novel highly thermostable xylanase stimulated by Ca2þ from Thermotoga thermarum: cloning, expression and characterization. Biotechnol. Biofuels 6 (1), 26. Singhania, R.R., Patel, A.K., Pandy, A., 2010. In: Soetaert, W., Vandamme, E.J. (Eds.), Industrial Biotechnology: Sustainable Growth and Economic Success. Wiley VCH Verlag GmbH, Weinheim, Germany, pp. 207e226. Stekhanova, T.N., Rakitin, A.L., Mardanov, A.V., Bezsudnova, E.Y., Popov, V.O., 2017. A Novel highly thermostable branched-chain amino acid aminotransferase from the crenarchaeon Vulcanisaeta moutnovskia. Enzyme Microb. Technol. 96, 127e134. Sukumaran, R.K., Singhania, R.R., Pandey, A., 2005. Microbial cellulases-production, applications and challenges. J. Sci. Ind. Res. 64, 832e844. Tengerdy, R.P., 1996. Cellulase production by solid substrate fermentation. J. Sci. Ind. Res. 55, 313e316. Zverlov, V., Piotukh, K., Dakhova, O., Velikodvorskaya, G., Borriss, R., 1996. The multidomain xylanase A of the hyperthermophilic bacterium Thermotoga neapolitana is extremely thermoresistant. Appl. Microbiol. Biotechnol. 45, 245e247.

Further reading Bouzas, T., Barros-Vel
azquez, J., Gonzalez Villa, T., 2006. Industrial applications of hyperthermophilic enzymes: a review. Protein Pept. Lett. 13 (7), 645e651.

Relevant websites https://amfep.org/about-enzymes/production. https://www.creative-enzymes.com/resource/application-of-enzymes-in-pulp-and-paper-industry_64. html.

CHAPTER 7

Enhancement of production/activity of thermophilic/hyperthermophilic enzymes In recent years, hyperthermophilic enzymes have shown successful industrial applications but still, there are many issues that require more R&D. The most noticeable is to increase the production and/or efficacy of enzymes under severe industrial conditions. The current trends used to increase the production and/or efficacy of thermophilic/hyperthermophilic enzymes are presented here.

7.1 Optimization of growth conditions and medium composition The traditional methods for increasing the production of certain metabolite involves optimization of the production medium and the fermentation conditions. Before the 1970s, optimization of media was performed by using conventional methods, which were expensive, tedious, and involved several experiments with compromised accuracy. However, with the introduction of the latest mathematical/statistical methods, medium optimization is becoming more efficient in giving results. To design a production medium, the appropriate medium components and the fermentation conditions must be identified and optimized to obtain the maximum product concentration (Wang et al., 2011; Franco-Lara et al., 2006; Gupte and Kulkarni, 2003). Margaritis and Merchant (1986) performed single factorial experiments for optimizing the pH, temperature, and 18 different carbon sources to maximize the production of cellulase enzyme in Thielavia terrestris, which is a thermophilic fungus. Under the optimized conditions, the cellulase activity was 0.17 IU/mL. A total of 18 carbon sources were studied. Out of which, solka floc induced the maximum production of cellulase by T. terrestris. The optimum temperature for growth was between 44 and 52
C. The effect of pH on the growth of the fungus and the production of cellulase was studied. The maximum volumetric productivity of filter paper activity was obtained when pH was controlled in the range of 4.5e5.0. The production of endoglucanase from Streptomyces sp. B14 PNG23 was optimized by Azzeddine et al. (2013). The maximum activity of endoglucanase was 1.21 IU/mL. It was achieved in a medium containing 2 g/L wheat bran, 2 g/L yeast extract, 2 g/L sodium chloride, 2.5 g/L ammonium chloride, and 0.4 g/L magnesium sulfate. The

Developments and Applications of Enzymes From Thermophilic Microorganisms ISBN 978-0-443-19197-8, https://doi.org/10.1016/B978-0-443-19197-8.00001-3

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enzyme was found to be active at a temperature and pH range of 40e70
C and 5e8, respectively. The optimum pH and optimum temperature were 6.0 and 50
C, respectively. When the metal ions manganese (2þ), copper (2þ), and ammonium ion were present, the activity of the enzyme was found to increase considerably. The enzyme retained 50% of its activity when incubated at a temperature of 50
C for 6 h. Response surface methodology was used for optimizing cellulase production. This method also showed encouraging results for the production of cellulases from Aspergillus niger using solid-state fermentation. Xue et al. (2012) used Eichhornia crassipes as a main substrate and natural seawater as a source of mineral salts, which increased the production of cellulases. Raw rice straw and corn cob showed a considerable positive effect on the production of cellulases. The optimized medium contained E. crassipesd76.9% (w/w), raw rice strawd3.5% (w/w), raw corn cobd8.9% (w/w), raw wheat brand10.7% (w/w), and natural seawater, which was 2.33 times the weight of the dry substrates. Maximum biomass and cellulase production were obtained in 96 and 144 h, respectively. Cellulase production was 17.80 U/g the dry weight of substrates. The seawater medium avoided the pretreatment of substrates and also the use of chemicals. This method looks promising for ecofriendly production of cellulases. Jang and Chang (2005) produced thermostable cellulase from Streptomyces sp. T3-1 in a fermenter of 50 L capacity. Maximum production of cellulases was achieved on the fourth day at an agitation speed of 300 rpm and an aeration rate of 0.75 vvm. Maximum CMCase activity was 148 IU per mL; Avicelase was 45 IU per mL; b-glucosidase was 137 IU per mL with a productivity of 326 IU/L/h. These values were higher by 10%e32% as compared to the values achieved in shake-flasks. Nevertheless, optimization of enzyme production by developing special bioprocesses and use of specific conditions is not pertinent on an industrial scale. Furthermore, the increase in enzyme activity obtained by optimizing the medium components and growth conditions is still considerably less required for the industry. So, other advanced methods were examined for increasing the enzyme yield and reducing the cost of production for making this process more appealing for the industry.

7.2 Chemical methods Several chemical methods have been reported for improving the properties of thermophilic/hyperthermophilic enzymes. Chemical modification of enzymes is found to be an effective method to stabilize the proteins (Tyagi and Gupta, 1998; O’Fagain, 2003; Davis, 2003) (Fig. 7.1). Chemical modifications are usually not site-directed, except using specific chemicals directed at typical groups of protein structure for example thiol exchange, DielseAlder cycloaddition, and reaction with terminal amino groups (Palomo, 2010). For instance,

Enhancement of production/activity of thermophilic/hyperthermophilic enzymes

Figure 7.1 Chemical modification versus genetic modification. (Reproduced with Permission from Cowan, D.A., Fernandez-Lafuente, R., 2011. Enhancing the functional properties of thermophilic enzymes by chemical modification and immobilization. Enzyme Microb. Technol. 49 (4), 326e346.)

site-specific chemical modifications have been obtained by the use of thiol-reactive compounds and proteins with only a single surface Cysdeither natural or introduced by sitedirected mutagenesis (Wynn and Richards, 1993; DeSantis et al., 1998; Chalker et al., 2009) (Fig. 7.2). There are benefits if the modification is done in the solid phase with the use of immobilized enzymes (Rodrigues et al., 2011). The chemical modification can be controlled in a better way. The interactions of proteineprotein are minimized (Fig. 7.3). Immobilized enzyme when used makes easier the use of an incompatible medium and makes simpler the step-by-step chemical modification of enzymes (Fernandez-Lafuente et al., 1992; Fernandez-Lafuente, 2009; Rodrigues et al., 2011). The enzymes stabilized through multisubunit or multipoint immobilization also limit the amount of enzyme inactivated, which can result using chemical modification processes. By chemically modifying the amino acid side chains greater, a variety of groups can be introduced. It can be used for changing the properties of the enzyme surface or to alter the major residues (Davis,

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Figure 7.2 Site-directed modification of proteins. (Reproduced with Permission from Cowan, D.A., Fernandez-Lafuente, R., 2011. Enhancing the functional properties of thermophilic enzymes by chemical modification and immobilization. Enzyme Microb. Technol. 49 (4), 326e346.)

2003; Rodrigues et al., 2011; O’Fagain, 2003). However, the reactions used to introduce them are generally nonspecific in nature. Therefore, despite the several potential benefits, numerous traditional methods used for modifying proteins produce protein mixtures as a result of weakly differentiating or not adequately efficient chemistry. This is also worsened by the limited variety and several copy numbers of chemical functional groups in proteins. The effects of calcium on native as well as chemically modified forms of mesophilic and thermophilic a-amylases were conducted by Khajeh et al. (2001). Circular dichroism spectroscopy and irreversible heat activation studies were conducted with and without calcium at a dose level of 10 mM. Circular dichroism studies confirmed changes in the tertiary structure of these enzymes, caused by modification. Moreover, these changes were found to be affected when calcium was present. Sorbitol provided protection against irreversible heat inactivation of the native as well as modified forms of the enzyme both in the presence and absence of calcium. This strategy, which involves a combination of media and chemical modifications to stabilize proteins and improve catalytic activity, has proven useful.

Enhancement of production/activity of thermophilic/hyperthermophilic enzymes

Figure 7.3 Advantages of chemical modification of proteins on solid phases. (Reproduced with Permission from Cowan, D.A., Fernandez-Lafuente, R., 2011. Enhancing the functional properties of thermophilic enzymes by chemical modification and immobilization. Enzyme Microb. Technol. 49 (4), 326e346.)

Enzyme catalysis in organic solvents containing little water is finding a growing number of applications in different areas. Gupta and Roy (2004) described various approaches for achieving higher efficacy and stability in such media. Ultrasonication and microwaveassisted reaction are two promising strategies to increase the rate of reaction in a medium containing little water. Medium engineering and controlling water activity are two essential strategies in optimizing catalytic behavior in nonaqueous enzymology. This can play an important role in the future in organometallics and synthesis/modification of polymer areas. A better understanding of enzyme behavior in nonaqueous media would lead to better and still greater different types of applications. Alternatively, modification with chemicals can be used to change catalytic properties. Cabrera et al. (2009) performed physical or chemical modifications of Novozym 435. The physical modification included the ionic exchange of ionic polymers. Chemical modification included hydroxyethylamidation, succinylation, and aminoethylamidation. The resulting biocatalysts were used in different reactions.

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Coating the immobilized enzyme with dextran sulfate using ion exchange increased the asymmetry factor of the biocatalyst from A ¼ 13e24 by hydrolyzing 3-phenylglutarate dimethyl diester to form (R)-monomethyl ester. Chemical succinylation of Novozym 435 could increase the enantiospecificity of the biocatalyst from E ¼ 1e13 in the hydrolysis of ()-methyl mandelate. For the hydrolysis of ()-2O-butyryl-2-phenylacetic acid, the enantiospecificity of Novozym 435 was higher compared to the S-enantiomer. However, it was reversed by the chemical hydroxyethylamidation of the immobilized enzyme. Better performance was observed when glutaraldehyde-crosslinked lipase enzyme was adsorbed on aminated supports in the presence of detergents (Fernandez-Lorente et al., 2006). But, the impact of chemical modification on the enzyme properties is usually not easy to guess. This method can be also used for creating covalent cross-linking bonds between different groups on the surface of the enzyme. Where such cross-linking takes place between different structural elements of a protein, it generally increases the structural firmness and thereby enhances the stability of the protein with respect to agents which encourage conformational changes (like heat or chaotropic agents). In actual fact, intramolecular cross-linking is not easy. The agent used for crosslinking should be of proper length (Torchilin et al., 1979). When homo-bifunctional reagents are used, there might be tough competition between intra- and intermolecular cross-linking and single-point modification (Rodrigues et al., 2011). When multifunctional polymers are used, it simplifies the creation of multiple interconnections and is not severely constrained by distance or single-point modification. But, the bendable structure and length of the polymer are found to limit the degree of protein stiffening, which can be obtained (Betancor et al., 2004). On the other hand, these polymeric multirole reagents have efficiently avoided the breakup of multiprotein complexes or multimeric proteins (Rodrigues et al., 2011; Fernandez-Lafuente, 2009; Betancor et al., 2013). Tanaka et al. (1996) and Gupta and Bisaria (2018) have reported that chemical crosslinking of enzymes may be utilized for avoiding the breakup of multimeric enzymes. This is the widespread purpose for increasing the stability of the enzyme. For example, glutaraldehyde was used for cross-linking a-glucosidase enzyme of Thermococcus AN1 hyperthermophilic microbe both homogenously or mixed with bovine serum albumin (Piller et al., 1996). After cross linking, the half-life time was found to increase from 5 to 8 min at a temperature of 110
C. Additionally, in the presence of 50% trehalose, the cross-linked enzyme was found to show higher stability. The half-life increased from 30 to 90 min at 110
C in the wild-type enzyme. EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) was used for crosslinking Bacillus stearothermophilus SD1 tetrameric D-hydantoinase (Cheon et al., 2000). Crosslinking conditions were optimized for catalytic activity and stabilization of the resulting enzyme.

Enhancement of production/activity of thermophilic/hyperthermophilic enzymes

Although the native enzyme was more or less completely inactivated, the crosslinked D-hydantoinase exhibited a fourfold longer half-life under operating conditions and was highly stable at higher temperatures. Additionally, intersubunit cross-linking of D-hydantoinase also stabilized the enzyme in the presence of 20% methyl alcohol and under acidic conditions. The cross-linked enzyme effectively converted the substrate. This could be ascribed to the improved stability of the enzyme. Cross-linked enzyme aggregates (CLEAs) from a mixture of commercial cellulases were produced by Gupta and Bisaria (2018). The CLEAs were found to be more stable in comparison to the native enzymes. The half-lives were 2.30, 1.56, 3.07, and 1.67 times higher at a temperature of 70
C for filter paper activity, endoglucanases, b-glucosidases, and xylanases, respectively. CLEAs were found to retain 85.9% of xylanase activity and 77.4% of endoglucanase after five cycles of hydrolysis of soluble substrates like xylan and CMC, respectively. Soluble enzymes showed 31.8% saccharification and CLEAs showed 32.9% saccharification when wheat straw pretreated with alkali was subjected to hydrolysis. CLEAs produced higher saccharification yield. The yield was 43.3% in comparison to 31.8% with soluble enzymes on repeated batch hydrolysis for five successive cycles of 24 h each. Pant et al. (2015) and Xu et al. (2015) immobilized thermostable protease enzymes, for the production of peptides as well as protein hydrolysis. Immobilized extracellular protease enzyme from Thermus strain Rt41A was immobilized on controlled pore glass beads (Wilson et al., 1994). The enzyme remained stable at a broader pH range of 5e11 and a temperature of 80
C. Cowan and Fernandez-Lafuente (2011) immobilized the hyperthermophilic enzyme caldolysin on carboxymethyl cellulose, controlled pore glass, and Sepharose 4B. Caldolysin is a protease produced by Thermus aquaticus T351. It is an extracellular metalchelator-sensitive enzyme. But chemical modification of enzymes has several problems, which are mostly related to their harmful effect on the environment. Even though genetic engineering is displacing the chemical modification of enzymes, the latter remains an efficient technique for protein stabilization (Davis, 2003). Chemical modification has several advantages in comparison to genetic engineering. These are listed in the following (Cowan and Fernandez-Lafuente, 2011; Basle et al., 2010): ➢ Faster ➢ Larger range of chemical groups can be introduced to the enzyme ➢ Previous knowledge of the protein structure is not required ➢ Conducted on the correctly folded enzyme

7.3 Genetic engineering Enzymes from hyperthermophilic organisms are encoded by the cellular genes of thermophilic microorganisms that are typically grown under severe conditions and so

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heterogeneous expression of the gene has been found as the major strategy for obtaining such enzymes (Ebaid et al., 2019; Fusco et al., 2018; Xu et al., 2017). Heterogeneous gene expression is effectively used for producing enzymes for molecular biotechnology and medicinal/clinical proteins. Heterologous expression is actually the introduction of either complementary DNA or RNA encoding for a protein of interest from one species into the cell of another species, in such a way that the cellular machinery of the host expresses the foreign protein. Cultured immortalized cells can be transfected with cDNA short term (transiently), or long term (stable), depending on whether the foreign DNA is integrated into the host genome. Transient DNA expression typically lasts 24e72 h, whereas stable DNA expression potentially allows permanent overexpression of the protein. The most commonly used approach for producing hyperthermostable enzymes is to clone the gene encoding the enzyme of interest into a medium with reduced doubling times and a simpler and cheaper medium compared to the original hyperthermophilic organism (Atalah et al., 2019). Several industries produce products of lower value like textiles, paper, and biofuels, so increasing the productivity of industrial enzymes at lower cost poses challenges to existing gene expression methods. On this point, E. coli is an established host for several available commercial vectors (Ebaid et al., 2019; Sarmiento et al., 2015; Cabrera and Blamey, 2017). Table 7.1 shows the benefits of using E. coli as the host for heterogeneous protein expression. Table 7.1 Advantages of using E. coli as the host for heterogeneous protein expression.

It has unparalleled fast growth kinetics. In glucose-salts media and given the optimal environmental conditions, its doubling time is about 20 min High cell density cultures are easily achieved. The theoretical density limit of an E. coli liquid culture is estimated to be about 200 g dry cell weight/L or roughly 1  1013 viable bacteria/mL Rich complex media can be made from readily available and inexpensive components. Transformation with exogenous DNA is fast and easy. Plasmid transformation of E. coli can be performed in as little as 5 min Ease of growth and manipulation using simple laboratory equipment Availability of dozens of vectors and host strains that have been developed for maximizing expression A wealth of knowledge about the genetics and physiology of E. coli Based on Rosano, G.L., Ceccarelli, E.A., 2014. Recombinant protein expression in Escherichia coli: advances and challenges. Front. Microbiol. 5, 172. Published 2014 Apr 17. https://doi.org/10.3389/fmicb.2014.00172; Sezonov, G., JoseleauPetit, D., D’Ari, R., 2007. Escherichia coli physiology in Luria-Bertani broth. J. Bacteriol. 189, 8746e8749. https://doi.org/ 10.1128/JB.01368-07; Lee, S.Y., 1996. High cell-density culture of Escherichia coli. Trends Biotechnol. 14, 98e105. https://doi.org/10.1016/0167-7799(96)80930-9; Shiloach, J., Fass, R., 2005. Growing E. coli to high cell density e a historical perspective on method development. Biotechnol. Adv. 23, 345e357. https://doi.org/10.1016/j.biotechadv. 2005.04.004; Pope, B., Kent, H.M., 1996. High efficiency 5 min transformation of Escherichia coli. Nucleic Acids Res. 24, 536e537. https://doi.org/10.1093/nar/24.3.536.

Enhancement of production/activity of thermophilic/hyperthermophilic enzymes

Fig. 7.4 shows the structure of an expression plasmid. The nature of the promoter in the expression element is one of the main factors affecting gene transcription rate, target protein toxicity to the host, efficacy/cost of gene induction, and target protein degradation by the host. During the past 30 years, several E. coli expression vectors have been developed using the operator genes and their repressors from various sources. The most popular expression vectors are those that contain the lac promoter or hybrids thereof, the bacteriophage pL promoter and the T7 promoter. But because of protein misfolding and/or ineffective host cell expression, the target gene sequence may result in inclusion bodies (Ebaid et al., 2019). Additionally, E. coli builds up lipopolysaccharides endotoxin. It is not able to take out introns and has codon bias (Gomes et al., 2016). For overcoming the previously mentioned problems, instead of E. coli, B. subtilis was used as a host. But, the major drawbacks of using this organism are as follows: ➢ Plasmid not stable ➢ Reduced expression of protein ➢ Production of extracellular protease enzymes capable of degrading target enzymes (Ebaid et al., 2019) Conversely, eukaryotic organisms, like Saccharomyces cerevisiae, were used as a host for producing genetically engineered enzymes. S. cerevisiae grows quickly on inexpensive media, can be scaled up easily, and has high-level expression system.

Figure 7.4 Structure of an expression plasmid. (Reproduced with Permission from Ebaid, R., Wang, H., Sha, C., Abomohra, A.E., Shao, W., 2019. Recent trends in hyperthermophilic enzymes production and future perspectives for biofuel industry: a critical review. J. Clean. Prod. 238. https://doi.org/10.1016/j. jclepro.2019.117925.)

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However, the major drawbacks of using yeast as a host are codon bias, hyperglycosylation, and the slow production rates of target enzymes (Gomes et al., 2016). Given that the target expression process can fail due to differences in the gene expression machinery of various taxa, the phylogenetic distance between the host and the hyperthermophiles should be taken into account (Ekkers et al., 2012). Nowadays, mostly because of the lack of effective gene transfer systems in most of the hyperthermophilic organisms, it was not easy to apply molecular engineering to multiple hosts. Efforts have therefore been made to develop efficient genetic tools in hyperthermophilic microorganisms with improved cell manipulation potential while studying gene function in vivo (Ebaid et al., 2019). Stedman et al. (1999) reported developing multiple transformation techniques using shuttle vectors and viral DNA in Sulfolobus, the first hyperthermophilic archaea. To create a shuttle vector-based transformation system in Pyrococcus abyssiw, a host strain lacking the pyre gene and a pyrE marker from S. acidocaldarius was used (Lucas et al., 2002). Waege et al. (2010) used a simvastatin/HMG-CoA reductase overexpression cassette system for developing a shuttle vector for P. furiosus. Presently, S. acidocaldarius, S. solfataricus, and Sulfolobus islandicus can be transformed using several SulfolobuseE. coli shuttle vectors without difficulty (Aucelli et al., 2006; Deng et al., 2009; Berkner et al., 2010). The LacS gene was investigated using host cells lacking this gene as a selectable marker. Using the S. solfataricus lacS gene as a selectable marker for mutant lacS gene host cells demonstrated the potential for disruption of the gene in S. solfataricus (Worthington et al., 2003). Also, disruption of the gene was proficiently conducted in several Sulfolobus, like S. solfataricus (Su et al., 2018; Rolfsmeier and Haseltine, 2018) and S. acidocaldarius (Wagner et al., 2009). Several inducible promoters were examined in S. acidocaldarius, using lacS as a reporter gene (Berkner et al., 2010). The constitutive examined promoters, in this case, were the genes for glutamate dehydrogenase (gdhA) and abundant DNA binding protein (Sac7d). Sac7d was found as a good constitutive promoter for the expression of the gene in S. acidocaldarius because it led to higher expression levels than gdhA (Atomi et al., 2011). Numerous inducible promoters were also researched for S. solfataricus’s homologous and heterologous gene expression. For example, the comparison was made of genes for lacS (-glycosidase), dps (DNA binding protein from starved cells), and mal (or mbp, for maltose binding protein) (Haseltine et al., 1999). Notably, when the maltose or dextrin was added the level of expression increased by 17-fold using the mal promoter. Additionally, the use of a heat-inducible promoter from the heat shock chaperonin gene tf55a resulted in an additional 10-fold increase in the expression level of S. solfataricus after giving heat shock. Additionally, after supplementing with arabinose, the gene expression was increased by 13 times using the

Enhancement of production/activity of thermophilic/hyperthermophilic enzymes

AraS (arabinose-binding protein) promoter. Despite this, the high cost of enzyme production prevented the productivity of enzymes using the aforementioned vectors from reaching the industrial scale (Albers et al., 2006; Jonuscheit et al., 2003). For the purpose of increasing the productivity of hyperthermophilic enzymes, novel expression systems were created. One of the expression systems, lac/tac/trc, uses vectors to carry the lac operon promoter or its hybrids, trc or tac. In the absence of isopropyl b-d1-thiogalactopyranoside or lactose, the lac operon repressor can inhibit the target gene’s transcription by controlling one of these promoters. In those tests, the repressor was released by the inducer, which enabled transcription to occur. By introducing lactose or isopropyl-b-D-thiogalactopyranoside (IPTG) as an inducer to the genetically modified E. coli, the expression of the target genes was induced (Shao et al., 2010). Despite being less expensive than IPTG, lactose can be metabolized, making it less efficient. However, IPTG’s industrial uses for producing enzymes are constrained by its toxicity and high price. The cI gene in bacteriophages encodes a repressor that controls the pL and pR early transcription promoters. A temperature-sensitive mutant of cI has so far been used in a few expression vectors with the pL expression system to control the expression of the target gene. Gene expression can be started by raising the growth temperature above 37
C because the repressor binds to pL in cells containing these vectors, causing repression of the transcription only at lower temperature. A promoter for heat shock controlled by E. coli’s alternative sigma factor 32 of E. coli was tasked with regulating the expression of foreign genes in several expression vectors (pHsh), designated as “Hsh system” (Wu et al., 2011; Shao et al., 2010). By exposing E. coli containing a pHsh vector to a rapid rise in temperature, heatshock proteins are expressed due to recognition of heat-shock promoters by s32 factor. Further research revealed significant differences between the DNA sequences of many heat-shock promoters and the common promoters that are recognized by s 70 (Debnath and Bhatta, 2005). While the regulatory mechanisms and physiological functions of the heat shock system in E. coli were well studied before the discovery of pHsh vector by Shao et al. (2010), regulation of gene expression for producing the target enzymes by heat-shock promoter regulation has not been successfully applied. This may be explained by the fact that the heat-shock reaction only lasts for 20 min after E. coli is exposed to a rapid temperature increase. In some E. coli expression systems, like pL/pR, where quick temperature changes are essential to boost expression efficiency, thermal induction may be used for driving the expression of the cloned gene. However, a rapid temperature increase is simple to achieve in a lab setting but difficult on an industrial scale. According to Shao et al. (2010), either a temperature change from 30
C to 42
C or a constant temperature of 30
C, the levels of 32 expression were significantly greater in cells carrying pHsh vectors than they were in cells carrying other plasmids, such as

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pLac. Additionally, expression of genes in pHsh vectors may last up to 8 h, whereas wild E. coli normally reaches its peak heat-shock protein synthesis rates after about 5 min of temperature upshift (Le et al., 2011). The use of pHsh vectors showed that Hsh systems present several benefits (Ebaid et al., 2019). These are listed below: ➢ No requirement of chemicals for inducing the expression ➢ Strict control of expression ➢ Higher production of the target protein ➢ Avoidance of inclusion body formation (Wang et al., 2016; Peng et al., 2011). Though developing new vectors may offer a potential gene overexpression, several genes of natural sequences are expressed in small amounts in the genetically modified cells. So, genetic mutagenesis is still needed for improving enzyme activity and/or production. Shao et al. (2017) developed a simpler and more effective method known as in situ error-prone PCR to improve proteins and genes through directed evolution. Furthermore, glmS was found to be an effective selection marker in eukaryotic as well as prokaryotic microorganisms (Sun et al., 2018). As a selective stress naturally takes place in the medium without adding chemicals, the use of glmS avoids using drug-resistant genes as a selection marker that might have a public health risk and also have an impact on the cost of waste treatment. The use and improvement of genes encoding hyperthermostable enzymes will continue to be a major area of R&D for satisfying the growing worldwide industry. A synergistic effect is produced by improving growth conditions, chemical modification, and genetic engineering (Im et al., 2009). For example, the stability and activity of the genetically engineered enzymes may be improved further by the use of chemical modification and vice versa (Atomi et al., 2011; Fukuda et al., 2008). On the whole, developing an effective strategy for the improvement of hyperthermostable enzymes will decrease the cost of production to the desired level for the commercial production of biofuels.

References Albers, S.V., Jonuscheit, M., Dinkelaker, S., Urich, T., Kletzin, A., Tampe, R., Driessen, A., Schleper, C., 2006. Production of recombinant and tagged proteins in the hyperthermophilic archaeon Sulfolobus solfataricus. Appl. Environ. Microbiol. 72 (1), 102e111. Atalah, J., Caceres-Moreno, P., Espina, G., Blamey, J.M., 2019. Thermophiles and the applications of their enzymes as new biocatalysts. Bioresour. Technol. 280, 478e488. Atomi, H., Sato, T., Kanai, T., 2011. Application of hyperthermophiles and their enzymes. Curr. Opin. Biotechnol. 22 (5), 618e626. Aucelli, T., Contursi, P., Girfoglio, M., Rossi, M., Cannio, R., 2006. A spreadable, nonintegrative and high copy number shuttle vector for Sulfolobus solfataricus based on the genetic element pSSVx from Sulfolobus islandicus. Nucleic Acids Res. 34 (17), e114.

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Gomes, A.R., Byregowda, S.M., Veeregowda, B.M., Balamurugan, V., 2016. An overview of heterologous expression host systems for the production of recombinant proteins. Adv. Anim. Vet. Sci. 4 (7), 346e356. Gupta, M.N., Roy, I., 2004. Enzymes in organic media forms, functions and applications. Eur. J. Biochem. 271, 2575e2583. Gupta, M.N., Bisaria, V.S., 2018. Effectiveness of cross-linked enzyme aggregates of cellulolytic enzymes in hydrolyzing wheat straw. J. Biosci. Bioeng. 126 (4), 445e450. Gupte, M., Kulkarni, P., 2003. A study of antifungal antibiotic production by Thermomonospora sp MTCC 3340 using full factorial design. J. Chem. Technol. Biotechnol. 78, 605e610. https://doi.org/10.1002/ jctb.818. Haseltine, C., Montalvo-Rodriguez, R., Bini, E., Carl, A., Blum, P., 1999. Coordinate transcriptional control in the hyperthermophilic archaeon Sulfolobus solfataricus. J. Bacteriol. 181 (13), 3920e3927. Im, Y.J., Ji, M., Lee, A., Killens, R., Grunden, A.M., Boss, W.F., 2009. Expression of Pyrococcus furiosus superoxide reductase in Arabidopsis enhances heat tolerance. Plant Physiol. 151, 893e904. Jang, H.D., Chang, K.S., 2005. Thermostable cellulases from Streptomyces sp.: scale-up production in a 50-l fermenter. Biotechnol. Lett. 27 (4), 239e242. Jonuscheit, M., Martusewitsch, E., Stedman, K.M., Schleper, C., 2003. A reporter gene system for the hyperthermophilic archaeon Sulfolobus solfataricus based on a selectable and integrative shuttle vector. Mol. Microbiol. 48 (5), 1241e1252. Khajeh, K., Naderi-Manesh, H., Ranjbar, B., Moosavi-Movahedi, A.A., Nemat-Gorgani, M., 2001. Chemical modification of lysine residues in Bacillus alpha-amylases: effect on activity and stability. Enzym. Microb. Technol. 28, 543e549. Le, Y., Peng, J., Wu, H., Sun, J., Shao, W., 2011. An approach to the production of soluble protein from a fungal gene encoding an aggregation-prone xylanase in Escherichia coli. PLoS One 6 (4), e18489. Lee, S.Y., 1996. High cell-density culture of Escherichia coli. Trends Biotechnol. 14, 98e105. https:// doi.org/10.1016/0167-7799(96)80930-9. Lucas, S., Toffin, L., Zivanovic, Y., Charlier, D., Moussard, H., Forterre, P., Prieur, D., Erauso, G., 2002. Construction of a shuttle vector for, and spheroplast transformation of, the hyperthermophilic archaeon Pyrococcus abyssi. Appl. Environ. Microbiol. 68 (11), 5528e5536. Margaritis, A., Merchant, R.F., 1986. Optimization of fermentation conditions for thermostable cellulase production by Thielavia terrestris. J. Ind. Microbiol. 1 (3), 149e156. O’Fagain, C., 2003. Enzyme stabilizationdrecent experimental progress. Enzym. Microb. Technol. 33, 137e149. Palomo, J.M., 2010. Diels-alder cycloaddition in protein chemistry. Eur. J. Org. Chem. 2010, 6303e6314. Pant, G., Prakash, A., Pavani, J., Bera, S., Deviram, G., Kumar, A., Panchpuri, M., Prasuna, R.G., 2015. Production, optimization and partial purification of protease from Bacillus subtilis. J. Taibah Univ. Sci. 9 (1), 50e55. Peng, J., Wang, W., Jiang, Y., Liu, M., Zhang, H., Shao, W., 2011. Enhanced soluble expression of a thermostable cellulase from Clostridium thermocellum in Escherichia coli. Curr. Microbiol. 63(6), 523. Piller, K., Daniel, R.M., Petach, H.H., 1996. Properties and stabilization of an extracellular a- glucosidase from the extremely thermophilic archaebacteria Thermococcus strain AN 1: enzyme activity at 130
C. Biochim. Biophys. Acta Protein Struct. Mol. Enzymol. 1292 (1), 197e205. Pope, B., Kent, H.M., 1996. High efficiency 5 min transformation of Escherichia coli. Nucleic Acids Res. 24, 536e537. https://doi.org/10.1093/nar/24.3.536.  Fernandez-Lafuente, R., 2011. Coupling chemical modification Rodrigues, R.C., Berenguer-Murcia A, and immobilization to improve the catalytic performance of enzymes. Adv. Synth. Catal. 353, 2216e2238. Rolfsmeier, M.L., Haseltine, C.A., 2018. The RadA recombinase and paralogs of the hyperthermophilic archaeon Sulfolobus solfataricus. Methods Enzymol. 600, 255e284. Rosano, G.L., Ceccarelli, E.A., 2014. Recombinant protein expression in Escherichia coli: advances and challenges. Front. Microbiol. 5, 172. https://doi.org/10.3389/fmicb.2014.00172. Sarmiento, F., Peralta, R., Blamey, J.M., 2015. Cold and hot extremozymes: industrial relevance and current trends. Front. Bioeng. Biotechnol. 3, 148. Sezonov, G., Joseleau-Petit, D., D’Ari, R., 2007. Escherichia coli physiology in Luria-Bertani broth. J. Bacteriol. 189, 8746e8749. https://doi.org/10.1128/JB.01368-07.

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Shao, W., Ma, K., Le, Y., Wang, H., Sha, C., 2017. Development and use of a novel random mutagenesis method: in situ error-prone PCR (is-epPCR). In: Reeves, A. (Ed.), In Vitro Mutagenesis. Methods in Molecular Biology. Humana Press, New York, NY, pp. 497e506. Shao, W., Wu, H., Pei, J., 2010. Expression vector system regulated by s32 and methods for using it to produce recombinant protein. USA Patents US7807460B2. Shiloach, J., Fass, R., 2005. Growing E. coli to high cell density e a historical perspective on method development. Biotechnol. Adv. 23, 345e357. https://doi.org/10.1016/j.biotechadv.2005.04.004. Stedman, K.M., Schleper, C., Rumpf, E., Zillig, W., 1999. Genetic requirements for the function of the archaeal virus SSV1 in Sulfolobus solfataricus: construction and testing of viral shuttle vectors. Genetics 152 (4), 1397e1405. Su, X., Wang, S., Su, G., Zheng, Z., Zhang, J., Ma, Y., Liu, Z., Zhou, H., Zhang, Y., Zhang, L., 2018. Production of microhomologous-mediated site-specific integrated LacS gene cow using TALENs. Theriogenology 119, 282e288. Sun, Y., Wang, H., Ma, K., Shao, W., 2018. Construction and characterization of the GFAT gene as a novel selection marker in Aspergillus nidulans. Appl. Microbiol. Biotechnol. 102 (18), 7951e7962. Tanaka, T., Inoue, M., Sakamoto, J., Sone, N., 1996. Intra-and inter-complex cross-linking of subunits in the quinol oxidase super-complex from thermophilic Bacillus PS3. J. Biochem. 119 (3), 482e486. Torchilin, V.P., Maksimenko, A.V., Smirnov, V.N., Berezin, I.V., Klibanov, A.M., Martinek, K., 1979. The principles of enzyme stabilization IV. Modification of ‘key’ functional groups in the tertiary structure of proteins. Biochem. Biophys. Acta 567, 1e11. Tyagi, R., Gupta, M.N., 1998. Chemical modification and chemical cross-linking for protein/enzyme stabilization. Biochemistry (Moscow) 63, 334e344. Waege, I., Schmid, G., Thumann, S., Thomm, M., Hausner, W., 2010. Shuttle vector-based transformation system for Pyrococcus furiosus. Appl. Environ. Microbiol. 76 (10), 3308e3313. Wagner, M., Berkner, S., Ajon, M., Driessen, A.J., Lipps, G., Albers, S.V., 2009. Expanding and understanding the genetic toolbox of the hyperthermophilic genus Sulfolobus. Biochem. Soc. Trans. 37, 97e101. Wang, H., Ma, K., Shao, W., Wang, Q., He, Y., 2016. Advanced techniques of heterologous gene expression for preparing pharmaceutical and clinical proteins. Int. J. Pharm. Rev. Res. 5 (8), 13e18. Wang, Y., Fang, X., An, F., Wang, G., Zhang, X., 2011. Improvement of antibiotic activity of Xenorhabdus bovienii by medium optimization using response surface methodology. Microb. Cell Factories 10, 1e15. Wilson, S.A., Peek, K., Daniel, R., 1994. Immobilization of a proteinase from the extremely thermophilic organism Thermus Rt41A. Biotechnol. Bioeng. 43 (3), 225e231. Worthington, P., Hoang, V., Perez-Pomares, F., Blum, P., 2003. Targeted disruption of the a- amylase gene in the hyperthermophilic archaeon Sulfolobus solfataricus. J. Bacteriol. 185 (2), 482e488. Wu, G., Sun, Y., Qu, W., Huang, Y., Lu, L., Li, L., Shao, W., 2011. Application of GFAT as a novel selection marker to mediate gene expression. PLoS One 6 (2), e17082. Wynn, R., Richards, F.M., 1993. Unnatural amino acid packing mutants of Escherichia coli thioredoxin produced by combined mutagenesis/chemical modification techniques. Protein Sci. 2, 395e403. Xu, J., Luo, H., L opez, C., Xiao, J., Chang, Y., 2015. Novel immobilization process of a thermophilic catalase: efficient purification by heat treatment and subsequent immobilization at high temperature. Bioproc. Biosyst. Eng. 38 (10), 1983e1991. Xu, X., Jiao, L., Feng, X., Ran, J., Liang, X., Zhao, R., 2017. Heterogeneous expression of DnaK gene from Alicyclobacillus acidoterrestris improves the resistance of Escherichia coli against heat and acid stress. AMB Express 7 (1), 36. https://doi.org/10.1186/s13568-017-0337-x. Xue, D.S., Chen, H.Y., Lin, D.Q., Guan, Y.X., Yao, S.J., 2012. Optimization of a natural medium for cellulase by a marine Aspergillus niger using response surface methodology. Appl. Biochem. Biotechnol. 167 (7), 1963e1972.

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Further reading Lermant, A., Magnanon, A., Silvain, A., Lubrano, P., Lhuissier, M.J., Dury, C., Follenfant, M., Guiot, Z., Christien, G., Coudert, P., 2018. A thermo-responsive plasmid for biconditional protein expression. bioRxiv 289264. Peng, J., Abomohra, A.E.F., Elsayed, M., Zhang, X., Fan, Q., Ai, P., 2019. Compositional changes of rice straw fibers after pretreatment with diluted acetic acid: towards enhanced biomethane production. J. Clean. Prod. 230, 775e782. Valdez-Cruz, N.A., Caspeta, L., Perez, N.O., Ramírez, O.T., Trujillo-Roldan, M.A., 2010. Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pL and/or pR promoters. Microb. Cell Factories 9 (1), 18. Wong, S.S., Wong, L.J.C., 1992. Chemical crosslinking and the stabilization of proteins and enzymes. Enzym. Microb. Technol. 14, 866e874.

CHAPTER 8

Industrial applications of thermophilic/ hyperthermophilic enzymes Over the last 20 years, researchers are investigating thermophilic and hyperthermophilic microorganisms because their enzymatic systems have special features. These detailed studies are now forthcoming to mastering the cloning and industrial application of a broad variety of genes encoding enzymes involved in protein and starch hydrolysis, biosynthesis of amino acids, etc. A lot of interest is being shown in using thermophiles as a source of industrially relevant thermostable enzymes. Thermophilic enzymes possess remarkable stability toward the denaturing action of heat and protein denaturants and hence can be used as biocatalyst under rather severe environmental conditions. Many thermophiles have properties appropriate for commercial and biotechnological applications. There is, undeniably, a substantial requirement for stable enzymes, which can resist rigorous conditions in industrial processes by substituting or augmenting the conventional processes. Most hyperthermophilic enzymes exhibit optimum activity at temperatures closer to the optimal growth temperature of the host, which is generally between 70 and 125
C (Hicks et al., 1999). From an applied perspective, two types of protein stability are important. These are thermodynamic thermal stability and long-term stability. Active enzymes, not one that is in a reversibly inactivated state, are required. In case of diagnostics enzymes, long-term stability must be improved (Mozhaev, 1993). Enhancing the thermodynamic thermal stability may have a favorable impact upon the long-term stability. Directed evolution is the most useful method in biology. This method is used to create biological entities with desired properties through repititive rounds of genetic diverseness and library screening or selection. Nowadays, directed evolution is mostly used for designing enzymes with improved thermostability (Schmidt-Dannert and Arnold, 1999). This technique has been also used for several other purpose, like development of enzymes, which are active in solvents or thermostable enzymes showing optimum activity at higher temperatures. Improvement of enzymes by directed evolution method has been commercialized (Schmidt-Dannert and Arnold, 1999; Bhattacharya and Pletschke, 2014). Today, many speciality and industrial enzymatic processes are using thermophilic/hyperthermophilic enzymes. The increased number of enzymes obtained from hyperthermophiles and the current introduction of effective protein engineering methods propose that such enzymes will see increasingly applications in several areas.

Developments and Applications of Enzymes From Thermophilic Microorganisms ISBN 978-0-443-19197-8, https://doi.org/10.1016/B978-0-443-19197-8.00016-5

© 2023 Elsevier Inc. All rights reserved.

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SUBCHAPTER 8.1

Amylases 8.1.1 Microbial sources and properties Microorganisms, particularly bacteria are one of the most important sources of a-amylases. These have shorter generation time. Mesophilic, thermophilic, and extremophilic bacteria are the important sources of thermostable a-amylase enzymes. These enzymes show optimum activity at high temperatures. Amylases are classified into endoamylases and exoamylases. - Endoamylase enzymes hydrolyze the a-1,4-glycosidic bonds of amylose in starch or amylopectin randomly. These produce linear and branched oligosaccharides of varying chain length - Exoamylase enzymes hydrolyze starch from the nonreducing end to form successively shorter end-products (Lim et al., 2019). There are three categories of amylases denoted as a-, b-, and g-amylases. These enzymes differently attack the bonds of the starch molecules. - a-amylase (E.C 3.2.1.1, glucan-1,4-a-glucanohydrolase) is widespread in living organisms and are found in microorganisms, plants, humans, and animals. - b-amylase (E.C 3.2.1.2, glucan-1,4-a-maltohydrolase; glycogenase; saccharogen amylase) is found in microorganisms and plants. - g-amylase (E.C 3.2.1.3, glucan-1,4-a-glucosidase; amyloglucosidase; exo-1,4a-glucosidase; glucohydrolase) is found in plants and animals. a-Amylase is a calcium-containing metalloenzyme, which hydrolyses starch into smaller units like glucose and maltose (Singh et al., 2016) (Fig. 8.1.1). These enzymes need calcium ions for their activity, stability, as well as structural integrity (Mehta et al., 2016). Fig. 8.1.2 presents the three-dimensional structures of amylases. A single polypeptide chain found in a-amylases is folded into the A, B, and C domains, which are three distinct domains (Fig. 8.1.3). Low-molecular-weight carbohydrate moieties like glucose, maltose, and a-limiting dextrin are produced by this endo-amylase (Singh and Guruprasad, 2014). The functional hydroxyl group in these hydrolyzed products is in the alpha-configuration, hence the name “amylase” for this enzyme. b-amylase is an exoamylase. This enzyme catalyzes the hydrolysis of a-1,4-glycosidic bonds in starch to produce b-maltose and b-limit dextrin (Oktiarni et al., 2015). This enzyme is present in the microbes in the gastrointestinal tract and is not synthesized in animal tissues.

Industrial applications of thermophilic/hyperthermophilic enzymes CH2OH H O

H

OH

H

H

OH

O CH2

CH2OH O

H O

Starch

Starch O

H

OH

H

H

OH

CH2OH O

H O

H

OH

H

H

OH

O

H O

H

OH

H

H

OH

O

Digestion

Glucose CH2OH O

H OH

OH

OH H

OH

O

H

H

HO H

CH2OH

CH2OH OH

H

OH H

HO H

OH

Glucose O

H

OH

H H

HO H

OH

Figure 8.1.1 Scheme for the hydrolysis of starch by amylase. Starch is a polysaccharide made up of simple sugars (glucose). Upon the action of amylase, either glucose (a monosaccharide) or maltose (a disaccharide with two glucose molecules) is released. (From Gopinath, S.C.B., Anbu, P., Md Arshad, M.K., Lakshmipriya, T., Voon, C.H., Hashim, U., Chinni, S.V., 2017. Biotechnological processes in microbial amylase production. BioMed Res. Int. 2017, Article ID 1272193, 9 pages, 2017. Distributed under the Creative Commons Attribution License.)

g-Amylases hydrolyze a-1,4 and a-1,6 glycosidic bonds, thus functioning as both exoamylases and endoamylases. But, the optimum pH of this enzyme is 3, and it is most effective in the environments of low pH (Lim et al., 2019; Saini et al., 2017). Table 8.1.1 shows the classification of amylases and their mode of action. a-Amylase enzymes belong to family 13 (GH-13) of the glycoside hydrolase group of enzymes (Hiteshi and Gupta, 2014; Bordbar et al., 2005). But few bacteria produce a-amylases, which are calcium independent. This property is desired in large scale when hydrolysis of starch is done on a large scale (Hailemarium et al., 2013). The three-dimensional structure of amylase enables to bind to substrate and helps in breaking the glycoside links due to the presence of catalytic groups (Iulek et al., 2000). The two-dimensional structure of the a-amylase prototype is composed of three domains: A, B, and C. The N-terminal TIM barrel structure is known as domain A. Domain B is made up of a protruding long loop between a helix 3 and a b strand 3,

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Figure 8.1.2 Three-dimensional structures of amylases. (A) a-Amylase (RCSB PDB accession code 1SMD; the calcium-binding regions are indicated). (B) b-Amylase (RCSB PDB accession code PDB 2xfr). (From Gopinath, S.C.B., Anbu, P., Md Arshad, M.K., Lakshmipriya, T., Voon, C.H., Hashim, U., Chinni, S.V., 2017. Biotechnological processes in microbial amylase production. BioMed Res. Int. 2017, Article ID 1272193, 9 pages, 2017. Distributed under the Creative Commons Attribution License.)

and domain C is connected to domain A by a b sheet structure. The (b/a)8 barrel has eight alternating b-strands and a-helices. The a-helices are on the outside of the cylinder, and the b strands are arranged parallel to each other. TIM barrel contains four highly conserved regions that are directly connected to the active site present in all amylases. The amino acid residues that make up thermostable a-amylase can be charged (Asp, Glu, Arg, Lys, His), polar (Thr, Cys, Asn, Gln, Ser), or hydrophobic (Trp, Pro, Phe, Tyr, Ile, Leu, Met, Ala, Val). His and Asp are less abundant among charged residues in thermophilic proteins when they are exposed as opposed to burried. While Leu is reduced and Val is increased at buried sites, among hydrophobic residues, Tyr, Trp, Pro, and Val are increased at exposed sites. The surfaces of thermophilic proteins have higher concentrations of charged residues.

Industrial applications of thermophilic/hyperthermophilic enzymes

Figure 8.1.3 (1A, 1B) Crystal structure of Bacillus amyloliquefaciens a-amylase PDB ID 3BH4 [207]; resolution: 1.40 Å. It consists of 483 amino acids. The A domain is shown in green, the B domain is shown in cyan, and the C domain is shown by reddish orange color. (1C) The blue sphere represents the single Naþ ion, and gray spheres represent the Ca2þ ions (front view). (1D) Blue spheres represent the Ca2þ ion (front view). (2A, 2B) Crystal structure of calcium (Ca2þ)-independent Bacillus sp. a-amylase KSMK38 PDB ID 1UD4; resolution: 2.15 Å [208]. It consists of 480 amino acids. The A domain is shown in green, the B domain is shown in cyan, and C domain is shown by reddish orange color. (2C) Blue spheres represent the three Naþ ions. (From Paul, J.S., Gupta, N., Beliya, E., Tiwari, S., Jadhav, S.K., 2021. Aspects and recent trends in microbial a-amylase: a review. Appl. Biochem. Biotechnol. 193 (8), 2649e2698. Reproduced with permission.)

Table 8.1.1 Classification of amylases and their mode of action. Name of the enzyme

EC classification

a-Amylase

3.2.1.1

b-Amylase

3.2.1.2

g-Amylase

3.2.1.3

Alternate names

Reaction catalyzed

1,4-a-D-glucan glucanohydrolase, glycogenase 1,4-a-D-glucan maltohydrolase, saccharogen amylase, glycogenase Glucoamylase, 1,4-a-Dglucan glucohydrolase, amyloglucosidase, lysosomal aglucosidase, exo-1,4-a-glucosidase

Hydrolysis of 1,4-a-glucosidic linkages Hydrolysis of terminal 1,4-linked a-D glucose residues Hydrolysis of terminal 1,4-linked a-D glucose residues

From Hiteshi, K., Gupta, R., 2014. Thermal adaptation of a-amylases: a review. Extremophiles 18 (6), 937e944. Reproduced with permission.

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In thermophiles, the proportions of Glu, Val, Lys, and Arg, are significantly higher than those of Ser, Asn, Gln, and Thr. Protein surface residues are often flexible and exhibit free intraprotein interactions, such as surface salt bridges that form as a result of a high concentration of charged surface residues. Proteins from thermophilic organisms are more thermotolerant as a result of these interactions. Bacillus licheniformis’ thermostable a-amylase exhibits exceptional heat stability. This enzyme offers a fascinating model for examining the structural underpinnings of proteins’ thermostability. It has 469 residues of amino acids and 294 water molecules. The polypeptide chain folds into three distinct domains, just like other a-amylases. A (b/a) 8-barrel structure is formed by the first domain, which consists of 291 residues (from residue 3e103 to 207e396) in total. Between domain A’s third beta strand and third ahelix is the second domain, which contains residues 104e206. Residues 397e482 of the third C-terminal domain fold into an eight-stranded antiparallel beta barrel. This region of the enzyme is crucial for thermostability because a large number of the stabilizing and destabilizing mutations discovered till now fall in domain B or at its border. Increased ionic and internal packing interactions, decreased surface area, may be the causes of this enzyme’s remarkable thermostability. Thermophilic B. licheniformis a-amylase has been found to have increased thermostability primarily as a result of additional salt bridges involving some selected lysine residues. Similarly, B. stearothermophilus a-amylase and mutant B. amyloliquefaciens a-amylase are stabilized against thermal denaturation by ionic interactions (Hiteshi and Gupta, 2014; Cambillau and Claverie, 2000; Chakravarty and Vardarajan, 2000; Hwang et al., 1997; Karshikoff and Ladenstein, 2001; Kraulis, 1991; Kuriki et al., 2005; Loladze et al., 1999; Perl et al., 2000; Reidhaar-Olson and Sauer, 1990; Spectar et al., 2000; Strop and Mayo, 2000; Takano et al., 2000). Thermostable a-amylases are extensively used in industry (Table 8.1.2) (Haki and Rakshit, 2003). Major benefits of thermostable amylases are listed below (Fincan and Enez, 2014): ➢ Cooling cost is reduced ➢ Solubility of the substrate is reduced ➢ Chance of contamination by microorganisms is lesser ➢ Resistance to proteases and denaturants Highly thermostable a-amylase enzymes have been characterized from several hyperthermophilic archaea. These enzymes show optimum performance between 80 and 100
C. These include Sulfolobus, Thermophilum, Desulfurococcus, Staphylothermus, Pyrococcus woesei, and Thermococcus profundus. Most of enzymes that hydrolyze starch belong to the a-amylase family. These enzymes contain a (b/a)8-barrel catalytic domain. The problem of insufficient expression in the host is avoided by molecular cloning of the corresponding genes and expression in heterologous hosts. The a-amylase from Pyrococcus furiosus has been cloned, and this enzyme is expressed in Escherichia coli and B. subtilis. The extremely

Industrial applications of thermophilic/hyperthermophilic enzymes

Table 8.1.2 Applications of a-amylases.

Paper Textile Laundary and detergents Brewing Baking Sugar Biofuel production Bioremediation Potent biological de-emulsifier Clinical and pharmaceutical Based on Haki, G.D., Rakshit, S.K., 2003. Developments in industrially important thermostable enzymes: a review. Bioresour. Technol. 89, 17e34 their enhanced stability. FEBS Lett. 304, 1e3.

higher thermostability of this extracellular a-amylase (thermoactive even at 130
C) makes this enzyme very appealing for commercial use. a-Amylase enzymes with inferior thermostability have been also obtained from the archaea (Pyrococcus kodakaraensis; Thermococcus profundus) and the bacteria (Thermotoga maritima, Dictioglomus thermophilum). The genes encoding these enzymes have been effectively expressed in E. coli. The T. maritima enzyme needs calcium ions for activity alike to the amylase from B. licheniformis. Pyrodictium abyss, a highly marine hyperthermophilic archaea, can grow on multiple polysaccharides and also produces a thermostable a-amylase (Bertoldo and Antranikian, 2009). Table 8.1.3 shows the characteristics of archaeal and bacterial a-amylases, and in Table 8.1.4, the characteristics of fungal a-amylases are shown.

8.1.2 Applications of a-amylases The use of amylolytic enzymes such as a-amylases has increased in several applications in industry in the recent years (Paul et al., 2021). Amylases have prospective application in numerous industrial processes. a-Amylases are used for producing syrups and sweeteners and in beverage industry. It is also used in starch saccharification and liquefaction, cheese ripening and flavoring, bioethanol production, animal feed, laundry, papermaking, and the textile industry (Afrisham et al., 2016; Xie et al., 2014; Song et al., 2016). Other applications of a-amylases include purification of wastewater, hydrogel formation, pharmaceuticals and medicine, and amylose cross-linked preparation of polymer for controlled drug release. It is a potential antibiofilm agent against biofilm-forming bacteria (Priya and Renu, 2018; Suribabu et al., 2014; Dumoulin et al., 1999; Vaikundamoorthy et al., 2018). Several microbial a-amylases are available commercially by leading manufacturers. For each application, a-amylase with specific characteristics is required. Table 8.1.5

111

112

Developments and Applications of Enzymes From Thermophilic Microorganisms

Table 8.1.3 Characteristics of bacterial and archaeal a-amylases.

Source

Mol. Wt.

pH opt.

Temp. opt. (8C)

Acyclobacillus acidocaldarius Alicyclobacillus sp.A4 Amphibacillus sp.NMRA2 Bacillus acidicola

160

3.0

75

e

64 e

4.2 8

75 54

e e

66

4

60

e

B. amyloliquefaciens

52

6.0

55

B. amyloliquifaciens

43

7

70

1.92 mgmL1, 351 UmL1 e

B. circulans

48

4.9

48

B. halodurans

10.5e11

60e65

B. licheniformis

90, 85, 70, 65, 58 31

11.66 mgml1, 68.97 U e

6.5

90

e

B. licheniformis

58

4e9

90

e

B. stearothermophilus B. stearothermophilus

59 56

5.6 5.6

80e82 80

e e

B. subtilis AS-S01a

21

9

55

B.subtilis KIBGE

56

7.5

50

Bacillus sp.

e

8

110

1.9 mgml1, 198.2 Uml1 2.68 mgml1, 1773 Uml1 e

Bacillus sp.A3-15 Bacillus sp.ANT-6

86 94.5

8.5 10.4

60 80

e e

Bacillus sp.KR8104

59

75e80

e

Bacillus sp.TS-23 Bacillus sp.WPD616 Bacillus sp.YX1

65 59 56

4.0e 6.0 9 6 5.0

50 40e50

e e e

Bacillus sp.

53

4,5

70

Km, Vmax

References

Matzke et al. (1997) Bai et al. (2012) Mesbah and Wiegel (2014) Sharma and Satyanarayana (2006) Demirkan et al. (2005) Kikani and Singh (2011) Dey et al. (2002) Murakami et al. (2008) Bozic et al. (2011) Hmidet et al. (2008) Ali et al. (2001) Khemakhem et al. (2009) Roy et al. (2012) Bano et al. (2011) Pancha et al. (2010) Burhan (2008) Burhan et al. (2003) Sajedi et al. (2005) Lo et al. (2001) Liu et al. (2006) Liu and Xu (2008) Asoodeh et al. (2010)

Industrial applications of thermophilic/hyperthermophilic enzymes

Table 8.1.3 Characteristics of bacterial and archaeal a-amylases.dcont’d

Source

Mol. Wt.

pH opt.

Temp. opt. (8C)

Bacillus subtilis JS-2004 Chromohalobacter sp.

e

7

50

e

62,72

9

65

e

Chryseobacterium taeanense Desulfurococcus mucosus

47

9

50

e

e

5.5

100

e

Km, Vmax

References

Asgher et al. (2007) Prakash et al. (2009) Wang et al. (2011) Canganella et al. (1994) Kolcuoglu et al. (2010)

Geobacillus caldoxylosilyticus TK4 G. thermodenitrificans

70

7

50

e

58

5.5

80

G. thermoleovorans

26

7

100

3.05 mgml1, 7.35 Uml1 e

Geobacillus sp.IIPTN

97

6.5

60

Geobacillus sp.LH8

52

5e7

80

Geobacillus sp.LH8

52

5e7

80

Halomonas meridiana

e

7

37

3 mgml1, 6.5 m molmin1 e

Lactobacillus manihotivorans Marinobacter sp.EMB8

135

5.5

5

e

72

6e11

80

36 mgml1, 222 Uml1

Nesterenkonia sp. strainF Pyrococcus furiosus

100

7.5

45

4.6 mgml1, 1.3 mgmin1 mL1 e

48

5.6

115

e

P. woesei

45

5.5

85

e

Saccharopolyspora sp.A9 Staphylothermusmarinus Sulfolobussolfataricus

66

11

55

e

82.5 120

5 3

100 80

e e

Ezeji and Bahl (2006) Rao and Satyanarayana (2007) Dheeran et al. (2010) Khajeh et al. (2009) Mollania et al. (2010) Coronado et al. (2000)

Kumar and Khare (2012) Shafiei et al. (2010) Savchenko et al. (2002) Frillingos et al. (2000) Chakraborty et al. (2011) Li et al. (2010) Haseltine et al. (1996) Continued

113

114

Developments and Applications of Enzymes From Thermophilic Microorganisms

Table 8.1.3 Characteristics of bacterial and archaeal a-amylases.dcont’d

Source

Mol. Wt.

pH opt.

Temp. opt. (8C)

Thermococcus sp.CL1 T. aggregans

e e

6 5.5

98 100

e e

T. celer

e

5.5

90

e

T. guaymagensis

e

6.5

100

e

T. hydrothermalis

e

75e85

e

T. hydrothermalis

53.5

5.0e 5.5 5.5

85

e

T. profundus

43

5.5

80

e

Thermotoga maritima

50

7

70

e

Km, Vmax

References

Jeon et al. (2014) Canganella et al. (1994) Canganella et al. (1994) Canganella et al. (1994) Legin et al. (1998) Horvathova et al. (2006) Kwak et al. (1998) Lim et al. (2003)

From Mehta, D., Satyanarayana, T., 2016. Bacterial and archaeal a-amylases: diversity and amelioration of the desirable characteristics for industrial applications. Front. Microbiol. 7, 1129. Distributed under the terms of the Creative Commons Attribution License (CC BY).

shows the list of commercially available a-amylases. Some of them are AmzymeTX, Aquazym 120L, Aquazym Ultra 250L, BANTM, Enzymex from B. amyloliquefaciens, Fructamyl, Liquozyme, Termamyl, Natalase, Stainzyme from B. licheniformis, etc. (Mehta and Satyanarayana, 2016). Novozymes (Denmark), Megazyme (USA), and AB Enzymes (USA) are just a few of the companies that produce these enzymes. These enzymes are used in fabric desizing, detergent and laundry industry, food industry, and paper industry. Novozyme has the highest market share for industrial enzymes (48% in 2016), and it will continue to produce the most amylases. A total of 400 small suppliers and 12 major producers supply the entire world’s demand for enzymes. The top producers of a-amylases are Novozymes, based in Denmark, DuPont, Roche, and AB enzymes based in the United States (Paul et al., 2021; Li et al., 2016; Silveira et al., 2019). By 2025, the worldwide a-amylase market size is expected to reach USD 382.4 million (https://paperzz.com/doc/6747135/global-alpha-amylase-market-trends–price–share-and -grow). Main factors driving market growth include improvement of food quality, ecofriendly production process, higher reaction specificity, improving the quality of food, and rising consciousness about nutritional and healthy food. Furthermore, development in biotechnology has witnessed a surge in demand for the worldwide a-amylase market. The increased usage of a-amylase in several applications listed above will push the growth of this enzyme in the next few years.

Table 8.1.4 Characteristics of fungal a-amylases. Fermentation

pH optimal/ stability

Temperature optimal/ stability (8C)

Mol. wt. (kDa)

Inhibitors

References

Thermomyces lanuginosus ATCC 58160 Thermomyces lanuginosus ATCC 2000 Aspergillus niger SSF Aspergillus sp. AS-2 Aspergillus niger UO-1 (32) Aspergillus niger ATCC 16404

SSF

6.0

50

e

e

6.0

50

e

e

Kunamneni et al. (2005) Jensen et al. (2002)

SSF SSF SmF

5.5 6.0 4.95

70 50 50

e e

SmF

5.0/6.0

30

e

e e Cu2þ, Hg2þ and Zn2þ e

SSF

5.0e9.0 7.0

25e35 35

e e

e e

Uguru et al. (1997) Soni et al. (2003) Hern
andez et al. (2006) DjekrifDakhmouche et al. (2006) Jin et al. (1998) Rahardjo et al. (2005)

30

e

e

Francis et al. (2003)

Aspergillus oryzae Aspergillus oryzae CBS570.64 Aspergillus oryzae NRRL 6270 Aspergillus oryzae CBS 125-59 Aspergillus fumigatus Aspergillus kawachii Cryptococcus flavus

SSF SSF

6.0

30

SmF

6.0 3.0 5.5

30 30 50

e 108 75

Penicillium fellutanum

SmF

6.5

30

e

Pycnoporus sanguineus

SmF

7.0

37

e

Pycnoporus sanguineus Mucor sp.

SSF

5.0 5.0

37 60

e e

Glucose, maltose e EDTA

5.0

30

e

e

Saccharomyces kluyveri YKM5

e e Hg2þ, Fe2þ and Cu2þ e

Goto et al. (1998) Kajiwara et al. (1997) Wanderley et al. (2004) Kathiresan and Manivannan (2006) Siqueira et al. (1997) Siqueira et al. (1997) Mohapatra et al. (1998) Moller et al. (2004)

115

From de Souza and de Oliveira Magalh~aes (2010).

Murado et al. (1997)

Industrial applications of thermophilic/hyperthermophilic enzymes

Microorganism

116

Developments and Applications of Enzymes From Thermophilic Microorganisms

Table 8.1.5 Commercially available bacterial a-amylases. Commercial name of a-amylase

Manufacturer

Amzyme TX

Parchema

Aquazym 1201

Novo Nordisk, Denmarkb Novo Nordisk, Denmarkb Novozymes

Aquazym Ultra 2501 BANTM Enzymex (Cocktail), Fructamyl FHT

Liquozyme SC DC

Exotic Biosolutions Pvt. Ltd.c ERBSLOEHd Novozymese

Natalase Stainzyme plus

Novozymese Novozymese

Thermamyl, Takaterm

Novo Nordisk, Denmarkb DSM Valley Researchf AB enzymesg

Validase BM VERON TENDER

Producer microorganism

Bacillus amyloliquifaciens

Application

Foods and feeds Desizing of textiles Desizing of textiles

B. amyloliquifaciens B. amyloliquifaciens

Genetically engineered from B. licheniformis Genetically engineered B. lichentformis B. amyloliquifaciens

Foods and feeds, paper industry Foods and feeds Starch saccharification Starch saccharification Detergent industry Detergent industry Detergent industry, paper industry Food industry Baking industry

a

www.parchem.com. www.novonordisk.com. www.exoticbiosolutions.com. d www.erbsloeh.com. e www.novozymes.com. f www.dsm.com. g http://www.abenzymes.com. From Mehta, D., Satyanarayana, T., 2016. Bacterial and archaeal a-amylases: diversity and amelioration of the desirable characteristics for industrial applications. Front. Microbiol. 7, 1129. Distributed under the terms of the Creative Commons Attribution License (CC BY). b c

8.1.2.1 Starch conversion The most important application of a-amylases is in the starch industry. It plays an important role in the hydrolysis of starch. The products of starch saccharification are glucose and fructose syrups (Radeloff and Beck, 2014). Following three steps are involved in the starch conversion process (Fig. 8.1.4) (Farooq et al., 2021): (1) gelatinization (2) liquefaction (3) saccharification

Industrial applications of thermophilic/hyperthermophilic enzymes

Figure 8.1.4 Three steps of starch conversion: 1 gelatinization, 2 liquefaction, and 3 saccharification. (From Farooq, M.A., Ali, S., Hassan, A., Tahir, H.M., Mumtaz, S., 2021. Biosynthesis and industrial applications of a-amylase: a review. Arch. Microbiol. 203, 1281e1292. Reproduced with permission.)

Starch granules dissolve in water during the first step, known as (1) gelatinization, and a viscous suspension is created. The water now contains both amylose and amylopectin as a result of this dissolution. The second liquefaction step reduces the viscosity of the starch solution by causing a-amylase to partially hydrolyze the starch into the short chain of dextrin. The formation of glucose and fructose syrup and maltose results from additional

117

118

Developments and Applications of Enzymes From Thermophilic Microorganisms

hydrolysis that takes place in the third and final step, known as saccharification. Glucoamylase is responsible for carrying out this reaction. Since it is an exoamylase, it catalyzes the a-1e4 glycosidic linkage of the nonreducing moiety. Fructose syrup is used as an artificial sweetener in the beverage industry. It is produced by the isomerization of high-glucose syrups by the action of glucose isomerase (Farooq et al., 2021; Uthumporn et al., 2010). a-Amylases of B. amyloliquefaciens, B. licheniformis, and B. stearothermophilus have been used (van der Maarel et al., 2002). B. licheniformis’ a-amylase can withstand a temperature of 90e95
C (Torabizadeh et al., 2014). For commercial applications, enzymes from the Bacillus species are particularly interesting due to the availability of efficient expression systems and their amazing thermostability (Prakash and Jaiswal, 2010). 8.1.2.2 Detergent industry The detergent industry is a main consumer of enzymes, both in terms of volume and value. Enzymes improve the ability of detergent for removing the stubborn stains, making it environment friendly. Amylases are added in 90% of liquid detergents. These enzymes are used in the formulation of laundry detergents and automatic dishwashing detergents to break down residues of starchy materials such as potatoes, chocolate, sauces, and puddings into dextrins and other small oligosaccharides. Amylases are active at alkaline pH and low temperatures. These enzymes maintain the required stability under detergent conditions. One of the most essential yardstick for using amylases in detergents is oxidative stability where the wash environment is highly oxidative. Removing the starch from the surface improves the whiteness. Amylases from Bacillus or Aspergillus species are used in the detergent industry (Mitidieri et al., 2006; Kirk et al., 2002; Mitidieri et al., 2006; Gupta et al., 2003; Hmidet et al., 2009; Olsen and Falholt, 1998; Mukherjee et al., 2009; de Souza and de Oliveira Magalh~aes, 2010). 8.1.2.3 Fuel alcohol production Ethanol is the most common liquid biofuel used across the world (de Souza and de Oliveira Magalh~aes, 2010). The most common substrate for producing ethanol is starch because its price is lower, and it is readily available in various parts of the world (Chi et al., 2009). Starch is first solubilized and is then subjected to two steps involving enzyme for obtaining fermentable sugars. Starches are converted to sugars using starch-degrading microorganisms or enzymes such as a-amylase. Microorganisms fermenting ethanol like € S. cerevisiae yeast are used to ferment sugars to ethanol (Oner, 2006; Moraes et al., 1999). Yeast-based ethanol production plays a very important role in the Brazilian economy (de Moraes et al., 1995). To obtain a yeast strain capable of producing ethanol directly from starch without the requirement for a separate saccharification step. Chi et al. (2009) performed protoplast fusion between the starch-degrading yeasts S. cerevisiae and S. fibuligera. a-Amylases from thermostable bacteria such as B. licheniformis or from

Industrial applications of thermophilic/hyperthermophilic enzymes

genetically modified E. coli or B. subtilis are used to hydrolyze starch (Sanchez and Cardona, 2008). Several studies were undertaken to design microbial cultures capable of producing more ethanol and improving the efficiency of ethanol production (Mobini-Dehkordi and Javan, 2012). S. cerevisiae possesses more specific characters physiologically, biotechnologically, and genetically, as compared to other microorganisms. Hence, it is considered the best strain for production of bioethanol. One of the best ways to improve ethanol efficiency is to isolate mutant yeast cells that can tolerate higher ethanol concentrations and produce more bioethanol. Mobini-Dehkordi et al. (2008) have isolated mutants of yeast, which show higher ethanol productivity. These mutants produced 7% (W/ V) ethanol and endured up to 12% (V/V) exogenous ethanol. However, these were not able to grow in the presence of other alcohols like 2-propanol and 1-butanol (MobiniDehkordi et al., 2011). Furthermore, S. cerevisiae is classified as GRAS. Developed countries have biosecurity regulations regarding the integration of other safer genes of yeast into the genome of S. cerevisiae genome and the design of recombinant Saccharomyces sp. by auxotrophic markers. A new recombinant strain of S. cerevisiae was engineered by overexpressing self-genes to improve ethanol efficiency (Mobini-Dehkordi and Javan, 2012). 8.1.2.4 Food industry Amylases are widely used in the processed food industry to make cakes, fruit juices, corn syrups, digestive supplements, bakeries, and breweries. a-Amylase enzymes are commonly used in the bakery industry. The starch in the flour is converted into tiny dextrins by the addition of enzymes to the bread dough. Yeast then ferments these dextrins. The volume and texture of the finished product are increased, and the fermentation is accelerated when a-amylase is mixed in the dough. As a result, the flavor, ability of bread to toast, and crust color are all improved. Aside from producing fermentable compounds, a-amylase also makes baked goods softer, increases their shelf life, and prevents staleness when baking bread (de Souza and de Oliveira Magalh~aes, 2010; Couto and Sanrom
an, 2006; Gupta et al., 2003; van der Maarel et al., 2002). A thermostable maltogenic amylase from B. stearothermophilus is used in the bakery industry. Amylases are also used to clarify beer and fruit juices and to pretreat animal feed to increase the digestibility of dietary fiber (van der Maarel et al., 2002; Gavrilescu and Chisti, 2005; Ghorai et al., 2009; van der Maarel et al., 2002; Ghorai et al., 2009). a-Amylase is also used to process cocoa slurries for producing chocolate syrup (Paul et al., 2021). a-Amylase treatment prevents chocolate syrup from thickening. B. subtilis strain US586 a-amylase was dissolved in distilled water after purification and added to wheat flour at a dose level of 0.04 and 0.06 U/g. a-Amylase at a concentration of 0.06 U/g showed better rheological properties of dough and improved bread quality

119

120

Developments and Applications of Enzymes From Thermophilic Microorganisms

(Trabelsi et al., 2019). Chryseobacterium taeanense TKU001 a-amylase has been proven to be a natural antioxidant. It supports the growth of probiotic bacteria Lactobacillus paracasei subsp. Paracasei TKU010. Therefore, it also functions as a prebiotic enhancer, indicating its potential as a health food (Wang et al., 2011). 8.1.2.5 Textile industry For sizing of fabrics, several diverse types of compounds have been used. The most common sizing agent has been starch. The sizing is removed for preparing the fabric for the final stage of bleaching or dyeing. Starch hydrolase is used to desize the fabrics. These enzymes are very efficient and remove thread size without damaging the threads. Desizing on Jigger is a simple process where the fabric from one roll is processed in a bath and rewound on another roll. The sized fabric is first washed with hot water (80e95
C) for gelatinizing the starch. Depending on the enzyme, the desizing solution’s pH is adjusted to 5.5e7.5 and its temperature to 60e80
C. The fabric is then impregnated before the addition of amylase enzyme. The starch is degraded to dextrin. This is removed by washing at a temperature of 90e95
C for 2 min. Jigger process is a batch process. In a fast continuous process, the enzyme is reacted for only 15 s. Desizing of pad rollers is done continuously in the flow of goods. However, if cold alpha-amylase is used, it should be held at 20e60
C for 2e16 h before size removal in the wash chamber. The demolding reaction is performed using high temperature amylase in a steam chamber at temperatures of 95e100
C or higher, allowing a fully continuous process (Bajpai, 2018). 8.1.2.6 Paper industry a-Amylase enzymes have long been used to modify starch for sizing and coating paper surfaces (Bajpai, 2005, 2012, 2015; Smook, 1992). If the enzymatic modification of starch is properly controlled, papermakers can obtain starch pastes of consistent quality as required. Papermakers can produce high-quality paper with minimal cost of starch content (Bajpai, 2018). Enzymatically modified starches possess all the properties needed for surface sizing of writing and printing papers (de Souza and de Oliveira Magalh~aes, 2010; Maurer, 2001a,b; Svenson, 2006). Various types of starch can be enzymatically modified. However, reaction times and enzyme dosages are different. Enzymatically modified starch can be produced on-site at a paper mill in a batch or continuous process. It is also sold by starch manufacturers. Capital investment is not needed to move from an oxidized starch process to an in situ enzymatically modified starch. AOX products are present in oxidized starch and impact their use in consumer products. These are generated from the reaction of sodium hypochlorite with residual lipids of native starch (Maurer, 2001a,b). In the enzymatic modification of starch, AOX products are not present because no chemicals are involved. Oxidized starch is produced at relatively low temperatures, but the reaction time is long. This reaction is not very selective,

Industrial applications of thermophilic/hyperthermophilic enzymes

resulting in the loss of 30 to 40 percent starch in the wastewater. This requires expensive processing, and the cost of oxidized starch is high. Conversely, enzymes are relatively selective, hydrolysis may be controlled, and formation of soluble materials is avoided. The viscosity drops to the preferred value. Chemical modification is used for producing the oxidized starch. This is done at the starch manufacturer’s site. So, the paper manufacturer cannot control the quality such as viscosity. The oxidized starch is only cooked for gelation and dispersion at the papermaker’s location. The papermaker modifies the material using enzymes, and the paper mill regulates the final viscosity. Enzymatically modified starch is much less expensive to surface size than oxidized starch. Enzymatically modified starch has a slightly lower brightness than oxidized starch because native starch still contains residual proteins. Optical brighteners can be used to correct brightness. Metals present in native starch cause staining, which is another issue. Therefore, the native starch that will be modified by enzymes must contain very little protein and ash. Process variables like reaction time, enzyme dose, and the pH of the starch slurry are crucial for regulating the quality of enzymatically modified starch. The price of oxidized starch and enzymatically modified starch differs significantly. As a result, using the enzymatically modified starch allows paper mills to save a lot of money (Bajpai, 2015). Combination of a-amylase enzyme with lipases and proteases in paper machine boilout has shown exceptional results in comparison to conventional treatment with sodium hydroxide. These enzymes remove slime and control the bacterial growth in paper machines. This method is being used by most of the mills particularly those using a starch based coating system. a-Amylase enzymes have been used for improving the flotation deinking of copy papers (Bajpai and Bajpai, 1998; Bajpai et al., 2003). The a-amylase pretreatment with BAN 120 L, from Novo Nordisk, enhanced the deinking efficiency of papers containing starch. For instance, the residual toner area decreased by 35% after treatment of the starch surface-sized paper with a-amylase enzyme (https://imisrise.tappi.org/TAPPI/ Products/REC/REC98261.aspx). Elegir et al. (2000) recycled the waste paper from xerographic offices using the enzymatic deinking. The a-amylase-cellulase mixture was used for removing laser and xerographic toners. Smaller ink particles were removed by amylases. Cellulase treatment was found to be effective in deinking of sorted, nonimpact papers from the xerographic offices. Highest efficiency (96%) was obtained when the treatment was done with cellulase IUZ342 (0.05%) along with amylase (BAN 240) (0.001%). The blend gained seven points when compared with the treatment with amylase alone (89%) and 3 points when treated with cellulase alone (93%). Compounds like starch, pitch, slime, glue, latex, and other synthetic binders, which hold the deposits together, can be removed by using enzyme-based boil-outs. It depends upon the type and amount of scale present in the system. The starch slurry contains microbial

121

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and/or starch protein-based deposits. Boil-outs with products containing stabilized protease enzymes have been shown to be effective in these systems. Cooked starch systems use a-amylase products for removing deposits that are primarily made up of cooked starch. In either case, a preboiling system flush is necessary. This gets rid of the heated starch and allows the enzymes to work especially tough on the deposits (Bajpai, 2015). 8.1.2.7 Bioremediation Starch is widely distributed in nature and is found abundantly in waste products generated from the processing of plant materials (Mobini-Dehkordi and Javan, 2012; Barbesgard et al., 1999). A large amount of starch processing waste is generated, causing environmental problems. Microorganisms or amylolytic enzyme are used for purifying starch pollutant materials (Wu et al., 2008). For this, strains producing high level of enzyme such as yeast can be used (Jurado-Alameda et al., 2003; Mobini-Dehkordi et al., 2011). a-Amylase has been used for bioremediation of n-alkanes (Pinto et al., 2020). B. subtilis TB1 a-amylase degraded n-alkanes ranging from 10 to 14 carbons (Karimi and Biria, 2016). Also, a-amylases have been found to degrade low-density polyethylene (Karimi and Biria, 2019). Starch was found to stimulate degradation of n-alkane. The addition of starch substantially helps in bacterial growth and thus increases the enzyme synthesis for degradation. US9650276B2 describes method for improving the dewaterability of sludge. Sludge was treated with enzyme comprising an a-amylase. 8.1.2.8 Clinical and pharmaceutical a-Amylase enzymes are used in pharmaceutical and fine chemical industries (Fogarty and Kelly, 1980). In 1894, the first enzyme produced on an industrial scale was a-amylase. This enzyme was obtained from a fungal source. It has been used to treat gastrointestinal disorders (Pandey et al., 2000). Natural and synthetic biodegradable polymers are of interest in pharmaceutical research. These polymers are used for controlling drug release from parenteral controlled delivery systems (Dumoulina et al., 1999). For partially soluble drugs, or for some drugs whose solubility may be affected by changes in gastrointestinal pH, drug release enhancement systems are required. Biodegradable polysaccharide matrices are of special interest as the degradation of natural products such as starch occurs naturally in the human body (Kost and Shefer, 1990). Cross-linked starch and alpha-starch (pregelatinized starch) have been used as hydrogels. When the degree of starch cross-linking is increased, the rate of drug release is reduced. The rate also depends on the a-amylase activity present in the eluate. Addition of a-amylase enzyme to cross-linked amylose tablets can modulate drug release (Dumoulin et al., 1999).

Industrial applications of thermophilic/hyperthermophilic enzymes

8.1.2.9 Other applications The a-amylase enzyme from B. cereus showed extraordinary antibiofilm activity against the marine biofilm-forming bacteria Staphylococcus aureus and Pseudomonas aeruginosa in the Congo red assay and microplate assay (Vaikundamoorthy et al., 2018). Light and confocal laser scanning microscopy (CLSM) analyses were used for confirming the potential biofilm activity of amylase enzymes. Complete inhibition of biofilm formation on glass surfaces treated with amylase enzyme was discovered by CLSM analysis. An in vivo toxicity assay of the amylase enzyme was performed against the marine organisms Artemia salina and Dioithona rigida. No morphological changes were seen because of negligible amylase enzyme. Amylase enzymes obtained from marine bacteria can be developed as potential antibiofilm agents.

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.S.M., Dorn, M., Feltes, B.C., 2020. The tale of a versatile enzyme: alpha-amylase evolution, strucPinto, E ture, and potential biotechnological applications for the bioremediation of n-alkanes. Chemosphere 126202. Prakash, O., Jaiswal, N., 2010. Alpha-Amylase: an ideal representative of thermostable enzymes. Appl. Biochem. Biotechnol. 160 (8), 2401e2414. Prakash, B., Vidyasagar, M., Madhukumar, M.S., Muralikrishna, G., Sreeramulu, K., 2009. Production, purification, and characterization of two extremely halotolerant, thermostable, and alkali-stable a-amylases from Chromohalobacter sp. TVSP 101. Process Biochem. 44, 210e215. Priya, F.S., Renu, A., 2018. Efficacy of amylase for wastewater treatment from Penicillium sp. SP2 isolated from stagnant water. Journal of Environmental Biology 39 (2), 189e195. Radeloff, M.A., Beck, R.H., 2014. Starch hydrolysisdnutritive syrups and powders. Sugar Ind. 139, 222e227. Rahardjo, Y.S.P., Weber, F.J., Haemers, S., Tramper, J., Rinzema, A., 2005. Aerial mycelia of Aspergillus oryzae accelerate a-amylase production in a model solid-state fermentation system. Enzyme Microb. Technol. 36, 900e902. Rao, J.L.U.M., Satyanarayana, T., 2007. Improving production of hyperthermostable and high maltoseforming a-amylase by an extreme thermophile Geobacillus thermoleovorans using response surface methodology and its applications. Bioresour. Technol. 98, 345e352. Reidhaar-Olson, J.F., Sauer, R.T., 1990. Functionally acceptable substitutions in two alpha-helical regions of lambda repressor. Proteins 7, 306e316. Roy, J.K., Rai, S.K., Mukherjee, A.K., 2012. Characterization and application of a detergent-stable alkaline alpha-amylase from Bacillus subtilis strain AS-S01a. Int. J. Biol. Macromol. 50, 219e229. Saini, R., Saini, H.S., Dahiya, A., 2017. Amylases: characteristics and industrial applications. J. Pharmacogn. Phytochem. 6 (4), 1865e1871. Sajedi, R.H., Naderi-Mahesh, H., Khajeh, K., Ahmadvand, R., Ranjbar, B.A., Asoodeh, A., Moradian, F., 2005. A calcium independent a-amylase that is active and stable at low pH from the Bacillus sp. KR8104. Enzyme Microb. Technol. 36, 666e671. Sanchez, O.J., Cardona, C.A., 2008. Trends in biotechnological production of fuel ethanol from different feedstocks. Bioresour. Technol. 99, 5270e5295.

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Savchenko, A., Vieille, C., Kang, S., Zeikus, G., 2002. Pyrococcus furiosus a-amylase is stabilized by calcium and zinc. Biochemistry 41, 6193e6201. Schmidt-Dannert, C., Arnold, F.H., 1999. Directed evolution of industrial enzymes. Trends Biotechnol. 17, 135e136. Shafiei, M., Ziaee, A.A., Amoozegar, M.A., 2010. Purification and biochemical characterization of a novel SDS and surfactant stable, raw starch digesting, and halophilic a-amylase from a moderately halophilic bacterium, Nesterenkonia sp. strain F. Process Biochem. 45, 694e699. Sharma, D.C., Satyanarayana, T., 2006. A marked enhancement in the production of a highly alkaline and thermostable pectinase by Bacillus pumilus dcsr1 in submerged fermentation by using statistical methods. Bioresour. Technol. 97, 727e733. Silveira, B.M., Barcelos, M.C., Vespermann, K.A., Pelissari, F.M., Molina, G., 2019. In: Molina, G., et al. (Eds.), An Overview of Biotechnological Processes in the Food Industry, Bioprocessing for Biomolecules Production. Wiley Online Library, pp. 1e19. Singh, S., Guruprasad, L., 2014. Structure and sequence-based analysis of a-amylase evolution. Protein Peptide Lett. 21 (9), 948e956. Singh, R., Kumar, M., Mittal, A., Mehta, P.K., 2016. Amylases: a note on current application. Int. Res. J. Biol. Sci. 5 (11), 27e32. Siqueira, E.M.A., Mizuta, K., Giglio, J.R., 1997. Pycnoporus sanguineus: a novel source of a-amylase. Mycol. Res. 101, 188e190. Smook, G.A., 1992. Handbook for Pulp & Paper Technologists, second ed. Angus Wilde Publications, Vancouver. 419 pp. Song, Q., Wang, Y., Yin, C., Zhang, X.H., 2016. LaaA, a novel high-active alkalophilic alpha-amylase from deep-sea bacterium Luteimonas abyssi XH031T. Enzyme Microb. Technol. 90, 83e92. Soni, S.K., Kaur, A., Gupta, J.K., 2003. A solid state fermentation based bacterial a-amylase and fungal glucoamylase system and its suitability for the hydrolysis of wheat starch. Process Biochem. 39, 185e192. Spectar, S., Wang, M., Carp, S.A., Robblee, J., Hendsch, Z.S., Fairman, R., Tidor, B., Raleigh, D.P., 2000. Rational modification of protein stability by the mutation of charged surface residues. Biochemistry 39, 872e879. Strop, P., Mayo, S.L., 2000. Contribution of surface salt bridges to protein stability. Biochemistry 39, 1251e1255. Suribabu, K., Govardhan, T.L., Hemalatha, K.P.J., 2014. Application of partially purified a-amylase produced by Brevibacillus borostelensis R1 on sewage and effluents of industries. Int. J. Curr. Microbiol. Appl. Sci. 3, 691e697. Svensson, G., 2006. Alternative Enzymatic Conversion of Surface Sizing Starch at Nymölla Mill. Department of Chemical Engineering, Lund Institute of Technology, Lund. www.chemeng.lth.se/exjobb/ E256.pdf. Takano, K., Tsuchimori, K., Yamagata, Y., Yutani, K., 2000. Contribution of salt bridges near the surface of a protein to the conformational stability. Biochemistry 39, 12375e12381. Torabizadeh, H., Tavakoli, M., Safari, M., 2014. Immobilization of thermostable a-amylase from Bacillus licheniformis by cross-linked enzyme aggregates method using calcium and sodium ions as additives. J. Mol. Catal. B: Enzymat. 108, 13e20. Trabelsi, S., Ben Mabrouk, S., Kriaa, M., Ameri, R., Sahnoun, M., Mezghani, M., Bejar, S., 2019. The optimized production, purification, characterization, and application in the bread making industry of three acid-stable alpha-amylases isoforms from a new isolated Bacillus subtilis strain US586. J. Food Biochem. 43 (5), e12826. Uguru, G.C., Akinyauju, J.A., Sani, A., 1997. The use of yam peel for growth of locally isolated Aspergillus niger and amylase production. Enzyme Microb. Technol. 21, 46e51. Uthumporn, U., Zaidul, I.S., Karim, A.A., 2010. Hydrolysis of granular starch at sub-gelatinization temperature using a mixture of amylolytic enzymes. Food Bioprod. Process 88, 47e54. Vaikundamoorthy, R., Rajendran, R., Selvaraju, A., Moorthy, K., Perumal, S., 2018. Development of thermostable amylase enzyme from Bacillus cereus for potential antibiofilm activity. Bioorgan. Chem. 77, 494e506.

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van der Maarel, M.J., van der Veen, B., Uitdehaag, J.C., Leemhuis, H., Dijkhuizen, L., 2002. Properties and applications of starch-converting enzymes of the alpha-amylase family. J. Biotechnol. 94, 137e155. Wanderley, K.J., Torres, F.A., Moraes, L.M., Ulhoa, C.J., 2004. Biochemical characterization of alphaamylase from the yeast Cryptococcus flavus. FEMS Microbiol. Lett. 231, 165e169. Wang, S.L., Liang, Y.C., Liang, T.W., 2011. Purification and characterization of a novel alkali-stable a-amylase from Chryseobacterium taeanense TKU001, and application in antioxidant and prebiotic. Process Biochem. 46 (3), 745e750. Wu, H., Mulchandani, A., Chen, W., 2008. Versatile microbial surface-display for environmental remediation and biofuels production. Trends Microbiol. 14 (4), 181e188. Xie, F., Quan, S., Liu, D., Ma, H., Li, F., Zhou, F., Chen, G., 2014. Purification and characterization of a novel a-amylase from a newly isolated Bacillus methylotrophicus strain P11-2. Process Biochem. 49 (1), 47e53.

Industrial applications of thermophilic/hyperthermophilic enzymes

SUBCHAPTER 8.2

Glucoamylases 8.2.1 Microbial sources and properties Glucoamylase (1, 4-a-D-glucan glucanohydrolase; EC 3.2.1.3) is also referred to as amyloglucosidase. This enzymes act in an exo manner and catalyzes the hydrolysis of a-1,4 and a-1,6 glycosidic linkages of starch and related oligosaccharides from the nonreducing ends and liberates b-D-glucose (Sauer et al., 2000). But the branching points are hydrolyzed rather slowly (Fierobe et al., 1998; Thorsen et al., 2006; Norouzian et al., 2006; Michelin et al., 2008). Quite the reverse to a-glucosidases, this enzyme produces glucose in the b-configuration. Glucoamylases are present in bacteria, fungi, and archaea, but these enzymes are mostly found in fungi. At present, commercial glucoamylases are mostly obtained from filamentous fungi, for instance Rhizopus niveus, R. delemar, and Aspergillus niger, showing reasonable thermostability and sluggish catalytic activities (Wang et al., 2020; Carrasco et al., 2017). In recent years, thermostable glucoamylases derived from thermophilic bacteria have attracted a great deal of attention (Tong et al., 2021). Until now, many thermostable glucoamylases have been obtained from Chaetomium thermophilum, Rhizomucor pusillus, Fomitopsis palustris, Thermoanaerobacter tengcongensis, A. wentii, A. oryzae, A. flavus, and Sulfolobus solfataricus, all of which show optimum activity above 60
C (Wang et al., 2020; Lago et al., 2021; Karim et al., 2019; Tanaka et al., 2020; Zheng et al., 2010; He et al., 2014; Chen et al., 2007; Kim et al., 2004). Glucoamylases are being used on a commercial scale for converting maltooligosaccharides in glucose (Pandey, 1995). These enzymes are generally considered safe (GRAS) by the Food and Drug Administration (FDA) and are most commonly derived from A. niger or Rhizopus sp. Glucoamylases from these microorganisms are preferred in the starch processing industry because of their excellent thermal stability and higher activity at near-neutral pH values (Norouzian et al., 2006; Reilly, 1999). Structurally, glucoamylases are classified as family 15 of the glycoside hydrolases. These enzymes have properties indicating the consistent presence of a catalytic domain containing the (a/a)6-fold, often linked to noncatalytic domains with varied functions and origins. Fungal glucoamylases are of great biotechnological importance due to their extensive use in many industries and are extensively researched over the last 30 years. Glucoamylases from prokaryotes are thermostable enzymes and show optimum activity at higher temperatures (https://infinitabiotech.com/blog/glucoamylase-enzyme/?).

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These glycosyl hydrolases have a reversal reaction mechanism involving the transfer of proton from a common acid catalyst to the glycosidic oxygen. This is followed by nucleophilic attack of a deprotonated water molecule by a common basic catalyst (McCarter and Withers, 1994). Mammalian a-glycosidases, which act on starch and maltooligosaccharides, are functionally related to glucoamylases. Despite the fact that some mammalian glycosidases are usually referred to as glucoamylases, these enzymes have distinct structural families (GH31), unique EC numbers (EC 3.2.1.20), and unique mechanisms of action (retaining instead of inverting). Strictly speaking, glucoamylase can hydrolyze a-1,6 bonds at the branch points of starch molecules though their efficiency varies depending on the source. They typically act on polysaccharides more quickly as compared to oligosaccharides. Like the majority of secreted eukaryotic proteins, fungal glucoamylases are glycosylated, with varying degrees of O and N glycosylation. An extreme example of glycosylation is the glucoamylase from Saccharomyces cerevisiae (var. diastaticus), where carbohydrates account for up to 80% of the enzyme mass (Marín-Navarro and Polaina, 2011; Adam et al., 2004; Sauer et al., 2000; Kumar and Satyanarayana, 2009). Hostinov
a and Gasperík (2010) have published an excellent review on yeast glucoamylases. Top producers of glucoamylase include various genera of yeast including Saccharomyces, Candida, Debaryomyces, Arxula, Aureobasidium, Lipomyces, Saccharomycopsis (Endomycopsis), Ambrosiozyma, and Schwanniomyces. The focus of the in-depth molecular research has been on glucoamylases from Saccharomyces cerevisiae, Saccharomycopsis fibuligera, S. cerevisiae var. diastaticus, and Arxula adeninivoran. These yeasts produce the real glucoamylases, which are members of family 15 glycoside hydrolases in the classification of carbohydrate-active enzymes based on sequence. Unlike another group of exo-acting yeast, starch hydrolases from Debaryomyces occidentalis, Schwanniomyces occidentalis, and Candida albicans share a common mechanism of action which involves reversal of the anomeric configuration formerly called glucoamylase. However, these enzymes are distinctive a-glucosidases and belong to the family of glycoside hydrolases 31 (Spencer-Martins and van Uden, 1979; McCann and Barnett, 1986; Chi et al., 2009a,b; Cantarel et al., 2009; Sauer et al., 2000; Sturtevant et al., 1999; Ghang et al., 2007; Sato et al., 2005). Table 8.2.1 shows microorganisms that produce glucoamylase. The threedimensional structures of glucoamylases from various fungal, yeast, and bacterial sources have been studied. Various structural types of characterized glucoamylases are shown schematically in Fig. 8.2.1. Fig. 8.2.2 shows the structure of various domains found in glucoamylase.

8.2.2 Applications of glucoamylases Glucoamylases have several applications in industry. These are mostly used for producing high-glucose syrup, high fructose corn syrup, and high-conversion syrup. Starch

Table 8.2.1 Microorganisms producing glucoamylase.

Bacteria Clostridium sp. G0005, Clostridium acetobutylicum, C. thermosaccharolyticum, C. thermoamylolyticum, C. thennohydrosulfuricum, Bacillus stearothermophilus, Flavobacterium sp., Halobacterium sodomense Bacillus sp. Lactobacillus amylovorus Thermoanaerobacterium Thermosaccharolyticum Streptosporangium sp. Sulfolobus solfataricus Thermoplasma acidophilum Picrophilus torridus P. oshimae Yeast Saccharomyces cerevisiae var. diastaticusm Saccharomycopsis fibuligera, Saccharomycopsis capsularis, Schwanniomyces castellii, S. occidentalis, Lipomyces starkeyi, Endomycopsis fibuligera, Pichia subpelliculosa, Arxula adeninivorans, Candida albicans, Kluyveromyces lactis, Aureobasidium pullulans N13d, Candida tsulcubaensis CBS 6389, Filobasidium capsuligenum, Lipomyces kononenkoae, Trichosporon pullulans Fungi Rhizopus oryzae, R. javanicu,s R. niveus, R. delemar, Rhizopus sp., Aspergillus awamori, A. foetidus, A. niger, A. oryzae, A. terreus, A. candidus, A. phoenicis, A. saitoi, A. hennebergi, Neurospora crassa, Arthrobotrys sp., Penicillium oxalicum, Penicillium sp., Cladosporium gossypiicola ATCC 38026, Sclerotinia sclerotiorum, Trichoderma sp., Curvularia lunata, Fusarium solani, A. fumigates, Neosartorya fischeri M-1, Mucor rouxianus, M. javanicus, Rhizomucor pusillus, Cephalosporium eichhorniae, Humicola lanuginose, H. grisea, H. grisea var thennoidea, Torula thermophilus, Talaromyces duponti, Talaromyces emersonii, Thermomyces lanuginosus, Scytalidium thermophilum, Thielavia terrestris, Thermomucor indicae-seudaticae, Chaetomium thermovhilum Based on Kumar, P., Satyanarayana, T., 2009. Microbial glucoamylases: characteristics and applications. Crit. Rev. Biotechnol. 29, 225e255.

Figure 8.2.1 Different modular arrangements found in glucoamylases. Representative species of each arrangement are indicated: CD GH15 catalytic domain, CBM20 carbohydrate-binding domain family 20, CBM21 carbohydrate-binding domain family 21, STRD Ser/Thr-rich domain found in Sta1 from Saccharomyces cerevisiae var. diastaticus, bS super-b-sandwich domain present in prokaryotic enzymes. (From Marín-Navarro, J., Polaina, J., 2011. Glucoamylases: structural and biotechnological aspects. Appl. Microbiol. Biotechnol. 89 (5), 1267e1273. Reproduced with permission.)

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Figure 8.2.2 A catalytic domain of Aspergillus niger glucoamylase (PDB code 3EQA), with the (a/a)6barrel-fold characteristic of GH15 enzymes. Catalytic residues acting as acid and base in the mechanism of reaction are highlighted in red. (From Marín-Navarro, J., Polaina, J., 2011. Glucoamylases: structural and biotechnological aspects. Appl. Microbiol. Biotechnol. 89 (5), 1267e1273. Reproduced with permission.)

conversion to sugars is one of the most important biotechnological processes. Other applications include the bakery industry, low-calorie beer brewing, and the hydrolysis of whole grain products for the alcohol industry (James and Lee, 1997). 8.2.2.1 Production of sugars Glucoamylase catalyzes the saccharification of starch to produce glucose that is used in fermentation and the food industry. Combined action of different amylases is required for producing glucose from starch with glucoamylases. Firstly about 30%e35% dry solid starch slurry of starch is gelatinized at a temperature of about 60e90
C, and afterward, it is liquefied at 95e105
C at a pH of 6.5 by the use of bacterial a-amylases to dextrins, which are short-chain glucose polymer. These dextrins are saccharified by glucoamylases to produce glucose in the next step. Fungal glucoamylases shows optimum activity at a pH 4.0 to 4.5. Saccharification is performed under acidic conditions at a temperature of 60
C for 3e4 days. Glucose yield of about 96% is obtained (Reilly, 1999; Crabb and Mitchinson, 1997). In addition, debranching enzymes (pullulanases or isoamylases) have been used to speed up the processing of starch by breaking a-1,6 glycosidic bonds. This enables to obtain an early peak in glucose yield with lesser production of byproducts (Reilly, 2006). The sweetness of glucose is about 75% as compared to sucrose, whereas the sweetness of fructose is 2 times as compared to sucrose. Thus, fructose is favored

Industrial applications of thermophilic/hyperthermophilic enzymes

particularly in low-calorie diet/health foods. It is metabolized without insulin. The sweetness of fructose is 2 times the sweetness of sucrose at 50% of the weight (Synowiecki, 2007). On a commercial scale, fructose is produced from glucose. Glucose is isomerized by using glucose/xylose isomerase at a temperature of 50e60
C and pH of 7e8. Glucose isomerase (E.C. 5.3.1.5, D-xylose-ketol isomerase) is the most costly enzyme among all the enzymes used in processing of starch, and therefore, it is reutilized until most of its activity is lost. Concentrated glucose syrup is passed through a column containing the immobilized glucose isomerase or cells producing glucose isomerase (Crabb and Mitchinson, 1997). About 40%e42% fructose is obtained. The glucose-fructose mixture is enriched by chromatographic technique to increase the fructose concentration to 55% in the final product (Reilly, 1999; Crabb and Shetty, 1999). 8.2.2.2 Ethanol production Several starchy raw materials have been used for production of ethanol. These include potato, corn, wheat, cassava, etc. (Lindeman and Rocchiccioli, 1979; Maisch et al., 1979; Maiorella et al., 1981; Wilke et al., 1981; Mojovic et al., 2006; Giordano et al., 2008). Though several yeasts are found to produce amylases, only a few of them have the ability to ferment. These include Schwanniomyces alluvius, Candida tropicalis and Saccharomycopsis fibuligera (Simoes-Mendes, 1984; Nakamura, 1970; Jamai et al., 2007). The most widely used yeast for fermenting glucose to ethanol is S. cerevisiae. This yeast lacks the amylolytic activity so it is not able to use starch directly. As a result, the conventional production of ethanol involves two steps. The starch is initially degraded to fermentable sugars, which are used by the yeast (Panchal et al., 1984). The rising demand for ethanol as a fuel additive has increased the usage of yeasts producing amylolytic enzymes in direct use of the leftover agricultural residues rich in starch and industrial wastes for formulating more cost-effective and easy one step process for producing bioethanol (Eksteen et al., 2003; Steyn and Pretorius, 1990; Pretorius et al., 1991). Several genes encoding for diverse amylases (a-amylases, b-amylases, glucoamylases, and pullulanases) have been cloned and expressed in S. cerevisiae for simplifying use of starch and improving ethanol production by yeasts producing amylolytic enzymes (Birol et al., 1998; Khaw et al., 2006). Among the glucoamylase, the encoding genes from R. oryzae, S. fibuligera, S. cerevisiae var. diastaticus, A. awamori have been expressed in S. cerevisiae (Ashikari et al., 1986; Yamashita et al., 1985; Erratt and Nasim, 1986; Lin et al., 1998; Innis et al., 1985; Cole et al., 1988). Expression of the H. resinae glucoamylase P gene in S. cerevisiae allowed the transformed yeast to grow on 5% soluble starch and produce ethanol. A Saccharomyces yeast producing S. diastaticus glucoamylase and mouse a-amylase was found to produce more than 90% starch in a single step process (Vainio et al., 1993, 1994; Kim et al., 1988).

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A recombinant strain of S. cerevisiae was developed (Janse and Pretorius, 1995). Klebsiella pneumoniae pullulanase genes, Bacillus amyloliquefaciens a-amylase, and S. cerevisiae var. diastaticus glucoamylase in diverse combinations were expressed. One recombinant strain showed 99% utilization of starch. The expression of Pseudomonas amyloderamosa isoamylase genes and A. awamori glucoamylase in S. cerevisiae under the control of the yeast alcohol dehydrogenase gene promoter was found to be quite helpful. The recombinant strains were found to utilize starch more effectively. The conversion rate was more than 95% (Ma et al., 2000). Rapid growth of S. cerevisiae, showing Bacillus stearothermophilus a-amylase and Rhizopus oryzae glucoamylase on its cell surface, on starch was reported by Murai et al. (1999). The recombinant S. cerevisiae YPB-G expressing the A. awamori glucoamylase and Bacillus subtilis a-amylase was found to produce large quantities of bioethanol in the medium, which contained 40 g/L of starch (Altintas et al., 2002). The addition of glucose during the initial phase of fermentation was found to support rapid growth and produced higher amount of ethanol. The transformants of S. cerevisiae, which contained Lipomyces kononenkoae a-amylase gene produced ethanol. The yield was 0.4 g per gram starch utilized (Knox et al., 2004). The recombinant S. cerevisiae containing Lentinula edodes glucoamylase and barley a-amylase were found to hydrolyze starch about three times faster in comparison to the individual enzymes. However, its ability to produce ethanol was not studied (Wong et al., 2007). In comparison to conventional culture using fermenting and an amylolytic microorganism, higher ethanol production by 1.5 times was obtained by Nakamura et al. (2000). Surface contact between yeast cells (exhibiting a-amylase and glucoamylase) and the starch granules is a crucial factor that enhances a-amylase activity and is a limiting factor for direct fermentation of ethanol by recombinant yeasts (Khaw et al., 2007). 8.2.2.3 Other applications Glucoamylases are also being used in beer, bread, textile, and pharmaceutical industries. For producing low-calorie beer, glucoamylases are used. By using immobilized glucoamylases, higher production of glucose is obtained, which could be efficiently fermented to ethanol (Synowiecki, 2007). Glucoamylases improves the bread crust color. It helps in the saccharification of starch to glucose, which is fermented easily by the yeast (Kumar and Satyanarayana, 2008). A recombinant yeast strain using glucoamylase of R. oryzae and two types of cellulose-binding domains of Trichoderma reesei for use in desizing of cotton cloth was constructed by Fukuda et al. (2008). Glucoamylases are also being used as a digestive aid. Several different types of enzyme formulations for this application are available from different producers. These

Industrial applications of thermophilic/hyperthermophilic enzymes

Table 8.2.2 Manufacturers of glucoamylases.

Novozymes, Bagsvaerd, Demmark Genecor International, Copenhagen, Denmark Amano Enzymes USA Co., Ltd. Elgin, IL China-America Technology Corp. New York, USA Jinzhu Tibet Co., Ltd., China Enzyme Development Corporation USA Sichuan Shan Ye BioTech Co., Ltd. China Wuxi Syder Bioproducts Co., Ltd China Shanghai Kaiquan Biotechnology Co., Ltd. China Sunson Industry Group Co., Ltd. China Qingdao Continent Industry Co., Ltd. China Advanced Enzyme Technologies Ltd. Thane, India Maps (India) Limited, India Based on Kumar, P., Satyanarayana, T., 2009. Microbial glucoamylases: characteristics and applications. Crit. Rev. Biotechnol. 29, 225e255.

formulations usually do not consist of pure glucoamylases but a mixture of enzymes including a-amylases, lipases, proteases, cellulases, peptidases, pancreatin, and others. Glucoamylases are sold in both liquid and solid forms with different trade names and compositions. Many companies are commercially producing glucoamylases for several applications like the production of bioethanol and starch saccharification. Some of the commercially available glucoamylases are presented in Table 8.2.2 (Kumar and Satyanarayana, 2009).

Bibliography Adam, A.C., Latorre-Garcia, L., Polaina, J., 2004. Structural analysis of glucoamylase encoded by the STA1 gene of Saccharomyces cerevisiae (var. diastaticus). Yeast 21, 379e388. Altintas, M.M., Ulgen, K., Kirdar, B., Onsan, Z.I., Oliver, S.G., 2002. Improvement of ethanol production from starch by recombinant yeast through manipulation of environmental factors. Enzym. Microb. Technol. 31, 640e647. Ashikari, T., Nakamura, N., Tanaka, Y., Kiuchi, N., ShiBano, Y., Tanaka, T., Amachi, T., Yoshizumi, H., 1986. Rhizopus raw-starch-degrading glucoamylase: its cloning and expression in yeast. Agric. Biol. Chem. 50, 957e964. Birol, G., Onsan, Z.I., Kirdar, B., Oliver, S.G., 1998. Ethanol production and fermentation characteristics of recombinant Saccharomyces cerevisiae strains grown on starch. Enzym. Microb. Technol. 22, 672e677. Cantarel, B.L., Coutinho, P.M., Rancurel, C., Bernard, T., Lombard, V., Henrissat, B., 2009. The carbohydrate-active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res. 37 (Database issue), D233eD238. Carrasco, M., Alcaíno, J., Cifuentes, V., Baeza, M., 2017. Purification and characterization of a novel cold adapted fungal glucoamylase. Microb. Cell Factories 16, 75. Chen, J., Zhang, Y.Q., Zhao, C.Q., Li, A.N., Zhou, Q.X., Li, D.C., 2007. Cloning of a gene encoding thermostable glucoamylase from Chaetomium thermophilum and its expression in Pichia pastoris. J. Appl. Microbiol. 103, 2277e2284.

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Chi, Z., Chi, Z., Liu, G., Wang, F., Ju, L., Zhang, T., 2009a. Saccharomycopsis fibuligera and its application in biotechnology. Biotechnol. Adv. 27, 423e431. Chi, Z.M., Wang, F., Chi, Z., Yue, L.X., Liu, G.L., Zhang, T., 2009b. Bioproducts from Aureobasidium pullulans, a biotechnologically important yeast. Appl. Microbiol. Biotechnol. 82, 793e804. Cole, G.E., McCabe, P.C., Inlow, D., Gelfand, D.H., Ben-Bassat, A., Innis, M.A., 1988. Stable expression of Aspergillus awamori glucoamylase in distiller’s yeast. Bio Technol. 6, 417e421. Crabb, W.D., Mitchinson, C., 1997. Enzymes involved in the processing of starch to sugars. TIBTECH 15, 349e352. Crabb, W.D., Shetty, J.K., 1999. Commodity scale production of sugars from starches. Curr. Opin. Microbiol. 2, 252e256. Eksteen, J.M., Rensburg, P., Cordero Otero, R.R., Pretorius, I.S., 2003. Starch fermentation by recombinant Saccharomyces cerevisiae strains expressing the alpha-amylase and glucoamylase genes from Lipomyces kononenkoae and Saccharomycopsis fibuligera. Biotechnol. Bioeng. 84, 639e646. Erratt, J.A., Nasim, A., 1986. Cloning and expression of a Saccharomyces diastaticus glucoamylase gene in Saccharomyces cerevisiae and Schizosaccharomyces pombe. J. Bacteriol. 166, 484e490. Fierobe, H.P., Clarke, A.J., Tull, D., Svensson, B., 1998. Enzymatic properties of cysteinesulfinic acid derivative of the catalytic base mutant Glu400!Cys of glucoamylase from Aspergillus awamori. Biochemistry 37, 3753e3759. Fukuda, T., Kato-Murai, M., Kuroda, K., Ueda, M., Suye, S.I., 2008. Improvement in enzymatic desizing of starched cotton cloth using yeast codisplaying glucoamylase and cellulose-binding domain. Appl. Microbiol. Biotechnol. 77, 1225e1232. Ghang, D.M., Yu, L., Lim, M.H., Ko, H.M., Im, S.Y., Lee, H.B., Bai, S., 2007. Efficient one-step starch utilization by industrial strains of Saccharomyces cerevisiae expressing the glucoamylase and a-amylase genes from Debaryomyces occidentalis. Biotechnol. Lett. 29, 1203e1208. Giordano, R.L.C., Trovati, J., Schmidell, W., 2008. Continuous production of ethanol from starch using glucoamylase and yeast co-immobilized in pectin gel. Appl. Biochem. Biotechnol. 147, 47e61. He, Z., Zhang, L., Mao, Y., Gu, J., Pan, Q., Zhou, S., Gao, B., Wei, D., 2014. Cloning of a novel thermostable glucoamylase from thermophilic fungus Rhizomucor pusillus and high-level co-expression with a-amylase in Pichia pastoris. BMC Biotechnol. 14, 114. Hostinov
a, E., Gasperík, J., 2010. Yeast glucoamylases: molecular genetic and structural characterization. Biol. Sect. Cell Mol. Biol. 65, 559e568. Innis, M.A., Holland, M.J., McCabe, P.C., Cole, G.E., Wittman, V.P., Tal, R., Watt, K.W.K., Gelfand, D.H., Holland, J.P., Meade, J.H., 1985. Expression, glycosylation, and secretion of an Aspergillus glucoamylase by Saccharomyces cerevisiae. Science 228, 21e26. Jamai, L., Ettayebi, K., Yamani, J.E., Ettayebi, M., 2007. Production of ethanol from starch by free and immobilized Candida tropicalis in the presence of alpha-amylase. Bioresour. Technol. 98, 2765e2770. James, J.A., Lee, B.H., 1997. Glucoamylases: microbial sources, industrial applications and molecular biologyda review. J. Food Biochem. 21, 1e52. Janse, B.J.H., Pretorius, I.S., 1995. One-step enzymatic hydrolysis of starch using a recombinant strain of Saccharomyces cerevisiae producing alpha-amylase, glucoamylase and pullulanase. Appl. Microbiol. Biotechnol. 42, 878e883. Karim, K.M.R., Husaini, A., Sing, N.N., Tasnim, T., Mohd Sinang, F., Hussain, H., Hossain, M.A., Roslan, H., 2019. Characterization and expression in Pichia pastoris of a raw starch degrading glucoamylase (GA2) derived from Aspergillus flavus NSH9. Protein Exp. Purif. 164, 105462. Khaw, T.S., Katakura, Y., Koh, J., Kondo, A., Ueda, M., Shioya, S., 2006. Evaluation of performance of different surface-engineered yeast strains for direct ethanol production from raw starch. Appl. Microbiol. Biotechnol. 70, 573e579. Khaw, T.S., Katakura, Y., Ninomiya, K., Moukamnerd, C., Kondo, A., Ueda, M., Shioya, S., 2007. Enhancement of ethanol production by promoting surface contact between starch granules and arming yeast in direct ethanol fermentation. J. Biosci. Bioeng. 103, 95e97.

Industrial applications of thermophilic/hyperthermophilic enzymes

Kim, K., Park, C.S., Mattoon, J.R., 1988. High-efficiency, one-step starch utilization by transformed Saccharomyces cells which secrete both yeast glucoamylase and mouse alpha-amylase. Appl. Environ. Microbiol. 54, 966e971. Kim, M., Park, J., Kim, Y., Lee, H., Nyawira, R., Shin, H., Park, C., Yoo, S., Kim, Y., Moon, T., Park, K., 2004. Properties of a novel thermostable glucoamylase from the hyperthermophilic archaeon Sulfolobus solfataricus in relation to starch processing. Appl. Environ. Microbiol. 70, 3933. Knox, A.M., Preez, J.C., Kilian, S.G., 2004. Starch fermentation characteristics of Saccharomyces cerevisiae strains transformed with amylase genes from Lipomyces kononenkoae and Saccharomycopsis fibuligera. Enzyme Microb. Technol. 34, 453e460. Kumar, P., Satyanarayana, T., 2009. Microbial glucoamylases: characteristics and applications. Crit. Rev. Biotechnol. 29, 225e255. Kumar, P., Satyanarayana, T., 2008. Potential applications of microbial enzymes in improving quality and shelf life of bakery products. In: Koutinas, A., Pandey, A., Larroche, C. (Eds.), Current Topics on Bioprocesses in Food Industry. Asiatech Publishers, New Delhi, India, pp. 132e142. Lago, M.C., dos Santos, F.C., Bueno, P.S.A., de Oliveira, M.A.S., Barbosa-Tessmann, I.P., 2021. The glucoamylase from Aspergillus wentii: purification and characterization. J. Basic Microbiol. 61, 443e458. Lin, L.L., Ma, Y.J., Chien, H.R., Hsu, W.H., 1998. Construction of an amylolytic yeast by multiple integration of the Aspergillus awamori glucoamylase gene into a Saccharomyces cerevisiae chromosome. Enzyme Microb. Technol. 23, 360e365. Lindeman, L.R., Rocchiccioli, C., 1979. Ethanol in Brazil; brief summary of the state of the industry in 1977. Biotechnol. Bioeng. 21, 1107e1119. Ma, Y.J., Lin, L.L., Chien, H.R., Hsu, W.H., 2000. Efficient utilization of starch by a recombinant strain of Saccharomyces cerevisiae producing glucoamylase and isoamylase. Biotechnol. Appl. Biochem. 31, 55e59. Maiorella, B., Wilke, C.H.R., Blanch, H.W., 1981. Alcohol production and recovery. Adv. Biochem. Eng. 20, 43e92. Maisch, W.F., Sobolov, M., Petricola, A.J., 1979. Distilled beverages. In: Peppler, H.J., Perlman, D. (Eds.), Microbial Technology. Academic Press, New York, p. 79. Marín-Navarro, J., Polaina, J., 2011. Glucoamylases: structural and biotechnological aspects. Appl. Microbiol. Biotechnol. 89 (5), 1267e1273. McCann, A.K., Barnett, J.A., 1986. The utilization of starch by yeasts. Yeast 2, 109e115. McCarter, J.D., Withers, S.G., 1994. Mechanisms of enzymatic glycoside hydrolysis. Curr. Opin. Struct. Biol. 4, 885e892. Michelin, M., Ruller, R., Ward, R.J., Moraes, L.A., Jorge, J.A., Terenzi, H.F., Polizeli Mde, L., 2008. Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus Paecilomyces variotii. J. Ind. Microbiol. Biotechnol. 35 (1), 17e25. Mojovic, L., Nikolic, S., Rakin, M., Vukasinovic, M., 2006. Production of bioethanol from corn meal hydrolyzates. Fuel 85, 1750e1755. Murai, T., Ueda, M., Shibasaki, Y., Kamasawa, N., Osumi, M., Imanaka, T., Tanaka, A., 1999. Development of an arming yeast strain for efficient utilization of starch by co-display of sequential amylolytic enzymes on the cell surface. Appl. Microbiol. Biotechnol. 51, 65e70. Nakamura, L.K., 1970. Influence of the acceptor during transglucosylation by transglucosylamylase of Candida tropicalis. Can. J. Biochem. 48, 1260e1267. Nakamura, Y., Kobayashi, F., Ohnaga, M., Sawada, T., 2000. Alcohol fermentation of starch by a genetic recombinant yeast having glucoamylase activity. Biotechnol. Bioeng. 53, 21e25. Norouzian, D., Akbarzadeh, A., Scharer, J.M., Moo Young, M., 2006. Fungal glucoamylases. Biotechnol. Adv. 24, 80e85. Panchal, C.J., Russell, I., Sills, A.M., Stewart, G.G., 1984. Genetic manipulation of brewing and related yeast strains. Food Technol 38, 99e111. Pandey, A., 1995. Glucoamylase research and overview. Starch 47 (11), 439e445. Pretorius, I.S., Lambrechts, M.G., Marmur, J., 1991. The glucoamylase multigene family in Saccharomyces cerevisiae var. diastaticus: an overview. Crit. Rev. Biochem. Mol. Biol. 26, 53e76.

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Reilly, P.J., 1999. Protein engineering of glucoamylase to improve industrial properties: a review. Starch 51, 269e274. Reilly, P.J., 2006. Glucoamylase. In: Whitaker, J.R., Voragen, A.G.J., Wong, D.W.S. (Eds.), Handbook of Enzymology. Marcel Dekker, Inc., New York, pp. 727e738. Sato, F., Okuyama, M., Nakai, H., Mori, H., Kimura, A., Chiba, S., 2005. Glucoamylase originating from Schwanniomyces ocidentalis is a typical a-glucosidase. Biosci. Biotechnol. Biochem. 69, 1905e1913. Sauer, J., Sigurskjold, B.W., Christensen, U., Frandsen, T.P., Migorodskaya, E., Harrison, M., Roepstorff, P., Svensson, B., 2000. Glucoamylase: structure/function relationships, and protein engineering. Biochim. Biophys. Acta 1543, 275e293. Simoes-Mendes, B., 1984. Purification and characterisation of the extracellular amylases of the yeast Schwanniomyces alluvius. Can. J. Microbiol. 30, 1163e1170. Spencer-Martins, I., van Uden, N., 1979. Extracellular amylolytic system of the yeast Lipomyces kononenkoae. Eur. J. Appl. Microbiol. Biotechnol. 6, 241e250. Steyn, A.J.C., Pretorius, I.S., 1990. Expression and secretion of amylolytic enzymes by Saccharomyces cerevisiae. Acta Varia 5, 76e126. Sturtevant, J., Dixon, F., Wadsworth, E., Latge, J.P., Zhao, X.J., Calderone, R., 1999. Identification and cloning of GCA1, a gene that encodes a cell surface glucoamylase from Candida albicans. Med. Mycol. 37, 357e366. Synowiecki, J., 2007. The use of starch processing enzymes in the food industry. In: Polaina, J., Mac Cabe, A.P. (Eds.), Industrial Enzymes, Structure, Function and Applications. Springer, the Netherlands, pp. 19e34. Tanaka, Y., Konno, N., Suzuki, T., Habu, N., 2020. Starch-degrading enzymes from the brown-rot fungus Fomitopsis palustris. Protein Exp. Purif. 170, 105609. Thorsen, T.S., Johnsen, A.H., Josefsen, K., Jensen, B., 2006. Identification and characterization of glucoamylase from fungus Thermomyces lanuginosus. Biochim. Biophys. Acta 1764, 671e676. Tong, L., Zheng, J., Wang, X., Wang, X., Huang, H., Yang, H., Tu, T., Wang, Y., Bai, Y., Yao, B., Luo, H., Qin, X., 2021. Improvement of thermostability and catalytic efficiency of glucoamylase from Talaromyces leycettanus JCM12802 via site-directed mutagenesis to enhance industrial saccharification applications. Biotechnol. Biofuels 14 (1), 202. Vainio, A.E.I., Torkkeli, H.T., Tuusa, T., Aho, S.A., Fagerstrom, B.R., Korhola, M.P., 1993. Cloning and expression of Hormoconis resinae glucoamylase P cDNA in Saccharomyces cerevisiae. Curr. Genet. 24, 38e44. Vainio, A.E.I., Lantto, R., Parkkinen, E.E.M., Torkkeli, H.T., 1994. Production of Hormoconis resinae glucoamylase P by a stable industrial strain of Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 41, 53e57. Wang, C., Yang, L., Luo, L., Tang, S., Wang, Q., 2020. Purification and characterization of glucoamylase of Aspergillus oryzae from Luzhou-flavour Daqu. Biotech. Lett. 42, 2345e2355. Wilke, C.R., Yang, R.D., Scamanna, A.F., Freitas, R.P., 1981. Raw material evaluation and process development studies for conversion of biomass to sugars and ethanol. Biotechnol. Bioeng. 23, 163e183. Wong, D.W., Robertson, G.H., Lee, C.C., Wagschal, K., 2007. Synergistic action of recombinant alphaamylase and glucoamylase on the hydrolysis of starch granules. Protein J. 26 (3), 159e164. Yamashita, I., Itoh, T., Fukui, S., 1985. Cloning and expression of the Saccharomycopsis fibuligera glucoamylase gene in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 23, 130e133. Zheng, Y., Xue, Y., Zhang, Y., Zhou, C., Schwaneberg, U., Ma, Y., 2010. Cloning, expression, and characterization of a thermostable glucoamylase from Thermoanaerobacter tengcongensis MB4. Appl. Microbiol. Biotechnol. 87, 225e233.

Relevant websites https://infinitabiotech.com/blog/glucoamylase-enzyme/?. https://www.novozymes.com/en/advance-your-business/food-and-beverage/starch/saccharification/ extenda. https://biosolutions.novozymes.com/en/distilling/saccharification/saczyme-plus-2x.

Industrial applications of thermophilic/hyperthermophilic enzymes

SUBCHAPTER 8.3

Glucosidases 8.3.1 Microbial sources and properties The enzyme b-glucosidase (b-D-glucopyrranoside glucohydrolase) [E.C.3.2.1.21] is a common enzyme and plays a significant role in a number of biological processes. It is produced by several bacteria, yeasts, fungi, plants, and animals including noncellulolytic organisms. This enzyme hydrolyzes the glycosidic bond of a carbohydrates and liberate nonreducing terminal glycosyl residues, glycosides as well as oligosaccharides (Li et al., 2013; Bhatia et al., 2022; Cairns and Esen, 2010; Morant et al., 2008). Several physiological functions are found to be linked with this enzyme. These actually depend upon the biological system in which they are found and also the position of the enzyme. In case of cellulolytic microbes, b-glucosidases are involved in the induction of cellulases and hydrolysis of cellulose whereas in plants, these enzymes are involved in the synthesis of b-glucans during the development of cell wall, cleavage of glycosylated flavonoids, fruit ripening, pigment metabolism, and defense mechanisms (Tomme et al., 1995; Esen, 1993). In mammals, b-glucosidases are involved in the hydrolysis of glucosyl ceramides (Lieberman et al., 2007). In humans, deficiency in b-glucosidase activity is associated with Gaucher disease. Certain fatty substances get buildup in certain organs, mainly spleen and liver. These enzymes show broader substrate specificity and are utilized in many biotechnological processes in liberation of aromas, flavors, and isoflavone aglycones and production of oligosaccharides and alkylglycosides. These enzymes have been extensively studied to saccharify cellulosic biomass for producing ethanol. Production of arylglycosides and oligosaccharides is also researched rigorously (Singhania et al., 2017). b-Glucosidases are a significant part of cellulolytic enzymes. These enzymes are important for completely hydrolyzing cellulose into glucose (Singh et al., 2016). Cellulases first hydrolyze the cellulose to cellobiose and other short chain oligosaccharides, which are then finally hydrolyzed to glucose by the action of b-glucosidases. The enzymes, involved in hydrolysis of cellulose, are generally grouped as cellulases, which consist of the following enzymes (Teeri, 1997; Bhat and Bhat, 1997): Endoglucanase[E.C.3.2.1.4] (endo-1, 4eb-glucanase) Exoglucanase (cellobiohydrolase) [E.C.3.2.1.91] (exo-1, 4-b-glucanase b-glucosidase[E.C.3.2.1.21] (b-D-glucoside glycohydrolase) These enzymes act synergistically in the breakdown of cellulose (Krogh et al., 2009). Endoglucanases and cellobiohydrolases degrade native cellulose for producing cellobiose, which inhibits these enzymes (Bhat and Bhat, 1997). Still, b-glucosidase can scavenge the end product, cellobiose, by slicing the b (1e4) bond to yield D-glucose. Thus,

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b-glucosidases produce glucose from cellobiose and reduce cellobiose inhibition, thus allowing cellulolytic enzymes to work more effectively (Saha et al., 1994). Furthermore, the addition of b-glucosidase to the lignocellulosic material increased the liberation of phenolic compounds, showing that cellulolytic enzymes may also be involved in the degradation of the polymeric phenolic matrix (Liu et al., 2012; Zheng and Shetty, 2000). Fig. 8.3.1 shows the action of b-glucosidases, along with the other enzyme components of cellulases (Srivastava et al., 2019). The classification of b-glucosidases has been done according to several criteria (Singhania et al., 2017). No single precise method exists for classifying these versatile enzymes. There are two methods for their classification. These are based on (Henrissat and Bairoch, 1996): (1) Substrate specificity (2) Nucleotide sequence identity These enzymes are classified as follows based upon their substrate specificity: (1) Aryl-b-glucosidases (act on arylglucosides) (2) True cellobiases (hydrolyze, cellobiose, and liberate glucose) (3) Broader substrate specificity

Figure 8.3.1 Hydrolysis of cellulose by the synergistic action of cellulases. (From Srivastava, N., Rathour, R., Jha, S., Pandey, K., Srivastava, M., Thakur, V.K., Sengar, R.S., Gupta, V.K., Mazumder, P.B., Khan, A.F., Mishra, P.K., 2019. Microbial beta glucosidase enzymes: recent advances in biomass conversion for biofuels application. Biomolecules 9 (6), 220. Distributed under the Creative commons Attribution License.)

Industrial applications of thermophilic/hyperthermophilic enzymes

Many of the b-glucosidases characterized so far are placed in the last group with different capacities to cleave b-1,4; b-1,6; b-1,2; a-1,3; a-1,4; and a-1,6 glycosidic bonds. The thermal stability of the enzyme is important because in the saccharification step, steam is used to create a more suitable substrate for enzymatic hydrolysis (Liu et al., 2011). Thermostable enzymes have been used directly and also simultaneously in the saccharification step with no precooling process. Extensive research is being conducted for obtaining efficient and thermostable b-glucosidases. At present, microorganisms are the major source for producing b-glucosidases on a commercial scale. Fungal b-glucosidases have been widely researched in case of few model organisms for instance Phanerochaete chrysosporium and Trichoderma reesei (Matai et al., 1992; Lymar et al., 1995). The b-glucosidases from Aspergillus species have been examined in detail. These include the b-glucosidases produced by A. terreus, A. niger, and A. oryzae (Workman and Day, 1982; Watanabe et al., 1992; Riou et al., 1998). A. oryzae b-glucosidases show a high tolerance to glucose. Research has focused on b-glucosidases from the thermophilic strain of A. fumigates (Rudick and Elbein, 1975). b-Glucosidases are intracellular, extracellular, or membrane bound. Intracellular b-glucosidase is normally synthesized when carbon sources in the medium are depleted. However, membrane-bound b-glucosidases are widespread in yeast. A. niger b-glucosidase, commonly used for supplementing the cellulolytic cocktail of T. reesei, is produced extracellularly, facilitating isolation and purification and significantly dropping the downstream processing cost. Microbial sources are widely used for producing b-glucosidase by solid-state fermentation and submerged fermentation methods. Among microorganisms, filamentous fungi are the most important sources of b-glucosidases. Phanerochaete chrysosporium, Paecilomyces sp. Penicillium brasilianum, P. decumbens, A. oryzae, and A. niger are the important producers of b-glucosidases (Chen et al., 2010; Riou et al., 1998; Tsukada et al., 2006; Yang et al., 2009; Gunata and Vallier, 1999; Krogh et al., 2010). b-glucosidase production has been also reported by yeast (mostly Candida) and some bacteria. Even Clostridium (Anaerobic bacteria) also produces b-glucosidase along with other cellulolytic components. Commercially b-glucosidase is produced using Aspergillus species. Aspergillus produces higher levels of the enzyme (Rajasree et al., 2013). Not much is published on large-scale production of b-glucosidases. b-Glucosidase genes from numerous bacterial, fungal, yeast, plant, and animal systems have been cloned and expressed in E. coli as well as eukaryotic microorganisms like Saccharomyces cerevisiae and filamentous fungi. Cloning can be either (1) selection of recombinant clones by generating a genomic DNA library followed by production of b-glucosidase or (2) starting from a cDNA (or genomic) library and selecting recombinant clones containing specific nucleotides. Probes have been developed from a previous knowledge of polypeptide sequences (Singhania et al., 2017; Bhatia et al., 2022).

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Table 8.3.1 Microorganisms producing b-glucosidases. Microorganism

Penicillium pinophilum

Production process

Application

References

Enhancing wine aroma

Villena et al. (2006)

Cellulosic bioconversion Biomass (corncob) conversion Biomass hydrolysis Cellobiose hydrolysis

Ng et al. (2010)

Penicillium decumbens

Shake-flask fermentation Solid-state fermentation SmF

Penicillium echinulatum Stachybotrys sp.

SmF Fed-batch SmF

Humicola insolens

Shake flask

Fomitopsis palustris

Shake flask

Cellulosic biomass hydrolysis Cellobiose hydrolysis

Fomitopsis sp.

Solid-state fermentation SmF, shake flask

Rice straw and wheat straw hydrolysis Biomass hydrolysis

SmF, shake flask Shake-flask fermentation Shake-flask fermentation SmF

Biomass hydrolysis Enhancing wine aroma

Singhania et al. (2011) Rajasree et al. (2013) Villena et al. (2006)

Biomass hydrolysis

Kovacs et al. (2008)

Biomass conversion

Harnpicharnchai et al. (2009)

Penicillium citrinum

Aspergillus niger Aspergillus unguis Debaryomyces pseudopolymorphus Trichoderma atroviride Periconia sp.

Chen et al. (2010) Martins et al. (2008) Amouri and Gargouri (2006) Souza et al. (2010) Okamoto et al. (2011) Deswal et al. (2011)

Based on Singhania, R.R., Patel, A.K., Saini, R., Pandey, A., 2017. Industrial enzymes b-glucosidases. In: Current Developments in Biotechnology and Bioengineering, Elsevier.

Fungal strains involved in b-glucosidase production and the bioprocess for their production is presented in Table 8.3.1 (Singhania et al., 2017). Sources and hosts of b-Glucosidases genes and properties of the recombinant b-glucosidases are presented in Table 8.3.2 (Singhania et al., 2017).

8.3.2 Industrial application of b-glucosidases b-Glucosidases are widely used in many biotechnological processes. b-Glucosidases are involved in the hydrolysis of b (1e4) glucosidic linkages of disaccharides, for instance, cellobiose, oligosaccharides, and glucose-substituted molecules, though few novel b-glucosidases are able to hydrolyze bonds for instance b (1e3), b (1e6), b (1e2) bonds. So, these can be used in several applications in pharmaceutical and food industries and for producing biofuels (Ahmed et al., 2017).

Industrial applications of thermophilic/hyperthermophilic enzymes

Table 8.3.2 Sources and hosts of b-glucosidases genes and properties of the recombinant b-glucosidases. Source

Host organism

Saccharomycopsis fibuligera Talaromyces emersonii

Saccharomyces cerevisiae Trichoderma reesei

Periconia sp.

Pichia pastoris

Penicillium decumbens

T. reesei

Aspergillus niger

T. reesei

Caldicellulosiruptor saccharolyticus

Escherichia coli

Paecilomyces thermophila

P. pastoris

Periconia sp.

T. reesei QM9414

Neocallimastix patriciarum

P. pastoris

A. niger Chaetomium thermophilum

Penicillium verruculosum P. pastoris

Fervidobacterium islandicum Aspergillus aculeateus Aspergillus fumigatus Z5

E. coli T. reesei P. pastoris X33

Properties of recombinant BGL

References

1.021 U/mg

Shen et al. (2008)

GH family 3, thermostable active at 71.5
C, Vmax ¼ 5121 U/mg, K ¼ 0.254 mM against glucose Thermotolerant BGL, optimal activity at 70
C and at pH 5e7 Six- to eight fold increased BGL activity compared to native strain 5.3 IU/mL (106) times higher than native BGL Thermostable with 13 U/ mg, having optimum activity at 70
C and pH 5.5 GH family 3, 274.4 U/mL, optimal at pH 6 and 60
C 10.5-fold BGL activity increased from 2.2 to 23.91 U/mg, thermotolerant and active in acidic pH GH family 3, 34.5 U/mg against cellobiose, optimally active at 40
C and pH 5.0 Specific activity of BGL increased by 22% Optimally active at pH 5.0 and 60
C GH family 1, thermostable 10 U/mg against cellobiose Active at pH 6.0 and 60
C with specific activity of about 1001 U/mg

Murray et al. (2004)

Harnpicharnchai et al. (2011) Ma et al. (2011)

Wang and Xia (2011) Hong et al. (2009)

Yan et al. (2012)

Dashtban and Qin (2012)

Chen et al. (2012)

Dotsenko-Gleb et al. (2015) Xu et al. (2011) Jabbour et al. (2012) Nakazawa et al. (2012) Liu et al. (2011)

From Singhania, R.R., Patel, A.K., Saini, R., Pandey, A., 2017. Industrial enzymes b-glucosidases. In: Current Developments in Biotechnology and Bioengineering, Elsevier. Reproduced with permission.

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8.3.2.1 Biofuel production Production of biofuels from lignocelluloses involves the usage of several enzymes, which act in a synergist manner to degrade the lignocelluloses to pentoses and hexoses. These sugars are then converted to bioethanol. Cellulases and xylanases are the major enzymes involved in the degradation of lignocelluloses (Yan et al., 2008). Ethanol is produced from cellulose through the degradation of cellulose to cellobiose and glucose, which is converted to ethanol by the microorganisms (Kotaka et al., 2008). Endoglucanases and exoglucanases degrade cellulose to cellobiose and some cello-oligosaccharides. These sugars get converted to glucose by the action of b-glucosidases. This reaction is the most essential step in cellulose degradation as it limits the efficacy of hydrolysis and relieves the inhibition of endoglucanases and exoglucanases caused by cellobiose (Zhenming et al., 2009). The yeast Clavispora NRRL Y-50464 uses cellobiose as the only carbon source and produces b-glucosidases for production of cellulosic ethanol (Liu et al., 2012b). This yeast is able to tolerate main inhibitors obtained from lignocellulosic biomass pretreatment for instance furfural and hydroxymethyl furfural and convert furfural into furan methanol in less than 12 h and hydroxymethyl furfural into furan-2,5-dimethanol in 24 h in the presence of 15 mM of furfural and hydroxymethyl furfural. Ethanol was produced at a concentration of 23 g/L without adding any external b-glucosidases by this culture. But, the most extensively used cellulases from T. viridae shows a low activity of b-glucosidases, and the buildup of cellobiose lead to product inhibition. When thermostable b-glucosidases were added to commercial preparation of cellulases, a synergistic effect and higher concentration of reducing sugars were observed (Krisch et al., 2010). Bacteria-producing ethanol is attracting a lot of attention because they show higher growth rate in comparison to S. cerevisiae, which is mostly used for commercially producing ethanol. Yanase et al. (2005) conducted study on Zymomonas mobilis, which is an ethanol-producing bacterium. This is an attractive candidate for production of ethanol as it shows higher growth rate as well as higher production of ethanol. However, it cannot ferment broad range of fermentable sugars. This reduces its use for production of ethanol. To solve this problem, genetic engineering in b-glucosidase gene was performed to enlarge its substrates range, which produced 0.49 g ethanol/g cellobiose by genetically engineered organism. Several studies have focused on using microbial b-glucosidases for production of biofuel from cellulosic waste (Singh et al., 2016). 8.3.2.2 Isoflavones glycoside hydrolysis Phenolic compounds are a class of plants secondary metabolites, which differ in their biological functions and chemical structure. Phenolic compounds include flavonoids, flavones, flavonones, and isoflavones. These compounds have been extensively researched particularly in the field of food technology and health. These have biological

Industrial applications of thermophilic/hyperthermophilic enzymes

activity as anti-inflammatory agents, antiallergic, anticancer, antioxidant, antihypertensive, etc. (Servili et al., 2013; Karimi et al., 2012; Kabera et al., 2014). Naturally, most of these compounds are present in the form of glycosides. This increases their stability and water solubility and limit their absorption from human gastrointestinal tract (Setchell et al., 2002). Generally, glycosides have monoglucose unit attached to other sugars like xylose, galactose, and arabinose. The liberation of aglycone moiety needs the action of specific enzymes like arabinosidase and b-glucosidases. Released aglycones can be readily absorbed, thereby increase their biological effectiveness (Day et al., 1998). Many b-glucosidases from microorganisms have been reported for hydrolyzing flavonoid compounds (Ahmed et al., 2017). 8.3.2.3 Flavor industry The majority of the flavor compounds in fruit tissues and plants are present in the form of glycoconjugates, which render them tasteless and nonvolatile compounds. Glycoside flavor compounds have been found in a variety of fruits like mango, strawberry, yellow plum grapes, etc. (Maicas and Mateo, 2005; Krammer et al., 1991; Sakho et al., 1997; Roscher et al., 1996; Williams et al., 1982; Gunata et al., 1985). In the flavor industry, b-glucosidases are extensively used. Hundreds of b-glucoside precursors are present in plants to improve taste. Hydrolysis by b-glucosidases improves the quality of foods and beverages made from them. These are the main enzymes involved in the release of aromatic compounds from glucoside precursors present in fruits and fermented products. b-Glucosidases are also being used for improving the organoleptic properties of juices from citrus fruits. Fruit bitterness is reduced after b-glucosidase treatment, which is due to the presence of the glucoside compound naringin. The gellan hydrolysis reduces the viscosity of fruit juices (Schroder et al., 2014; Keerti et al., 2014; Roitner et al., 1984). The isolation and characterization of a thermostable b-glucosidase from Bacillus subtilis were done by Keerti et al. (2014). They used it for improving the quality of the sugarcane juice. The enzyme was immobilized on alginate beads. 8.3.2.4 Cassava detoxification b-Glucosidases can be used for detoxifying cassava. Cassava grows in many parts of the world and is rich in carbohydrates, but the use of raw cassava is detrimental to human health as it contains cyanogenic glycoside for instance linamarin and lotaustralin. Furthermore, a correlation has been established between human central nervous system syndrome “Konzo” and long-lasting use of cassava products. Detoxification of cassava is done during processing and grating by b-glucosidases and linamarase present in the root. But these enzymes are not expressed sufficiently, which leaves a part of cyanogenic glycosides in the processed foods. So, b-glucosidases from microorganisms and exogenous linamarase can be used to improve the hydrolysis of cyanogenic glycosides (Vasconcelos et al., 1990; Maduagwu, 1983; Etsuyankpa et al., 2015; Ugwuanyi et al., 2007).

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8.3.2.4 Deinking of waste paper Waste paper is a main environmental pollutant. Recycling of the waste paper gives dual advantage. The, consumption of forest wood as well as the landfill pollution is reduced. Two methods are used for paper recycling paper. These involve chemical or the enzymatic process. The use of chemicals is not safe environmentally, so the use of enzymes such as cellulases, b-glucosidases, and hemicellulases is recommended (Lee et al., 2013; Prasad et al., 1992; Pathak et al., 2011). 8.3.2.5 Other applications b-Glucosidases are used for synthesizing the alkyl-glycosides and oligosaccharides (Bankova et al., 2006). Oligosaccharides are used to promote growth and as diagnostic tools and therapeutic agents. These have important roles in embryogenesis, fertilization, and cell proliferation. Alkyl-glycosides show antimicrobial properties and higher biodegradability. These are nonionic surfactants and find application in food, detergents, chemicals, cosmetics, and pharmaceutical industries as these can be hydrolyzed by b-glucosidases (Bankova et al., 2006). For improving the food quality, enzymes can be added to beverages and foods before, during, or after processing. Thus, these enzymes with advantageous properties may be used for tissue culture, plant breeding programs, and recombinant techniques for increasing their overproduction in genetically engineered plants or microbial hosts, and their catalytic properties that improve the stability and palatability. Apart from enhancing the flavor, beverages, foods and feeds can be enhanced nutritionally by releasing the vitamins, antioxidants, and other useful compounds from their glycosides. b-glucosidase release vitamin B6 in rice (Opassiri et al., 2004). Other vitamins are also present as glucosides in different plants and liberation of their aglycones improves the availability of nutrients. This enzyme hydrolyzes anthocyanins into sugars and anthocyanidins.

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rez, I.B., 2006. Characterization of an exocellular beta-glucosidase from Debaryomyces pseudopolymorphus. Enzyme Microb. Technol. 39, 229e34. Wang, B., Xia, L., 2011. High efficient expression of cellobiase gene from Aspergillus niger in the cells of Trichoderma reesei. Bioresour. Technol. 102, 4568e4572. Watanabe, T., Sato, T., Yoshioka, S., Koshijima, T., Kuwahara, M., 1992. Purificication and properties of Aspergillus niger b-glucosidase. Eur. J. Biochem. 209, 651e659. Williams, P.J., Strauss, C.R., Wilson, B., Massy-Westropp, R.A., 1982. Use of C18 reversed-phase liquid chromatography for the isolation of monoterpene glycosides and nor-isoprenoid precursors from grape juice and wines. J. Chromatogr. A 235 (2), 471e480. Workman, W.E., Day, D.F., 1982. Purification and properties of beta-glucosidase from Aspergillus terreus. Appl. Environ. Microbiol. 44, 1289e1295. Xu, R., Teng, F., Zhang, C., Li, D., 2011. Cloning of a gene encoding beta-glucosidase from Chaetomium thermophilum CT2 and its expression in Pichia pastoris. J. Microbiol. Biotechnol. 20, 16e23. Yan, Q., Hua, C., Yang, S., Li, Y., Jiang, Z., 2012. High level expression of extracellular secretion of a betaglucosidase gene (PtBglu3) from Paecilomyces thermophila in Pichia pastoris. Protein Expr. Purif. 84, 64e72. Yan, Q., Zhou, X.-W., Zhou, W., Li, X.-W., Feng, M.-Q., Zhou, P., 2008. Purification and properties of a novel beta-glucosidase, hydrolyzing ginsenoside Rb1 to CK, from Paecilomyces bainier. J. Microbiol. Biotechnol. 18 (6), 1081e1089. Yanase, H., Nozaki, K., Okamoto, K., 2005. Ethanol production from cellulosic materials by genetically engineered Zymomonas mobilis. Biotechnol. Lett. 27, 259e263. Yang, S., Wang, L., Yan, Q., Jiang, Z., Li, L., 2009. Hydrolysis of soybean isoflavone glycosides by a thermostable b-glucosidase from Paecilomyces thermophila. Food Chem. 115 (4), 1247e1252. Zheng, Z., Shetty, K., 2000. Solid-state bioconversion of phenolics from cranberry pomace and role of Lentinus edodes beta-glucosidase. J. Agric. Food Chem. 48, 895e900. Zhenming, C., Zhe, C., Guanglei, L., Fang, W., Liang, J., Tong, Z., 2009. Saccharomycopsis fibuligera and its applications in biotechnology. Biotechnol. Adv. 27, 423e431.

Further reading Baffi, M., Martin, N., Tobal, T., Ferrarezi, A., Lago, J., Boscolo, M., Gomes, E., Da-Silva, R., 2013. Purification and characterization of an ethanol-tolerant b-glucosidase from Sporidiobolus pararoseus and its potential for hydrolysis of wine aroma precursors. Appl. Biochem. Biotechnol. 171 (7), 1681e1691. Baffi, M.A., Tobal, T., Lago, J.H.G., Boscolo, M., Gomes, E., Da-Silva, R., 2013. Wine aroma improvement using a b-glucosidase preparation from Aureobasidium pullulans. Appl. Biochem. Biotechnol. 169 (2), 493e501. Gonz
alez-Pombo, P., Fari~ na, L., Carrau, F., Batista-Viera, F., Brena, B.M., 2011. A novel extracellular b-glucosidase from Issatchenkia terricola: Isolation, immobilization and application for aroma enhancement of white Muscat wine. Process Biochemistry 46 (1), 385e389. Gunata, Z., Bitteur, S., Brillouet, J.-M., Bayonove, C., Cordonnier, R., 1988. Sequential enzymic hydrolysis of potentially aromatic glycosides from grape. Carbohydr. Res. 184, 139e149. Mateo, J., Jim
enez, M., 2000. Monoterpenes in grape juice and wines. J. Chromatogr. A 881 (1), 557e567. Michlmayr, H., Sch€ umann, C., Wurbs, P., Da Silva, N.M.B.B., Rogl, V., Kulbe, K.D., Andr
es, M., 2010. A b-glucosidase from Oenococcus oeni ATCC BAA-1163 with potential for aroma release in wine: cloning and expression in E. coli. World J. Microbiol. Biotechnol. 26 (7), 1281e1289. Vervoort, Y., Herrera-Malaver, B., Mertens, S., Guadalupe Medina, V., Duitama, J., Michiels, L., Derdelinckx, G., Voordeckers, K., Verstrepen, K.J., 2016. Characterization of the recombinant Brettanomyces anomalus b-glucosidase and its potential for bioflavouring. J. Appl. Microbiol. 121 (3), 721e733. Whitaker, J.R., Voragen, A.G., Wong, D.W., 2003. Handbook of Food Enzymology. Marcel Dekker.

Industrial applications of thermophilic/hyperthermophilic enzymes

SUBCHAPTER 8.4

Pullulanase 8.4.1 Microbial sources and properties Pullulanase (pullulan a-glucano hydrolase; EC 3.2.1.41) is an extracellular carbohydrase. Bender and Wallenfels in 1961 first discovered this enzyme from Klebsiella pneumonia, which is a mesophilic organism. It has since become an indispensable enzyme in starch processing. Pullulanases are also called debranching enzyme. Pullulanase has been broadly used for hydrolyzing the a-1,6 glucosidic linkages in starch, pullulan, amylopectin, and other related oligosaccharides. This allows complete and an effective conversion of the branched polysaccharides to smaller sugars during the saccharification process. Pullulan contains repeating units of a-maltotriose. These are joined “head to tail” by 1, 6 linkage. Pullulanase specially attacks a-1, 6-glycosidic linkage and also attacks the a-1, 4-glycosidic linkage with other residues. Due to these properties, pullulanase enzymes have become an important enzyme in structural studies of oligosaccharides and polysaccharides (Saha et al., 1988; Drummond et al., 1969; Ling et al., 2009). Several thermostable pullulanase enzymes with double specificities have been studied. Pullulanase obtained from thermophilic microbes, attacks on a-1, 4-glycosidic linkage as well as a-1, 6-glycosidic linkage in amylopectin and maltooligosaccharides (Rudiger et al., 1995; Kriegshauser and Liebl, 2000; Koch et al., 1997). Pullulanase (EC. 3.2.1.41), isopullulanase (EC.3.2.1.57), and neopullulanase (EC.3.2.1.35) cleave three types of bond. Based upon the substrate specificities and the reaction products, enzymes degrading pullulan are classified into the following four groups (Bertoldo et al., 1999): (i) Pullulan hydrolase type I attacks a-1,4 glycosidic linkages in pullulan and forms panose (ii) Pullulan hydrolase type II attacks a-1,4 glycosidic linkages in pullulan and forms isopanose. (iii) Pullulanase type I exclusively hydrolyzes a-1,6 glycosidic linkages in pullulan forming maltotriose or branched oligosaccharides. (iv) Pullulanase type II attacks a-1,6 glycosidic linkages in pullulan and branched substrates and also attacks the a-1,4 glycosidic linkages in polysaccharides other than pullulan. Table 8.4.1 presents reaction specificities of pullulan-degrading enzymes. Thermostable enzymes are needed in several industrial processes including starch processing. In the processing of starch, the amylolytic enzymes, which are mostly used are a-amylase, b-amylase, glucoamylase, and pullulanase. Glucoamylases and pullulanases

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Enzyme

EC number

Bonds processed

Pullulanase type I

3.2.1.41

Pullulanase type II (amylopullulanase)

Preferred substrate

End products

References

a-(1,6)

Oligo- and polysaccharides

Pullulan trimer (maltotriose)

3.2.1.41

a-(1,6) a-(1,4)

Pullulan, poly-, and oligosaccharide (starch)

Trimer (maltotriose) Mixture of glucose, maltose, and maltotriose

Pullulan hydrolase type I (neopullulanase)

3.2.1.135

a-(1,4)

Pullulan panose

Panose

Pullulan hydrolase type II (isopullulanase) Pullulan hydrolase type III

3.2.1.57

a-(1,4)

Pullulan

Isopanose

3.2.1

a-(1,4) and a-(1,6)

Pullulan Starch, amylose, and amylopectin

Mixture of panose, maltose, and maltotriose Maltotriose and maltose

Bertoldo and Antranikian (2002), Kim et al. (1996) Roy et al. (2003), Duffner et al. (2000), L
ev^eque et al. (2000), Tulasi and Mohanan (2018) Ara et al. (1995), Kuriki et al. (1988), Sunna et al. (1997) Van Der Maarel et al. (2002) Niehaus et al. (2000)

From Hii, S.L., Tan, J.S., Ling, T.C., Ariff, A.B., 2012. Pullulanase: role in starch hydrolysis and potential industrial applications. Enzyme Res. 2012, 921362. Distributed under Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/).

Developments and Applications of Enzymes From Thermophilic Microorganisms

Table 8.4.1 Reaction specificities of pullulan-degrading enzymes.

Industrial applications of thermophilic/hyperthermophilic enzymes

show superior thermostability in comparison with the enzyme used in the saccharification process (Madi et al., 1987). Clostridium thermohydrosulfuricum strain E-39 has been studied for degradation of starch. This bacteria is anaerobic thermophilic and is able to produce ethanol from starch. Pullulanase as well as a glucoamylases from cell extract of this strain have been reported (Melashiemi, 1988). Hobson et al. (1950) identified an enzyme from broad beans which hydrolyses the a-1, 6 linkages of amylopectin, amylopectin a-limit dextrin, and a-amylopectin b-limit dextrin. It was named r-enzyme afterward. Purification and characterization of Pullulanase enzymes from different microorganismsdthermophilic Bacillus cereus FDTA-13, Bacillus sp. AN-7, Geobacillus stearothermophilus. B. acidopullulyticus, B. deramificans, and K. planticoladhave been reported by several researchers (Teague and Brumm, 1992; Uhlig, 1998; Zareian et al., 2010; Nair et al., 2007; Kunamneni and Singh, 2006). Mesophilic bacteria like Streptomyces sp., Aerobacter aerogenes, B. acidopullulyticus, and K. pneumonia have been shown to produce pullulanase type I enzyme. This enzyme is secreted by Gram-positive bacteria that are moderately thermophilic, including Thermos caldophilus, B. flavocaldarius, B. thermoleovorans, and Clostridium sp. Fervidobacterium pennavorans hyperthermophilic bacterium also produces pullulanase type I enzyme. Pullulanase type II, in contrast to pullulanase type I, is widely present in hyperthermophilic archaea and extreme thermophilic bacteria. The hyperthermophilic archaeon Pyrococcus furiosus and P. woesei was found to produce pullulanase type II enzyme, which was found to be the most thermostable and thermoactive to date (Prabhu et al., 2018; Ben Messaoud et al., 2002; Bender and Wallenfels, 1961; Kim et al., 1996; Klingeberg et al., 1990; Koch et al., 1997; Kornacker and Pugsley, 1990; Ohba and Ueda, 1973; Rudiger et al., 1995; Sunna et al., 1997; Suzuki et al., 1991; Takasaki et al., 1993; Jensen and Norman, 1984). Microbial pullulanases are attracting a lot of interest because it specifically acts on a-1,6 linkages in pullulan, which is a linear a-glucan containing maltotriosyl units connected by 1,6-a-bonds. From an industrial perspective, the highly thermostable pullulanase type-II enzyme is very interesting. These enzymes could undoubtedly make possible an important change in the present approach of starch processing (Chang-Pi-Hin et al., 2002). Fig. 8.4.1 shows degradation of pullulan by pullulanase. The enzyme producers and starch processors are continuously searching for better enzymes for improving the standard processes and new products from pullulanase. Nowadays, debranching enzymes, for instance, pullulanase of Bacillus species and K. pneumoniae species are gaining escalating interest as they show excellent activity and stability at the high temperatures required for best amylolytic enzyme.

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Figure 8.4.1 Schematic presentation of the action of amylolytic and pullulytic enzymes. Pullulanase type I also attacks a-1,6-glycosidic linkages in oligosaccharides and polysaccharides. Pullulanase type II also attacks a-1,4-linkages in various oligosaccharides and polysaccharides [3•]. Black circles indicate reducing sugars. (From Bertoldo, C., Antranikian, G., 2002. Starch-hydrolyzing enzymes from thermophilic archaea and bacteria. Curr. Opin. Chem. Biol. 6 (2), 151e160. Reproduced with permission.)

Industrial applications of thermophilic/hyperthermophilic enzymes

8.4.2 Industrial application of pullulanase The industrial application of pullulanase has recently increased in starch based industries for production of glucose and also in ethanol biorefineries that use starch as a raw material. The use of crude oil as an energy source has seriously damaged the ozone layer and also leads to climate change, which is the major cause of disease outbreaks and pandemic. Pullulanase is a debranching enzyme, which specifically hydrolyzes a-1, 6 glucosidic linkages in starch, amylopectin, pullulan, and other related oligosaccharides. The branched polysaccharides are completely and efficiently converted into smaller fermentable sugars. The industrial production of glucose is performed in two steps. The first step is liquefaction, and the second step is saccharification. Pullulanase is being used for increasing the final glucose concentration with lesser amount of glucoamylases during the saccharification step, thus preventing the reverse reaction involving the resynthesis of saccharides from glucose. 8.4.2.1 Starch processing industry Earlier conversion of starch to glucose was performed using the traditional acid method but since 1960s, enzymatic process is used for converting starch to glucose (Olsen et al., 2000). Nowadays, most of the starch hydrolysates which are commercially available are enzymatically produced products of higher dextrose equivalent (DE). DE is a measure of the amount of reducing sugars on a dry basis present in sugar product. This is expressed as percentage on a dry basis relative to glucose. The DE of pure glucose being 100 and DE of starch being close to DE 0. DE gives an idea about the average degree of polymerization for starch sugars. Starch is enzymatically converted into glucose, fructose, and maltose, which are used as food sweeteners. This is an important growing area in the industrial application of enzymes. Pullulanase enzymes are used in producing sugar syrup. These are used along with a-amylases for completing the hydrolysis of starch. Use of pullulanase in combination with other amylolytic enzymes increases the quality of sugar syrups. Treatment of starch with amylases and pullulanases increases the effectiveness of saccharification reaction. This method has a benefit of producing higher yield of a desired end product from starch. Acidophilus and thermostable pullulanase from B. stearothermophilus are useful for this application (Silva et al., 2005; Saha et al., 1988; Nair et al., 2006; Michaelis et al., 1985). In the food industry, pullulanase enzyme is used as a processing aid. Particularly pullulanase debranches corn starch for producing certain corn sweeteners (Modderman and Foley, 1995; Haga et al., 2003; Bertoldo and Antranikian, 2002). The most important use of pullulanase is in the production of high glucose or high maltose syrups (Van Der Maarel et al., 2002; Gomes et al., 2003). High-glucose syrup contains 30%e50% glucose and 30%e40% maltose. High-maltose syrup contain 30%e50% maltose and 6%e10% glucose. Pullulanase is generally used along with glucoamylases or b-amylases

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in the saccharification process (Haki and Rakshit, 2003; Chaplin, 2002; Gomes et al., 2003). High-maltose syrups have good thermal stability, reduced hygroscopicity, mild sweetness, and lower viscosity in solution. It has several applications in food processing. High-maltose syrups are used for manufacturing ice cream and high-quality candies (Shaw and Sheu, 1992). Recently, the pharmaceutical industry has shown great interest in the use of pure maltose. Maltose can be used instead of glucose for intravenous feeding. Higher doses can be given without raising blood sugar levels (Shaw and Sheu, 1992). Pure maltose can be used for producing maltitol and crystalline maltitol (Jensen and Norman, 1984). Crystalline glucose and high-fructose syrups are produced from high-glucose syrup (Nigam and Singh, 1995; Olsen et al., 2000). High-fructose syrup is produced from high-dextrose syrup, specially high-glucose syrup (DE 95e96) with the use of immobilized glucose isomerase (Van Der Maarel et al., 2002). High fructose corn syrup (HFCS) is produced from corn. It is a high-quality, clean-tasting caloric sweetener. HFCS having high DE value is needed for producing crystalline glucose. HFCS is inexpensive and less caloric as compared to sucrose. It is 1.2e1.8 times sweeter than sucrose on a dry weight basis (Guzman-Maldonado and Paredes-Lopez, 1995). HFCS can be used by diabetics as it can be metabolized without insulin just like high-maltose syrup (Dziezak, 1989). 8.4.2.2 High-amylose starch High-amylose starch has enormous market demand and is of great interest. Pullulanase has been used to produce such starches (Vorwerg et al., 2002). High-amylose starches are used in adhesive products and for producing corrugated boards and paper (Jobling, 2004). High-amylose starches can be processed into “resistant starch,” which shows nutritional benefits (Bird et al., 2000). Resistant starch is not digested in the small intestine and is fermented in the large intestine by intestinal bacteria. Short-chain fatty acids such as butyric acid are formed and advantageous for gut health. 8.4.2.3 Detergents Few alkaline pullulanase enzymes are being used as effective additives in laundry and dishwashing detergents for removing starches under the alkaline conditions (Hatada et al., 2001; Schallmey et al., 2004). The effectiveness of these alkaline pullulinases is demonstrated by the fact that when used together with alkaline a-amylases, as one single amylopullulanase can perform debranching (a-1,6-hydrolysis) and liquefaction (a-1,4hydrolysis) reactions (Ara et al., 1995). The amylopullulanase of G. thermoleovorans NP33, which is alkalitolerant, is used as an additive in detergents (Nisha and Satyanarayana, 2014). This enzyme shows considerable

Industrial applications of thermophilic/hyperthermophilic enzymes

stability in various detergents and can effectively remove starch stains from the stained cotton fabrics. Washing with a combination of enzymes and detergents increases reflectance compared to detergent alone. 8.4.2.4 Production of cyclodextrins The pullulanase enzyme has also been used to increase the yield of cyclodextrin by reacting cyclodextrin glycosyltransferase with gelatinized starch and maltodextrin syrup in the presence of a cyclodextrin complexing agent (Rendleman, 1997). Cyclodextrins have multiple uses in agricultural products as antioxidants, emulsifiers, and stabilizers. They are also used as complexing agents in pharmaceuticals, foods, and plastics (Rendleman, 2000). 8.4.2.5 Antistaling agent In the baking industry, staling effect is the major problem. “Staling” refers to changes, which occur in bread after baking causing it to become “stale,” which results in spoilage and reduced consumer acceptance. The staling effect includes loss of crust crispness, decreased moisture content in crumb, and loss of flavor resulting in worsening of quality (van der Maarel et al., 2002). The use of enzymes is a preferred method because enzymes are natural and highly efficient. Pullulanase plays an important role in the antistaling treatment (Noorwez et al., 2006; Bertoldo and Antranikian, 2002; Kulp and Ponte, 1981; Champenois et al., 1999; De Stefanis and Turner, 1981; Carroll et al., 1987). 8.4.2.6 Preparation of maltooligosaccharides Malto-oligosaccharides are extensively used in the beverage, pharmaceutical, fine chemical industries, food, and cosmetics. Malto-oligosaccharides are characterized by higher solubility, lower sweetness, low calorie content, low water activity, high water content, high acid resistance, and high heat stability. From malto-oligosaccharides, several nutritional and healthcare foods like baby foods, confectionary items, health candies, beverages, beer, ice cream, etc. can be produced. Maltotriose, maltotetraose, and maltopentaose prevent moisture migration from starch granules, reduce retrogradation by inhibiting rearrangement of amylose and amylopectin chains, and disrupt starche gluten interactions. These properties make it useful as an antistaling agent in the bakery industry (Fogarty and Kelly, 1990; Min et al., 1998; Nagarajan et al., 2006; Pl
acido Moore et al., 2005). Malto-oligosaccharide syrup serves as a highly nutritious food for toddlers and the elderly people (Stahl et al., 2013). Malto-oligosaccharides belong to the soluble indigestible carbohydrates that stimulate the intestinal flora, paticularly bifidobacteria, and produces fermentation products in the large intestine (Roberfroid and Slavin, 2000). Digestible oligosaccharides, termed prebiotics (Mussatto and Mancilha, 2007), are

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classified as functional food ingredients that can escape low-pH gastric juices and digestive enzymes and are digested in the colon. Malto-oligosaccharides are gradually absorbed by the human body and provide a longlasting supply of energy. In addition, maltose and malto-oligosaccharides are low in calories and thus regulate blood sugar levels and lower postprandial blood sugar levels (Kayode et al., 2009). So, it is advantageous for heart patients, diabetics, and obese patients. Furthermore, malto-oligosaccharides enhance physical endurance and immunity and have excellent therapeutic effects in individuals with abnormally low blood sugar and encephalopathy associated with excessive energy expenditure (Zhu et al., 2011). Malto-oligosaccharides are available on a commercial scale as Fuji Oligosyrup, a product of Nippon Shokuhin Kako Kogyo Co., Ltd. (Tokyo, Japan) (Satyanarayana and Nisha, 2018). 8.4.2.7 Others Pullulanase enzymes also find some application in the production of low-calorie beer and as a dental plaque control agent (Olsen et al., 2000; Marotta et al., 2002).

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Industrial applications of thermophilic/hyperthermophilic enzymes

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ev^eque, E., Janecek, S., Haye, B., Belarbi, A., 2000. Thermophilic archaeal amylolytic enzymes. Enzyme Microb. Technol. 26, 3e14. Ling, H.S., Ling, T.C., Rosfarizan, M., Ariff, A.B., 2009. Characterization of pullulanase type II from Bacillus cereus H1.5. Am. J. Biochem. Biotechnol. 5, 170e179. Madi, E., Antranikian, G., Ohmiya, K., Gottschalk, G., 1987. Thermostable amylolytic enzymes from a new Clostridium isolate. Appl. Environ. Microbiol. 53, 1661e1667.

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Marotta, M., Martino, A., De Rosa, A., Farina, E., Carteni, M., De Rosa, M., 2002. Degradation of dental plaque glucans and prevention of glucan formation using commercial enzymes. Process Biochem. 38 (1), 101e108. Melasniemi, H., 1988. Purification and some properties of the extracellular a-amylase pullulanase produced by Clostridium thermohydrosulfuricum. Biochem. J. (250), 813e818. Michaelis, S., Chapon, C., D’Enfert, C., Pugsley, A.P., Schwartz, M., 1985. Characterization and expression of the structural gene for pullulanase, a maltose-inducible secreted protein of Klebsiella pneumoniae. J. Bacteriol. 164 (2), 633e638. Min, B.C., Yoon, S.H., Kim, J.W., Lee, Y.W., Kim, Y.B., Park, K.H., 1998. Cloning of novel maltooligosaccharide-producing amylases as antistaling agents for bread. J. Agric. Food Chem. 46, 779e782. Modderman, J.P., Foley, H.H., 1995. Safety evaluation of pullulanase enzyme preparation derived from Bacillus licheniformis containing the pullulanase gene from Bacillus deramificans. Regul. Toxicol. Pharmacol. 21 (3), 375e381. Mussatto, S.I., Mancilha, I.M., 2007. Non-digestible oligosaccharides: a review. Carbohydr. Polym. 68, 587e597. Nagarajan, D.R., Rajagopalan, G., Krishnan, C., 2006. Purification and characterization of a maltooligosaccharide-forming a-amylase from a new Bacillus subtilis KCC103. Appl. Microbiol. Biotechnol. 73, 591e597. Nair, S.U., Singhal, R.S., Kamat, M.Y., 2007. Induction of pullulanase production in Bacillus cereus FDTA13. Bioresour. Technol. 98 (4), 856e859. Nair, S.U., Singhal, R.S., Kamat, M.Y., 2006. Enhanced production of thermostable pullulanase type-I using Bacillus cereus FDTA and its mutant. Food Technol. Biotechnol. 44 (2), 275e282. Nakakuki, T., 2002. Present status and future of functional oligosaccharide development in Japan. Pure Appl. Chem. 74, 1245e1251. Niehaus, F., Peters, A., Groudieva, T., Antranikian, G., 2000. Cloning, expression and biochemical characterisation of a unique thermostable pullulan-hydrolysing enzyme from the hyperthermophilic archaeon Thermococcus aggregans. FEMS Microbiol. Lett. 190 (2), 223e229. Nigam, P., Singh, D., 1995. Enzyme and microbial systems involved in starch processing. Enzyme Microb. Technol. 17 (9), 770e778. Nisha, M., Satyanarayana, T., 2014. Characterization and multiple applications of a highly thermostable and Ca2þ-independent amylopullulanase of the extreme thermophile Geobacillus thermoleovorans. Appl. Biochem. Biotechnol. 174, 2594e2615. Nisha, M., Satyanarayana, T., 2019. Amylases. Encyclopedia of Microbiology. Elsevier. Noorwez, S.M., Ezhilvannan, M., Satyanarayana, T., 2006. Production of a high maltose-forming hyperthermostable and Ca2þ independent amylopullulanase by an extreme thermophil Geobacillus thermoleovorans in submerged fermentation. Indian J. Biotechnol. 5, 337e345. Ohba, R., Ueda, S., 1973. Purification, crystallization and some properties of intracellular pullulanase from Aerobacter aerogenes. Agricul. Biol. Chem. 37 (12), 2821e2826. Olsen, H.S., Goddard, P., Novo Nordisk, A.S., 2000. Enzymes at Work: A Concise Guide to Industrial Enzymes and Their Uses. Novo Nordisk A/S. Pl
acido Moore, G.R., Rodríguez do Canto, L., Amante, E.R., 2005. Cassava and corn starch in maltodextrin production. Quim Nova 28, 596e600. Prabhu, N., Maheswari, R.U., Singh, M.V.P., Karunakaran, S., Kaliappan, C., Gajendran, T., 2018. Production and purification of extracellular pullulanase by Klebsilla aerogenes NCIM 2239. Afr. J. Biotechnol. 17 (14), 486e494. Rendleman Jr., J.A., 1997. Enhancement of cyclodextrin production through use of debranching enzymes. Biotechnol. Appl. Biochem. 26 (1), 51e61. Rendleman Jr., J.A., 2000. Enzyme modification of starch granules: formation and retention of cyclomaltodextrins inside starch granules by reaction of cyclomaltodextrin glucanosyltransferase with solid granules. Carbohydr. Res. 328 (4), 509e515. Roberfroid, M., Slavin, J., 2000. Nondigestible oligosaccharides. Crit. Rev. Food Sci. Nutr. 40, 461e480.

Industrial applications of thermophilic/hyperthermophilic enzymes

Roy, A., Messaoud, E.B., Bejar, S., 2003. Isolation and purification of an acidic pullulanase type II from newly isolated Bacillus sp. US149. Enzyme Microb. Technol. 33 (5), 720e724. Saha, B.C., Mathupala, S.P., Zeikus, J.G., 1988. Purification and characterization of a highly thermostable novel pullulanase from Clostridium thermohydrosulfuricum. Biochem. J. 252, 343e348. Satyanarayana, T., Nisha, M., 2018. Archaeal and bacterial thermostable amylopullulanases: characteristic features and biotechnological applications. Amylase 2 (1), 44e57. Schallmey, M., Singh, A., Ward, O.P., 2004. Developments in the use of Bacillus species for industrial production. Can. J. Microbiol. 50 (1), 1e17. Shaw, J.F., Sheu, J.R., 1992. Production of high-maltose syrup and high-protein flour from rice by an enzymatic method. Biosci., Biotechnol., Biochem. 56 (7), 1071e1073. Silva, T.M., Attili-Angeli, D., Carvalho, A.F., Da Silva, R., Boscolo, M., Gomes, E., 2005. Production of saccharogenic and dextrinogenic amylases by Rhizomucor pusillus A 13.36. J. Microbiol. 43 (6), 561e568. Stahl, B., Alles, M.S., Maria, B.A., 2013. Cereal Based Infant Nutrition with Fibre. European Patent EP2124624B1: 2013, p. 2013. Sunna, A., Moracci, M., Rossi, M., Antranikian, G., 1997. Glycosyl hydrolases from hyperthermophiles. Extremophiles 1 (1), 2e13. Suzuki, Y., Hatagaki, K., Oda, H., 1991. A hyperthermostable pullulanase produced by an extreme thermophile, Bacillus flavocaldarius KP 1228, and evidence for the proline theory of increasing protein thermostability. Appl. Microbiol. Biotechnol. 34 (6), 707e714. Takasaki, Y., Hayashida, A., Ino, Y., Ogawa, T., Hayashi, S., Imada, K., 1993. Cell-bound pullulanase from Streptomyces sp. No. 27. Biosci. Biotechnol. Biochem. 57 (3), 477e478. Teague, W.M., Brumm, P.J., 1992. Commercial enzymes for starch hydrolysis products. In: Schenck, F.W., Hebeda, R.E. (Eds.), Starch Hydrolysis Products: Worldwide Technology, Production and Applications, 79. VCH, New York, NY, USA, p. 45. Tulasi, S., Mohanan, N., 2018. Archaeal and bacterial thermostable amylopullulanases: characteristic features and biotechnological applications. Amylase 2 (1), 44e57. Uhlig, H., 1998. Industrial Enzymes and Their Applications. Wiley-Interscience, New York, NY, USA. van der Maarel, M.J., van der Veen, B., Uitdehaag, J.C., Leemhuis, H., Dijkhuizen, L., 2002. Properties and applications of starch-converting enzymes of the alpha-amylase family. J. Biotechnol. 94 (2), 137e155. Vorwerg, W., Radosta, S., Leibnitz, E., 2002. Study of a preparative-scale process for the production of amylose. Carbohydr. Polym. 47 (2), 181e189. Yamasaki, Y., Nakashima, S., Konno, H., 2008. Pullulanase from rice endosperm. Acta Biochim. Polonica 55, 507e510. Zareian, S., Khajeh, K., Ranjbar, B., Dabirmanesh, B., Ghollasi, M., Mollania, N., 2010. Purification and characterization of a novel amylopullulanase that converts pullulan to glucose, maltose, and maltotriose and starch to glucose and maltose. Enzyme Microb. Technol. 46 (2), 57e63. Zebardast Roodi, F., Aminzadeh, S., Farrokhi, N., Karkhane, A., Haghbeen, K., 2017. Cohnella amylopullulanases: biochemical characterization of two recombinant thermophilic enzymes. PLoS ONE 12, e0175013. Zhu, A., Romero, R., Huang, J.B., Clark, A., Petty, H.R., 2011. Maltooligosaccharides from JEG-3 trophoblast-like cells exhibit immunoregulatory properties. Am. J. Reprod. Immunol. 65, 54e64. Zona, R., Chang-Pi-Hin, F., O’Donohue, M.J., Janecek, S., 2004. Bioinformatics of the glycoside hydrolase family 57 and identification of catalytic residues in amylopullulanase from Thermococcus hydrothermalis. Eur. J. Biochem. 271, 2863e2872.

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SUBCHAPTER 8.5

Amylopullulanases 8.5.1 Microbial sources and properties Amylopullulanase (EC 3.2.1.1/41) is a pullulanase type II enzyme. It attacks the a-1,4 and a-1,6 glycosidic bonds in branched and linear polysaccharides and produces glucose, maltose, and maltotriose as degradation products (Tonkova, 2006). Amylopullulanase is able to hydrolyze a-1,6 bonds in pullulan and produces maltotriose. This enzyme is also able to hydrolyze a-1,4-bonds in linear and branched oligosaccharides, for instance, amylose and amylopectin. Amylopullulanase has numerous applications in the food processing industry and also in the distilleries. During liquefaction of starch, this enzyme is used to replace a-amylase enzyme for producing glucose, maltose, and fructose syrups (Roy and Gupta, 2004). The amylopullulanases are multidomain enzymes and have one catalytic domain with an active site for hydrolyzing the a-1,4- and a-1,6-glycosidic linkages, whereas amylopullulanases having two catalytic domains separate active sites for hydrolyzing the a-1,4and a-1,6-glycosidic linkages. These are named alpha-amylase-pullulanases (Kim and Kim, 1995; Hatada et al., 1996; Coleman et al., 1987; Mathupala et al., 1993). Geobacillus thermoleovorans NP33 amylopullulanase is a thermostable enzyme and has the ability to hydrolyze raw starch. This enzyme has a single active site for hydrolyzing the a-1,4 and a-1,6 glycosidic bonds in starch and pullulan. It is able to hydrolyze starch and other related oligosaccharides and produces maltooligosaccharides of varying DP ranging from DP2 to DP4 and produces maltotriose from pullulan. This amylopullulanase has five conserved domain structures and six conserved regions around the strands b-2 to b-5 and b-7 and b-8 (Nisha and Satyanarayana, 2013a). Amylopullulanases are produced mostly from bacteria, Bacillus sp. DSM405, B. cereus, Geobacillus thermoleovorans NP33, Lactobacillus amylophilus GV6, and Clostridium thermosulfurogenes SV9 (Bakshi et al., 1992; Satyanarayana et al., 2004; Vishnu et al., 2006; Brunswick et al., 1999; Swamy and Seenayya, 1996). Actinomycetes species are not found to produce amylopullulanases. Amylopullulanases, because of their specific debranching capacity, are very important in starch processing industry. These enzymes are capable of converting polysaccharides, for instance, amylopectin into smaller sugars like glucose and maltose. These are produced from bacteria as well as hyperthermophilic archaea, which belong to the genera Pyrococcus (Schwerdtfeger et al., 1999); Thermococcus (Han et al., 2013; Erra-Pujada et al., 1988); Desulfurococcus (Dufner et al., 2000); Staphylothermus (Li et al., 2013); and in the halophilic archaeon Halorubrum (Moshfegh et al., 2013). Tables 8.5.1 and 8.5.2 show characteristics of thermophilic bacterial and archaeal amylopullulanases (Satyanarayana and Nisha, 2018).

Table 8.5.1 Characteristics of thermophilic bacterial amylopullulanases. Molecular weight

Source

pH

Temperature

References

140 126

5.0 5.0 9.0 6.0e6.5

60 62 55 70

224

9.0

70

5.9

60

Thermophilic bacteria

Bacillus Bacillus Bacillus Bacillus

sp. sp. sp. sp.

US149 3183 TS-23 DSM 405

70

6.0

60

Desulfucoccus mucosus

74

5.8

85

7.0

80

100

5.5

65

105 120

5.6

70

Coleman et al. (1987) Plant et al. (1987a,b)

150

5.0e5.5

65

450

5.0

75

133

5.5

e

5.0e6.0

75

e 96

5.0e6.0

75

Clostridium sp. strain EM1

Geobacillus thermoleovorans NP33 Geobacillus stearothermophilus L14 Therrnoanaerobium brockii Thermoanaerobium Tok6B1 Thermoanaerobacterium thermosaccharolyticum Therrnoanaerobacter strain B6A Thermoanaerobacter ethanolicus 39E Thermobacteroides acetoethylicus Thermoanaerobacter finni Thermotoga maritima

Ganghofner et al. (1998) Saha et al. (1990) Mathupala and Zeikus (1993) Koch et al. (1987), Koch and Antranikian (1990) Koch et al. (1987) Bibel et al. (1998)

165

From Satyanarayana, T., Nisha, M., 2018. Archaeal and bacterial thermostable amylopullulanases: characteristic features and biotechnological applications. Amylase 2, 44e57. Reproduced with permission.

Industrial applications of thermophilic/hyperthermophilic enzymes

Cohnella sp. A10

Roy et al. (2003) Shen et al. (1990) Lin et al. (1996) Gibson and Roberfroid (1995) Mussatto and Mancilha (2007) Antranikian et al. (1987) Zebardast et al. (2017) Canganella et al. (1994) Noorwez et al. (2006) Zareian et al. (2010)

Bacillus sp XAL 601

200

166

Developments and Applications of Enzymes From Thermophilic Microorganisms

Table 8.5.2 Characteristics of hyperthermophilic archaeal amylopullulanases. Source

Molecular weight

pH

Temperature

References

71

5.0

95

Naumoff (2011)

89

5.5

110

90

6.0

100

119

5.5

110

Thermococcus hydrothermalis

128

5.5

95

Thermococcus profundus Thermococcus celer

43

5.5

80e90

5.5e6.0

90

3.5

95e100

Dong et al. (1997), Brown and Kelly (1993) R€ udiger et al. (1995) Brown and Kelly (1993) Erra-Pujada et al. (1999), Zona et al. (2004) Kwak et al. (1998) Canganella et al. (1994) Li et al. (2018)

Desulfurococcus amylolyticus JCM9188 Pyrococcus furiosus

Pyrococcus woesei Thermococcus litoralis

Thermofilum pendens

e 65

From Satyanarayana, T., Nisha, M., 2018. Archaeal and bacterial thermostable amylopullulanases: characteristic features and biotechnological applications. Amylase 2, 44e57. Reproduced with permission.

Amylopullulanases produced from hyper/thermophilic archaea do not need calcium ions for activity, which is a needed for their industrial application. Amylopullulanases are endoacting bifunctional and debranching enzymes. These enzymes have attracted a wide range of interest for saccharifying starch on an industrial scale. Amylopullulanase-encoding genes have been cloned and overexpressed for achieving higher enzyme yields. Attempts have been also made to characterize these proteins. The truncation study on several amylopullulanases made possible to improve the activity of specific enzyme and also thermostability.

8.5.2 Industrial applications of amylopullulanases Amylopullulanases are excellent enzymes for use in the starch processing industries. The bifunctionality, debranching ability, and also the calcium independence of amylopullulanases make it very useful for the existing starch conversion processes (Fig. 8.5.1). This enzyme is used to catalyze the one step starch liquefactionesaccharification process and so is able to replace other amylolytic enzymes such as a-amylase and b-amylase thereby

Industrial applications of thermophilic/hyperthermophilic enzymes

Figure 8.5.1 Scanning electron micrographs of the untreated and treated raw rice starch granules with the amylopullulanase of G. thermoleovorans NP33. (A) Untreated raw rice starch granule. (B) Hydrolyzed granules in 30 min of reaction with the enzymes; (C) hydrolyzed portion of the granule in 1 h; and (D) almost completely hydrolyzed starch granules in 2 h. (From Nisha, M., Satyanarayana, T., 2013b. Recombinant bacterial amylopullulanases: developments and perspectives. Bioengineered 4, 1e13. Reproduced with permission.)

reducing the cost of production of sugars (Nisha and Satyanarayana, 2013b,c; Jiao et al., 2011; Jensen and Norman, 1984). In the starch industry, it is used for producing various sugar syrups including maltose, maltotriose, and maltotetraose. When the enzyme is added in the starch liquefaction process, the yield of maltose increases, and the quantity of branched oligosaccharides is reduced. The a-amylases used in the starch conversion process require calcium and do not function at a pH below 5.9. So, the calcium is added, and pH is adjusted to that of the starch slurry (pH 4.5). The reversion products produced by glucoamylases at the expense of glucose are required to be reduced. So, the use of amylopullulanases would be beneficial in starch processing because the maltose production is increased, the reaction time is reduced, substrate concentration is increased, and also the use of glucoamylases is reduced, thus making the process cost-effective. Maltose syrup and maltooligosaccharide syrup are used in beverage, food, chemical, and pharmaceutical industries. Syrups containing maltose are used in soft drinks,

167

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Developments and Applications of Enzymes From Thermophilic Microorganisms

confectionery, canning, baking, and other food industries. Maltotriose syrup has lower depression of freezing point and lower viscosity, better heat stability, milder sweetness, avoids retrogradation of starch in foodstuffs, keeps in moisture, and generates less color in comparison to glucose, maltose, and sucrose syrups. These properties are vital for the food and pharmaceutical industry (Zoebelein and Böllert, 2001). High maltotriose syrups are used in the food industry and also for baking and brewing. In the food industry, high maltotriose syrup is used for making desserts. In the pharmaceutical industry, the maltose and maltotriose syrups are used to substitute glucose in intravenous feeding. Transgenic rice seeds have been developed. These have a thermostable and bifunctional amylopullulanases from T. ethanolicus 39E enzyme. This would make possible the industrial production of fermentation products and the sweeteners. The granule-bound amylopullulanase activity is associated with the reduction of amylose in the transgenic rice. This enzyme has also been used for producing slowly digestible starch (Miao et al., 2009; Guraya et al., 2001). This type of starch impacts the human health. It has a low glycemic index for treating and preventing many diseases, like heart diseases, obesity, and diabetes, and provides stable and sustained energy for athletes (Eliasson, 2004; Wolf et al., 1999; Ells et al., 2005; Shin et al., 2005). Amylopullulanases also increase the resistant starch content (Zhang and Jin, 2011). During hydrolysis of starch, the gelatinized starch gets reverted to a form that is very much resistant to hydrolysis with a-amylase enzyme. This is called resistant starch (Annison and Topping, 1994). This type of starch has potential application in the food industry and is now attracting a lot of attention of the nutritionists. The level of plasma glucose and insulin is reduced, fecal bulk is increased, and the production of shorter chain fatty acids in the large intestine is reduced. Resistant starch is produced by chemical modification and a heatingecooling process (Mun and Shin, 2006). But, the chemical modification might not be safer, and the heatingecooling process alone might reduce the content of resistant starch because of the structure of starch. Branched cyclodextrins (CDs) have been also produced with this enzyme (Watanabe et al., 1997). These CDs possess one or more saccharide chains like glucose, maltose, and other saccharides linked to CDs by an a-1,6 linkage (French et al., 1965). The branched CDs are more soluble in water and organic solvents as compared to CDs, which have no branches and therefore may produce more soluble inclusion complexes with a variety of chemicals. CDs have application in the food, cosmetics, pharmaceutical, and plastic industries. These are used as antioxidants, emulsifiers, stabilizing agents, and traps for volatiles. In the baking industry, amylopullulanases find potential application as antistaling agents. Staling is the unwanted changes, which take place when baked products are stored. The crispness of the crust is lost, the firmness of the crumb is increased, and the flavor of the bread is lost. Staling takes place due to the retrogradation of the amylopectin fraction of the starch (Champenois et al., 1999; Kulp and Ponte, 1981). The

Industrial applications of thermophilic/hyperthermophilic enzymes

thermostable amylopullulanases reduce the higher adhesiveness of the a-amylase enzyme-treated bread associated with the production of branched maltodextrins and so, can be utilized as a substitute of a-amylases as an antistaling agent (De Stefanis and Turner, 1981). Amylopullulanases are also used along with glucoamylases in the brewing and alcohol industries. These enzymes are able to increase the quantity of fermentable sugars and might ease the filtration step. Amylopullulanases are also used for producing low-calorie lite beer. The enzyme is mixed with fungal a-amylases or glucoamylases to the wort during fermentation as a substitute of pullulanases. The amylopullulanases and a-amylase-pullulanase are also used in the detergent industry. The use of an alkaline pullulanase from alkaliphilic Bacillus sp. KSM-1876 in laundry detergents and dishwashing has been reported by Ara et al. (1992).

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Dufner, F., Bertoldo, C., Andersen, J.T., Wagner, K., Antranikian, G., 2000. A new thermoactive pullulanase from Desulfurococcus mucosus: cloning, sequencing, purifcation, and characterization of the recombinant enzyme after expression in Bacillus subtilis. J. Bacteriol. 182, 6331e6338. Eliasson, A.C., 2004. Starch in Food: Structure, Function and Applications. Woodhead Publishing Limited, Cambridge, pp. 477e505. Ells, L.J., Seal, C.J., Kettlitz, B., Bal, W., Mathers, J.C., 2005. Postprandial glycaemic, lipaemic and haemostatic responses to ingestion of rapidly and slowly digested starches in healthy young women. Br. J. Nutr. 94, 948e955. Erra-Pujada, M., Debeire, P., Duchiron, F., O’Donohue, M.J., 1999. The type II pullulanase of Thermococcus hydrothermalis: molecular characterization of the gene and expression of the catalytic domain. J. Bacteriol. 181, 3284e3287. French, D., Pulley, A.O., Effenberger, J.A., Rougvie, M.A., Abdullah, M., 1965. Studies on the Schardinger dextrins. XII. The molecular size and structure of the delta-, epsilon-, zeta-, and eta-dextrins. Arch. Biochem. Biophys. 111, 153e160. Ganghofner, D., Kellermann, J., Staudenbauer, W.L., Bronnenmeier, K., 1998. Purification and properties of an amylopullulanase, a glucoamylase, and an a-glucosidase in the amylolytic enzyme system of Thermoanaerobacterium thermosaccharolyticum. Biosci. Biotechnol. Biochem. 62, 302e308. Gibson, G.R., Roberfroid, M.B., 1995. Dietary modulation of the colonic microbiota: introducing the concept of prebiotics. J. Nutr. 25, 1401e1412. Guraya, H.S., James, C., Champagne, E.T., 2001. Effect of enzyme concentration and storage temperature on the formation of slowly digestible starch from cooked debranched rice starch. Starch 53, 131e139. Han, T., Zeng, F., Li, Z., Liu, L., Wei, M., Guan, Q., Liang, X., Peng, Z., Liu, M., Qin, J., Zhang, S., Jia, B., 2013. Biochemical characterization of a recombinant pullulanase from Thermococcus kodakarensis KOD1. Lett. Appl. Microbiol. 57 (4), 336e343. Hatada, Y., Igarashi, K., Ozaki, K., Ara, K., Hitomi, J., Kobayashi, T., Kawai, S., Watabe, T., Ito, S., 1996. Amino acid sequence and molecular structure of an alkaline amylopullulanase from Bacillus that hydrolyzes a-1,4 and a-1,6 linkages in polysaccharides at different active sites. J. Biol. Chem. 271, 24075e24083. Jiao, Y., Wang, S., Lv, M., 2011. Structural and functional analysis of GH57 family thermostable amylopullulanase-a review. Wei Sheng Wu Xue Bao 51, 21e28. Jensen, B.D., Norman, B.E., 1984. Bacillus acidopullyticus pullulanase: applications and regulatory aspects for use in food industry. Proc. Biochem. 1, 397e400. Kim, C.H., Kim, Y.S., 1995. Substrate specificity and detailed characterization of a bifunctional amylasepullulanase enzyme from Bacillus circulans F-2 having two different active sites on one polypeptide. Eur. J. Biochem. 227, 687e693. Koch, R., Zablowski, P., Antranikian, G., 1987. Highly active and thermostable amylases and pullulanases from various anaerobes. Appl. Microbiol. Biotechnol. 27, 192e198. Koch, R., Antranikian, G., 1990. The action of amylolytic and pullulytic enzymes from various anaerobic thermophiles on linear and branched glucose polymers. Starch/Staerke 42, 397e403. Kulp, K., Ponte Jr., J.G., 1981. Staling white pan bread: fundamental causes. Crit. Rev. Food Sci. Nutr. 15, 1e48. Kwak, Y.S., Akiba, T., Kudo, T., 1998. Purification and characterization of a-amylase from hyperthermophilic archaeon Thermococcus profundus, which hydrolyzes both a-1,4 and a-1,6-glucosidic linkages. J. Ferment. Bioeng. 86, 363e367. Li, X., Li, D., Park, K.H., 2013. An extremely thermostable amylopullulanase from Staphylothermus marinus displays both pullulan- and cyclodextrin degrading activities. Appl. Microbiol. Biotechnol. 97, 5359e5369. Li, X., Zhao, J., Fu, J., Pan, Y., Li, D., 2018. Sequence analysis and biochemical properties of an acidophilic and hyperthermophilic amylopullulanase from Thermofilum pendens. Int. J. Biol. Macromol. 114, 235e243. Lin, L.L., Tsau, M.R., Chu, W.S., 1996. Purification and properties of a 140 kDa amylopullulanase from thermophilic and alkaliphilic from Bacillus sp. strain TS-23. Appl. Biochem. Biotechnol. 24, 101e107.

Industrial applications of thermophilic/hyperthermophilic enzymes

Mathupala, S.P., Zeikus, J.G., 1993. Improved purification and biochemical characterization of extracellular amylopullulanase from Thermoanaerobacter ethanolicus 39E. Appl. Microbiol. Biotechnol. 39, 487e493. Mathupala, S.P., Lowe, S.E., Podkovyrov, S.M., Zeikus, J.G., 1993. Sequencing of the amylopullulanase (apu) gene of Thermoanaerobacter ethanolicus 39E, and identification of the active site by site-directed mutagenesis. J. Biol. Chem. 268, 16332e16344. Miao, M., Jiang, B., Zhang, T., 2009. Effect of pullulanase debranching and recrystallization on structure and digestibility of waxy maize starch. Carbohydr. Polym. 76, 214e221. Moshfegh, M., Shahverdi, A.R., Zarrini, G., Faramarzi, M.A., 2013. Biochemical characterization of an extracellular polyextremophilic a-amylase from the halophilic archaeon Halorubrum xinjiangense. Extremophiles 17, 677e687. Mun, S.H., Shin, M., 2006. Mild hydrolysis of resistant starch from maize. Food Chem. 96, 115e121. Mussatto, S.I., Mancilha, I.M., 2007. Non-digestible oligosaccharides: a review. Carbohydr. Polym. 68, 587e597. Naumoff, D.G., 2011. Hierarchical classification of glycoside hydrolases. Biochemistry (Moscow) 76, 622e635. Nisha, M., Satyanarayana, T., 2013a. Characterization of recombinant amylopullulanase (Gt-apu) and truncated amylopullulanase (gtapuT) of the extreme thermophile Geobacillus thermoleovorans NP33 and their action in starch saccharification. Appl. Microbiol. Biotechnol. 97, 6279e6292. Nisha, M., Satyanarayana, T., 2013b. Recombinant bacterial amylopullulanases: developments and perspectives. Bioengineered 4, 1e13. Nisha, M., Satyanarayana, T., 2013c. Thermostable archaeal and bacterial pullulanases and amylopullulanases. In: Satyanarayana, T., Littlechild, J., Kawarabayasi, Y. (Eds.), Thermophilic Microbes in Environmental and Industrial Biotechnology. Springer, New York, pp. 535e587. Noorwez, S.M., Ezhilvannan, M., Satyanarayana, T., 2006. Production of a high maltose-forming hyperthermostable and Ca2þ independent amylopullulanase by an extreme thermophil Geobacillus thermoleovorans in submerged fermentation. Indian J. Biotechnol. 5, 337e345. Plant, A.R., Clemens, R.M., Daniel, R.M., Morgan, H.W., 1987a. Purification and preliminary characterization of an extracellular pullulanase from Thermoanaerobium Tok6-B1. Appl. Microbiol. Biotechnol. 26, 427e433. Plant, A.R., Clemens, R.M., Morgan, H.W., Daniel, R.M., 1987b. Active-site and substrate-specificity of Thermoanaerobium Tok6-B1 pullulanase. Biochem. J. 246, 537e541. Roy, A., Messaoud, E.B., Bejar, S., 2003. Isolation and purification of an acidic pullulanase type II from newly isolated Bacillus sp. US149. Enzyme Microb. Technol. 33 (5), 720e724. Roy, I., Gupta, M.N., 2004. Hydrolysis of starch by a mixture of glucoamylase and pullulanase entrapped individually in calcium alginate beads. Enzyme Microb. Technol. 34, 26e32. Rudiger, A., Jorgensen, P.L., Antranikian, G., 1995. Isolation and characterization of a heat-stable pullulanase from the hyperthermophilic archaeon Pyrococcus woesei after cloning and expression of its gene in Escherichia coli. Appl. Environ. Microbiol. 61 (2), 567e575. Saha, B.C., Lamed, R., Lee, C.Y., Mathupala, S.P., Zeikus, J.G., 1990. Characterization of an endo-acting amylopullulanase from Thermoanaerobacter strain B6A. Appl. Environ. Microbiol. 56, 881e886. Satyanarayana, T., Noorwez, S.M., Kumar, S., Rao, J.L.U.M., Ezhilvannan, M., Kaur, P., 2004. Development of an ideal starch saccharification process using amylolytic enzymes from thermophiles. Biochem. Soc. Trans. 32, 276e278. Satyanarayana, T., Nisha, M., 2018. Archaeal and bacterial thermostable amylopullulanases: characteristic features and biotechnological applications. Amylase 2, 44e57. Schwerdtfeger, R.M., Chiaraluce, R., Consalvi, V., Scandurra, R., Antranikian, G., 1999. Stability, refolding and Ca2þ binding of pullulanase from the hyperthermophilic archaeon Pyrococcus woesei. Eur. J. Biochem. 264, 479e487. Shen, G.J., Srivastava, K.C., Saha, B.C., Zeikus, J.G., 1990. Physiological and enzymatic characterization of a novel pullulan degrading thermophilic Bacillus strain 3183. Appl. Microbiol. Biotechnol. 33, 340e344. Shin, S.I., Kim, H.J., Ha, H.J., Lee, S.H., Moon, T.W., 2005. Effect of hydrothermal treatment on formation and structural characteristics of slowly digestible nonpasted granular sweet potato starch. Starch 57, 421e430.

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Swamy, M.V., Seenayya, G., 1996. Thermostable pullulanase and a-amylase activity from Clostridium thermosulfurogenes SV9-optimization of culture conditions for enzyme production. Process Biochem. 31, 157e162. Tonkova, A., 2006. Microbial starch converting enzymes of the a-amylase family. In: Ray, R.C., Word, O.P. (Eds.), Microbial Biotechnology in Horticulture, vol. 1. Science Publishers, Enfield, pp. 421e472. Vishnu, C., Naveena, B.J., Altaf, M.D., Venkateshwar, M., Reddy, G., 2006. Amylopullulnaseda novel enzyme of L. amylophilus GV6 in direct fermentation of starch to L(þ) lactic acid. Enzyme Microb. Technol. 38, 545e550. Watanabe, N., Yamamoto, K., Tsuzuki, W., Ohya, T., Kobayashi, S., 1997. A novel method to produce branched a-cyclodextrins: pullulanase-glucoamylase-mixed method. J. Ferment. Bioeng. 83, 43e47. Wolf, B.W., Bauer, L.L., Fahey Jr., G.C., 1999. Effects of chemical modification on in vitro rate and extent of food starch digestion: an attempt to discover a slowly digested starch. J. Agric. Food Chem. 47, 4178e4183. Zareian, S., Khajeh, K., Ranjbar, B., Dabirmanesh, B., Ghollasi, M., Mollania, N., 2010. Purification and characterization of a novel amylopullulanase that converts pullulan to glucose, maltose, and maltotriose and starch to glucose and maltose. Enzyme Microb. Technol. 46 (2), 57e63. Zebardast Roodi, F., Aminzadeh, S., Farrokhi, N., Karkhane, A., Haghbeen, K., 2017. Cohnella amylopullulanases: biochemical characterization of two recombinant thermophilic enzymes. PLoS ONE 12, e0175013. Zhang, H., Jin, Z., 2011. Preparation of resistant starch by hydrolysis of maize starch with pullulanase. Carbohydr. Polym. 83, 865e867. Zoebelein, H., Böllert, V., 2001. Dictionary of Renewable Resources. Wiley-VCH, Weinheim, p. 181. Zona, R., Chang-Pi-Hin, F., O’Donohue, M.J., Janecek, S., 2004. Bioinformatics of the glycoside hydrolase family 57 and identification of catalytic residues in amylopullulanase from Thermococcus hydrothermalis. Eur. J. Biochem. 271, 2863e2872.

Relevant websites https://www.pfionline.com/pullulanase-enzyme-in-food-industry/. http://www.sunsonenzyme.com/. https://www.brenda-enzymes.org/.

Industrial applications of thermophilic/hyperthermophilic enzymes

SUBCHAPTER 8.6

Cyclodextrin glucanotransferases 8.6.1 Microbial sources and properties Cyclodextrin glycosyl transferase (CGTase; EC 2.4.1.19) enzyme belongs to the amylase family. This enzyme converts starch into CDs, which are closed-ring structures and have 6 or more glucose units joined together by a-1, 4 glucosidic linkages (Feng et al., 2011). CGTases are produced by bacteria. These enzymes are able to catalyze four different types of transferase reactions: ➢ Cyclisation ➢ Coupling ➢ Disproportionation ➢ Hydrolysis Depending upon the number of glucose units, three major types of CDsda, b, and g are produced by CGTases. Cyclodextrins are also known as cycloamyloses, cyclomaltoses, and Schardinger dextrins (Martin Del Valle, 2009; Eastburn and Tao, 1994). Fig. 8.6.1 shows chemical structure of a, b, and g CD (Ali et al., 2021). Fig. 8.6.2 shows schematic view of CD formation by CGTase. CGTases produced by Bacillus macerans have the maximum market share (Biwer et al., 2002). CGTases are used for commercial production of CDs, which have several industrial applications. CGTases show certain functional resemblance to amylase enzymes, which hydrolyze starch or derivatives of starch into linear products. Therefore, thermostable CGTases can be used to solubilize starch (Biwer et al., 2002). Most of the CGTases are produced by bacteria and also by hyperthermophilic archaea, which belong to the genera Thermococcus, Archaeoglobus fulgidus strain 7324, Pyrococcus, and also in Thermococcus and from Haloferax mediterranei, which is haloalkaliphilic archaeon (Bautista et al., 2012; Cabrera and Blamey, 2018; Egorova and Antranikian, 2007; Park et al., 2007; Rashid et al., 2002; Labes and Schönheit, 2007). Table 8.6.1 shows sources and the optimum growth conditions of CGTases. The bacteria used for producing CGTase for obtaining CDs are chosen based upon the type of CD to be produced. For example, CGTase obtained from Bacillus pseudalcaliphilus 8SB does not show any a activity but show higher b activity and lower g activity (Zhang et al., 2019). Thermostable CGTases are produced by Thermoanaerobacter sp. and Thermoanaerobacterium thermosulfurogenes (Wind et al., 1995; Pedersen et al., 1995; Norman and J urgensen, 1992). Thermostable and alkali-stable CGTase showing optimal activity temperature of

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Figure 8.6.1 Chemical structure of a-CD, b-CD, and g-CD. CD, Cyclodextrin. (From Ali, K.A., Roy, P., Maity, A., Chakraborty, P., 2021. Chapter: 6 Tailor-made cyclodextrin-based nanomaterials as drug carriers. In: Tailor-Made and Functionalized Biopolymer Systems, Elsevier. Reproduced with permission.)

65
C and pH of 10 and was obtained from Anaerobranca bogoriae. This bacterium was isolated from the Lake Bogoria, Kenya (Prowe et al., 1996). A CGTase has been characterized in a Thermococcus species. This enzyme is found to be very stable at 100e105
C and shows optimal activity under the acidic conditions at 90e100
C. This enzyme also shows higher alpha-amylase activity. This enzyme can possibly be used for developing a one-step method for producing CDs. It would replace alpha-amylase enzyme for liquefaction of starch (Vieille and Zeikus, 2001). Cyclodextrins were first isolated in 1891 and structurally characterized as cyclic oligosaccharides. The a, b, and g CDs are the most common CDs. These have 6, 7, and 8 glucose residues, respectively, though, a lot of bigger cyclic glucans are produced in the initial phase of the reaction (Leemhuis et al., 2010; Qi et al., 2007; Terada et al.,

Figure 8.6.2 Schematic view of cyclodextrin (CD) formation by cyclodextrin glycosyltransferase (CGTase). (From Leemhuis, H., Kelly, R.M., Dijkhuizen, L., 2010. Engineering of cyclodextrin glucanotransferases and the impact for biotechnological applications. Appl. Microbiol. Biotechnol. 85 (4), 823e835. Reproduced with permission.)

Table 8.6.1 Cyclodextrin glycosyltransferase (CGTase) sources and optimum growth conditions. Bacteria

Type of CGTase

Optimum condition

References

Bacillus Bacillus Bacillus Bacillus

a-CGTase b-CGTase b-CGTase b-CGTase

40
C, 56
C, 55
C, 55
C,

pH pH pH pH

6.0e8.0 6.4 5.0 9.0

Bacillus megaterium Bacillus subtilis Bacillus firmus strain 290-3 Paenibacillus macerans

b-CGTase g-CGTase b/g-CGTase a-CGTase

60
C, 65
C, 60
C, 45
C,

pH pH pH pH

7.2 8.0 6e8 6.0e10

Thermoanaerobacterium thermosulfurigenes Geobacillus thermoglucosidans Brevibacillus brevis strain CD162 B. macorous strain WSH02-06 Brevibacterium sp. strain 9605

a-CGTase

80e85
C, pH 45e7.0

Jamil et al. (2017) Szerman et al. (2007) Mora et al. (2012) Martins and Hatti-Kaul. (2002) Pishtiyski et al. (2007) Kato et al. (1986) Ebglbrecht (1990) Li et al. (2010) Wind et al. (1995)

b-CGTase b/g CGTase

65e70
C, pH 5.5 55
C, pH 8.0

Jia et al. (2018) Kim et al. (1998)

g-CGTase g-CGTase

50
C, pH 6.5 45
C, pH 10

Wang et al. (2004) Mori et al. (1994)

licheniformis circulans sp. agaradhaerens

From Ogunbadejo, B., Al-Zuhair, S., 2021. MOFs as potential matrices in cyclodextrin glycosyltransferase immobilization. Molecules 26 (3), 680. Distributed under Creative Common CC BY license 4.0 License.

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1997; Zheng et al., 2002). Certain types of bacteria and archaea most probably produce CGTases in nature for controlling the starch, converting it into CDs, which is not used by competing microbes (Hashimoto et al., 2001; Pajatsch et al., 1999). The first 3D structure of CGTases showed that these are five domain proteins (Klein and Schulz, 1991). The active site is placed at the bottom of a (b/a)8-barrel in the domain A. Substrate binds across the surface of enzyme in a long groove produced by the domain A and domain B, which are able to hold about seven glucose residues at the donor subsites and three at the acceptor subsites, which are shown by the kinetic experiments and crystal structures of substratee/inhibitore/producteCGTase complexes. The C-terminal region of CGTases is produced by domains C, D, and E. The role of domain D is not known, domain C binds substrate, and domain E binds raw starch. The domain E has been classified as a family 20 carbohydrate binding module. CGTases break the a-1,4-glycosidic linkages between the subsites 1 and þ1 in a-glucans and yield a covalent glycosyl-intermediate (stable) bound at the donor subsites. Then, the glycosyl-intermediate is transferred to the 4-hydroxyl of its own nonreducing end and forms a new a-1,4-glycosidic bond and yields a cyclic product. CGTases are able to transfer the glycosyl-intermediate to a second a-glucan producing a linear product or to water. Furthermore, CGTase are able to degrade the CDs. These open up the CD ring and transfer the linear CD to a sugar acceptor and produce a linear oligosaccharide. The sitedirected mutagenesis studies and structural information were used for explaining the mechanistic function of the residues at the catalytic center of CGTases (Bender, 1990; Cantarel et al., 2009; Chang et al., 1998; Dalmia et al., 1995; Haga et al., 2003; Kanai et al., 2001; Klein et al., 1992; Leemhuis et al., 2003a,b; Machovic and Janecek, 2006; Nakamura et al., 1993; Schmidt et al., 1998; Wind et al., 1998; Penninga et al., 1996; Uitdehaag et al., 1999b).

8.6.2 Application of CGTases CGTases are mostly used for producing cyclodextrins. The ability of cyclodextrins to form inclusion complexes with a variety of organic molecules means that they improve the solubility of hydrophobic compounds in aqueous solutions. Cyclodextrin production occurs in a multistage process in which, in the first step, starch is liquefied by a heat-stable amylase and, in the second step, the cyclization reaction with a CGTase from Bacillus sp. takes places. Because of the low stability of the latter enzyme, the process must run at two different temperatures. The finding of heat-stable and more specific CGTases from extremophiles will solve this problem. The application of heat-stable CGTase in jet cooking, where temperatures up to 105
C are used, will allow the liquefaction and cyclization to take place in one step (Niehaus et al., 1999). Several manufactures are producing cyclodextrins on a commercial scale from starch in thousands of tons. The demand is continuously increasing. In the first step, the starch is

Industrial applications of thermophilic/hyperthermophilic enzymes

Figure 8.6.3 Flow scheme of cyclodextrin (CD) production. Highlighted are the steps where protein engineers and process controllers can influence the process efficiency. (From Leemhuis, H., Kelly, R.M., Dijkhuizen, L., 2010. Engineering of cyclodextrin glucanotransferases and the impact for biotechnological applications. Appl. Microbiol. Biotechnol. 85 (4), 823e835. Reproduced with permission.)

first liquefied, generally using jet-cooking step. This step consumes a lot of energy (Buchholz and Seibel, 2008) (Fig. 8.6.3). But, the conversion of starch into CDs is only about 50%. The reason for this may be that CGTases have problem in bypassing the a-1,6branches in amylopectin producing CGTase limit dextrins (van der Maarel et al., 2002). The accessibility of the amylopectin fraction of starch is increased by adding isoamylase or pullulanase debranching enzymes thereby increasing the yield of CD (Rendleman, 1997). Yields of cyclodextrin yields are also not higher because of the product inhibition by enzyme and breakdown of CDs by CGTases into oligosaccharides in the coupling reaction. The effects of product inhibition as well as CD degradation are reduced by keeping the concentrations of CD in the reactor low, which is usually obtained by the addition of complexing agents, which lead to the precipitation of the CDs. Furthermore, complexing agent used has an important effect on the ratio of a, b, and g CD produced (Zhekova et al., 2009; Gaston et al., 2009; Leemhuis et al., 2003a; Biwer et al., 2002; Blackwood and Bucke, 2000). The disruption of CDs is further reduced by limiting the buildup of shorter oligosaccharides by using CGTases with lower

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hydrolytic activity. When the higher concentrations of saccharides are used, CGTases are not able to produce CDs from starch (Martin et al., 2001). Another main problem in the production of CD is that CGTase produces a mixture of CDs. Therefore, a purification step is, needed for obtaining pure a, b, and g CD, by using the complexing agents during synthesis of CD and the dissimilarity in solubility of the different CDs for allowing selective precipitation. The source of CGTase is an important factor in the type of CDs produced (Table 8.6.1). Also, the parameters like the type of starch used, reaction pH, and temperature, the buffer composition are also very important (Alves-Prado et al., 2008; Kamaruddin et al., 2005; Lee and Kim, 1992; Matioli et al., 2000; Qi et al., 2004; Son et al., 2008). Cyclodextrins are able to encapsulate hydrophobic molecules within their hydrophobic cavity (Leemhuis et al., 2010). These have several applications in the food, pharmaceutical, cosmetics, textile industry, etc. (Biwer et al., 2002; Li et al., 2007; Martin Del Valle, 2009). Encapsulation has been used for solubilizing hydrophobic molecules in water (CDs have a hydrophilic outer surface). This is mainly helpful because some drug molecules dissolve weekly in water or to shield guest molecules from heat, light, or oxidative conditions (Loftsson and Duchene, 2007; Astray et al., 2009). Cyclodextrins have been also used for lowering odor in perfumes and room fresheners for the release of the odor in a controlled manner. CDs are used for separating the enantiomers for extraction of toxic chemicals from chemical industry waste streams and in bioremediation (Fava and Ciccotosto, 2002; Martin Del Valle, 2009). CDs are also used to suppress objectionable tastes and to extract compounds such as cholesterol from foods (Szente and Szejtli, 2004, 2005).

Bibliography Ali, K.A., Roy, P., Maity, A., Chakraborty, P., 2021. Chapter 6: Tailor-made cyclodextrin-based nanomaterials as drug carriers. In: Tailor-Made and Functionalized Biopolymer Systems. Elsevier. Alves-Prado, H.F., Carneiro, A.A., Pavezzi, F.C., Gomes, E., Boscolo, M., Franco, C.M., da Silva, R., 2008. Production of cyclodextrins by CGTase from Bacillus clausii using different starches as substrates. Appl. Biochem. Biotechnol. 146, 3e13. Astray, G., Gonzalez-Barreiro, C., Mejuto, J.C., Rial-Otero, R., Simal-Gandara, J., 2009. A review on the use of cyclodextrins in foods. Food Hydrocolloids 23, 1631e1640. Bautista, V., Esclapez, J., P
erez-Pomares, F., Martínez-Espinosa, R.M., Camacho, M., Bonete, M.J., 2012. Cyclodextrin glycosyltransferase: a key enzyme in the assimilation of starch by the halophilic archaeon Haloferax mediterranei. Extremophiles 16, 147e159. Bender, H., 1990. Studies of the mechanism of the cyclisation reaction catalysed by the a-cyclodextrin glycosyltransferase from Klebsiella pneumoniae M5a1, and the b-cyclodextrin glycosyltransferase from Bacillus circulans 8. Carbohydr. Res. 206, 257e267. Biwer, A., Antranikian, G., Heinzle, E., 2002. Enzymatic production of cyclodextrins. Appl. Microbiol. Biotechnol. 59, 609e617. Blackwood, A.D., Bucke, C., 2000. Addition of polar organic solvents can improve the product selectivity of cyclodextrin glycosyltransferase. Solvent effects on CGTase. Enzyme Microb. Technol. 27, 704e708. Buchholz, K., Seibel, J., 2008. Industrial carbohydrate biotransformations. Carbohydr. Res. 343, 1966e1979.

Industrial applications of thermophilic/hyperthermophilic enzymes


Blamey, J.M., 2018a. Biotechnological applications of archaeal enzymes from extreme Cabrera, M.A., environments. Biol. Res. 51, 37. Cantarel, B.L., Coutinho, P.M., Rancurel, C., Bernard, T., Lombard, V., Henrissat, B., 2009. The carbohydrate-active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res. 37, D233eD238. Chang, H.Y., Irwin, P.M., Nikolov, Z.L., 1998. Effects of mutations in the starch-binding domain of Bacillus macerans cyclodextrin glycosyltransferase. J. Biotechnol. 65, 191e202. Dalmia, B.K., Schutte, K., Nikolov, Z.L., 1995. Domain E of Bacillus macerans cyclodextrin glucanotransferase: an independent starch-binding domain. Biotechnol. Bioeng. 47, 575e584. Eastburn, S.D., Tao, B.Y., 1994. Applications of modified cyclodextrins. Biotechnol. Adv. 12, 325e339. Ebglbrecht, A., 1990. Biochemical and genetic characterization of a CGTase from an alkalophilic bacterium forming primary-cyclodextrin. In: Proceeding of the 5th International Symposium on Cyclodextrins, Paris, France, 28e30 March 1990. Egorova, K., Antranikian, G., 2007. Biotechnology. In: Garrett, R., Klenk, H. (Eds.), Archaea Evol Physiol Mol Biol, first ed. Blackwell Publishing, Malden. Fava, F., Ciccotosto, V.F., 2002. Effects of randomly methylatedbeta-cyclodextrins (RAMEB) on the bioavailability and aerobic biodegradation of polychlorinated biphenyls in three pristine soils spiked with a transformer oil. Appl. Microbiol. Biotechnol. 58, 393e399. Feng, T., Zhuang, H., Ran, Y., 2011. The application of cyclodextrin glycosyltransferase in biological science. J. Bioequiv. Availab. 3, 202e206. Gaston, J.A.R., Szerman, N., Costa, H., Krymkiewicz, N., Ferrarotti, S., 2009. Cyclodextrin glycosyltransferase from Bacillus circulans DF 9R: activity and kinetic studies. Enzyme Microb. Technol. 45, 36e41. Haga, K., Kanai, R., Sakamoto, O., Aoyagi, M., Harata, K., Yamane, K., 2003. Effects of essential carbohydrate/aromatic stacking interaction with Tyr100 and Phe259 on substrate binding of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp. 1011. J. Biochem. 134, 881e891. Hashimoto, Y., Yamamoto, T., Fujiwara, S., Takagi, M., Imanaka, T., 2001. Extracellular synthesis, specific recognition, and intracellular degradation of cyclomaltodextrins by the hyperthermophilic archaeon Thermococcus sp. B1001. J. Bacteriol. 183, 5050e5057. Jamil, N., Man, R.C., Shaarani, S.M., Sulaiman, S.Z., Mudalip, S.K.A., Arshad, Z.I.M., 2017. Characterization of a-cyclodextrin glucanotransferase from Bacillus licheniformis. Indian J. Sci. Technol. 10. Jia, X., Ye, X., Chen, J., Lin, X., Vasseur, L., You, M., 2018. Purification and biochemical characterization of a cyclodextrin glycosyltransferase from Geobacillus thermoglucosidans CHB1. Starch St€arke 70, 1700016. Kamaruddin, K., Illias, R.M., Aziz, S.A., Said, M., Hassan, O., 2005. Effects of buffer properties on cyclodextrin glucanotransferase reactions and cyclodextrin production from raw sago (Cycas revoluta) starch. Biotechnol. Appl. Biochem. 41, 117e125. Kanai, R., Haga, K., Yamane, K., Harata, K., 2001. Crystal structure of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011 complexed with 1-deoxynojirimycin at 2.0 A resolution. J. Biochem. 129, 593e598. Kato, T., Horikoshi, K., 1986. A new-cyclodextrin forming enzyme produced by Bacillus subtilis no. 313. J. Jpn. Soc. Starch Sci. 33, 137e143. Kim, M.H., Sohn, C.B., Oh, T.K., 1998. Cloning and sequencing of a cyclodextrin glycosyltransferase gene from Brevibacillus brevis CD162 and its expression in Escherichia coli. FEMS Microbiol. Lett. 164, 411e418. Klein, C., Hollender, J., Bender, H., Schulz, G.E., 1992. Catalytic centre of cyclodextrin glycosyltransferase derived from X-ray structure analysis combined with site-directed mutagenesis. Biochemistry 31, 8740e8746. Klein, C., Schulz, G.E., 1991. Structure of cyclodextrin glycosyltransferase refined at 2.0 A resolution. J. Mol. Biol. 217, 737e750. Labes, A., Schönheit, P., 2007. Unusual starch degradation pathway via cyclodextrins in the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324. J. Bacteriol. 189, 8901e8913. Lee, Y.D., Kim, H.S., 1992. Effect of organic solvents on enzymatic production of cyclodextrins from unliquefied corn starch in an attrition bioreactor. Biotechnol. Bioeng. 39, 977e983.

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Leemhuis, H., Kelly, R.M., Dijkhuizen, L., 2010. Engineering of cyclodextrin glucanotransferases and the impact for biotechnological applications. Appl. Microbiol. Biotechnol. 85 (4), 823e835. Leemhuis, H., Dijkstra, B.W., Dijkhuizen, L., 2003a. Thermoanaerobacterium thermosulfurigenes cyclodextrin glycosyltransferase: mechanism and kinetics of inhibition by acarbose and cyclodextrins. Eur. J. Biochem. 270, 155e162. Leemhuis, H., Rozeboom, H.J., Dijkstra, B.W., Dijkhuizen, L., 2003b. The fully conserved Asp residue in conserved sequence region I of the alpha-amylase family is crucial for the catalytic site architecture and activity. FEBS Lett. 541, 47e51. Li, Z., Wang, M., Wang, F., Gu, Z., Du, G., Wu, J., Chen, J., 2007. g-Cyclodextrin: a review on enzymatic production and applications. Appl. Microbiol. Biotechnol. 77, 245e255. Li, Z., Li, B., Gu, Z., Du, G., Wu, J., Chen, J., 2010. Extracellular expression and biochemical characterization of a-cyclodextrin glycosyltransferase from Paenibacillus macerans. Carbohydr. Res. 345, 886e892. Loftsson, T., Duchene, D., 2007. Cyclodextrins and their pharmaceutical applications. Int. J. Pharm. 329, 1e11. Machovic, M., Janecek, S., 2006. Starch-binding domains in the post-genome era. Cell Mol. Life Sci. 63, 2710e2724. Martin Del Valle, E.M.M., 2009. Cyclodextrins and their uses: a review. Process Biochem. 39, 1033e1046. Martin, M.T., Alcalde, M., Plou, F.J., Dijkhuizen, L., Ballesteros, A., 2001. Synthesis of maltooligosaccharides via the acceptor reaction catalyzed by cyclodextrin glycosyltransferases. Biocatal. Biotransform. 19, 21e35. Martins, R.F., Hatti-Kaul, R., 2002. A new cyclodextrin glycosyltransferase from an alkaliphilic Bacillus agaradhaerens isolate: purification and characterisation. Enzym. Microb. Technol. 30, 116e124. Matioli, G., Zanin, G.M., de Moraes, F.F., 2000. Enhancement of selectivity for producing g-cyclodextrin. Appl. Biochem. Biotechnol. 84e86, 955e962. Mora, M.M.M., S
anchez, K.H., Santana, R.V., Rojas, A.P., Ramírez, H.L., Torres-Labandeira, J.J., 2012. Partial purification and properties of cyclodextrin glycosiltransferase (CGTase) from alkalophilic Bacillus species. SpringerPlus 1, 61. Mori, S., Hirose, S., Oya, T., Kitahata, S., 1994. Purification and properties of cyclodextrin glucanotransferase from Brevibacterium sp. No. 9605. Biosci. Biotechnol. Biochem. 58, 1968e1972. Nakamura, A., Haga, K., Yamane, K., 1993. Three histidine residues in the active center of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011 effects of the replacement on pH dependence and transition-state stabilization. Biochemistry 32, 6624e6631. Niehaus, F., Bertoldo, C., K€ahler, M., Antranikian, G., 1999. Extremophiles as a source of novel enzymes for industrial application. Appl. Microbiol. Biotechnol. 51 (6), 711e729. Norman, B.E., J urgensen, S.T., 1992. Thermoanaerobacter sp. CGTase: its properties and application. Depun Kagaku 39, 101e108. Ogunbadejo, B., Al-Zuhair, S., 2021. MOFs as potential matrices in cyclodextrin glycosyltransferase immobilization. Molecules 26 (3), 680. Pajatsch, M., Andersen, C., Mathes, A., Bock, A., Benz, R., Engelhardt, H., 1999. Properties of a cyclodextrin-specific, unusual porin from Klebsiella oxytoca. J. Biol. Chem. 274, 25159e25166. Park, H.S., Park, J.T., Kang, H.K., Cha, H., Kim, D.S., Kim, J.W., Park, K.H., 2007. TreX from Sulfolobus solfataricus ATCC 35092 displays isoamylase and 4-a-glucanotransferase activities. Biosci. Biotechnol. Biochem. 71, 1348e1352. Pedersen, S., Jensen, B.F., Dijkhuizen, L., J urgensen, S.T., Dijkstra, B.W., 1995. A better enzyme for cyclodextrins. Chemtech 12, 1925. Penninga, D., van der Veen, B.A., Knegtel, R.M., van Hijum, S.A., Rozeboom, H.J., Kalk, K.H., Dijkstra, B.W., Dijkhuizen, L., 1996. The raw starch binding domain of cyclodextrin glycosyltransferase from Bacillus circulans 251. J. Biol. Chem. 271, 32777e32784. Pishtiyski, I., Popova, V., Zhekova, B., 2007. Characterization of cyclodextrin glucanotransferase produced by Bacillus megaterium. Appl. Biochem. Biotechnol. 144, 263e272. Prowe, S., Vossenberg, J van de, Driessen, A., Antranikian, G., Konings, W., 1996. Sodium-coupled energy transduction in the newly isolated thermoalkaliphic strain LBS3. J. Bacteriol. 178, 4099.

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Qi, Q., Mokhtar, M.N., Zimmermann, W., 2007. Effect of ethanol on the synthesis of large-ring cyclodextrins by cyclodextrin glucanotransferases. J. Incl. Phenom. Macrocycl. Chem. 57, 95e99. Qi, Q., She, X., Endo, T., Zimmermann, W., 2004. Effect of the reaction temperature on the transglycosylation reactions catalyzed by the cyclodextrin glucanotransferase from Bacillus macerans for the synthesis of large-ring cyclodextrins. Tetrahedron 60, 799e806. Rashid, N., Cornista, J., Ezaki, S., Fukui, T., Atomi, H., Imanaka, T., 2002. Characterization of an archaeal cyclodextrin glucanotransferase with a novel C-terminal domain. J. Bacteriol. 184, 777e784. Rendleman Jr., J.A., 1997. Enhancement of cyclodextrin production through use of debranching enzymes. Biotechnol. Appl. Biochem. 26 (Pt 1), 51e61. Schmidt, A.K., Cottaz, S., Driguez, H., Schulz, G.E., 1998. Structure of cyclodextrin glycosyltransferase complexed with a derivative of its main product b-cyclodextrin. Biochemistry 37, 5909e5915. Son, Y.J., Rha, C.S., Park, Y.C., Shin, S.Y., Lee, Y.S., Seo, J.H., 2008. Production of cyclodextrins in ultrafiltration membrane reactor containing cyclodextrin glycosyltransferase from Bacillus macerans. J. Microbiol. Biotechnol. 18, 725e729. Szejlti, J., Szente, L., 2005. Elimination of bitter, disgusting tastes of drugs and foods by cyclodextrins. Eur. J. Pharm. Biopharm. 61, 115e125. Szente, L., Szejtli, J., 2004. Cyclodextrins as food ingredients. J. Food. Sci. Technol. 15, 137e142. Szerman, N., Schroh, I., Rossi, A.L., Rosso, A.M., Krymkiewicz, N., Ferrarotti, S.A., 2007. Cyclodextrin production by cyclodextrin glycosyltransferase from Bacillus circulans DF 9R. Bioresour. Technol. 98, 2886e2891. Terada, Y., Yanase, M., Takata, H., Takaha, T., Okada, S., 1997. Cyclodextrins are not the major cyclic a-1, 4-glucans produced by the initial action of cyclodextrin glucanotransferase on amylose. J. Biol. Chem. 272, 15729e15733. Uitdehaag, J.C., Mosi, R., Kalk, K.H., van der Veen, B.A., Dijkhuizen, L., Withers, S.G., Dijkstra, B.W., 1999a. X-ray structures along the reaction pathway of cyclodextrin glycosyltransferase elucidate catalysis in the a-amylase family. Nat. Struct. Biol. 6, 432e436. Uitdehaag, J.C.M., Kalk, K.H., van der Veen, B.A., Dijkhuizen, L., Dijkstra, B.W., 1999b. The cyclization mechanism of cyclodextrin glycosyltransferase (CGTase) as revealed by a g-cyclodextrin-CGTase complex at 1.8-A resolution. J. Biol. Chem. 274, 34868e34876. van der Maarel, M.J.E.C., van der Veen, B.A., Uitdehaag, J.C.M., Leemhuis, H., Dijkhuizen, L., 2002. Properties and applications of starch converting enzymes of the alpha-amylase family. J Biotechnol 94, 137e155. Vieille, C., Zeikus, G.J., 2001. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65, 1e43. Wang, F., Du, G., Li, Y., Chen, J., 2004. Optimization of cultivation conditions for the production of gcyclodextrin glucanotransferase by Bacillus macorous. Food Biotechnol. 18, 251e264. Wind, R., Liebl, W., Buitlaar, R., Penninga, D., Spreinat, A., Dijkhuizen, L., Bahl, H., 1995. Cyclodextrin formation by the thermostable a-amylase of Thermoanaerobacterium thermosulfurigenes EM1 and reclassification of the enzyme as a cyclodextrin glycosyltransferase. Appl. Environ. Microbiol. 61, 1257e1265. Wind, R.D., Uitdehaag, J.C., Buitelaar, R.M., Dijkstra, B.W., Dijkhuizen, L., 1998. Engineering of cyclodextrin product specificity and pH optima of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1. J. Biol. Chem. 273, 5771e5779. Zhang, J., Mao, H., Li, M., Su, E., 2019. Cyclodextrin glucosyltransferase immobilization on polydopaminecoated Fe3O4 nanoparticles in the presence of polyethyleneimine for efficient b-cyclodextrin production. Biochem. Eng. J. 150, 107264. Zhekova, B., Dobrev, G., Stanchev, V., Pishtiyski, I., 2009. Approaches for yield increase of b-cyclodextrin formed by cyclodextrin glucanotransferase from Bacillus megaterium. World J. Microbiol. Biotechnol. 25, 1043e1049. Zheng, M., Endo, T., Zimmermann, W., 2002. Synthesis of large-ring cyclodextrins by cyclodextrin glucanotransferases from bacterial isolates. J. Incl. Phenom. Macrocycl. Chem. 44, 387e390.

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SUBCHAPTER 8.7

Cellulases 8.7.1 Microbial sources and properties Cellulase enzymes are hydrolyzing enzymes. These enzymes hydrolyze the b-1, 4-Dglucan bonds in cellulose and produce glucose, cellobiose, and oligosaccharides. Three types of cellulases are mostly involved in the hydrolysis of native cellulose. These are cellobiohydrolase (CBH), endo-b-1, 4-glucanase (EG), and b-glucosidase (BGL) (Prasad et al., 2018). Several microorganisms are known as potential producers of cellulases. Fungi are known to secrete cellulases more efficiently. Trichoderma reesei is the most studied fungus for production of cellulase enzymes. This fungus is found to produce two CBH components, eight EG components, and seven BGL components (Aro et al., 2005). For efficient hydrolysis of cellulose, synergistic action of CBH, EG, and BGL is required. EG makes a nick in the cellulose polymer, thereby opens the reducing as well as nonreducing ends. CBH is able to act on noreducing as well as reducing ends and releases cellobiose and cello-oligosaccharides, whereas BGL finally slices the cellobiose, frees the glucose, and completes the hydrolysis (Sukumaran et al., 2005). Thus, a complete cellulolytic system consisting of EG, CBH, and BGL components acts synergistically for converting crystalline cellulose into glucose. Cellulase enzymes have several commercial applications as they are able to convert lignocellulosic biomass into monosaccharides by the action of enzyme. These monosaccharides can then be used to produce many value added products in the industry (Patel et al., 2019). Cellulases are used for several other applications, which include extraction of pigments and bioactive molecules from the plant materials and waste management (Ghosh et al., 2020). Thermostable cellulases are used to depolymerize the complex lignocellulosic polymers. Biomass pretreated with chemicals can be hydrolyzed by cellulases from thermophilic microorganisms. These enzymes are produced as free enzymes or consolidated into complexes of multienzymes termed as cellulosome. These are able to liberate sugars efficiently when directly used on a cellulosic substrate (Azadian et al., 2017; Singh et al., 2021a; Thomas et al., 2014). Many microorganisms including fungi, bacteria, and archaea are able to produce cellulase enzymes, but the thermostable cellulolytic enzymes, which are used in industry, are obtained from extreme ecological niches for instance hydrothermal vents, hot springs, glaciers, and so forth. Moreover, the genetic engineering methods can be used for producing thermostable cellulase enzymes for industrial applications. Not much attention is paid to cellulase enzymes obtained from bacteria. Both the aerobic as well as anaerobic bacteria are found to produce cellulases. There are prominent

Industrial applications of thermophilic/hyperthermophilic enzymes

resemblances and dissimilarities between bacteria growing aerobically and anaerobically concerning their cellulases, biomass yield, and yield of lignocellulosic polymer hydrolysis (Kuhad et al., 2016). Several thermophilic bacteria produce thermostable cellulases. These include Clostridium, Caldocellum, Caldibacillus, Geobacillus, Bacillus, and Acidothermus (Ghosh et al., 2020). The bacterium Caldicellulosiruptor bescii contains highly active cellulases. This is a hyperthermophilic anaerobe isolated from a Kamchatka hot spring and was found to depolymerize cellulose without chemical treatment and this was due to the reason that it produced CelA which is a multifunctional and multimodular enzyme better than commercial cellulase enzymes (Singh et al., 2021b). Cellulase enzymes of C. bescii were utilized for building a “designer cellulosome” that was active and stable at a temperature of 75
C (Kahn et al., 2019). Dictyoglomus turgidum, an extremely thermophilic bacterium has a gene which encodes BGL was expressed in E. coli. This enzyme shows optimal activity at a pH of 5.4 and temperature of 80
C. This enzyme is very stable at a pH of 5e8. It retained 70% of its original activity after 2 h at a temperature of 70
C. It was found to be appropriate for commercial production of ethanol as the enzyme was highly tolerant to ethanol and glucose (Fusco et al., 2018). From hyperthermophilic bacteria Thermotoga naphthophila RKU-10T, a cellulolytic gene was isolated and overexpressed in E. coli. The purified TnCel12B showed optimum activity at temperature of 90
C and pH 6.0. After incubation at temperature of 85
C, it was found to maintain full activity after 8 h with exceptional stability over a broader temperature and pH range of 50e85
C and 5.0e9.0, respectively (Akram and Haq, 2020). A gene from Sulfolobus shibatae, which is a hyperthermophilic archaea, encodes endo-1,4-b-D-glucanase. This gene was cloned and expressed in E. coli. This genetically engineered enzyme showed optimum activity at 95e100
C. The enzyme showed exceptional resistance to higher temperatures, and full activity was observed after 60 min at temperature of 75e85
C. After 120 min at a temperature of 75
C, 80
C, and 85
C, about 98%, 90%, and 84% of original activity were observed, respectively (Boyce and Walsh, 2018). Very few reports on thermostable cellulases from archaea are available. Cellulase F1, obtained from function-based screening, contained two dissimilar cellulase modules, possibly resulting from fusion of two archaeal cellulases and with a novel protein structure resulting in improved thermostability and activity (Lewin et al., 2017). Table 8.7.1 shows characteristics of thermostable cellulases from several thermophilic microorganism. Several commercial applications need thermostable enzymes which are produced by thermophilic fungi. The thermostable enzymes, which are found in their habitat, are receiving considerable attention, particularly degradation of lignocellulosic biomass. Several thermophilic fungi are found to produce highly thermostable cellulases, including Thermoascus aurantiacus, Talaromyces emersonii, Sporotrichum sp., and Syncephalastrum racemosum. These fungi show optimum activity at 70e80
C. Cellulase enzymes produced by these eukaryotes have undergone structural and functional characterization (Gomes

183

Table 8.7.1 Thermostable cellulases from various thermophilic microorganism and their characteristics. Microorganism

Enzyme

Optimum pH

Optimum temperature (8C)

Specific activity

Thermostability/half-life

References

Thermotoga naphthophila RKU-10T Dictyoglomus turgidum

Endo-1,4-b-glucanase

6

90

1664 U mg

Half-life of 180 min at 95
C

Akram and Hag (2020)

b-Glucosidase

5.4

80

160 U mg1

Fusco et al. (2018a)

Dictyoglomus thermophilum

Endo-1,4-b-glucanase

5.0

50e85

7.47 t 0.06 U/mg

Sulfolobus shibatae

Endo-1,4-b-glucanase

3e5

95e100

ND

Bacillus licheniformis A5

Cellulase

6.0

50

ND

Aspergillus heteromorphic

Cellulase

4.5

60

ND

Sporothrix carnis

Cellulase

5.0

80

ND

Paecilomyces thermophila

b-glucosidase

After incubation at 70
C for 2 h, it retained 70% of its activity It retained 80% of its relative activity after incubation at 50
C for 135 days Retained 98%, 90%, and 84% of its activity at 75
C, 80
C, and 85
C, respectively, after 120 min Retained 82% of its activity after 120 min at 80
C Retained 60.0% of its activity after 1 h at 90
C Retained 75% of its activity after 300 min at 80
C

6.0

65

ND

Shi et al. (2013)

Boyce and Walsh (2018)

Yang et al. (2021)

Singh et al. (2009)

Olajuyigbe and Ogunyewo (2016) Yan et al. (2012)

Putranjiva roxburghii Thermoascus aurantiacus

b-glucosidase b-glucosidase

5.0 5.0

65 70

ND 23.3 U/mg

Geobacillus sp. HTA426 Talaromyces emersonii Talaromyces emersonii Talaromyces emersonii CBS394.64

Cellulase

7.0

60

ND

71.5

482.8 U/mg

b-glucosidase Cellobiohydrolase

5.0

68

ND

Endo-1,4-b-glucanase

4.5

90

ND

75
C for 60 min It maintained 70% of its relative activity after incubation at 60
C for 1 h Stable at 50e70
C for 300 min Half-life of 62 min at 65
C Half-life of 68 min at 80
C Highly thermostable at 70
C

Kar et al. (2017) Hong et al. (2007)

Potprommanee et al. (2017) Murray et al. (2004) Grassick et al. (2004) Wang et al. (2014)

From Ajeje, S.B., Hu, Y., Song, G., Peter, S.B., Afful, R.G., Sun, F., Asadollahi, M.A., Amiri, H., Abdulkhani, A., Sun, H., 2021. Thermostable cellulases/xylanases from thermophilic and hyperthermophilic microorganisms: current perspective. Front. Bioeng. Biotechnol. 9, 794304. Distributed under the terms of the Creative Commons Attribution License (CC BY).

186

Developments and Applications of Enzymes From Thermophilic Microorganisms

et al., 2000; Ishihara et al., 1999; Murray et al., 2001; Patel et al., 2019; Thapa et al., 2020; Wonganu et al., 2008). A gene TeEgl5A encoding an extremely thermostable EG was obtained from a Talaromyces emersonii CBS394.64 (a thermophilic fungi). This gene was overexpressed in Pichia pastoris. The genetically engineered EG showed maximum activity at pH of 4.5 and a temperature of 90
C after purification. It showed extremely high stability at a temperature of 70
C and over a broader pH range of 1.0e10.0. It was found to be highly resistant to most of the metal ions, protease enzymes, and sodium dodecyl sulfate. TeEgl5A was found to possess a broad range of substrate specificity and showed enhanced activity against polymers which contained b-1,3 and b-1,4 glycosidic bonds (Wang et al., 2014). The fungus Thermoascus aurantiacus secretes an extremely thermostable cellulase enzyme for deconstruction of biomass (Mohsin et al., 2019). Trichoderma sp. was found to be the best cellulase producing fungi, but it is susceptible to product inhibition (Akram et al., 2018). Both novel thermostable b-glucosidases from filamentous fungi were expressed in T. reesei. These enzymes showed very high thermostability and were optimally active at 60
C and pH 5.0. Enzymes derived from CEL3a and CEL3b were incubated at 60
C and pH 5.0. After 6 h of treatment, these enzymes retained 98% and 88% of their original activity (Colabardini et al., 2016). The cellulase activity of Talaromyces emersonii and Sporotrichum thermophile is almost the same as that of mesophilic T. reesei (Coutts and Smith, 1976; Folan and Coughlan, 1978). Several thermophiles, T. Aurantiacus, C. Thermophile, and S. thermophile, have been found to produce cellulases with two- or three-fold higher activity when compared with the relative cellulase activity of T. viridie (Akram et al., 2018). In comparison to thermophilic fungi, more important heat-stable proteins were found in thermophilic bacteria and hyperthermophilic archaea (Ajeje et al., 2021).

8.7.2 Application of cellulases Cellulase enzymes account for a substantial share of the global industrial enzyme market. The demand of cellulase enzymes is continuously increasing because of potential applications in many industrial processes, for instance, in pulp and paper industry, textile industry, chemical industry, food industry, detergent industry, and jeans biostoning, improving the nutritional value of animal feeds and pretreatment of industrial and agricultural wastes (Sharma et al., 2019). Cellulase enzymes stand second in the global industrial enzyme market, just after protease enzymes. The thermostable cellulase enzymes, active at high temperatures, are needed in the detergent industry (assisting in brightening of the colors and softening), production of bioethanol from lignocellulosic biomass, and in the pulp and paper industry (Obeng et al., 2017; Sharma and Bajaj, 2014; Nizamudeen and Bajaj, 2009).

Industrial applications of thermophilic/hyperthermophilic enzymes

8.7.2.1 Textile and detergent industry Thermostable cellulase enzymes are one of the main enzymes used in the textile industry for biopolishing of cotton and other cellulosic fabrics and creating the stone-washed look in jeans. The action of thermostable cellulase enzymes on the cotton fabric during stonewashing disrupts the smaller fiber ends on the surface of yarn. This loosens the dye, which is removed during washing without any problem. Therefore, the stone-washed jeans after treatment with thermostable cellulase enzymes appears faded. Additionally, the appliance of thermostable cellulase enzymes in the detergents results in reduced discoloration and the fuzzing effects caused by several washes (Gupta et al., 2017). The incompletely separated microfibrils on the surface make cotton garments dull and fluffy during repetitive washing. The cellulase enzymes, when added to the detergents, bring back a smoother surface and original color to the clothes by the removal of these microfibrils. Cellulase enzymes were used by Verenich et al. (2008) for treating the nonwoven fabrics. Cellulases are used in washing of denims. Cellulases actually loosen the indigo dye on the denim. This process is known as “Bio-stonewashing.” A small amount of enzyme is able to replace several kilograms of pumice stones. Usage of less pumice stones results in lesser pumice dust, lesser harm to garment and machine. A big benefit of bioblasting is that it avoids pilling. A ball of fluff is called a pill in the textile industry. These pills can cause severe quality problems as they result in an unsightly knotty fabric appearance. Cellulase hydrolyzes the microfibrils that protrude from the surface of the thread. This is due to the reason that they are most prone to enzymatic attack. This weakens the microfibrils and tends to detach them from the body of the fiber, leaving a smoother thread surface. The tissue is much less prone to pilling after bioblasting. Other advantages of lint removal include smoother and softer feel and better color brightness. While traditional fabric softeners tend to wash off and usually result in a sticky feel, the softening effect of BioBlast is washable and nonsticky. For cotton fabrics, the use of bioblasting to improve the fabric is optional. But, bioblasting is urgently needed for novel regenerated cellulose fiber lyocell. It is made of cellulose and is characterized by a higher propensity to fibrillate when wet. Briefly, fibrils separate from the fiber surface. If not removed, the finished lyocell garment will have unacceptable pilling. For this reason, the lyocell fabric is treated with cellulase during finishing. Cellulase enzymes additionally enhance the attractive, silky look of lyocell (Bajpai, 2018). 8.7.1.2 Pulp and paper industry Treating the pulp with thermostable cellulase enzymes serves as a good option to mechanical pulping of lignocellulosic biomass. Enzymatic treatment results in improved mechanical strength of the pulp and saves energy. This is quite the reverse to bulky and stiffer pulp produced during mechanical pulping of lignocelluloses (Bajpai, 1999, 2018).

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Additionally, the bleaching of the pulp and deinking of waste papers of different types are also improved by the use of cellulase enzymes. The bleaching of paper pulp by the use of cellulase enzymes leads to an additional advantage of lesser fine particles in pulp, reduced usage of alkali, and better pulp brightness (Dienes et al., 2004). The bleaching of pulp can be improved when cellulase enzymes are used alone or in combination with xylanase enzymes. These combinations can be used for manufacturing of paper towels and sanitary paper and easily biodegradable cardboard. Cellulase enzymes have been also used for deinking of waste paper. On an industrial scale, enzymatic deinking is found to be effective and economical. Cellulases and xylanases are found to be very effective for deinking of old newsprint and improve the deinking efficiency (Bajpai, 2018; Wang and Kim, 2005). One patent application reports a cellulase preparation containing nonionic surfactants combined with endoglucanases obtained from zygomycetes. Other patents on enzymatic deinking deal with the treatment of nonimpact-printed waste paper using an enzyme mixture containing higher ratio of b-glucosidase activity to filter paper activity. A Japanese patent covers a novel endoglucanase enzyme containing fully defined sequence of 266 amino acids in which one or more sequences are substituted, deleted, added, or inserted. This enzyme is found useful for processing cellulose fibers including deinking of waste paper, improving pulp drainage, and reducing the fiber stiffness (Selin, 2004). Treatment with cellulase enzymes in the presence of surfactant and mechanical agitation can improve the efficacy of enzymatic deinking. This is attributed to the increased flotation efficiency, which results from better detachment of ink particles from the fiber surface. Cellulase enzymes have a cellulose-binding domain (CBD) and catalytic domain. CBD plays a significant role in degrading the cellulose. It has negative impact on deinking. The weak hydrolytic ability to pulp cellulose is an advantageous characteristic for an enzyme used in deinking. Endoglucanase lacking CBD is superior for deinking. Desired physical contact between enzyme, and substrate is a precondition. Enzyme dose and consistency of pulp are both of the greatest importance in enzymatic deinking (Bajpai and Bajpai, 1998; Bajpai, 1999, 2018). 8.7.2.3 Animal feed industry Cellulase enzymes are finding application in the feed industry as these enzymes can improve the feed utilization and performance of animals. These enzymes improve fiber degradation in vitro (Dhiman et al., 2002). Cellulase enzymes degrade the antinutritional components, for instance, lignin, cellulose, pectins, glucan, oligosaccharides, inulin, arabinoxylans, and dextrins, which finally improve the nutritional value of feed and animal health (Kuhad et al., 2011; Mori et al., 2014; Asmare, 2014; Rastogi et al., 2010; Ali et al., 1995; Godfrey and West, 1996).

Industrial applications of thermophilic/hyperthermophilic enzymes

Cellulase enzymes can be used for improving silage production for livestock feed. This includes improving the digestibility of grasses that contain large quantity of potentially fully digestible nutrients and energy value, in addition to smaller quantity of watersoluble carbohydrates. Ruminant diets that contain cellulose, hemicellulose, pectin, and lignin are more complex as compared to grain-based diets for pigs and poultry. Enzyme mixtures containing high amount of cellulases, hemicellulases, and pectinases have been used for improving the nutritional quality of feed (Graham and Balnave, 1995; Lewis et al., 1996). However, the results of adding enzyme mixtures containing cellulases, hemicellulases, and pectinases to ruminant diets are rather contradictory. Animal feed production processes commonly include heat treatment for inactivating contaminants both viral and microbial. Use of thermstable cellulases in feed production may reduce pathogens and improve feed digestibility and nutrition, facilitating the combination of heat treatment and feed conversion in a single step. Cellulase and hemicellulase enzymes are involved in partial hydrolysis of lignocelluloses, grain dehulling, b-glucan hydrolysis, and better emulsification and flexibility of feed ingredients, improving the nutritional quality of animal feeds. In addition, these enzymes may cause partial hydrolysis of plant cell walls during silage and feed storage (Kuhad et al., 2011; Bhat, 2000; Galante et al., 1998; Cowan, 1996). 8.7.2.4 Food industry Cellulases from bacteria (Paenibacillus and Bacillus) and fungi (Trichoderma and Aspergillus) have potential uses in the food industry (Sukumaran et al., 2005). Cellulases have several applications in the food industries. Cellulase enzymes are used to clarify the fruit juices and improve the filterability of vanilla extracts and can tenderize fruits, extract flavoring materials and essential oils (Fig. 8.7.1). Thermophilic cellulases hemicellulases play an essential role in food biotechnology (Gupta et al., 2015). Because of the increasing health awareness among the public, there is a rise in demand of fruit juices. But, the presence of cellulosic polysaccharides hampers the conventional methods of extraction of the fruit juices. The synergistic complex of mashing enzymes, which include cellulases, xylanases, and pectinases are used for clarifying the vegetable and fruit juices. Moreover, the use of b-glucosidase and pectinase enzymes results in enhanced flavor, texture, and smell properties of vegetables and fruits. When the cellulase enzymes are added during the processing of fruits, the strength of the cell wall is reduced. The cellulosic polysaccharides are also solubilized, which result in nearly total liquefaction. Cellulases are able to reduce the viscosity of puree and nectar from fruits, for instance, mango, apricot, pear, peach, plum, and papaya. Cellulase enzymes are also used for extracting the flavonoids from the seeds and flowers. Fibers present in juice are insoluble and denser. This creates problem for industries as the fibers may block the manufacturing line and cause enormous loss to industry (Kumar et al., 2019). The fibers

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Figure 8.7.1 Application of cellulases in the food industry. (From Ejaz, U., Sohail, M., Ghanemi, A., 2021. Cellulases: from bioactivity to a variety of industrial applications. Biomimetics (Basel) 6 (3), 44. https://doi. org/10.3390/biomimetics6030044. Distributed under the terms and conditions of the Creative commons Attribution (CC BY) license.)

are cellulosic in nature, so the addition of cellulase enzymes removes the fibers. Because of this, juices are filtered easily. The turbidity of orange juice was reduced, and significant reduction in viscosity and acidity was achieved by using combination of cellulases and xylanases (Shariq and Sohail, 2019). Cellulase enzymes increase the taste and aroma of citrus fruits. Food containing fibers can also provide health benefits like lessening the danger of certain types of cancer, heart diseases, and diabetes and also help in maintaining a proper body weight (Shariq and Sohail, 2019; Sajith et al., 2016; Dhingra et al., 2012). Baking gets also influenced by pure cellulose, so addition of cellulase enzymes converts the cellulosic polymers to monomeric sugars; this reduces the roughage in dough. Chandrasekaran (2012) used purified cellulase enzymes from T. reesi, A. niger, and Humicola insolens, for reducing roughage in dough. Cellulases can also be used for hydrolyzing the roasted coffee. This process uses lesser energy and is inexpensive in comparison to thermal hydrolysis of coffee (Baraldi et al., 2016). Cellulases are used for extracting the oil from olives (Kuhad et al., 2011). This results in reduced wastage, increase in antioxidant components, poorer tendency to rancidity, higher extraction yield, and better quality (Kumar et al., 2019). Olivex, a commercial enzyme, containing a mixture of xylanases, cellulases, and pectinases from A. aculeatus was used for extracting the olive oil (Sharada et al., 2014).

Industrial applications of thermophilic/hyperthermophilic enzymes

The market for carotenoids is constantly growing. Carotenoids are chemically diverse group of yellow to red colored polyenes. Generally, a mixture of cellulolytic and pectinolytic enzymes are used for extracting the carotenoids. Neagu et al. (2014) used cellulases for extracting carotenoids from tomatoes, which is used in the beverage and food industries as coloring agents. Cinar (2005) performed extraction of carotenoids from carrot, sweet potato, and orange peels, by using a mixture of pectinases and cellulases. 8.7.2.5 Biofuels Cellulase enzymes hydrolyze the biomass into simple sugars, either pentoses or hexoses, which are subsequently fermented to fuel ethanol. Cellulase enzymes are primarily involved in the conversion of lignocelluloses. Degradation of biomass consists of following steps: (1) Biomass pretreatment (2) Enzymatic degradation of polysaccharides into fermentable sugars (3) Fermentation of sugars to bioethanol Processing of biomass by cellulase producing microbes can reduce 40% of the processing cost (Menendez et al., 2015). Presently, several countries are adopting policies regarding cellulosic ethanol and have planned to shift to cellulosic materials in place of starchy or sugar cane materials for production of ethanol (De Farias and Bonato, 2002, 2003). For the consolidated bioprocessing of plant materials, a single microbial strain is not available yet. Chung et al. (2014) observed that Caldicelluloseruptor bescii is an exceptionally thermophilic cellulolytic bacterium with great potential for consolidated bioprocessing of renewable plant biomass. This bacterium has the ability to convert biomass in to ethanol directly and can be used to commercially produce bioethanol. Many industries are focusing on using cellulase and xylanase enzymes in producing biofuels. Ethanol, butanol, 2,3-butanediol, and acetoin are produced by degradation of celluloses and hemicelluloses by cellulases and xylanases (Gusakov, 2013). To date, bioethanol production is not only required due to oil crisis reasons but also for reducing greenhouse gas emissions significantly. Because of these reasons, bioethanol is the most common and extensively used renewable fuel today. Bioethanol obtained from lignocelluloses is environment friendly (Akram et al., 2018; Bala and Singh, 2019; Pereira et al., 2016). Saccharification of biomass with enzymes is a very important step, which contributes significantly to the overall production cost. The saccharification process can be improved by the use of thermostable enzymes produced from thermophiles (Kuhad et al., 2016). Thermostability is a very important and wanted property required to expedite the process of saccharification. In the CBP process, genetically engineered microbes produce enzyme, hydrolyzes, and ferment simultaneously. Cellulolytic microorganisms can be genetically modified for producing ethanol (CBP-I), or ethanol-producing organisms can be genetically modified for producing cellulases (CBP-II). Most consolidated bioprocesses prefer thermophilic microbes as compared to mesophilic microbes as thermophilic microbes

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convert biomass to biofuel at higher temperatures. Trichoderma has low amount of b-glucosidase. Because of this, the cellulose is not efficiently hydrolyzed. Few thermophilic fungi, for instance, Scytalidium thermophillum, and Thielavia terrestris Sporotrichum thermophile, Thermoascus aurantiacus can be used for replacing it because these fungi efficiently degrade lignocelluloses through the production of secretary enzymes (Candido et al., 2019; Antonov et al., 2017; Jiang et al., 2017; Houfani et al., 2020; Mohanram et al., 2013; Bala and Singh, 2017; Kaur et al., 2004; Berka et al., 2011). So, these microbes have been recommended as exceptional candidates for degrading the lignocellulosic polymers to simple fermentable sugars, which can be converted to bioethanol. Molecular biology and metabolic engineering have allowed the advent of effective promoters and regulatory factors that improve expression of cellulases and xylanases (Beier et al., 2020). The cellulose activity and yield of T. reesei were found to increase in an exponential manner after overexpression of the Cbh2 gene. The maximum yield was 119.49 IU/l/h (Li et al., 2017). Combining genetic engineering to enzymatically saccharify complex polymers with the generation of potent inducers of cellulose enzyme, a versatile cellulase system has been constructed (Gao et al., 2017). The production of biobutanol by old acetone-butanol-ethanol (ABE) fermentation process has been proposed for addressing some key drawbacks of bioethanol. The production process of butanol is facing significant economic penalties despite butanol’s great potential as a liquid fuel. On this point, efficient use of lignocelluloses was proposed based on the important contribution of carbon sources to the economics of biobutanol production (Ajeje et al., 2021).

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Kuhad, R.C.G., Singh, R.A., 2011. Microbial cellulases and their industrial applications. Enzyme Res. 2011, 280696. Kumar, V.A., Kurup, R.S.C., Snishamol, C., Prabhu, G.N., 2019. Role of cellulases in food, feed, and beverage industries. In: Green Bio-Processes. Springer, Singapore, pp. 323e343. Lewin, A., Zhou, J., Pham, V., Haugen, T., Zeiny, M.E., Aarstad, O., Liebl, W., Wentzel, A., Liles, M.R., 2017. Novel archaeal thermostable cellulases from an oil reservoir metagenome. AMB Express 7 (1), 183. Lewis, G.E., Hunt, C.W., Sanchez, W.K., Treacher, R., Pritchard, G.T., Feng, P., 1996. Effect of direct-fed fibrolytic enzymes on the digestive characteristics of a forage-based diet fed to beef steers. J Anim. Sci. 74 (12), 3020e3028. Li, Y., Zhang, X., Xiong, L., Mehmood, M.A., Zhao, X., Bai, F., 2017. On-site cellulase production and efficient saccharification of corn stover employing Cbh2 overexpressing Trichoderma reesei with novel induction system. Bioresour. Technol. 238, 643e649. Menendez, E., Paula, G., Rivas, R., 2015. Biotechnological applications of bacterial cellulases. AIMS Bioeng. 2, 163e182. Mhiri, S., Bouanane-Darenfed, A., Jemli, S., Neifar, S., Ameri, R., Mezghani, M., Bouacem, K., Jaouadi, B., Bejar, S., 2020. A thermophilic and thermostable xylanase from Caldicoprobacter algeriensis: recombinant expression, characterization and application in paper biobleaching. Int. J. Biol. Macromol. 164, 808e817. Mohanram, S., Amat, D., Choudhary, J., Arora, A., Nain, L., 2013. Novel perspectives for evolving enzyme cocktails for lignocellulose hydrolysis in biorefineries. Sustain. Chem. Process. 1, 15. Mohsin, I., Poudel, N., Li, D.C., Papageorgiou, A.C., 2019. Crystal structure of a GH3 b-glucosidase from the thermophilic fungus Chaetomium thermophilum. Int. J. Mol. Sci. 20, 5962. Mori, T., Kamei, I., Hirai, H., Kondo, R., 2014. Identification of novel glycosyl hydrolases with cellulolytic activity against crystalline cellulose from metagenomic libraries constructed from bacterial enrichment cultures. Springerplus 3, 1e7. Murray, P.G., Grassick, A., Laffey, C.D., Cuffe, M.M., Higgins, T., Savage, A.V., Planas, A., Tuohy, M.G., 2001. Isolation and characterization of a thermostable endo-beta-glucanase active on 1,3-1,4-beta-Dglucans from the aerobic fungus Talaromyces emersonii CBS 814.70. Enzyme Microb. Technol. 29 (1), 90e98. Murray, P., Aro, N., Collins, C., Grassick, A., Penttil€a, M., Saloheimo, M., Tuohy, M., 2004. Expression in Trichoderma reesei and characterisation of a thermostable family 3 beta-glucosidase from the moderately thermophilic fungus Talaromyces emersonii. Protein Expr. Purif. 38 (2), 248e257. Neagu, D., Leopold, L.F., Thonart, P., Destain, J., Socaciu, C., 2014. Enzyme-assisted extraction of carotenoids and phenolic derivatives from tomatoes. Bull. Univ. Agric. Sci. Vet. Med. Cluj-Napoca-Anim. Sci. Biotechnol. 71, 20e26. Nizamudeen, S., Bajaj, B.K., 2009. A novel thermo-alkalitolerant endoglucanase production using costeffective agricultural residues as substrates by a newly isolated Bacillus sp. NZ. Food Technol. Biotechnol. 47, 435e440. Obeng, E.M., Adam, S.N., Budiman, C., Ongkudon, C.M., Maas, R., Jose, J., 2017. Lignocellulases: a review of emerging and developing enzymes, systems, and practices. Bioresour. Bioprocess. 4, 1e22. Olajuyigbe, F.M., Ogunyewo, O.A., 2016. Enhanced production and physicochemical properties of thermostable crude cellulase from Sporothrix carnis grown on corn Cob. Biocatal. Agric. Biotechnol. 7, 110e117. Patel, A.K., Singhania, R.R., Sim, S.J., Pandey, A., 2019. Thermostable cellulases: current status and perspectives. Bioresour. Technol. 279, 385e392. Pelach, M.A., Pastor, F.J., Puig, J., Vilaseca, F., Mutje, P., 2003. Enzymic deinking of old newspapers with cellulose. Process. Biochem. 38, 1063e1067. Pereira, S.C., Maehara, L., Machado, C.M.M., Farinas, C.S., 2016. Physicalechemicalemorphological characterization of the whole sugarcane lignocellulosic biomass used for 2G ethanol production by spectroscopy and microscopy techniques. Renew. Energy 87, 607e617. Potprommanee, L., Wang, X.Q., Han, Y.J., Nyobe, D., Peng, Y.P., Huang, Q., Liu, J.Y., Liao, Y.L., Chang, K.L., 2017. Characterization of a thermophilic cellulase from Geobacillus sp. HTA426, an efficient cellulase-producer on alkali pretreated of lignocellulosic biomass. PLoS ONE 12 (4), e0175004. Prasad, R.K., Chatterjee, S., Sharma, S., Mazumder, P.B., Vairale, M.G., Raju, P.S., 2018. Insect gut bacteria and their potential application in degradation of lignocellulosic biomass: a review BTd bioremediation: applications for environmental protection and management. In: Varjani, S.J.,

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Agarwal, A.K., Gnansounou, E., Gurunathan, B. (Eds.), Bioremediation: Applications for Environmental Protection and Management. Springer, Singapore, pp. 277e299. Rastogi, G., Bhalla, A., Adhikari, A., Bischoff, K.M., Hughes, S.R., Christopher, L.P., Sani, R.K., 2010. Characterization of thermostable cellulases produced by Bacillus and Geobacillus strains. Bioresour. Technol. 101 (22), 8798e8806. Sajith, S., Priji, P., Sreedevi, S., Benjamin, S., 2016. An overview on fungal cellulases with an industrial perspective. J. Nutr. Food Sci. 06, 1e13. Selin, R., 2004. Production of Fiber Suspension from Waste Paper. Publication number US 20040140069 (July 22, 2004). Sharada, R., Venkateswarlu, G., Venkateswar, S., Anandrao, M., 2014. Applications of cellulasesdreview. Int. J. Pharm. Chem. Biol. Sci. 4, 424e437. Shariq, M., Sohail, M., 2019. Citrus limetta peels: a promising substrate for the production of multienzyme preparation from a yeast consortium. Bioresour. Bioprocess. 6, 1e15. Sharma, M., Bajaj, B.K., 2014. Cellulase production from Bacillus subtilis MS 54 and its potential for saccharification of biphasic-acid-pretreated rice straw. J. Biobased Mater. Bioenergy 8, 449e456. Sharma, S., Surbhi, V., Bhat, B., Singh, S., Bajaj, B.K., 2019. Thermostable enzymes for industrial biotechnology. In: Advances in Enzyme Technology. Elsevier, pp. 469e495. Shi, R., Li, Z., Ye, Q., Xu, J., Liu, Y., 2013. Heterologous expression and characterization of a novel thermo-halotolerant endoglucanase Cel5H from Dictyoglomus thermophilum. Bioresour. Technol. 142, 338e344. Singh, A., Bajar, S., Devi, A., Pant, D., 2021a. An overview on the recent developments in fungal cellulase production and their industrial applications. Bioresour. Technol. Rep. 14, 100652. Singh, N., Mathur, A.S., Gupta, R.P., Barrow, C.J., Tuli, D.K., Puri, M., 2021b. Enzyme systems of thermophilic anaerobic bacteria for lignocellulosic biomass conversion. Int. J. Biol. Macromol. 168, 572e590. Singh, R., Kumar, R., Bishnoi, K., Bishnoi, N.R., 2009. Optimization of synergistic parameters for thermostable cellulase activity of Aspergillus heteromorphus using response surface methodology. Biochem. Eng. J. 48, 28e35. Sukumaran, R.K., Singhania, R.R., Pandey, A., 2005. Microbial cellulases-production, applications and challenges. J. Sci. Ind. Res. 64, 832e844. Thapa, S., Mishra, J., Arora, N., Mishra, P., Li, H., O’Hair, J., Bhatti, S., Zhou, S., 2020. Microbial cellulolytic enzymes: diversity and biotechnology with reference to lignocellulosic biomass degradation. Rev. Environ. Sci. Bio/technol. 19, 621e648. Thomas, L., Joseph, A., Gottumukkala, L.D., 2014. Xylanase and cellulase systems of Clostridium sp.: an insight on molecular approaches for strain improvement. Bioresour. Technol. 158, 343e350. Tong, X., Qi, Z., Zheng, D., Pei, J., Li, Q., Zhao, L., 2021. High-level expression of a novel multifunctional GH3 family b-xylosidase/a-arabinosidase/b-glucosidase from Dictyoglomus turgidum in Escherichia coli. Bioorg. Chem. 111, 104906. Toushik, S.H., Lee, K.-T., Lee, J.-S., Kim, K.-S., 2017. Functional applications of lignocellulolytic enzymes in the fruit and vegetable processing industries. J. Food Sci. 82, 585e593. Uzuner, S., Cekmecelioglu, D., 2019. Chapter 3dEnzymes in the beverage industry. In: Enzymes in Food Biotechnology. Academic Press, pp. 29e43. Verenich, S., Arumugam, K., Shim, E., Pourdeyhimi, B., 2008. Treatment of raw cotton fibers with cellulases for nonwoven fabrics. Text. Res. J. 78, 540e548. Wang, K., Luo, H., Bai, Y., Shi, P., Huang, H., Xue, X., Yao, B., 2014. A thermophilic endo-1,4-b-glucanase from Talaromyces emersonii CBS394.64 with broad substrate specificity and great application potentials. Appl. Microbiol. Biotechnol. 98 (16), 7051e7060. Wang, S., Kim, M., 2005. Study on old newsprint deinking with cellulases and xylanase. In: 59th Appita Annual Conference and Exhibition. ISWFPC, Auckland. Wonganu, B., Pootanakit, K., Boonyapakron, K., Champreda, V., Tanapongpipat, S., Eurwilaichitr, L., 2008. Cloning, expression and characterization of a thermotolerant endoglucanase from Syncephalastrum racemosum (BCC18080) in Pichia pastoris. Protein Expr. Purif. 58, 78e86. Wu, H., Cheng, X., Zhu, Y., Zeng, W., Chen, G., Liang, Z., 2018. Purification and characterization of a cellulase-free, thermostable endo-xylanase from Streptomyces griseorubens LH-3 and its use in biobleaching on eucalyptus kraft pulp. J. Biosci. Bioeng. 125, 46e51.

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Yan, Q., Hua, C., Yang, S., Li, Y., Jiang, Z., 2012. High level expression of extracellular secretion of a b-glucosidase gene (PtBglu3) from Paecilomyces thermophila in Pichia pastoris. Protein Expr. Purif. 84, 64e72. Yang, G., Yang, D., Wang, X., Cao, W., 2021. A novel thermostable cellulase-producing Bacillus licheniformis A5 acts synergistically with Bacillus subtilis B2 to improve degradation of Chinese distillers’ grains. Bioresour. Technol. 325, 124729. Yang, J., Ma, T., Shang-guan, F., Han, Z., 2020. Improving the catalytic activity of thermostable xylanase from Thermotoga maritima via mutagenesis of non-catalytic residues at Glycone subsites. Enzyme Microb. Technol. 139, 109579. Yu, J., Liu, X., Guan, L., Jiang, Z., Yan, Q., Yang, S., 2021. Highelevel expression and enzymatic properties of a novel thermostable xylanase with high arabinoxylan degradation ability from Chaetomium sp. suitable for beer mashing. Int. J. Biol. Macromol. 168, 223e232.

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SUBCHAPTER 8.8

Xylanases 8.8.1 Microbial sources and properties Xylan is the most prevalent hemicellulose in plant cell walls and second most common polysaccharide in nature after cellulose. It is a main component of the plant cell wall. It has a backbone made up of b-1,4-xylose units that are frequently embellished with arabinofuranose side chains. Xylanase enzymes catalyze the hydrolysis of xylans (Bajpai, 2022; Lu et al., 2004). Microbial xylanases have several uses in the industry (Mamo et al., 2009; Dwivedi et al., 2010; Magnuson, 1996; Valls and Roncero, 2009; Bajpai, 2022; Nagar et al., 2010). Combined action of several main and side-chain-cleaving enzymes, including endo-b-1,4-xylanases, b-d-xylosidases, a-1-arabinofuranosidases, a-glucuronidases, and acetyl xylan esterases, are required for completely hydrolyzing the xylan. However, hydrolysis by endo-b-1,4-xylanases is considered as one of the major reactions in the process (Wang et al., 1997; Ahmed et al., 2009). This enzyme catalyzes the hydrolysis of the bonds between xylose subunits in the polymer of xylan and produces oligosaccharides, which get converted to xylose by the action of b-xylosidase enzyme (Dwivedi et al., 2010; Choi et al., 2000). With an estimated annual market of USD 500 million, thermophilic fungal and bacterial xylanases are widely accepted on the commercial market for use in food, feed, pulp, and paper industry and the conversion of lignocelluloses. The analysis of thermophilic fungi’s entire genome demonstrates the presence of complex genetic material that codes for multiple xylanases, mainly GH10 and GH11 and also GH7 and GH30 xylanases. Transcriptomics and proteome profiling have shown that these xylanases are expressed differently in some thermophilic fungi. The discovery of novel thermophilic xylanases through bioprospecting has received support from commercial companies for heterologous overexpression and formulations (Chadha et al., 2019). Because of their use in biorefineries, xylanases are anticipated to see an exponential rise in usage in the future. The mainstay for meeting the rising demand for thermostable xylanases is anticipated to be the discovery of novel and enhancement of existing xylanases using tools like directed molecular evolution. Thermotolerant/thermophilic fungi, yeast, and thermophilic/extremely thermophilic bacteria are just a few examples of the various microorganisms, which can produce xylanases. Several thermophilic/hyperthermophilic bacteria such as Rhodothermus marinus, Caldicoprobacter algeriensis, Bacillus sp., Sulfolobus solfataricus, Pyrodictium abyssi, and Thermococcus

Industrial applications of thermophilic/hyperthermophilic enzymes

zilligii have been studied in detail for producing extremely thermostable xylanase enzymes (Mhiri et al., 2020; Karlsson et al., 2004; Raj et al., 2018). Most of the xylanases obtained from these thermophilic bacteria were found to be the members of family 10 and family 11 of glycoside hydrolases (Ghosh et al., 2020). Geobacillus sp. Strain WSUCF1 has attracted awareness in the recent years as it produces xylanase having an excellent thermostability. It showed half-lives of 18 days at a temperature of 60
C and 12 days at a temperature of 70
C (Bhalla et al., 2015). In another study by Bhalla et al. (2013), a gene encoding GH10 endo-xylanase was isolated from Geobacillus sp. WSUCF1 and expressed in E. coli. This endo-xylanase was purified and examined on birchwood. It exhibited optimum activity at pH 6.5 and a temperature of 70
C. It was found to be extremely thermostable at 60
C and retained 50% activity. At a temperature of 50
C, it retained 82% of its original activity after 60 h. Likewise, a gene for xylanase xynBCA encoding a polypeptide of 439 residues (XynBCA) was obtained from Caldicoprobacter algeriensis and expressed heterologously in E. coli BL21 (DE3) by Mhiri et al. (2020). The purified thermostable xylanase showed exceptional thermostability. It showed optimal activity at a temperature of 80
C and pH 6.5. The half-life was 20 min at a temperature of 80
C. A xylanase from B. licheniformis showed optimal activity at a pH of 9.0 and temperature of 60
C (Raj et al., 2018). It retained 80% of its original activity when treatment was done for 1 h at a 60
C. Few thermostable xylanases have been obtained from thermoalkaliphiles, thermohalophiles, and thermoacidophiles. For instance, thermostable xylanase from thermoacidophilic Alicyclobacillus sp. was expressed heterologously in E. coli. It showed a broader pH range from 3.8 to 9.4. It was found to retain 90% of its original activity when incubated for 1 h at a temperature of 60
C (Bai et al., 2010). Thermostable xylanase from Thermoanaerobacterium saccharolyticum NTOU1, which is a halophilic bacterium, was found to be resistant, to higher salt concentration. When treated with 2M NaCl for 24 h, it retained 71% of its original activity. Furthermore, 50% of the original activity of this xylanase was maintained when treatment was done at a temperature of 65
C for 0.91 h (Hung et al., 2011). From Enterobacter sp. MTCC 5112, thermo-alkalophilic xylanase was cloned by (Khandeparkar and Bhosle, 2006). It was found to maintain 90% of its original activity when treatment was done at pH 9.0 and at a temperature of 80
C for 0.66 h. It retained 85% of its original activity at a temperature of 60
C and 64% of its original activity at a temperature of 70
C after 18 h. Different thermophilic fungi such as Corynascus thermophiles, Thielavia terrestris, and Rhizomucor pusillus have been found to be very good sources of thermostable xylanases (van den Brink et al., 2013; García-Huante et al., 2017; H€ uttner et al., 2018). Thielavia terrestris Co3Bag1 is found to produce an extremely thermostable xylanase having a molecular weight of 82 kDa. This enzyme retained maximum activity at a

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temperature of 85
C and pH 5.5. It showed a half-life of 23.1 days after incubation at temperature of 65
C (García-Huante et al., 2017). R. pusillus obtained from maize silage was found to produce 824 (U/g) of xylanase, which was very stable at temperature of 75
C (Robledo et al., 2016). H€ uttner et al. (2018) identified genes encoding putative xylan-degrading enzymes in the thermophilic fungus R. pusillus. Achaetomium sp. Xz-8, a thermophilic fungus, produced thermophilic xylanase (XynC01). It was overexpressed in P. pastoris. It showed optimum activity at a temperature of 75
C and pH 5.5 with very good stability over a broad pH range of pH 4.0 to 10.0 and at temperature of 55
C and lower. This enzyme showed very good tolerance to chemical reagents and metal. The performance improved (38.50%) when this enzyme was combined with commercial b-glucanase. These characteristics make it appropriate for industrial uses especially in breweries (Zhao et al., 2013). Thermostable xylanase (Xyn11A) from Corynascus thermophilus was expressed in P. pastoris. This enzyme showed optimum activity at a pH of 7.4 and temperature of 70
C. When treatment was done at a temperature of 50
C and 60
C for 60 min, Xyn11A retained more than 90% of its original activity. Xyn11A was found to show higher stability over a wider pH range of 2.0e11.0. This enzyme was not inhibited by the presence of metal ions, making it appropriate for industrial uses (Yang and Zhang, 2017). A gene encoding an extremely thermostable xylanase in Paecilomyces thermophile was expressed in P. pastoris (Fan et al., 2012). XynA showed optimum activity at a temperature of 75
C and was extremely stable when incubated at 80
C for 30 min. XynA produced xylobiose and xylotriose as its major products after hydrolyzing beechwood, birchwood xylan, and xylooligosaccharides. The fungus Thermoascus aurantiacus M-2 produced xylanase with molecular weight of about 31.0 kDa. It showed maximum activity at a temperature of 75
C and pH of 5.0. It was active over a broader range of pH 2.0 to 10.0 and showed very good stability over a wider temperature range of 30
Ce80
C for 120 min. The xylanase activity was improved by manganese ions to 120.0% and silver ions to 119.6% (Ping et al., 2018). Table 8.8.1 presents a summary of thermostable xylanases from thermophilic fungi.

8.8.2 Application of xylanases Xylanases are industrially active enzymes because of their exceptional capability to carry out hemicellulosic biomass degradation (of which xylan is the major one). Due to their hydrolytic action, xylanases make it possible to liberate sugars for the industrial production of numerous commercial goods. Xylanolytic enzymes are required for the production of commercially viable products. Hemicellulose is the second-most abundant renewable biomaterial on Earth after cellulose (Sudan and Bajaj, 2007; Bajaj et al., 2012). Due to

Table 8.8.1 Thermostable xylanases from various thermophilic microorganism.

Enzyme

Opt. pH

Caldicoprobacter algeriensis Streptomyces griseorubens LH-3 Chaetomium sp. CQ31

xynBCA

6.5

80

117 U/mg

20 min at 80
C

Endoxylanase Xylanase

5.0

60

6.5

85

767.2 U/ mg 2489 U/mg

Retained 60% of its activity at 50
C for 1 h It maintained over 90% of its relative activity at 60
C for half an hour

Thermotoga maritima, TmxB Thermotoga neapolitana

Endo-b-1,4xylanase hemicellulytic

5.0

100

ND

6.0

90

ND

Dictyoglomus turgidum

Endomannase

5.4

70

ND

Dictyoglomus turgidum

b-xylosidase

5.0

98

ND

Dictyoglomus turgidum Acinetobacter Johnsonii

b-xylosidase

5.0

75

6.79 U/mg

Xylanase

6.0

55

ND

Xylanase

5.0

75

ND

Thermoascus aurantiacus M-2

Specific activity

Thermostability/half-life

References

Mhiri et al. (2020) Wu et al. (2018) Yu et al. (2021)

It retained 50% of its relative activity when incubated for 2 h at 70
C ND retained over 90% of its relative activity within this range of temperature 80e95
C Retaining 88% activity at 65
C for 60 Retained 80% relative activity after 60 min at 65
C Highly stable for 2 h from 30 to 80
C temperature range

Yang et al. (2020b) Benedetti et al. (2019) Fusco et al. (2018a)

Tong et al. (2021)

Li et al. (2020b) Xue et al. (2019) Ping et al. (2018)

From Ajeje, S.B., Hu Y, Song G, Peter SB, Afful RG, Sun F, Asadollahi MA, Amiri H, Abdulkhani A and Sun H (2021) Thermostable cellulases/xylanases from thermophilic and hyperthermophilic microorganisms: current perspective. Front. Bioeng. Biotechnol. 9, 794304. Distributed under the terms of the Creative Commons Attribution License (CC BY).

Industrial applications of thermophilic/hyperthermophilic enzymes

Microorganism

Opt. temp (8C)

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Developments and Applications of Enzymes From Thermophilic Microorganisms

their extensive application potential in numerous industrial processes, xylanases have received a great deal of research attention (food, feed, paper, textile, pharmaceuticals, and chemical industries) (Bajpai, 1997, 1999, 2004, 2009, 2012, 2013a, 2018a,b,c,d, 2019; Basit et al., 2020; Beg et al., 2001; Butt et al., 2008; Dhiman et al., 2008a; Gangwar et al., 2014; Goswami and Rawat, 2015; Harris and Ramalingam, 2010; Juturu and Wu, 2012; Malhotra and Chapadgaonkar, 2018; Polizeli et al., 2005; Sharma and Kumar, 2013; Sharma and Sharma, 2017; Sharma, 2017). The use of xylanases began in the 1980s. They were initially used in animal feed and later in the food, paper, textile, pharmaceutical, and chemical industries. Polizeli et al. (2005) reported that 20% of the world’s total enzyme production is derived from biomass hydrolyzing enzymesdxylanases, cellulases, and pectinases. 8.8.2.1 Pulp and paper industry Xylanase has a wide range of alkaline stability and can withstand temperatures around 90
C and is the preferred choice for paper industry (Verma and Satyanarayana, 2012; Bajaj and Manhas, 2012). The use of xylanases in pulp bleaching has received considerable attention in recent years (Bajpai, 1997, 1999, 2004, 2009, 2012, 2013a; Bajpai, 2018a,b,c,d; Bajpai and Bajpai, 1996, 1997, 1998; Bajpai and Bajpai, 2001a; Bajpai et al., 1993, 1994, 1999; Gangwar et al., 2014; Khonzue et al., 2011; Manji, 2006; Ojanpera, 2004; Paice and Zhang, 2005; Shatalov and Pereira, 2008, 2009; Viikari et al., 1986, 1990, 1991, 1993, 1994, 2002, 2009). “Bleaching of chemical pulps with xylanases is the most broadly used biotechnical application in pulp and paper industry. Presently, a significant number of European, North American, South American, and Japanese mills are bleaching full time with xylanases. Xylanase use is more common in Canada than in the United States because of more stringent AOX levels. Most full-time applications focus on cost reductions using O2-ECF bleaching. Recent developments involve use of enzymes to eliminate the first chlorine dioxide stage and thereby help reduce water usage. The main enzyme that enhances the delignification of kraft pulp is found to be endo-b-xylanase but if xylanases are enriched with other hemicellulolytic enzymes then the effect of enzymatic treatment is improved. The results from laboratory studies and mill trials show about a 35%e41% reduction in active chlorine at the chlorination stage for hardwoods and 10%e20% for softwoods” (Bajpai, 2009; Atkinson et al., 1993; Saleem et al., 2009; Bajpai et al., 1993, 1994, 1999). In ECF bleaching, when chlorine dioxide production is a limiting factor, xylanases increase the productivity of the bleach plant. This is usually the case when elemental chlorine is not used in the process. In TCF bleaching, xylanases increase the final brightness of the pulp, which is a crucial factor in the marketing of chlorine-free pulps. Furthermore, the reduction in the cost in TCF bleaching is also important.

Industrial applications of thermophilic/hyperthermophilic enzymes

Use of xylanase pretreatment results in reduction in AOX in the effluent and also dioxin concentration because of reduced amount of chlorine needed for achieving desired brightness. There is no adverse effect on the strength properties when the pulps are treated with xylanases. In some cases, xylanase pulps show better strength properties. These pulps can be refined easily as compared to reference pulps. Xylanase treatment also improves the pulp viscosity. Xylanases when combined with cellulase enzymes are used for the deinking of waste paper. Enzymatic deinking method is found to be cost-effective on an industrial scale. Combination of cellulase and xylanase enzymes significantly influences the deinking efficiency of old newsprint. Treatment of chemical pulps with xylanase enzymes improves the beatability of pulps and treatment of thermomechanical pulps with xylanase enzymes reduces the energy requirement and improves the fiber properties. The pulp properties are comparable to those of the slightly beaten pulps (Bajpai, 2018d). Xylanases enzymes also remove shives effectively. Xylanases have been also used for purifying cellulose and processing of plant fiber sources like hemp and flax. 8.8.2.2 Food industry Xylanase enzymes are used in the food industry in the baking sector to enhance the stability of dough and crumb structure, leading to a more softer crumb, which is more uniform and the bread volume is bigger. A. niger produces a xylanase that is particularly good at increasing the specific volume of breads without having an undesirable impact on dough handling, as is the case with xylanases obtained from other fungal and bacterial sources. The water gets distributed from the pentosan phase to the gluten phase. This is the cause of xylanase’s impact on bread volume improvement. Better ovenspring is the end-result of the gluten’s increased extensibility due to the rise in gluten volume fraction. Additionally, xylanases have been discovered to delay staling, enhance the texture of high fiber bread, and balance out the variable quality of flour used in baking wheat bread. Additionally, they are helpful in the production of low-fat biscuits. Xylanases help fruits and vegetables produce more juice. The process of maceration, or chewing, is aided by xylanase. Additionally, the addition of xylanases can lessen the viscosity of juice and increase its filterability. Xylanases also help to separate starch from gluten in grains like wheat or other cereals. Xylanases are used to process spent barley into animal feed and to extract more fermentable sugar from barley for brewing beer. Xylanases can make the brewing liquid less viscous and thus improve its filterability. In order to speed up filtration and increase yield, xylanases can also be used to process soybean protein, starch, and other materials. Additionally, xylanase enhances the extraction of oil from plant materials rich in oil, like corn oil. By lowering the viscosity of the juice, xylanase enhances the juice’s filterability (Bajpai, 2009).

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8.8.2.3 Feed industry Nowadays, poultry and pig diets based on wheat, triticale, and rye are frequently supplemented with xylanase-based enzyme products all over the world (Bajpai, 2009). Xylanase enzymes enhance the nutritional value and digestion of various feed diets. In broiler diets, significant quantities of wheat, barley, sunflower and rye, must be added with enzymes. The addition of enzymes ensures that nutrients locked inside plant cells are utilized to their full potential while also lessening the viscosity created by nonstarch polysaccharides in the digestive tract of the bird. The addition of b-glucanases and xylanases to animal feed has been shown to change the milk fat content of ruminants and increase beef cattle productivity. Additionally, it was discovered that adding these enzymes to the total mixed feed ration, as well as to corn silage or alfalfa hay, improved animal health, particularly in young animals. Increased xylanase content in pig feedd600 g of the commercial enzyme per tonne of feed containing 20%e35% barley and 30%e35% wheatdimproved performance as measured by increases in live weight and feed conversion ratio. Due to the higher overall digestive efficiency of pigs compared to poultry, pig feed typically contains more by-products than poultry feed. By adding the enzyme, these by-products can be improved, and in some cases, they have more potentials than the raw materials from which they were produced. Since these substrates were not found to be degraded by the xylanase used, the presence of noncereal feed raw materials in the complex diet, such as sugar beet pulp, rapeseed, and sunflower meal, has been found to reduce the effect of enzyme as these substrates were not found to be degraded by the xylanase used. So for achieving greatest enzyme effect, attention should be given to the composition of the raw material of pig diets. Xylanase enzymes can also be used for improving the silage (Bajpai, 2009). Treatment of forages with combination of xylanase and cellulase enzymes results in superior-quality silage and enhances the digestion rate of plant cell wall by the ruminants. Substantial amount of sugars is sequestered in the xylan of plant biomass. Xylanases in addition to converting hemicelluloses into nutritive sugars that the ruminants can digest; these enzymes also produce compounds that might be a good source of nutrients for the ruminal microflora. Xylanase enzymes improve the degradability of plant waste material, for example, agricultural wastes. Thus the disposal of organic waste in landfill sites is reduced. There is much interest in the production of xylose, xylobiose, and xylooligomers. These sugars are produced by the hydrolysis of xylan by xylanolytic enzymes, while other sugar residues are added using the transglycosylation activity of enzymes such as b-xylosidase. These xylose-containing sugars are used for rheological properties and research. 8.8.2.4 Detergents Xylanases are also used in the detergent industry. These enzymes help to remove various stains on clothes (Juturu and Wu, 2012). The thermostable and alkaline xylanases are

Industrial applications of thermophilic/hyperthermophilic enzymes

typically appropriate for use in the detergent industry, as washing the clothes in warm water is mostly preferred for removing the stains. The stain removal potential of cellulase enzymes by chemically linking the clan 11 endo-xylanases to the cellulose binding domain of cellulases has been reported (Smets et al., 2002). Moid et al. (2021) studied xylanases as detergent additive for improving laundry application. The effectiveness of xylanases as a plant stain remover was found to be very effective in comparison to the commercial detergent. 8.8.2.5 Textiles Xylanases have been used for desizing and scouring. In the textile industry, the xylanolytic enzymes are used for processing of plant fibers. For this type of application, the xylanase enzymes must not contain any cellulolytic enzymes. The dried ramee stems were treated with xylanases for freeing the longer cellulose fibers intact. The use of the strong bleaching step is not required in this process, as the lignin does not get oxidized and the fibers do not get darkened (Dhiman et al., 2008a; Br€ uhlmann et al., 2000; Losonczi et al., 2005; Prade, 1995; Csiszar et al., 2001). Desizing of cotton and micropoly fabrics by the use of thermostable xylanases from Bacillus pumilus ASH was studied by Battan et al. (2012). Better desizing with micropoly fabric in comparison to cotton under similar conditions was observed. 8.8.2.6 Pharmaceuticals and chemicals Xylanase enzymes are sometimes combined with hemicellulases, proteases, and other enzymes as a dietary supplement for the treatment of poor digestion. There are very few medicinal products available with this formulation. Xylanase enzymes can be used with mannanases, ligninases, xylosidases, glucanases, glucosidases enzymes, etc. and can be used for producing xylitol from lignocellulose. Xylitol is a sweetener and a polyalcohol. Its sweetening power is comparable to that of sucrose (Kuhad and Singh, 1993; Paraj
o et al., 1998). Xylitol prevents tooth decay. It is suitable for diabetics and overweight people and is recommended for the prevention of osteoporosis, respiratory disease, dyslipidemia, renal, and parenteral lesions. Two steps are involved in the production of xylitol from xylose. In the first step, a chemically pretreated lignocellulose-containing aqueous phase is converted to xylose using immobilized xylanase enzymes and b-xylosidase at a temperature of 40
C for 15 h. In the second step xylose is hydrogenated to xylitol (Sinner et al., 1988). Xylitol has received a lot of attention due to its use in food and confectionery applications. Many commercial products containing xylitol are available, such as chewing gum (Saha and Bothast, 1997). Most of the xylitol produced is consumed in various foods such as soft drinks, ice creams, candies, chewing gums, etc. Due to its high negative heat of

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solution, it provides a pleasant cooling sensation. Xylitol recovery from the xylem fraction is approximately 50%e60% and 8%e15% of the raw material input (Winkelhausen and Kuzmanova, 1998). Degradation of xylan requires several enzymatic activities such as endo-1,4-b-xylanase, xylosidase and arabinosidase. The end-products are xylose, arabinose, and methylglucuronic acid containing oligosaccharides, xylooligosaccharides (XOs) (Sharma and Kumar, 2013). XO is a sugar oligomer that has demonstrated practical application potential in multiple fields. These include feed formulations, food and agricultural applications, and pharmaceuticals (Vazquez et al., 2000). Presently, XO is mainly produced by enzymatic hydrolysis because enzymes are highly specific. The by-product formation is very low and also the substrate loss is lower (Tan et al., 2008). At the University of Belgium, researchers have isolated family 8 cold-active xylanases. This enzyme is used in the production of xylo-oligosaccharides. 8.8.2.7 Biofuel industry Xylanase enzymes can be used together with other hydrolases to produce biofuels such as bioethanol from lignocellulose (Bajpai, 2012, 2013b; Sreenath and Jeffries, 2000; Olsson and Hahn-H€agerdal, 1996; Beg et al., 2001). Researchers at INL discovered Xtreme Xylanase, the most heat- and acid-stable xylanase enzyme from Alicyclobacillus acidocaldarius. The hemicellulose and cellulose components of biomass can be efficiently converted into energy-rich sugars. These sugars are building blocks used to replace petroleum to make fuels and valuable chemicals (www.inl.gov/research/xtreme-xylanase/d/xtremexylanase.pdf). By virtue of its incredible acid and heat stability, Xtreme Xylanase is expected to revolutionize biorefineries in many ways. 8.8.2.8 Other applications Xylanases play a very important role in inducing plant defense mechanisms (Bailey et al., 1995). Biosynthesis of ethylene and two other pathogen-associated proteins in tobacco was induced by xylanase purified from T. viride. Cellular elongation and fruit softening are also significantly influenced by xylanases (Kulkarni et al., 1999). Additionally, it plays a role in fruit softening and cell elongation in addition to performing crucial physiological tasks. Xylanase enzymes are also involved in protoplastation of plant cells, liquefaction of coffee mucilage for making liquid coffee, the maceration of vegetable matter, extraction of flavors, plant oils, pigments, and starch, and recovery of oil from underground mines (Beck and Scoot, 1974; Biely, 1985; McCleary, 1986).

Industrial applications of thermophilic/hyperthermophilic enzymes

Transgenic endoglucanases and xylanases increase the biomass conversion and digestibility of tobacco (Pappan et al., 2009). Few xylanases have been used to enhance the maceration of cell walls when making plant protoplast. Transgenic tobacco plants’ rhizosecretion contained the bacterial xylanase gene from Clostridium thermocellum that had been truncated (Borisjuk et al., 1999).

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Pappan, K.L., Corredor, D., Gerdes, B., Lee, D.A., Yelundur, S.A., Wu, X., Wang, D., 2009. Transgenic expression of endoglucanase and xylanase genes increases tobacco digestibility and biomass conversion. In: The 31st Symposium on Biotechnology for Fuels and Chemicals. Paraj
o, J.C., Domíngues, H., Domíngues, J.M., 1998. Biotechnological production of xylitol. Part 1, interest of xylitol and fundamentals of its biosynthesis. Bioresour. Technol. 65, 191e201. Ping, L., Wang, M., Yuan, X., Cui, F., Huang, D., Sun, W., Zou, B., Huo, S., Wang, H., 2018. Production and characterization of a novel acidophilic and thermostable xylanase from Thermoascus aurantiacu. Int. J. Biol. Macromol. 109, 1270e1279. Polizeli, M.L., Rizzatti, A.C., Monti, R., Terenzi, H.F., Jorge, J.A., Amorim, D.S., 2005. Xylanases from fungi: properties and industrial applications. Appl. Microbiol. Biotechnol. 67, 577e591. Prade, R.A., 1995. Xylanases, from biology to biotechnology. Biotechnol. Genet. Eng. Rev. 13, 101e131. Raj, A., Kumar, S., Singh, S.K., Prakash, J., 2018. Production and purification of xylanase from alkaliphilic Bacillus licheniformis and its pretreatment of eucalyptus kraft pulp. Biocatal. Agric. Biotechnol. 15, 199e209. Robledo, A., Aguilar, C.N., Belmares-Cerda, R.E., Flores-Gallegos, A.C., Contreras-Esquivel, J.C., Monta~ nez, J.C., Mussato, S.L., 2016. Production of thermostable xylanase by thermophilic fungal strains isolated from maize silage. Cytal. J. Food 14, 302e308. Saha, B., Bothast, R.J., 1997. Microbial production of xylitol. In: Saha, B.C., Woodward, J. (Eds.), Fuels and Chemicals from Biomass. American Chemical Society, Washington, DC, pp. 307e319. Saleem, M., Tabassum, M.R., Yasmin, R., Imran, M., 2009. Potential of xylanase from thermophilic Bacillus sp. XTR-10 in biobleaching of wood kraft pulp. Int. Biodeterior. Biodegr. 33 (8), 1119e1124. Sharma, M., Kumar, A., 2013. Xylanases: an overview. Br. Biotechnol. J. 3 (1), 1e28. Sharma, N., Sharma, N., 2017. Microbial xylanases and their industrial applications as well as future perspectives: a review. G.J.B.A.H.S. 6 (3), 5e12. Sharma, P.K., 2017. Xylanases current and future perspectives: a review. JNBR 6 (1), 12e22. Shatalov, A.A., Pereira, H., 2008. Effect of xylanases on peroxide bleachability of eucalypt (E. globulus) kraft pulp. Biochem. Eng. J. 40, 19e26. Shatalov, A.A., Pereira, H., 2009. Impact of hexenuronic acids on xylanase-aided bio-bleaching of chemical pulps. Bioresour. Technol. 100 (12), 3069e3075. Sinner, M., Dietrichs, H.H., Puls, J., Schweers, W., Brachthauser, K.H., 1988. US Patent No. 4,742,814. US Patent and Trademark Office, Washington, DC. Smets, J., Bettiol, J.L.P., Boyer, S.L., Busch, A., 2002. US Patent No. 6,468,955. Washington, DC. Sreenath, H.K., Jeffries, T.W., 2000. Production of ethanol from wood hydrolysate by yeasts. Bioresour Technol 72, 253e260. Sudan, R., Bajaj, B.K., 2007. Production and biochemical characterization of xylanase from an alkalitolerant novel species Aspergillus niveus RS2. World J. Microbiol. Biotechnol. 23, 491e500. Tan, S.S., Li, D.Y., Jiang, Z.Q., Zhu, Y.P., Shi, B., Li, L.T., 2008. Production of xylobiose from the autohydrolysis explosion liquor of corncob using Thermotogamaritima xylanase B (XynB) immobilized on nickel-chelated Eupergit C. Bioresour. Technol. 99, 200e204. Valls, C., Roncero, M.B., 2009. Using both xylanase and laccase enzymes for pulp bleaching. Bioresour. Technol. 100 (6), 2032e2039. van den Brink, J., van Muiswinkel, G.C.J., Theelen, B., Hinz, S.W.A., de Vries, R.P., 2013. Efficient plant biomass degradation by thermophilic fungus Myceliophthora heterothallica. Appl. Environ. Microbiol. 79, 1316e1324. Vazquez, M.J., Alonso, J.L., Dominguez, H., Parajo, J.C., 2000. Xylooligosaccharides: manufacture and applications. Trends Food Sci. Technol. 11, 387e393. Verma, D., Satyanarayana, T., 2012. Molecular approaches for ameliorating microbial xylanases. Bioresour. Technol. 117 (2012), 360e367.

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Viikari, L., Kantelinen, A., Poutanen, K., Ranua, M., 1990. Characterization of pulps treated with hemicellulolytic enzymes prior to bleaching. In: Kirk, T.K., Chang, H.M. (Eds.), Biotechnology in Pulp and Paper Manufacture. Butterworth-Heinemann, Boston, MA, p. 145. Viikari, L., Kantelinen, A., R€attö, M., Sundquist, J., 1991. Enzymes in pulp and paper processing. In: Leatham, G.F., Himmel, M.E. (Eds.), Enzymes in Biomass Conversion, ACS Symp Ser 460. American Chemical Society, Washington, pp. 426e436. Viikari, L., Kantelinen, A., Sundquist, J., Linko, M., 1994. Xylanases in bleaching: from an idea to industry. FEMS Microbial. Rev. 13, 335e350. Viikari, L., Poutanen, K., Tenkanen, M., Tolan, J.S., 2002. Hemicellulases. In: Flickinger, M.C., Drew, S.W. (Eds.), Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis, and Bioseparation. Wiley, Chichester, West Sussex (Update. Electronic release). Viikari, L., Ranua, M., Kantelinen, A., Sundquist, J., Linko, M., 1986. Bleaching with enzymes. In: Proceedings of the 3rd International Conference on Biotechnology in the Pulp and Paper Industry, Stockholm, Sweden, pp. 67e69. Viikari, L., Ranua, M., Kantelinen, A., Linko, M., Sundquist, J., 1987. Application of enzymes in bleaching. In: Proceedings of 4th International Symposium on Wood and Pulping Chemistry, vol. 1. Paris, p. 151. Viikari, L., Rato, M., Kantelinen, A., 1989. Finish Patent Appl 896291. Viikari, L., Tenkanen, M., Buchert, J., Ratto, M., Bailey, M., Siika-aho, M., Linko, M., 1993. Hemicellulases for industrial applications. In: Saddler, J.N. (Ed.), Bioconversion of Forest and Agricultural Plant Residues. CAB International, Wallingford, UK, pp. 131e182. Viikari, L., Suurna kki, A., Gronqvist, S., Raaska, L., Ragauskas, A., 2009. Forest products: biotechnology in pulp and paper processing. In: Schaechter, M. (Ed.), Encyclopedia of Microbiology, third ed. Academic Press, New York, NY, pp. 80e94. Viikari, L., Vehmaanper€a, J., Koivula, A., 2012. Lignocellulosic ethanol: from science to industry. Biomass Bioenergy 1e12. Wang, M.Q., Saricks, C., Wu, M., 1997. Fuel-Cycle Fossil Energy Use and Greenhouse Gas Emissions of Fuel Ethanol Produced from U.S. Midwest Corn. Prepared for Illinois Department of Commerce and Community Affairs, Center for Transportation Research, Argonne National Laboratory, Argonne, IL, USA. Winkelhausen, E., Kuzmanova, S., 1998. Microbial conversion of D-xylose to xylitol. J. Ferment. Bioeng. 86, 1e14. Yang, Z., Zhang, Z., 2017. Codon-optimized expression and characterization of a pH stable fungal xylanase in Pichia pastoris. Process. Biochem. 53, 80e87. Zhang, G.M., Huang, J., Huang, G.R., Ma, L.X., Zhang, X.E., 2007. Molecular cloning and heterologous expression of a new xylanase gene from Plectosphaerella cucumerina. Appl. Microbiol. Biotechnol. 74 (2), 339e346. Zhao, L., Meng, K., Shi, P., Bai, Y., Luo, H., Huang, H., Wang, Y., Yang, P., Yao, B., 2013. A novel thermophilic xylanase from Achaetomium sp. Xz-8 with high catalytic efficiency and application potentials in the brewing and other industries. Process Biochem. 48, 1879e1885.

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SUBCHAPTER 8.9

Pectin-degrading enzymes 8.9.1 Microbial sources and properties Pectins are the third major structural polysaccharides in plant cell walls. These are highmolecular-weight polysaccharides composed mainly of a-1 / 4-linked D-galacturonic acid residues with a few rhamnose residues in the backbone and xylose, galactose, and arabinose, in the side chains. Pectin is present in the middle lamellae and primary cell walls of the higher plants in the form of magnesium and calcium pectate. Pectin provides rigidity to plants. The amount of pectin material present in the plant depends on the source of plant materials (Favela-Torress et al., 2003; Rangarajan et al., 2010; Khatri et al., 2015; Sakai et al., 1993; Jayani et al., 2005). During plant development, pectinase activity changes the structure of these pectin substances. These enzymes promote cell wall expansion and softening of some tissues during maturation of plant parts such as fruits. Pectin is abundant in sugar beet pulp and fruit (Spagnuolo et al., 1999). In citrus fruits and apples, it generally accounts for up to half of the cell wall polymer content (Brummell, 2006). Composed of homogalacturonic acid regions (sometimes methylated) and both rhamnose and galacturonic acid regions, the pectin backbone contains galactose, arabinose, L-rhamnose, and xylose. It has a neutral sugar side chain (Kashyap et al., 2001). The backbone L-rhamnose residues have side chains consisting of galactose and arabinose. There is also a single xylogalacturonan side chain (Brummell, 2006; Turner et al., 2007). Pectinases, particularly polygalacturonases, play an important role in pectin degradation during the last stages of fruit ripening. Pectinases act as carbon recyclers in nature. These enzymes break down pectic substances into saturated and unsaturated galacturonans. This is further converted to 5-keto-4-deoxy-uronic acid and ultimately to pyruvate and 3-phosphoglyceraldehyde. Pectinases from plant pathogens like Fusarium oxysporum, Botrytis cinerea, and Aspergillus flavus play an important role in plant pathogenicity or virulence by degradation of the pectic compounds present in the cell (Gummadi et al., 2007). Pectinases are classified into polygalcuturonases (PG), pectinesterases (PE), and pectin lyases (PL) based on their mechanism of action on substrates (Jayani et al., 2005) (Table 8.9.1). Production of pectinase enzymes by microorganisms has been widely studied (Favela-Torres et al., 2003). Various microorganisms are found to produce multiple sets of pectin-degrading enzymes. Pectinase enzymes are found in fungi, yeast, bacteria, and some actinomycetes (Blanco et al., 1999; Bruhlman et al., 1994;

Industrial applications of thermophilic/hyperthermophilic enzymes

Table 8.9.1 Enzymes involved in the degradation of pectin.

Polygalacturonase It hydrolyzes the O-glycosyl bond of homogalacturonan to form monomeric units. Acts on 1,4-a-D-galactosyluronic acid linkages between galacturonic acid residues. Most polygalacturonases are endoenzymes that act randomly on bonds to depolymerize chains or cut down the length of polymers. The natural substrate for endo-polygalacturonase is homogalacturonan, but other compounds such as oligogalacturonides can also act as substrates, depending upon the nature of the substrate. A class of exopolygalacturonases that degrade polygalacturonates to digalacturonates and monogalacturonates are also known. Pectin esterase It catalyzes the hydrolysis of methylated carboxylic acid esters of pectin to produce pectic acid and methanol. The natural substrate for pectinesterase is pectin but other compounds such as methyl pectate and methylated oligogalacturonides also serve as substrates. Pectin esterase activity is induced by ammonium sulfate, magnesium, and sodium chloride and is inhibited by the presence of copper and mercury ions. Most of the pectin esterases are produced from plants. However, pectin esterases of bacterial and fungal origin have also been recently discovered. Most pectinases are specific for esterified pectins and may not show activity on pectates. Pectin lyases Randomly degrades the pectin material to produce unsaturated oligomethylgalacturonates in a 4:5 ratio. Cleavage of the glycosidic bond, preferably by a transelimination reaction to polygalacturonic acid. Requirement for calcium ions; inhibited by chelators such as EDTA. Exopectin lyases catalyze the cleavage of substrates from the nonreducing ends of polymers.

Hayashi et al., 1997; Huang and Mahoney, 1999; Kapoor et al., 2000; Patil and Dayanand, 2006; Takao et al., 2000). In fact, most of commercial microbial pectinases are mainly obtained from fungal sources of which Aspergillus species predominate. Bacteria are a main source of pectinases, producing a different set of enzymes, which help in degrading the pectin. Common bacteria degrading the pectins include Staphylococcus, Bacillus, and Pseudomonas. Most of the pectinolytic activity of bacteria is seen under aerobic conditions by aerobic organisms, but some activity can be observed under anaerobic conditions also. Some bacteria, such as B. badius, B. asahin, B. psychrosaccharolyticus, and Pseudomonas aeruginosa, use their pectinolytic activity for the pathogenesis of various diseases. Few common thermophilic bacteria such as Anoxybacillus sp, Geobacillus sp, and Bacteroides also show pectinase activity that helps recycle carbon compounds in the biosphere. The most common groups of fungi involved in pectin degradation are species

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belonging to the Ascomycota and Deuterium fungi. The basidiomycetes Phanerochaete chrsosporium has been extensively studied. It is a white rot fungus. This fungus degrades the most complex polysaccharides like cellulose, pectin, and chitin. Other fungi involved in pectin degradation include A. clavatus, A. nidulans, Botrytis fuckeliana, Giberella zeae, Podospora anserine, Rhizopus oryzae, Sclerotinia sclerotiorum, Trichoderma virens, and Magnaporthe oryzae. The types of enzymes produced and their functions differ depending on the type of bacteria.

8.9.2 Applications of pectin-degrading enzymes Pectinase is one of the most important industrial enzymes. Pectinases are widely used in the industrial sector, particularly in the food industry for extraction of fruit juices, fermentation of coffee and tea, extraction of oils, and enhancement of color and stability of red wine (Jayani et al., 2005). In addition to the food industry, pectinase enzymes are commonly used in the textile, paper and pulp industries, and wastewater treatment (Ahlawat et al., 2014; Solbak et al., 2005). In the last decade, this enzyme has been used with cellulosic enzymes to produce ethanol from lignocellulosic biomass (Li et al., 2014). 8.9.2.1 Food industry Acid pectinases are commonly used for the extraction, clarification, and removal of pectin in fruit juices, maceration of vegetables for making pastes and purees, and winemaking, and is mostly produced by fungi, particularly A. niger. Crushing the pectin-rich fruit leaves the highly viscous juice attached to the pulp in a jelly-like structure, impeding the juice extraction process by pressing. The addition of pectinase in the extraction process improves the juice yield through a simpler process, reduces juice viscosity, breaks down the gel structure, and improves juice thickening ability (Kashyap et al., 2001; Alkorta et al., 1998). In the case of fruit juice, extraction with enzymatic maceration increases the yield by more than 90% in comparison to traditional mechanical juicing and further improve organoleptic (taste, color) and nutritional (vitamins) properties and technical efficiency (simple filtration). In some processes, pectinolytic enzymes are used in combination with other cell walledegrading enzymes such as cellulases and hemicellulases (Bhat, 2000). A mixture of pectinase and cellulase enzymes has been found to increase the extraction yield of fruit juice by more than 100% (Alkorta et al., 1998; Kashyap et al., 2001). A three- to four-fold improvement in juice yield from pear, papaya, and banana by the use of enzymatic extraction instead of conventional pressing was reported by Soares et al. (2001). Enzymatic treatment can reduce the viscosity of apple juice by 62%. When depectinized apple juice is ultrafiltered, the flux of the permeate is much higher as compared to when nondepectinized juice is processed. The increased rate of permeation

Industrial applications of thermophilic/hyperthermophilic enzymes

is attributed to both the reduced viscosity of apple juice and the total pectin content. Pectin is a fibrous colloid and causes rigorous fouling of ultrafiltration membranes (Alvarez et al., 1998). Commercial pectinases used in food processing are usually a combination of polygalacturonase, pectin lyase, and pectin methylesterase. These preparations are generally obtained from fungi, mainly Aspergillus (Semenova et al., 2006; Gummadi and Panda, 2003). Pectinase also plays a role in stabilizing turbidity and making cloudy juices, especially of citrus fruits, purees, and nectars. Pure PL has proven to be the most effective for extracting cloudy juices (Kashyap et al., 2001). Pasteurization is used to extract the cloudy juice and inactivate the remaining enzymes. Larger residues are removed by centrifugation, leaving smaller particles in suspension. Pectin esterase occurs naturally in oranges and only acts on partially methylated pectin. The viscosity of orange juice can be reduced by treating 100 kg of orange pulp with 0.5e2.0 g of pectinase at room temperature (Rebeck, 1990; Kubra et al., 2018). Pectinase is used for fermenting coffee, cocoa, and tea. During tea fermentation, fungal pectinase enzymes break down the pectin present in the cell walls of tea leaves, but excessive amounts of these enzymes can damage the tea leaves, so a constant concentration should be maintained during fermentation. It also acts as an antifoaming agent that destroys the pectin in instant tea powder (Carr, 1986). During fermentation of coffee, pectinase removes the mucus layer from the coffee beans. The cocoa beans are sprayed with a pectinase preparation and fermented. Fermentation of cocoa is important to produce the flavor of chocolate. Cocoa fermentation involves many different microorganisms, including pectin-degrading microorganisms. These microorganisms are responsible for degrading the cocoa pulp by releasing pectinase, ultimately obtaining the highest quality cocoa beans with the finest cocoa taste (Kubra et al., 2018). The enzyme pectin esterase is used for preparing jams, jellies, sauces, and soups (Grassin and Fauquembergue, 1996). Pectinase is also used for hardening. Pectin methylesterase is used with calcium in the curing industry where softening takes place during fermentation and storage (Baker and Wicker, 1996). 8.9.2.2 Agriculture Pectinase enzymes and other cell walledegrading enzymes are used to extract oils from plants, but olive oil extraction is the most common. Commercial enzyme for extracting olive oil contains cellulase and hemicellulase enzymes. When olives are crushed, these enzymes are added to make it easier to extract the oil. Pectin interferes with the collection of oil droplets from the peel extract that aids in oil extraction, as is the case with lemons thus resisting the emulsification process (Scott, 1978). Vegetable oils from palm, sunflower, coconut, lemon, or canola can also be extracted using organic solvents such as hexane, preferably with an essentially alkaline pectinase to make easier the extraction

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of the oil in an aqueous process. Olivex is an enzyme supplement made from the fungus A. aculatus. This commercial preparation has desirable pectinolytic, hemicellulose, and cellulolytic activities for high oil yield and better stability during storage. The Olivex treatment increases the polyphenol and vitamin E content and also improves the organoleptic qualities of the oil. This has a decisive impact on oil consumption (Iconomou et al., 2010). Pectinase enzymes play a very important role in the degumming of fibrous plants. The fibers obtained from these crops contain gums, and ramie fiber is an excellent natural fiber, but it contains 20%e35% ramie gum, which mainly contains pectins and hemicelluloses, making it difficult for fiber processing. It should be removed for further processing. Degumming is done either by chemical or enzymatic treatment. Chemical processing is not very efficient. Enzymatic treatments are more efficient and pectinases in combination with xylanases can be used for degumming in an environmentally friendly and biodegradable manner (Jayani et al., 2005). The use of enzymes also reduces energy and chemical consumption. Among pectinases, actinomycete pectate lyase is the most effective in removing gums and separating bast fibers. Pectinase is used in roasting flax fibers. Roasting is a fermentation process in which microorganisms break down the pectins, releasing a pure quality fiber. The bacteria most commonly used in roasting are Clostridium and Bacillus, whereas the most common fungi are Aspergillus and Penicillium. Water roasting of linseed involves bacterial-derived pectinase enzymes, which separates the fibers (Chesson, 1978). Pectinases are used for biological cleaning of cotton fibers. Natural cotton fibers contain impurities of noncellulosic substances that can be removed using specific enzymes. This process is known as bio-scouring. Commercial formulations containing pectinase, lipase, amylase, cellulose, and hemicellulase are used to efficiently and safely remove noncellulosic contaminants from cotton. This process also has an ecofriendly effect due to the biodegradability of the enzymes. Pectinase is also used in animal feed production. It has the ability to reduce feed viscosity, directly enhancing the animal’s ability to absorb nutrients. These nutrients get released from the fibers by hydrolysis process, and it also reduces animal defecation (Hoondal et al., 2002). 8.9.2.3 Wine industry Many enzymes are involved in the wine making process. Commercial formulations contain pectinases, hemicellulases, glucanases, and glycosidases, and among these, pectinases play an important role in the winemaking. They increase methanol levels in wine due to PME activity (Servili et al., 1992). High levels of methanol can prove toxic, so methanol levels should be regulated. So, commercial mixtures contain pectinases with low pectin methylesterase activity. Pectinase is involved in wine extraction, increasing juice yield, accelerating filtration, and enhancing the taste and color of wine. Pectinases

Industrial applications of thermophilic/hyperthermophilic enzymes

have a mechanism of enhancing and stabilizing wine color by increasing secondary metabolites, phenolic compounds (Pinelo et al., 2006; Busse-Valverde et al., 2011). Pectinase treatment of Australian wines involves a range of commercial preparations of pectin-degrading enzymes that result in faster and better wine color at various stages such as maceration, pressing, and fermentation. This treatment also results in improved clarification of wine (Van Rensburg and Pretorius, 2000). Pectinase enhances the aroma and flavor of wine. Various aromatic components exist in free form in grapes, and when these components are in a combined form or in high concentration, they emit fragrance. 8.9.2.4 Paper and pulp industry In papermaking, the addition of pectinase enzymes can reduce the need for cations in peroxide brightening mechanical pulp furnishes. Alkaline peroxide dissolves pectin, which accounts for a considerable portion of the cationic demand. Demand decreases when treatment is done with pectinases. This improves the effectiveness of some cationic polymers and improves retention of microparticles and filler particles (Thornton et al., 1992). Alkaline peroxide dissolves pectin, which accounts for a significant portion of the cationic demand. Demand decreases when hydrolyzed by pectinase. Experiments using a dynamic dehydration vessel have shown that treatment with pectinases improves the effectiveness of a number of cationic polymers and increases retention of microparticles and filler particles. In the absence of retention aids or with nonionic polymers, retention is not increased, and the strength properties of the pulp are unaffected. Pectinases can be easily incorporated into paper pulping systems to reduce the amount of cationic retention aid addition required for furnishes containing peroxide-bleached mechanical pulps. Work by Paprican researchers using pectinase from Novozyme led to the application of this technology in North America. Saving in retention aid and white water drainage was achieved (Reid and Ricard, 2000). Application of pectinase can be effective in reducing anionic waste levels in mills using peroxide bleached pulp (Thornton et al., 1996; Thornton, 1994). Pectinases were found to reduce the cationic demand of the dissolved fraction of peroxide-bleached thermomechanical pulp by up to 60% and improved the efficacy of cationic retention aids (Ricard et al., 2005a,b; Reid and Ricard, 2000, 2002; Thornton, 1994; Thornton et al., 1996). 8.9.2.5 Waste water treatment Pectinases are used to treat wastewater from various industries (Tanabe et al., 1987). Various methods have been investigated to treat wastewater from the citrus processing industry, including physical dewatering, spray irrigation, chemical coagulation, direct activated sludge treatment, chemical hydrolysis followed by methane fermentation. These processes are less efficient because of the chemical resistance of pectic substances,

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high processing costs, long processing times, and process-first complexity (Shet et al., 2018). It releases waste water as a by-product. These waste products contain pectin substances. Pretreatment of these wastewaters with pectinases facilitate the removal of pectic substances and makes them suitable for digestion by activated sludge treatment process (Praveen and Suneetha, 2014; Haile and Ayele, 2022). 8.9.2.6 Prebiotics/functional foods Pectin and pectin-derived oligosaccharides (PDOs) have emerged as good candidates for a new generation of prebiotics. Gut bacteria have been observed to ferment methylated pectin to produce short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate with health benefits (Saharan and Sharma, 2018). Pectinases are used not only to produce functional food ingredients and dietary supplements, but also to enhance the antioxidant potential of the food. Fig. 8.9.1 shows various applications of pectinases (Haile and Ayele, 2022).

Figure 8.9.1 Various applications of pectinases. (From Haile, S., Ayele, A., 2022. Pectinase from microorganisms and its industrial applications. Sci. World J. 2022, 1881305. Distributed under the Creative commons Attribution License.)

Industrial applications of thermophilic/hyperthermophilic enzymes

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Kubra, K.T., Ali, S., Walait, M., Sundus, H., 2018. Potential applications of pectinases in food. Agric. Environ. Sect. 6 (2), 23e34. Li, J., Zhou, P., Liu, H., Lin, J., Gong, Y., Xiao, W., Liu, Z., 2014. Monosaccharides and ethanol production from superfine ground sugarcane bagasse using enzyme cocktail. Bioresources 9 (2), 2529e2540. Patil, R., Dayanand, A., 2006. Exploration of regional agrowastes for the production of pectinase by Aspergillus niger. Food Technol. Biotechnol. 44, 289e292. Pinelo, M., Arnous, A., Meyer, A.S., 2006. Upgrading of grape skins: significance of plant cell-wall structural components and extraction techniques for phenol release. Tren. Food Sci. Tech. 17, 579e590. Praveen, K.G., Suneetha, V., 2014. A cocktail enzyme-pectinase from fruit industrial dump sites: a review. Res. J. Pharmaceut., Biol. Chem. Sci. 5 (2), 1252e1258. Rangarajan, V., Rajasekharan, M., Ravichandran, R., Sriganesh, K., Vaitheeswaran, V., 2010. Pectinase production from orange peel extract and dried orange peel solid as substrate using Aspergillus niger. Int. J. Biotechnol. Biochem. 6 (3), 445e453. Ravindran, R., Sharma, S., Jaiswal, A., 2016. Enzymes in processing of functional foods ingredients and nutraceuticals. In: Martirosyan, D.M. (Ed.), Functional Foods for Chronic Diseases, vol. 1. D&A Inc., Dallas, TX, USA, pp. 360e385. Rebeck, H., 1990. Processing of citrus juices. In: Hick, D. (Ed.), Production and Packaging of Noncarbohydrate Fruit Juices and Fruit Beverages. Van Nosrand Reinhold, New York. Reid, I., Ricard, M., 2002. Effectiveness of Low Pectinase Doses to Increase Fines and Filler Retention in Peroxide Bleached Mechanical Pulp. Paprican PRR 1599, Montreal. Reid, I.D., Ricard, M., 2000. Pectinase in papermaking: solving retention problems in mechanical pulps bleached with hydrogen peroxide. Enzyme Microb. Technol. 26, 115e123. Ricard, M., Orccotoma, J.A., Ling, J., Watson, R., 2005a. Pectinase reduces cationic chemical costs in peroxide-bleached mechanical grades. In: 91st Annual Meeting of the Pulp and Paper Technical Association of Canada, Feb 8-10 2005, Canada, pp. 1115e1120. Ricard, M., Reid, I., Orccotoma, J.A., 2005b. Pectinase reduces the cationic demand of peroxide-bleached TMP: a paper machine trial. Pulp. Pap. Can. 106 (12), T258eT263. Rombouts, F.M., Pilnik, W., 1980. Pectic enzymes. In: Rose, A.H. (Ed.), Microbial Enzymes and Bioconversions, 5. Academic Press, London, pp. 227e272. Saharan, R., Sharma, K.P., 2018. Industrial applications of thermophilic pectinase: a review. Int. J. Curr. Res. 10, 70762e70770. Sakai, T., Sakamoto, T., Hallaert, J., Vandamme, J., 1993. Pectin, pectinase and protopectinase: production, properties and applications. Adv. Appl. Microbiol. 39, 213e294. Scott, D., 1978. Enzymes, industrial. In: Grayson, M., Ekarth, D., Othmer, K. (Eds.), Encyclopedia of Chemical Technology. Wiley, New York, p. 173. Semenova, M., Sinitsyna, O., Morozova, V., et al., 2006. Use of a preparation from fungal pectin lyase in the food industry. Appl. Biochem. Microbiol. 42, 598e602. Servili, M., Begliomini, A.L., Montedoro, G., Petruccioli, M., Federic, F., 1992. Utilisation of a yeast pectinase in olive oil extraction and red wine making processes. J. Sci. Food Agric. 58, 253e260. Shet, A.R., Desai, S.V., Achappa, S., 2018. Pectinolytic enzymes: classification, production, purification and applications. RJLBPCS 4, 337. Singh, S.A., Ramakrishna, M., Rao, A.G.A., 1999. Optimization of downstream processing parameters for the recovery of pectinase from the fermented broth of Aspergillus carbonarious. Process Biochem. 35, 411e417. Soares, M.M.C.N., da Silva, R., Carmona, E.C., Gomes, E., 2001. Pectinolytic enzyme production by Bacillus species and their potential application on juice extraction. J. Microbiol. Biotechnol. 17, 79e82. Solbak, A.I., Richardson, T.H., McCann, R.T., Kline, K.A., Bartnek, F., Tomlinson, G., Tan, X., ParraGessert, L., Frey, G.J., Podar, M., Luginbuhl, P., Gray, K.A., Mathur, E.J., Robertson, D.E., Burk, M.J., Hazlewood, G.P., Short, J.M., Kerovuo, J., 2005. Discovery of pectin degrading enzymes and directed evolution of a novel pectatelyase for processing cotton fabric. J. Biol. Chem. 280, 9431e9438.

Industrial applications of thermophilic/hyperthermophilic enzymes

Spagnuolo, M., Crecchio, C., Pizzigallo, M.D.R., Ruggiero, P., 1999. Fractionation of sugar beet pulp into pectin, cellulose, and arabinose by arabinases combined with ultrafiltration. Biotechnol. Bioeng. 64, 685e691. Takao, M., Nakaniwa, T., Yoshikawa, K., Terashita, T., Sakai, T., 2000. Purification and characterization of thermostable pectate lyase with protopectinase activity from thermophilic Bacillus sp. TS 47. Biosci. Biotechnol. Biochem. 64, 2360e2367. Tanabe, H., Yoshihara, K., Tamura, K., Kobayashi, Y., Akamatsu, I., et al., 1987. Pretreatment of pectic wastewater from orange canning process by an alkalophilic Bacillus sp. J. Ferment. Technol. 65 (2), 243e246. Thornton, J.W., 1994. Enzymatic degradation of polygalacturonic acids released from mechanical pulp during peroxide bleaching. Tappi J. 77 (3), 161e167. Thornton, J.W., Eckerman, C.S., Ekman, R.O., Holmbom, B.R., 1996. Treatment of alkaline bleached mechanical pulp with pectinase. U.S. Patent 5,487,812. Thornton, J.W.C., Eckerman, R., Ekman, R., Holmbom, B., 1992. Treatment of alkaline treated pulp for use in papermaking. Eur Pat Appl. #92304028.1. Turner, P., Mamo, G., Karlsson, E.N.(, 2007. Potential and utilization of thermophiles and thermostable enzymes in biorefining. Microb. Cell Fact. 6, 9. Van Rensburg, P., Pretorius, I.S., 2000. Enzymes in winemaking: harnessing natural catalysts for efficient biotransformation: a review. S. Afr. J. Enol. Vitic. 21, 52e73.

Relevant websites https://microbenotes.com/microbial-degradation-of-pectin/?. https://www.marketwatch.com/press-release/pectinase-market-in-2022-demand-outlook-top-keyplayers-analysis-current-trends-development-status-cagr-value-industry-share-and-forecast-till-20282022-08-. https://dataintelo.com/report/pectinase-market/.

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SUBCHAPTER 8.10

Chitinases 8.10.1 Microbial sources and properties Chitin is the second most abundant polysaccharide in nature after cellulose (Lee et al., 2000). This linear polymer is hydrolyzed by acids, bases, or enzymes, for instance, lysozyme, some chitinases and glucanases. Chitinases (EC 3.2.1.14) catalyze the conversion of chitin to monomeric or oligomeric components (low-molecular-weight products) and are found in a variety of organisms including plants, insects, crustaceans, bacteria, and fungi (Kuzu et al., 2012; Dahiya et al., 2006; Matsumoto et al., 2006; Kasprzewska, 2003; Bhattacharya et al., 2007; Merzendorfer and Zimoch, 2003; Boot et al., 1995). Bacteria, insects, and plants have large families of chitinases, which perform different functions, including digestion, cuticle turnover, defense against pathogens, and cell differentiation. Due to their function, these enzymes act as biological control agents against pathogens and persistent pests in comparison to traditional fungicides and insecticides. These enzymes have other important roles in the disposal of crustacean waste, production of single-cell protein, and generation of fungal proptoplasts. In the recent years, among chitinases, thermophilic chitinases and thermostable chitinases are attracting attention because of their ability to tolerate higher temperature and maintain enzyme stability for long periods of time. All the chitinases are not found to be thermostable. Therefore, engineered thermophilic chitinases have been designed to improve thermostability through genetic engineering with mutagenesis, direct evolution and proteomics approaches (Mathew et al., 2021). Chitinase is a glycosyl hydrolase characterized by hydrolysis of the b-1,4-linkage of N-acetylglucosamine in chitin chains varying in size from 20 kDa to about 90 kDa (Bhattacharya et al. al., 2007). Chitin is tightly bound to lipids, proteins, and minerals such as calcium carbonate in its natural state. Therefore, chitin production involves deproteinization and desalting of chitin waste by strong acids or strong bases. These processes are associated with higher costs, lower yields, and corrosion problems that make the oligomers costly. So, chitinases are important component in use of chitinous waste and solving the environmental issues as these are decomposable and low-priced (Rathore and Gupta, 2015). The classification of chitinases is based on the mode of action as recommended by the International Union of Biochemistry and Molecular Biology (1992) and CAZy (http:// www.cazy.org/). They are classified as follows (Karthik et al., 2017; Matsumoto et al., 2006; Chen et al., 2010): - Endochitinases (EC 3.2.1.14)

Industrial applications of thermophilic/hyperthermophilic enzymes

Randomly hydrolyze the chitin polymer to produce soluble low molecular-weight polymers - Exochitinases Divided into two categories: Chitobiosidase (beta-D-acetylglucosaminidase, EC 3.2.1.30; now included in EC 3.2.1.52). Catalyze the progressive release of diacetylchitobiose from the nonreducing end of the chitin. Beta-N-acetylglucosaminidase (EC 3.2.1.52). Sequentially remove the NAG units from the nonreducing end of the products produced by endochitinases and chitobiosidases Based on the similarity of amino acid sequence, chitinases can be grouped into glycosyl hydrolase families (GH) 18, 19, and 20. These are structurally not related (Henrissat and Davies, 1997). Figure 8.10.1 shows the action of chitinase enzyme.

Figure 8.10.1 Action of chitinase enzyme. (From Karthik, N., Binod, P., Pandey, A., 2017. Chitinases. In: Current Developments in Biotechnology and Bioengineering, Elsevier, Amsterdam, pp. 335e368. Reproduced with permission.)

Chitinases are widely distributed in bacteria. ActinomycetesdS. albocinaceus S-22, S. griseus MG3, S. lydicus WYEC108, S. griseus HUT 6037, S. violaceus Niger XL-2, Bacillus, Paenibacillus sp. Arthrobacter etc.dare also found to have good chitinase activity (Chernin et al., 1995; Divatar et al., 2016; Jahromi and Barzkar, 2018; Kusaoke et al., 2017; Le and Yang, 2018; Pan et al., 2019; Wu et al., 2001; Bhattacharya et al., 2007; Susuki et al., 2002; Watanabe et al., 1997). These actinomycetes and bacterial species originate from a variety of natural habitats of aquatic and terrestrial organisms, including seawater, soil, sediment, hot springs, and crustacean excreta (Le and Yang, 2019). Chitinases from these culturable chitinolytic microbes have been isolated using a variety of solid media containing chitin substrates (Howard et al., 2003).

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Table 8.10.1 presents the list of chitinase-producing bacteria. Table 8.10.1 Chitinase-producing bacteria.

Streptomyces violaceusniger Streptomyces venezuelae Streptomyces sporovirgulis Streptomyces sp. Streptomyces rimosus Streptomyces griseus Stenotrophomonas maltophilia Sphingomonas sp. Serratia sp. Serratia marcescens Sanguibacter Vibrio sp. Rhodothermus marinus Pseudomonas sp. Paenibacillus thermoaerophilus Paenibacillus sp. Paenibacillus pasadenensis Micrococcus sp. Microbiospora sp. Massilia timonae Laceyella putida Halobacterium salinarum Glaciozyma antarctica Enterobacter sp. Citrobacter freundii Chromobacterium violaceum Alcaligenes faecalis Aeromonas sp. Aeromonas hydrophila Bacillus subtilis Bacillus sp. Bacillus licheniformis Bacillus cereus Based on Karthik, N., Binod, P., Pandey, A., 2017. Chitinases. In: Current Developments in Biotechnology and Bioengineering, Elsevier, Amsterdam, pp. 335e368.

Fungal chitinases are known on the basis of their similarity to the GH18 family from plants and bacteria. There are various species of chitinase-producing fungi associated with pathogens and plants like mycorrhizae such as Aspergillus, Beauveria, Candida albicans,

Industrial applications of thermophilic/hyperthermophilic enzymes

Conidiobolus. Kluyveromyces lactis, Lycoperdon, Metharhizium, Mucor, Neurospora, Penicillium, Saccharomyces cerevisiae, Stachybotrys, and Trichoderma, have also been confirmed to have chitinase genes. All the chitinases from fungi belong to the GH18 family, except the Nosema bombycis chitinase, which belongs to the GH19 family. Based on the amino acid sequences of the GH18 modules, these are further classified into three subgroups, namely A, B, and C, which differ in the construction of their substrate-binding cleft, hence, enzymatic activities, and carbohydrate binding modules, which allow them to get tightly bound to the insoluble substrates (Bougoure and Cairney, 2006; Duo Chuang, 2006; Duo-Chuan, 2006; Eijsink et al., 2008; Gruber and Seidl-Seiboth, 2012; Hamid et al., 2013; Han et al., 2016; Islam and Datta, 2015; Karthik et al., 2014; Li et al., 2009; Lopez-Mondejar et al., 2012; Sharma and Shanmugam, 2012; Takaya et al., 1998). Table 8.10.2 presents the list of chitinase-producing fungi.

Table 8.10.2 Chitinase-producing fungi.

Penicillium sp. Penicillium ochrochloron Penicillium aculeatum Paecilomyces thermophila Thermomyces lanuginosus Thermoascus aurantiacus Talaromyces flavus Thermomyces lanuginosus Rhizopus oryzae Orpinomyces sp. Monascus purpureus Gliocladium catenulatum Chaetomium thermophilum Aspergillus niger Aspergillus fumigatus Anaeromyces sp. Based on Karthik, N., Binod, P., Pandey, A., 2017. Chitinases. In: Current Developments in Biotechnology and Bioengineering, Elsevier, Amsterdam, pp. 335e368.

8.10.2 Applications of chitinases Chitinases are of interest for several applications in biotechnology because these enzymes can degrade chitin in fungal cell walls and insect exoskeletons (Karthik et al., 2017). This leads to its use as an antibacterial or insecticide used in the biological control of plant pathogens. Chitinases are also used in the bioconversion of chitin to pharmacologically active products, specifically N-acetyl glucosamine and chitooligosaccharides. These are

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useful as antibacterial agents, immune enhancers, activation of host defense systems, antioxidants, hemostasis and wound healing, drug carriers, regulation of blood cholesterol, and food preservation (Synowiecki and Al-Khateeb, 2003; Chen et al., 2010; Khoushab and Yamabhai, 2010). Other application is the production of protoplasts from filamentous fungi (Dahiya et al., 2006). Various applications of chitinases are shown in Fig. 8.10.2.

Figure 8.10.2 Various applications of chitinase. (From Rathore, A.S., Gupta, R.D., 2015. Chitinases from bacteria to human: properties, applications, and future perspectives. Enzyme Res. 2015, Article ID 791907, 8 pages. Distributed under the Creative Commons Attribution License (https://creativecommons.org/ licenses/by/4.0/).)

8.10.2.1 Waste management Recombinant chitinases are used to transform chitin biomass. Chitinous wastes from marine organisms are transformed into simpler beneficial depolymerizing components, thereby reducing water pollution (Rathore and Gupta, 2015). Chito-oligomers obtained by the action of chitinases have applications in biochemical, food, and several chemical industries. Chitinases can also be used to convert chitin waste into biofertilizers (Sakai et al., 1998). Another approach to effectively use chitin waste is the production of single-cell proteins (Revah-Moiseev and Carroad, 1981). In this case, chitinase-

Industrial applications of thermophilic/hyperthermophilic enzymes

degraded chitin waste is used as a carbon source or food source for biomass production. Chitinase-producing yeast and bacteria (Pichia kudriavzevii and S. marcescens) can be used in aquaculture for producing single-cell proteins. 8.10.2.2 Biocontrol agents The adverse effects of chemical pesticides, along with developing tolerant strains of pathogenic microorganisms, today focus attention on biological control methods. Microorganisms such as biological control agents have attracted attention because of their lower cost and ability to increase plant survival. Chitinase provides defense against a wide range of fungal, viral, and entomopathogens in plants. Therefore, it can be effectively utilized as a bioinsecticide and biopesticide. The antifungal properties of chitinases are based on their hydrolytic effects on chitin. Chitinases are one of the pathogenassociated proteins secreted by plants. Postharvest diseases can also be combated with the help of chitinase enzymes because of the strong antifungal effects of certain microbes (Beygmoradi et al., 2018; Bhattacharya et al., 2007; Castillo et al., 2016; Chalutz and Droby, 1998; Hayes et al., 2008; Hoster et al., 2005; Kurita, 2006; Le and Yang, 2018; Revathi et al., 2012; Sharma et al., 2009; Shekhar et al., 2006; Wang et al., 2002). Preservation of food can also be done with chitinases, which damage cell walls, prevent spore germination, and reduce deterioration of food. Genetic engineering techniques have been used to make plants tolerant to pathogens such as production of engineered strains of extracellular chitinases as biocontrol agents (Baek et al., 1999; Mathivanan et al., 1998). Transgenic plants such as potato and tobacco have been made tolerant to foliar pathogens such as Alternaria alternata, A. solani and Botryis cinerea by overexpressing the chitinase Chit 42 from T. harzianum (Howell, 2003). Trichoderma employs a variety of defense mechanisms against pathogens, including colonization of soil and plant parts, evasion of pathogen growth, production of enzymes and antibiotics, that degrade pathogen cell walls (Saba et al., 2012). A chitinase from Bacillus cereus has biocontrol potential against Botrytis leaf blight in lilies (Stojkov et al., 2015; Huang et al., 2005). Salinivibrio chitinase shows antifungal activity against Rhizoctonia solani and Fusarium oxysporum (Le and Yang, 2018). 8.10.2.3 Pharmaceutical and medical uses Chitinases are used as antifungal agents along with antifungal drugs in the treatment of several fungal infections (Oranusi and Trinci, 1985). Human AMCase has been found to enhance Th2 inflammation and is involved in asthma and allergic reactions and in the IL-13 effector pathway (Zhu et al., 2004). The use of chitinases to detect persistent fungal infections in humans has also been proposed (Vega and Kalkum, 2012). Chitooligosaccharides also have huge pharmaceutical potential for use in human medicine due to their antitumor activity (displayed by chitohexaose and chitoheptaose), antihypertensive activity, and wound-healing properties (Park et al., 2000). It has also been reported

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that N-acetylglucosamine, the monomeric unit of chitin polymers, is an antiinflammatory agent (Aloise et al., 1996). Plasmodium falciparum has also been found to produce a chitinase enzyme during the spore cycle. Chitinase is produced by the pathogen in the mid-gut of the Anopheles vector and disrupts the peritoneal trophoblast, allowing the parasite to enter the salivary glands (Giansanti et al., 2007). Chitinase inhibition can be considered a good target because it can stop the sporulation cycle. Plasmodium falciparum forms a membranous sac around ingested blood meal that is chitin containing peritrophic matrix, and thereby adding exogenous chitinases to blood meal avoids the development of peritrophic matrix (Li et al., 2005). 8.10.2.4 Other applications Chitinase has been used to isolate fungal protoplasts and as a laboratory tool for studying cell wall synthesis, enzyme synthesis and secretion, and strain improvement for biotechnology applications (Dahiya et al., 2005). Chitinase levels can also be used to indirectly determine the fungal biomass present in the soil. Tannase, an enzyme used in the food industry, is produced by A. niger, but tannase binds to its cell wall, resulting in lower yields. Chitinases are used to degrade fungal cell walls, releasing tannases from the cell walls and increasing yields (Barthomeuf et al., 1994).

Bibliography Aloise, P.A., Lumme, M., Haynes, C.A., 1996. N-Acetyl-D-glucosamine production from chitin-waste using chitinases from Serratia marcescens. In: Muzzarelli, R.A.A. (Ed.), Chitin Enzymology. European Chitin Society, Grottammare, Italy, pp. 581e594. Baek, J.M., Howell, C.R., Kenerley, C.M., 1999. The role of an extracellular chitinase from Trichoderma virens Gv29-8 in the biocontrol of Rhizoctonia solani. Curr. Genet. 35 (1), 41e50. Barthomeuf, C., Regerat, F., Pourrat, H., 1994. Improvement in tannase recovery using enzymatic disruption of mycelium in combination with reverse micellar enzyme extraction. Biotechnol. Tech. 8 (2), 137e142. Beygmoradi, A., Homaei, A., Hemmati, R., Santos-Moriano, P., Hormigo, D., Fern_andez-Lucas, J., 2018. Marine chitinolytic enzymes, a biotechnological treasure hidden in the ocean? Appl. Microbiol. Biotechnol. 102 (23), 9937e9948. Bhattacharya, D., Nagpure, A., Gupta, R.K., 2007. Bacterial chitinases: properties and potential. Crit. Rev. Biotechnol. 27 (1), 21e28. Boot, R.G., Renkema, G.H., Strijland, A., van Zonneveld, A.J., Aerts, J.M., 1995. Cloning of a cDNA encoding chitotriosidase, a human chitinase produced by macrophages. J. Biol. Chem. 270 (44), 26252e6. Bougoure, D.S., Cairney, J.W., 2006. Chitinolytic activities of ericoid mycorrhizal and other root-associated fungi from Epacris pulchella (Ericaceae). Mycol. Res. 110 (Pt 3), 328e334. Castillo, B.M., Dunn, M.F., Navarro, K.G., Mel_endez, F.H., Ortiz, M.H., Guevara, S.E., Palacios, G.H., 2016. Antifungal performance of extracellular chitinases and culture supernatants of Streptomyces galilaeus CFFSUR-B12 against Mycosphaerella fijiensis Morelet. World J. Microbiol. Biotechnol. 32 (3), 44. Chalutz, E., Droby, S., 1998. Biological control of postharvest disease. In: Plantemicrobe Interactions and Biological Control. Marcel Dekker, Inc., New York, pp. 157e170.

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Chen, J.K., Shen, C.R., Liu, C.L., 2010. N-acetylglucosamine: production and applications. Mar. Drugs 8 (9), 2493e516. Chernin, L., Ismailov, Z., Haran, S., Chet, I., 1995. Chitinolytic Enterobacter agglomerans antagonistic to fungal plant pathogens. Appl. Environ. Microbiol. 61 (5), 1720e1726. Dahiya, N., Tewari, R., Hoondal, G.S., 2006. Biotechnological aspects of chitinolytic enzymes: a review. Appl. Microbiol. Biotechnol. 71 (6), 773e82. Dahiya, N., Tewari, R., Tiwari, R.P., Hoondal, G.S., 2005. Chitinase production in solid-state fermentation by Enterobacter sp. NRG4 using statistical experimental design. Curr. Microbiol. 51 (4), 222e228. Deeba, F., Abdullah, S.H., Irfan, M., Iqbal, Q.J., 2016. Chitinase production in organisms: a review. Punjab Univ. J. Zoo 31 (1), 101e106. Divatar, M., Ahmed, S., Lingappa, K., 2016. Isolation and screening of soil microbes for extracellular chitinase activity. J. Adv. Sci. Res. 7 (2). Duo-Chuan, L., 2006. Review of fungal chitinases. Mycopathologia 161 (6), 345e360. Eijsink, V.G., Vaaje-Kolstad, G., Varum, K.M., Horn, S.J., 2008. Towards new enzymes for biofuels: lessons from chitinase research. Trends Biotechnol 26 (5), 228e235. https://doi.org/10.1016/j.pep/ j.tibtech.2008.02.004. Giansanti, A., Bocchieri, M., Rosato, V., Musumeci, S., 2007. A fine functional homology between chitinases from host and parasite is relevant for malaria transmissibility. Parasitol. Res. 101 (3), 639e645. Gruber, S., Seidl-Seiboth, V., 2012. Self versus non-self: fungal cell wall degradation in Trichoderma. Microbiology (Reading) 158 (Pt 1), 26e34. https://doi.org/10.1099/mic.0.052613-0. Hamid, R., Khan, M.A., Ahmad, M., Ahmad, M.M., Abdin, M.Z., Musarrat, J., Javed, S., 2013. Chitinases: an update. J. Pharm. Bioallied Sci. 5 (1), 21e29. Han, B., Zhou, K., Li, Z., Sun, B., Ni, Q., Meng, X., Pan, G., Li, C., Long, M., Li, T., Zhou, C., Li, W., Zhou, Z., 2016. Characterization of the first fungal glycosyl hydrolase family 19 chitinase (NbchiA) from Nosema bombycis (Nb). J. Eukaryot. Microbiol. 63 (1), 37e45. Hayes, M., Carney, B., Slater, J., Br€ uck, W., 2008. Mining marine shellfish wastes for bioactive molecules: chitin and chitosan-Part B: applications. Biotechnol. J. 3 (7), 878e889. Henrissat, B., Davies, G., 1997. Structural and sequence-based classification of glycoside hydrolases. Curr. Opin. Struct. Biol. 7 (5), 637e44. Hoster, F., Schmitz, J.E., Daniel, R., 2005. Enrichment of chitinolytic microorganisms: isolation and characterization of a chitinase exhibiting antifungal activity against phytopathogenic fungi from a novel Streptomyces strain. Appl. Microbiol. Biotechnol. 66 (4), 434e442. Howard, M.B., Ekborg, N.A., Weiner, R.M., Hutcheson, S.W., 2003. Detection and characterization of chitinases and other chitin-modifying enzymes. J. Ind. Microbiol. Biotechnol. 30 (11), 627e635. Howell, C.R., 2003. Mechanisms employed by Trichoderma species in the biological control of plant diseases: the history and evolution of current concepts. Plant Dis. 87 (1), 4e10. Huang, C., Wang, T., Chung, S., Chen, C., 2005. Identification of an antifungal chitinase from a potential biocontrol agent, Bacillus cereus 28-9. J. Biochem. Mol. Biol. 38 (1), 82e88. Islam, R., Datta, B., 2015. Diversity of chitinases and their industrial potential. Int. J. Appl. Res. 1, 55e60. Jahromi, S.T., Barzkar, N., 2018. Marine bacterial chitinase as sources of energy, eco-friendly agent, and industrial biocatalyst. Int. J. Biol. Macromol. 120 (Pt B), 2147e2154. Karthik, N., Akanksha, K., Binod, P., Pandey, A., 2014. Production, purification and properties of fungal chitinasesda review. Indian J. Exp. Biol. 52, 1025e1035. Karthik, N., Binod, P., Pandey, A., 2017. Chitinases. In: Current Developments in Biotechnology and Bioengineering. Elsevier, Amsterdam, pp. 335e368. Kasprzewska, A., 2003. Plant chitinases-regulation and function. Cell. Mol. Biol. Lett. 8 (3), 809e24. Khoushab, F., Yamabhai, M., 2010. Chitin research revisited. Mar. Drugs 8 (7), 1988e2012. Kurita, K., 2006. Chitin and chitosan: functional biopolymers from marine crustaceans. Mar. Biotechnol. (NY) 8 (3), 203e226. Kusaoke, H., Shinya, S., Fukamizo, T., Kimoto, H., 2017. Biochemical and biotechnological trends in chitin, chitosan, and related enzymes produced by Paenibacillus Ik-5 strain. Int. J. Biol. Macromol. 104 (Pt B), 1633e1640.

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Kuzu, S.B., G€ uvenmez, H.K., Denizci, A.A., 2012. Production of a thermostable and alkaline chitinase by Bacillus thuringiensis subsp. kurstaki strain HBK-51. Biotechnol. Res. Int. 2012, 135498. Le, B., Yang, S., 2018. Characterization of a chitinase from Salinivibrio sp. BAO-1801 as an antifungal activity and a biocatalyst for producing chitobiose. J. Basic Microbiol. 58 (10), 848e856. Le, B., Yang, S.H., 2019. Microbial chitinases: properties, current state and biotechnological applications. World J. Microbiol. Biotechnol. 35 (9), 144. Lee, H.S., Han, D.S., Choi, S.J., Choi, S.W., Kim, D.S., Bai, D.H., Yu, J.H., 2000. Purification, characterization, and primary structure of a chitinase from Pseudomonas sp. YHS-A2. Appl. Microbiol. Biotechnol. 54 (3), 397e405. Lee, Y., Chung, K., Wi, S., Lee, J., Bae, H., 2009. Purification and properties of a chitinase from Penicillium sp. LYG 0704. Protein Expr. Purif. 65 (2), 244e250. Li, F., Patra, K.P., Vinetz, J.M., 2005. An anti-Chitinase malaria transmission-blocking single-chain antibody as an effector molecule for creating a Plasmodium falciparum-refractory mosquito. J. Infect. Dis. 192 (5), 878e887. Lopez-Mondejar, R., Blaya, J., Obiol, M., Ros, M., Pascual, J., 2012. Evaluation of the effect of chitin-rich residues on the chitinolytic activity of Trichoderma harzianum: in vitro and greenhouse nursery experiments. Pestic Biochem. Physiol. 103 (1), 1e8. Mathew, G.M., Madhavan, A., Arun, K.B., Sindhu, R., Binod, P., Singhania, R.R., Sukumaran, R.K., Pandey, A., 2021. Thermophilic chitinases: structural, functional and engineering attributes for industrial applications. Appl. Biochem. Biotechnol. 193 (1), 142e164. Mathivanan, N., Kabilan, V., Murugesan, K., 1998. Purification characterization, and antifungal activity of chitinase from Fusarium chlamydosporum a mycoparasiteto groundnut rust, Puccinia arachidis. Can. J. Microbiol. 44 (7), 646e651. Matsumoto, K.S., Guevara-Gonza
lez, R.G., Torres-Pacheco, I., 2006. Fungal chitinases. Adv. Agric. Food Biotechnol. 289e304. Merzendorfer, H., Zimoch, L., 2003. Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases. J. Exp. Biol. 206 (24), 4393e412. Oranusi, N.A., Trinci, A.P.J., 1985. Growth of bacteria on chitin, fungal cell walls and fungal biomass, and the effect of extracellular enzymes produced by these cultures on the antifungal activity of amphotericin B. Microbios 43 (172), 17e30. Pan, M., Li, J., Lv, X., Du, G., Liu, L., 2019. Molecular engineering of chitinase from Bacillus sp. DAU101 for enzymatic production of chitooligosaccharides. Enzyme Microb. Technol. 124, 54e62. Park, S.H., Lee, J.H., Lee, H.K., 2000. Purification and characterization of chitinase from a marine bacterium, Vibrio sp. 98CJ11027. J. Microbiol. Seoul. 38 (4), 224e229. Rathore, A.S., Gupta, R.D., 2015. Chitinases from bacteria to human: properties, applications, and future perspectives. Enzyme Res. 2015. Article ID 791907, 8 pages. Revah-Moiseev, S., Carroad, P.A., 1981. Conversion of the enzymatic hydrolysate of shellfish waste chitin to single-cell protein. Biotechnol. Bioeng. 23 (5), 1067e1078. Revathi, M., Saravanan, R., Shanmugam, A., 2012. Production and characterization of chitinase from Vibrio species, a head waste of shrimp Metapenaeus dobsonii (Miers, 1878) and chitin of Sepiella inermis Orbigny. ABB 03 (04), 392e339. Saba, H., Vibhash, D., Manisha, M., Prashant, K.S., Farhan, H., Tauseef, A., 2012. Trichodermaea promising plant growth stimulator and biocontrol agent. Mycosphere 3 (4), 524e531. Sakai, K., Yokota, A., Kurokawa, H., Wakayama, M., Moriguchi, M., 1998. Purification and characterization of three thermostable endochitinases of a noble Bacillus strain, MH-1, isolated from chitincontaining compost. Appl. Environ. Microbiol. 64 (9), 3397e3402. Sharma, R.R., Singh, D., Singh, R., 2009. Biological control of post harvest diseases of fruits and vegetables by microbial antagonists: a review. Biol. Control 50 (3), 205e221. Sharma, V., Shanmugam, V., 2012. Purification and characterization of a 24 kDa chitobiosidase from the mycoparasitic fungus Trichoderma saturnisporum. J. Basic Microbiol. 52, 324e331. Shekhar, N., Bhattacharya, D., Kumar, D., Gupta, R.K., 2006. Biocontrol of wood-rotting fungi with Streptomyces violaceusniger XL-2. Can. J. Microbiol. 52 (9), 805e808.

Industrial applications of thermophilic/hyperthermophilic enzymes

Stoykov, Y.M., Pavlov, A.I., Krastanov, A.I., 2015. Chitinase biotechnology: production, purification, and application. Eng. Life Sci. 15 (1), 30e38. Suzuki, K., SuGAwARA, N., Suzuki, M., Uchiyama, T., Katouno, F., Nikaidou, N., Watanabe, T., 2002. Chitinases A, B, and C1 of Serratia marcescens 2170 produced by recombinant Escherichia coli: enzymatic properties and synergism on chitin degradation. Biosci. Biotechnol. Biochem. 66 (5), 1075e1083. Synowiecki, J., Al-Khateeb, N.A., 2003. Production, properties, and some new applications of chitin and its derivatives. Crit. Rev. Food Sci. Nutr. 43 (2), 145e71. Takaya, N., Yamazaki, D., Horiuchi, H., Ohta, A., Takagi, M., 1998. Cloning and characterization of a chitinase-encoding gene (chiA) from Aspergillus nidulans, disruption of which decreases germination frequency and hyphal growth. Biosci. Biotechnol. Biochem. 62 (1), 60e65. Vega, K., Kalkum, M., 2012. Chitin, chitinase responses, and invasive fungal infections. Int. J. Microbiol. 2012. Article ID 920459, 10 pages. Wang, S.L., Hsiao, W.J., Chang, W.T., 2002. Purification and characterization of an antimicrobial chitinase extracellularly produced by Monascus purpureus CCRC31499 in a shrimp and crab shell powder medium. J. Agric. Food Chem. 50 (8), 2249e2255. Watanabe, T., Kimura, K., Sumiya, T., Nikaidou, N., Suzuki, K., Suzuki, M., Taiyoji, M., Ferrer, S., Regue, M., 1997. Genetic analysis of the chitinase system of Serratia marcescens 2170. J. Bacteriol. 179 (22), 7111e7117. Wu, M.L., Chuang, Y.C., Chen, J.P., Chen, C.S., Chang, M.C., 2001. Identification and characterization of the three chitin binding domains within the multidomain chitinase Chi92 from Aeromonas hydrophila JP101. Appl. Environ. Microbiol. 67 (11), 5100e5106. Zhu, Z., Zheng, T., Homer, R.J., Kim, Y.K., Chen, N.Y., Cohn, L., Hamid, Q., Elias, J.A., 2004. Acidic mammalian chitinase in asthmatic Th2 inflammation and IL-13 pathway activation. Science 304 (5677), 1678e1682.

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SUBCHAPTER 8.11

Proteases 8.11.1 Microbial sources and properties Proteases catalyze the hydrolysis of peptide bonds between amino acid residues of proteins. Protease is one of the best-selling enzymes in the world. These enzymes are widely used in leather, textile, pharmaceutical, and food industries (Razzaq et al., 2019). Applications will continue to expand, as will the requirement for stable biocatalysts that can withstand severe operating conditions. Thermostability of industrial enzymes remains a wanted property to obtain faster conversion rates, higher catalytic efficiencies, and protection from microbial contaminants while operating at high temperatures. Proteases with these properties are required for bakery and textile applications. Generally, most industrial proteolytic enzymes are derived from Bacillus sp. Thermostability of proteases is conferred by genetic engineering or appropriate immobilization techniques. Proteases from thermophiles and hyperthermophiles are normal choices for investigating their intrinsic thermostability. Several traditional thermostable proteases, particularly those from Pyrococcus and Thermococcus, have attracted considerable interest. Thermal stability in these cases could be due to a high proportion of hydrophobic residues, substantial hydrogen bonding, and a higher proportion of disulfide bonds (Sinha and Khare, 2013). Proteases usually fall into two categories: - an exopeptidase that cleaves amino acids from the ends of protein chains - an endopeptidase that cleaves peptide bonds within proteins Proteases are found in prokaryotic microorganisms, fungi, plants, and animals. These enzymes catalyze the hydrolysis of proteins into smaller peptides or free amino acids. Proteases have been classified as cysteine, serine, asparagine, or metalloproteases. Microbial proteolytic enzymes are of great interest due to their all-round properties and multiple applications in different fields. Endo (1962) was the first to characterize thermolysin, a thermostable proteolytic enzyme from Bacillus thermoproteolyticus. Since then, several thermoactive proteases from various microorganisms have been identifieddPyrococcus and Thermococcus. Several proteases have been identified from thermophilic archaea. These belong to the genera Sulfolobus, Pyrobaculum, Desulfurococcus, and Staphylothermus. Archaeal proteolytic enzymes are highly stable, with optimal activity between 90 and 110
C. A record of a highly thermostable serine protease produced by the hyperthermophilic archaea strain Desulfurococcus is also available. The thermophilesdRhizopus, Rhizomucor, Sporotrichum, Torula, Achaetomium, Chaetomium, and Penicilliumdhave been found to produce thermostable proteases. A few of them are characterized by high thermal stability and faster rate of reaction. One

Industrial applications of thermophilic/hyperthermophilic enzymes

of the most thermostable acidic fungal proteases was found in Penicillium duponti K1014 isolated from compost. In contrast, an alkaline thermostable protease from Humicola lanuginosa and Malbranchea pulchella var. sulfurea has been reported. Among bacteria, Bacillus sp. has been a most important source of thermostable proteases. The first isolate belonged to B. stearothermophilus (Hanzawa et al., 1996; Hashimoto et al., 1973; Emi et al., 1976; Ong and Gaucher, 1973, 1976; Salleh et al., 1977). Proteases from different strains of B. stearothermophilus were found to differ in their thermostability, for instance, protease from one of the B. stearothermophilus sp. showed optimum activity at a temperature 85
C whereas that from B. stearothermophilus TP26 remained active at 75
C (Rahman et al., 1994; Gey and Unger, 1995). A thermostable alkaline protease was produced by alkaliophilic Bacillus species JB-99 (Johnevelsy and Naik, 2001). Thermostable proteases can be used at higher processing temperatures, which resulted in rapid reaction rate, better solubility of nongaseous reactants and products, and lower rates of microbial contamination by mesophiles, thus reducing the occurrence of microbial contamination (Nascimento and Martins, 2004). Proteolytic enzymes produced by Bacillus have attractive properties for various industrial applications and are recognized to account for about 60% of total enzyme sales worldwide. Proteases produced by Bacillus have been widely used in the detergent industry because of their activity and stability over a broad range of temperature and pH (Annamalai et al., 2013). In addition, proteolytic enzymes produced by Bacillus may be used in the food industry for obtaining bioactive peptides and process various foods (Bougatef et al., 2012; Ozcan and Kurdal, 2012). An additional characteristic of the proteolytic enzymes produced by Bacillus is their stability in organic solvents and thus have been used during organic synthesis (Caille et al., 2002). Proteases obtained from Bacillus are mostly alkaline, with optimal pH-activity levels above 7.0; the molecular weight in the range of 27e71 kDa, an optimum pH range between 6 and 10, and an optimum temperature between 37 and 60
C. Additionally, the proteolytic enzymes show considerable stability over a wider range of pH and temperatures. The activity of the halophilic serine proteases tested in the range pH 8e12 did not show large fluctuations in the pH range 8e11 but declined rapidly at pH values below 8 or above 11 (Raval et al., 2014). The enzyme retained its activity at pH 8e11 for 24 h without significant loss (Dhillon et al., 2017).

8.11.2 Applications of proteases Microbial proteases are considered to be the most important hydrolytic enzymes and have applications in several industries. The alkaline proteases rank highest in the enzyme market (Mahajan et al., 2016).

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8.11.2.1 Food and feed industry When making cheese from milk, protease enzymes are added to hydrolyze kappa casein, stabilizing micelle formation and preventing clotting. In the bakery industry, the gluten is partly hydrolyzed by thermolabile fungal proteases during subsequent baking for initial inactivation for preparing the dough more quickly. The addition of microbial alkaline protease has enabled a highly nutritious protein hydrolyzate formulation. Bioactive peptides play important roles in various pharmaceuticals (Fig. 8.11.1). Protein hydrolysates are found to be important in the regulation of blood pressure. Alkaline proteases have been used to generate hydrolysates from a variety of natural protein substrates. Commercial protein hydrolysates are obtained from soy protein, whey, and casein. The application of proteases in meat tenderization has long been known. When performing at 40e60
C, it may be better to use a thermostable protease. Wilson et al. (1992) have reported the benefits of using bacterial proteolytic thermostable enzyme E A.1 protease (from Bacillus strain E A.1) and 4-1.A protease (from Thermus strain Rt4-1.A) in meat tenderization.

Figure 8.11.1 Food-protein-derived peptides and their roles. (From Razzaq, A., Shamsi, S., Ali, A., Ali, Q., Sajjad, M., Malik, A., Ashraf, M., 2019. Microbial proteases applications. Front. Bioeng. Biotechnol. 7, 110. Distributed under the terms of the Creative commons Attribution License (CC BY).)

Industrial applications of thermophilic/hyperthermophilic enzymes

8.11.2.2 Waste management Poultry feathers have a very stiff keratin structure and account for 5% of body weight. These are a good source of protein in forage and food. Poultry waste can be converted into food and feed through keratinolysis processes (Lasekan et al., 2013; Neklyudov et al., 2000). A formulation consisting of hydrolytic enzymes isolated from B. subtilis, B. amyloliquefaciens, and Streptomyces sp. has been produced and patented as Genex for hair removal and cleaning of blocked pipes and drains (Lasekan et al., 2013). 8.11.2.3 Leather industry In the leather industry, microbial alkaline proteolytic enzymes are very popular (Brandelli et al., 2010; Takami et al., 1992). The increasing use of alkaline proteases in the emerging leather industry is because of their elastolytic and keratinolytic activities. These important properties of alkaline proteases make them very useful in the leather processing industry. A particular use of proteases has been found to be associated with the soaking, bating, and dehairing of hide and skin preparation. These destroy unwanted pigments through enzymatic action, resulting in clear skin. The enzymatic process of pancreatic protease is based on a bating system. 8.11.2.4 Detergent industry Proteases are extensively used in the detergent industry. Several products in the detergent industry that contain proteases as necessary ingredients are used for cleaning household linens, contact lenses and dentures. In the detergent industry, use of proteases accounts for about 20% of total enzyme sales. In 1913, the first enzyme preparation, “Brunus” containing raw pancreatic extract and sodium carbonate, was produced. This enzyme was first commercialized in 1956 under the trade name BIO-40. Novo Industry A/S in 1960 introduced B. licheniformis (Jacobson et al., 1985). The protease produced by B. cereus BM1 has been described as an excellent detergent ingredient, showing good activity in 10% (w/v) solutions of commercial detergents (Illanes, 2008). Isoelectric point is important in the selection of proteolytic enzymes for detergent manufacturing. These enzymes work well when the pH and isoelectric points of these enzymes are about the same. Compatibility with surfactants, bleaches, and fragrances, good activity, optimal pH and temperature, stability, ionic strength, and stain removal potential were also considered in the selection of detergent proteases. Detergents traditionally function at high temperatures, but there is increasing interest in discovering and identifying alkaline proteases that function over a broader temperature range (Breuer et al., 2004). In general,

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commercial proteases are not stable in the presence of bleach or oxidizing agents. Recombinant DNA technology has been used for creating bioengineered detergent proteases with superior stability and durability. Proteases are also used in dishwashing and cleaning products (Bornscheuer et al., 2012). 8.11.2.5 Photographic industry Alkaline proteolytic enzymes produced by Conidiobolus coronatus, B. subtilis, and S. avermectnus have been found to recover silver from X-ray films (Yang et al., 2000). Efficient use of a thermostable mutant alkaline proteases from Bacillus sp. B21-2 to recover silver has been reported (Araujo et al., 2008). 8.11.2.6 Chemical industry Several alkaline proteases producing microorganisms like Pseudomonas aeruginosa PseA B. pseudofirus SVB1 and Aspergillus flavus have shown remarkable results in peptide synthesis because of their stability in organic solvents (Shankar et al., 2010; Ahmed et al., 2008). Several alkaline proteases producing species of the genera Bacillus and Streptomyces in aquatic systems are found to be good candidates for peptide and organic synthesis (Yadav et al., 2015). 8.11.2.7 Silk degumming Alkaline proteases are used to degumm silk. Traditionally, silk gum should be removed by degumming the raw silk with an alkaline soap solution. Alkaline proteases are best for removing sericin without damaging the fibers. Fibers do not break and silk threads have proven to be much stronger than conventional treatments (da Silva et al., 2017; Radha et al., 2017). 8.11.2.8 Medical area Proteases are successfully used in the medical field. In medicine, various formulations like gauzes, nonwoven fabrics and ointment compositions containing alkaline proteases of B. subtilis show good therapeutic properties (Razzaq et al., 2019). Certain lytic enzyme deficiency syndromes have been identified as supported by oral administration of alkaline proteolytic enzymes (Joshi and Satyanarayana, 2013). Fibrin degradation was obtained by an alkaline fibrinolytic protease. This enzyme can be used as an anticancer agent and for thrombolytic therapy (Razzaq et al., 2019). Sustained-release formulations containing collagenase and alkaline proteases are widely used for therapeutic applications. Enzymatic hydrolysis of collagen releases low-

Industrial applications of thermophilic/hyperthermophilic enzymes

molecular-weight peptides without releasing therapeutic amino acids. Elastosterase preparations immobilized in bandages are used to treat a variety of ailments like burns, carbuncles, boils, and wounds.

Bibliography Ahmed, S.A., Al-Domany, R.A., El-Shayeb, N.M., Radwan, H.H., Saleh, S.A., 2008. Optimization, immobilization of extracellular alkaline protease and characterization of its enzymatic properties. Res. J. Agric. Biol. Sci. 4, 434e446. Annamalai, N.R., Rajeswari, M.V., Thavasi, R., Vijayalakshmi, S., Balasubramanian, T., 2013. Optimization, purification and characterization of novel thermostable, haloalkaline, solvent stable protease from Bacillus halodurans CAS6 using marine shellfish wastes: a potential additive for detergent and antioxidant synthesis. Bioprocess. Biosyst. Eng. 36, 873e883. Araujo, R., Casal, M., Cavaco-Paulo, A., 2008. Application of enzymes for textile fibres processing. Biocatal. Biotransform. 26, 332e349. Bornscheuer, U., Huisman, G., Kazlauskas, R., Lutz, S., Moore, J., Robins, K., 2012. Engineering the third wave of biocatalysis. Nature 485, 185. Bougatef, A.B., Balti, R., Haddar, A., Jellouli, K., Souissi, N., Nasri, M., 2012. Protein hydrolysates from bluefin tuna (Thunnus thynnus) heads as influenced by the extent of enzymatic hydrolysis. Biotechnol. Bioprocess Eng. 17, 841e852. Brandelli, A., Daroit, D.J., Riffel, A., 2010. Biochemical features of microbial keratinases and their production and applications. Appl. Microbiol. Biotechnol. 85, 1735e1750. Breuer, M., Ditrich, K., Habicher, T., Hauer, B., Kesseler, M., St€ urmer, R., Zelinski, T., 2004. Industrial methods for the production of optically active intermediates. Angew. Chem. Int. Ed. Engl. 43 (7), 788e824. Caille, J.C., Govindan, C.K., Junga, H., Lalonde, J., Yao, Y., 2002. Hetero alder-biocatalysis approach for the synthesis of (s)-3-[2-{(methylsulfonyl) oxy} ethoxy]-4-(triphenylmethoxy)-1-butanol methanesulfonate, a key intermediate for the synthesis of the pkc inhibitor. Org. Process. Res. Dev. 6, 471e476. da Silva, O.S., Gomes, M.H.G., de Oliveira, R.L., Porto, A.L.F., Converti, A., Porto, T.S., 2017. Partitioning and extraction protease from Aspergillus tamarii URM4634 using PEG-citrate aqueous two-phase systems. Biocatal. Agric. Biotechnol. 91, 68e73. Dhillon, A., Sharma, K., Rajulapati, V., Goyal, A., 2017. Proteolytic enzymes. In: Pandey, A., Negi, S., Soccol, C. (Eds.), Current Developments in Biotechnology and Bioengineering. Elsevier. Emi, S., Meyers, D.V., Iacobucci, G.A., 1976. Purification and properties of the thermostable acid protease of Penicillium duponti. Biochem. J. 15, 842e848. Endo, S., 1962. Studies on protease produced by thermophilic bacteria. J. Ferment. Technol. 40, 346e353. Gey, M.H., Unger, K.K., 1995. Calculation of the molecular masses of two newly synthesized thermostable enzymes isolated from thermophilic microorganisms. J. Chromatogr. B Biomed. Appl. 666 (1), 188e193. Hanzawa, S., Hoaki, T., Jannasch, H.W., Maruyama, T., 1996. An extremely thermostable serine protease from a hyperthermophilic archaeon Desulfurococcus strain SY, isolated from a deep-sea hydrothermal vent. J. Mar. Biotechnol. 4, 121e126. Hashimoto, H., Kaneko, Y., Iwaasa, T., Wokotsuka, T., 1973. Production and purification of acid protease from the thermophilic fungus, Penicillium duponti K1014. Appl. Microbiol. 25 (4), 584e588. Illanes, A., 2008. Enzyme Biocatalysis Principles and Applications. Springer-Verlag New York Inc., New York, NY. Jacobson, J.W., Glick, J.L., Madello, K.L., 1985. Composition for cleaning drains clogged with deposits containing hair. Google Patents. Johnvesly, B., Naik, G.R., 2001. Studies on production of thermostable alkaline protease from thermophilic and alkaliphilic Bacillus sp. JB-99 in a chemically defined medium. Process Biochem. 37, 139e144. Joshi, S., Satyanarayana, T., 2013. Characteristics and applications of a recombinant alkaline serine protease from a novel bacterium Bacillus lehensis. Bioresour. Technol. 131, 76e85.

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Lasekan, A., Bakar, F.A., Hashim, D., 2013. Potential of chicken byproducts as sources of useful biological resources. Waste Manag. 33, 552e565. Mahajan, R., Chaudhari, G., Chopadaa, M., 2016. Report on Biotechnological applications of proteolytic enzymes from lattices of euphorbian plants. J. Appl. Biotechnol. Rep. 2, 333e337. https://doi.org/ 10.21276/ijlssr.2016.2.4.7. M
oty
an, J., T
oth, F., Tozs
er, J., 2013. Research applications of proteolytic enzymes in molecular biology. Biomolecules 3, 923e942. Nascimento, W.C.A., Martins, M.L.L., 2004. Production and properties of an extracellular protease from thermophilic Bacillus sp. Braz. J. Microbiol. 35, 91e96. Neklyudov, A., Ivankin, A., Berdutina, A., 2000. Properties and uses of protein hydrolysates. Appl. Biochem. Microbiol. 36, 452e459. Ong, P.S., Gaucher, G.M., 1976. Productiou, purification and characterization of thermomycolase, the extracellular serine protease of the thermophilic fungus Malbranchea pulchella var. sulfurea. Can. J. Microbiol. 22, 165e176. Ong, P.S., Gaucher, G.M., 1973. Protease production by thermophilic fungi. Can. J. Microbiol. 19, 129e133. Ozcan, T.K., Kurdal, E., 2012. The effects of using a starter culture, lipase, and protease enzymes on ripening of Mihalic cheese. Int. J. Dairy Technol. 65, 585e593. Radha, S., Sridevi, A., Himakiran Babu, R., Nithya, V., Prasad, N., Narasimha, G., 2017. Medium optimization for acid protease production from Aspergillus sps under solid state fermentation and mathematical modelling of protease activity. J. Microbiol. Biotechnol. Res. 2, 6e16. Rahman, R., Razak, C., Ampon, K., Basri, M., Yunus, W., Salleh, A., 1994. Purification and characterization of a heat-stable alkaline protease from Bacillus stearothermophilus F1. Appl. Microbiol. Biotechnol. 40, 822e827. Raval, V.H., Pillai, S., Rawal, C.M., Singh, S.P., 2014. Biochemical and structural characterization of a detergent stable serine alkaline protease from seawater haloalkaliphilic bacteria. Process Biochem. 49, 955e62. Razzaq, A., Shamsi, S., Ali, A., Ali, Q., Sajjad, M., Malik, A., Ashraf, M., 2019. Microbial proteases applications. Front. Bioeng. Biotechnol. 7, 110. Salleh, A.B., Basri, M., Razak, C., 1977. The effect of temperature on the protease from Bacillus stearothermophilus strain F1. Mal. J. Biochem. Mol. Biol. 2, 37e41. Shankar, S., More, S., Laxman, R.S., 2010. Recovery of silver from waste X-ray film by alkaline protease from Conidiobolus coronatus. Kathmandu Univ. J. Sci. Eng. Technol. 6, 60e69. Singh, R., Mittal, A., Kumar, M., Mehta, P.K., 2016. Microbial protease in commercial applications. J. Pharm. Chem. Biol. Sci. 4, 365e374. Sinha, R., Khare, S., 2013. Thermostable Proteases in Book: Thermophilic Microbes in Environmental and Industrial Biotechnology. Springer, pp. 859e880. Takami, H., Nakamura, S., Aono, R., Horikoshi, K., 1992. Degradation of human hair by a thermostable alkaline protease from alkaliphilic Bacillus sp. no. AH-101. Biosci. Biotechnol. Biochem. 56, 1667e1669. Wilson, S.A., Young, O.A., Coolbear, T., Daniel, R.M., 1992. The use of proteases from extreme thermophiles for meat tenderization. Meat. Sci. 32, 93e103. Yadav, S.K., Bisht, D., Tiwari, S., Darmwal, N.S., 2015. Purification, biochemical characterization and performance evaluation of an alkaline serine protease from Aspergillus flavus MTCC 9952 mutant. Biocatal. Agric. Biotechnol. 4, 667e677. Yang, J.K., Shih, L., Tzeng, Y.M., Wang, S.L., 2000. Production and purification of protease from a Bacillus subtilis that can deproteinize crustacean wastes. Enzyme Microb. Technol. 26, 406e413.

Industrial applications of thermophilic/hyperthermophilic enzymes

SUBCHAPTER 8.12

Glucose (xylose) isomerase 8.12.1 Microbial sources and properties D-glucose/xylose

isomerase (D-xylose ketol isomerase; EC 5.3.1.5), also known as glucose isomerase, is one of the three highest tonnage enzymes, with amylases and proteases make up the remaining two (Lee et al., 2019). Glucose isomerase is also known as D-xylose ketoisomerase. The systematic name for this class of enzymes is D-xylose aldose ketose isomerase. Glucose isomerase catalyzes the reversible isomerization of glucose to fructose, which is much sweeter than glucose. This transformation represents the major commercial application of immobilized biocatalysts in industry. The glucose isomerase enzyme is an intramolecular oxidoreductase, which is able to interconvert aldoses and ketoses. Glucose isomerase is central to its physiological role and commercial applications (Deshpande and Rao, 2006). Particularly, this enzyme is widely used for producing high fructose corn syrup (HFCS) on a commercial scale (Kilara and Shahani, 1979). Furthermore, the conversion of xylose to ethanol by glucose isomerase is important for producing ethanol from hemicelluloses (Singh et al., 2019). Isomerization of glucose to fructose is of industrial importance in the manufacturing process of HFCS. Sucrose, obtained from sugar cane (60%), and sugar beets (40%) was the world’s most important sweetener until 1976. Production of HFCS using glucose isomerase was first commercialized in Japan and afterward in the United States. Due to sucrose shortages following the Cuban Revolution in 1958, glucose isomerase became commercially significant in the United States and is still one of the most crucial industrial enzymes today (Bhosale et al., 1996). Glucose isomerase is widely present in fungi, actinomycetes, bacteria, and plants (Young et al., 1975). Among heterolactic acid bacteria, Lactobacillus brevis produces the maximum enzyme yield. The genus Streptomyces and many Bacillus species are excellent producers of glucose isomerase. Aspergillus oryzae is the only fungus found to have glucose isomerase activity. The presence of glucose isomerase in malted barley and wheat germ has been observed. Not much information is available on extracellular secretion of glucose isomerase. Extracellular glucose isomerase has been found to be produced by S. glaucescens and S. flavogriseus (Weber, 1976; Chen et al., 1979) and release of the enzyme from the cell depends on cell wall permeability and partially believed to be due to changes in lysis of cells. The occurrence of glucose isomerase in several yeasts such as Candida utilis and C. boidinii has been reported (Wang et al., 1980; Vongsuvanlert and Tani, 1988). The only fungus reported to have glucose isomerase activity is Aspergillus oryzae. Presence of glucose isomerase has been reported in barley malt and wheat germ.

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Table 8.12.1 Glucose isomeraseeproducing microorganisms.

Actinomyces olivocinereus Aerobacter aerogenes Bacillus stearothermophilus Brevibacterium incertum Brevibacterium pentosoaminoacidicum Chainia spp. Corynebacterium spp. Escherichia spp. Flavobacterium spp. Lactobacillus spp. Leuconostoc mesenteroides Microbispora rosea Mycobacterium spp. Pseudonocardia spp. Streptococcus spp. Streptosporangium album Thermopolyspora spp. Thermus spp. Xanthomonas spp. Zymononas mobilis

Table 8.12.1 shows glucose isomerase-producing microorganisms. Commercial producers of glucose isomerase include Actinoplanes misouriensis, Bacillus coagulans, S. rubiginosus, Pheochromogenes, Arthrobacter sp., and S. olivaceus (Bhosale et al., 1996). Glucose isomerase is a topic of immense commercial importance. A lot of information regarding newly produced organisms and developed processes can be found in the form of patents (Bhosale et al., 1996; Weber, 1976). Brown et al. (1993) reported that glucose isomerase from the thermophilic eubacterium Thermotoga maritima (TmaGI) is produced when grown using xylose as a carbon source. The enzyme is most active at pH 6.5e7.5. TmaGI exhibits maximal activity at a temperature of 105e110
C, with a half-life of about 10 min at 120
C and pH 7.0. Deng et al. (2014) produced glucose isomerase from Thermobifida fusca WSH03-11 (TfuGI). TfuGI exhibited maximal activity at a temperature of 80
C, with a half-life of about 2 h at 80
C and 15 h at 70
C. TfuGI converts glucose (45% w/v) to fructose. A maximum conversion yield of 53% was obtained at pH 7.5 and temperature of 70
C. Jia et al. (2017) characterized the glucose isomerases of Thermoanaerobacter siderophilus (TsiGI) Geobacillus thermocatenulatus (GthGI), Thermoanaerobacterium xylanolyticum (TxyGI), and Thermus oshimai (TosGI). Among these enzymes, TosGI showed the highest catalytic performance for D-glucose with excellent thermostability. TosGI had a temperature optimum of 95
C and was found to retain more than 80% activity after 48 h at 85
C when 20 mM manganese was present.

Industrial applications of thermophilic/hyperthermophilic enzymes

8.12.2 Applications of glucose isomerase 8.12.2.1 High fructose corn syrup HFCS is an equilibrium blend of glucose and fructose with the following benefits (Bhasin and Modi, 2012). ➢ Sweetness ➢ Lower cost ➢ Higher solubility Depending upon their fructose content, HFCS can be classified as follows (SernaSaldivar, 2016): ➢ HFCS-42 (42% fructose, 53% glucose, 5% polysaccharides) ➢ HFCS-55 (55% fructose, 42% glucose, 3% polysaccharides) ➢ HFCS-90 (90% fructose, 9% glucose, 1% polysaccharides) Among them, HFCS-55 is the most widely used. Its production on a commercial scale involves multiple processes such as chromatographic purification and concentration. HFCS is extensively used as a food detergent and pharmaceutical industries (Singh et al., 2017). Conversion of glucose to fructose is performed using a chemical process that produces HFCS, but this chemical reaction is not specific. This results in formation of sugars, which are not metabolizable (Singh et al., 2019). In contrast, glucose isomerase catalyzes the isomerization of glucose to fructose with exceptional specificity. This is essential for commercial production and application of HFCS (Singh et al., 2019). 8.12.2.2 Ethanol production The conversion of renewable biomass to bioethanol is attractive in terms of fossil fuel exhaustion at a rapid rate (Canilha et al., 2012). Glucose isomerase catalyzes the isomerization of xylose from hemicellulosic biomass to xylulose, which is fermented to ethanol by common yeasts like Candida tropicalis, Saccharomyces cerevisiae, and Schizosaccharomyces pombe (Ko et al., 2016; Gong et al., 1981; Chiang et al., 1981). This property of glucose isomerase makes it attractive for application in production of bioethanol as it does not require coenzymes, and no intermediates are formed during the reaction. But, typical ethanol production processes suffer from reduced efficiency due to lower conversion yields of xylose to ethanol. Enzyme engineering of glucose isomerase and strain improvement strategies have been investigated for improving the conversion of xylose to ethanol. Engineering of the yeast strains exhibiting rapid xylose metabolism is important for strain improvement for production of ethanol (Bracher et al., 2019; Tran Nguyen Hoang et al., 2018).

Bibliography Bartfay, J., 1960. Glucose isomerase in barley malt. Nature (London) 185, 924. Bhasin, S., Modi, H.A., 2012. Optimization of fermentation medium for the production of glucose isomerase using Streptomyces sp. SB-P1 Biotechnol. Res. Int. 2012, 874152.

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Bhosale, S.H., Rao, M.B., Deshpande, V.V., 1996. Molecular and industrial aspects of glucose isomerase. Microbiol. Rev. 60 (2), 280e300. https://doi.org/10.1128/mr.60.2.280-300.1996. Bracher, J.M., Martinez-Rodriguez, O.A., Dekker, W.J.C., Verhoeven, M.D., van Maris, A.J.A., Pronk, J.T., 2019. Reassessment of requirements for anaerobic xylose fermentation by engineered, non-evolved Saccharomyces cerevisiae strains. FEMS Yeast Res. 19 (1), foy104. Brown, S.H., Sjoholm, C., Kelly, R.M., 1993. Purification and characterization of a highly thermostable glucose isomerase produced by the extremely thermophilic eubacterium, Thermotoga maritima. Biotechnol. Bioeng. 41, 878e886. Canilha, L., Kumar Chandel, A., dos Santos Milessi, T.S., Fernandes Antunes, F.A., da Costa Freitas, W.L., das Gracas Almeida Felipe, M., da Silva, S.S., 2012. Bioconversion of sugarcane biomass into ethanol: an overview about composition, pretreatment methods, detoxification of hydrolysates, enzymatic saccharification, and ethanol fermentation. J. Biomed. Biotechnol. 2012, 989572. Chen, W.P., Anderson, A.W., Han, Y.W., 1979. Production of glucose isomerase by Streptomyces flavogriseus. Appl. Environ. Microbiol. 37 (2), 324e331. Chiang, L.C., Gong, C.S., Chen, L.F., Tsao, G.T., 1981. D-xylulose fermentation to ethanol by Saccharomyces cerevisiae. Appl. Environ. Microbiol. 42 (2), 284e289. Deng, H., Chen, S., Wu, D., Chen, J., Wu, J., 2014. Heterologous expression and biochemical characterization of glucose isomerase from Thermobifida fusca. Bioprocess Biosyst. Eng. 37, 1211e1219. Deshpande, V., Rao, M., 2006. Glucose isomerase. In: Enzyme Technology. Springer, New York, NY, USA, pp. 239e252. Gong, C.S., Chen, L.F., Flickinger, M.C., Chiang, L.C., Tsao, G.T., 1981. Production of ethanol from Dxylose by using D-xylose isomerase and yeasts. Appl. Environ. Microbiol. 41, 430e436. Jia, D.X., Zhou, L., Zheng, Y.G., 2017. Properties of a novel thermostable glucose isomerase mined from Thermus oshimai and its application to preparation of high fructose corn syrup. Enzyme Microb. Technol. 99, 1e8. Kilara, A., Shahani, K.M., 1979. The use of immobilized enzymes in the food industry: a review. CRC Crit. Rev. Food Sci. Nutr. 12, 161e198. Ko, J.K., Um, Y., Woo, H.M., Kim, K.H., Lee, S.M., 2016. Ethanol production from lignocellulosic hydrolysates using engineered Saccharomyces cerevisiae harboring xylose isomerase-based pathway. Bioresour. Technol. 209, 290e296. Lee, D., Baek, S., Park, J., Lee, K., Kim, J., Lee, S.J., Chung, W.K., Lee, J.L., Cho, Y., Nam, K.H., 2019. Nylon mesh-based sample holder for fixed-target serial femtosecond crystallography. Sci. Rep. 9, 6971. Liu, Z.Q., Zheng, W., Huang, J.F., Jin, L.Q., Jia, D.X., Zhou, H.Y., Xu, J.M., Liao, C.J., Cheng, X.P., Mao, B.X., Zheng, Y.G., 2015. Improvement and characterization of a hyperthermophilic glucose isomerase from Thermoanaerobacter ethanolicus and its application in production of high fructose corn syrup. J. Ind. Microbiol. Biotechnol. 42 (8), 1091e1103. Nam, K.H., 2022. Glucose isomerase: functions, structures, and applications. Appl. Sci. 12 (1), 428. Pubols, M.H., Zahnley, J.C., Axelrod, B., 1963. Partial purification & properties of xylose & ribose isomerase in higher plants. Plant Physiol. 38 (4), 457e461. Serna-Saldivar, S.O., 2016. Maize: foods from maize. In: Reference Module in Food Science. Elsevier, Amsterdam, the Netherlands. Singh, R.S., Singh, T., Pandey, A., 2019. Microbial enzymesdan overview. In: Advances in Enzyme Technology. Elsevier, Amsterdam, the Netherlands, pp. 1e40. Singh, R.S., Chauhan, K., Singh, R.P., 2017. Enzymatic approaches for the synthesis of high fructose syrup. In: Plant Biotechnology: Recent Advancements and Developments. Springer, Singapore, pp. 189e211. Tran Nguyen Hoang, P., Ko, J.K., Gong, G., Um, Y., Lee, S.M., 2018. Genomic and phenotypic characterization of a refactored xylose utilizing Saccharomyces cerevisiae strain for lignocellulosic biofuel production. Biotechnol. Biofuels 11, 268. Vongsuvanlert, V., Tani, Y., 1988. Purification and characterization of xylose isomerase of a methanol yeast, Candida boidinii, which is involved in sorbitol production from glucose. Agric. Biol. Chem. 52, 1817e1824. Wang, P.Y., Johnson, B.F., Schneider, H., 1980. Fermentation of D-xylose by yeasts using glucose isomerase in the medium to convert D-xylose to D-xylulose. Biotechnol. Lett. 2, 273e278. Weber, P., 1976. Fructose by isomerisation of glucose. U.K. patent 1,496,309. Young, J.M., Schray, K.J., Mildvan, A.S., 1975. Proton magnetic relaxation studies of the interaction of Dxylose and xylitol with D-xylose isomerase. Characterization of metal-enzyme-substrate interactions. J. Biol. Chem. 250, 9021e9027.

Industrial applications of thermophilic/hyperthermophilic enzymes

SUBCHAPTER 8.13

Lipase 8.13.1 Microbial sources and properties Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are carboxylesterases that catalyze the hydrolysis and also synthesis of long-chain acylglycerols (Jaeger et al., 1999). As suggested by their wide range of applications in several industrial sectors, lipases are undoubtedly important from both a research and industrial point of view. They catalyze the hydrolysis of oils and fats and release free fatty acids, monoglycerides, diglycerides, and glycerol. Thermostable lipases are among the cheapest enzymes used in the food and pharmaceutical industries and have been actively investigated as potential biocatalysts for producing biodiesel and other biotechnological applications. Lipases are widespread in plants and animals around the world. But, they are more commonly found in yeast, fungi, and bacteria. Many Bacillus spp. have been described as a major source of lipolytic enzymes (Luisa et al., 1997; Kim et al., 1994; Schmidt et al., 1994; Wu et al., 1996). The majority of these enzymes are found to be active at a temperature of 60
C and a pH of 7.0, but lipases from B. thermoleovorans and thermophilic strains of Rhizopus oryzae show moderate performance at high pH and temperature (Abel et al., 2000; Dong-Woo et al., 1999). The optimal temperature and pH for catalytic activity of several thermostable lipase enzymes have been reported by Haki and Rakshit (2003). However, there are few reports of thermostable lipases derived from archaea. Phospholipase A2 produced by Archaea Pyrococcus horikoshii is involved in refining of crude oil (Yan et al., 2000). The enzyme shows optimum performance at 95
C and pH 7.0. Phospholipase A2 performs well in refinery degumming processes, reducing effluent problems and the operating costs (Klaus, 1998). Thermostability and alkali tolerance are the most important properties that a commercially important lipase should possess. To achieve this goal, sources of lipase enzymes with specific stability to temperature, pH, organic solvents, and ionic strength are continually sought. Few lipases are able to function at 100
C. However, their half-lives are shorter (Lee and Rhee, 1993; Rathi et al., 2000). Table 8.13.1 shows thermostable lipase-producing bacteria and their properties (Haki and Rakshit, 2003). Lipases are used for various kinds of biocatalyzed reactions. Lipases can catalyze different reactions such as esterification, transesterification, alcoholysis, acidolysis, and aminolysis in addition to its hydrolytic activity on triglycerides (Aravindan et al., 2007). As a hydrolytic enzyme, lipase does not need cofactors. Most regioselective lipase enzymes preferentially act on ester bonds at the sn-1 and sn-3

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Table 8.13.1 Thermostable lipaseeproducing microorganisms and their properties. Enzyme properties Organism

Optimal temperature (8C)

Bacillus acidocaldariusa (esterase) Bacillus sp. RSJ-1 Bacillus strain J 33a Bacillus stearothermophilusa Bacillus thermocatenletusa Bacillus thermoleovorans ID-1 Geobacillus sp. Pseudomonas sp.

70

Pseudomonas sp. Pyrobaculum calidifontis Pyrococcus furiosusa (esterase) Pyrococcus horikoshii Pyrococcus horikoshii

Optimal pH

References

Manco et al. (1998)

50 60 68

8.0e9.0 8.0

Sharma et al. (2002) Nawani et al. (1998) Gupta et al. (1999)

60e70

8.0e9.0

Klibanov (1983)

70e75

7.5

Dong-Woo et al. (1999)

70 65

9.0 9.6

90 90

11.0

Abdel-Fattah (2002) Kulkurani and Gadre (1999) Rathi et al. (2000) Hotta et al. (2002)

100 97 95

Ikeda and Clark (1998) 5.6 7.0

Ando et al. (2002) Yan et al. (2000)

a Active at water immiscible solvents. From Haki, G.D., Rakshit, S.K., 2003. Developments in industrially important thermostable enzymes: a review. Bioresour. Technol. 89 (1), 17e34. Reproduced with permission.

positions of the triglyceride structure, while few lipase enzymes are active at the sn-2 position. Lipase enzymes exhibit optimal activity over a wide temperature range.

8.13.2 Applications of lipases Lipases are expected to become one of the fastest growing enzymes because of their expanding applications in pharmaceuticals, organic synthesis, and increasing penetration into the detergent industry and for production of biofuels (Salihu and Zahangir Alam, 2012; Chandra et al., 2020; Guerrand, 2017; Salihu and Alam, 2014; Houde et al., 2004; Hasan et al., 2006; Aravindan et al., 2007). Lipases are widely used for producing a variety of industrially important product as presented below.

Industrial applications of thermophilic/hyperthermophilic enzymes

8.13.2.1 Food industry The reactions with lipases can occur in aqueous as well as nonaqueous media. This has been proposed as a new strategy for the manufacturing of many products important in the food industry. Products derived from lipase catalysis are believed to be widely used in flavor synthesis, wine, emulsifiers, baked goods, dairy products, and dietary supplements. Additionally, lipase-catalyzed reactions have been used for modifying and upgrading inexpensive oils to nutritionally important triacylglycerols like low-calorie triacylglycerol, PUFA-enriched oil, and oleic acideenriched oils (Rajendran et al., 2009; Gupta et al., 2003). Some food processing applications of microbial lipase enzymes are presented in Table 8.13.2. 8.13.2.2 Detergents Use of lipase enzymes in the detergent industry is the most important application of these enzymes. Different enzyme formulations for different purposes are produced using different enzymatic systems such as lipase, protease, amylase, and cellulase. For using the enzymes in detergents, stability at higher pH and temperature is needed. Therefore, alkaline lipase and protease enzymes are used in formulations (Khoo and Ibrahim, 2003). A lipase from the Humicola strain that can break down oily stains has been identified by Novo Nordisk.

Table 8.13.2 Lipase applications in the food industry. Food industry

Action

Application

Bakery foods Dairy foods

Improvement in flavor Hydrolysis of milk fat Ripening of cheese modification of butter fat Improvement in aroma Improvement in quality Tranesterification Improvement in flavor

Shelf-life propagation Development of flavoring agents in milk, cheese, and butter

Beverages Food dressings Health food Meat and fish Oils and fats

Tranesterification Hydrolysis

Alcoholic beverages, e.g., wine and sake Mayonnaise, dressings whippings Health food Meat and fish product, fat removal Cocoa butter Margarine Fatty acids Glycerol Mono- and diglycerides

Based on Aravindan, R., Anbumathi, P., Viruthagiri, T., 2007. Lipase applications in food industry. Indian J. Biotechnol. 6, 141e158.

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Although many strategies for increasing yield using traditional and conventional methods have proven unsuccessful, molecular techniques have led to the cloning of the gene encoding this lipase (Hasan et al., 2006). In 1988, Novozymes launched Lipolase, the first commercial lipase enzyme developed for use in the detergents. The optimum pH for lipolase is 10.5e11.0 and is effective over a wider temperature range. It shows oxidation stability and stable to many other ingredients added in detergents, including surfactants, and is stable in proteolytic wash. This enzyme is commonly used in detergents for removing greasy stains such as fried foods, salad oils, soups, butters, fat-based sauces, human sebum, and lipstick. This lipase is now being added to many leading detergent brands around the world. Three lipolase variants: Lipolase Ultra, LipoPrime, and Lipex were later introduced by Novozymes (Houde et al., 2004). 8.13.2.3 Textile industry Lipase enzymes are extensively used in the textile industry for removing sizing lubricants, thus improving fabric absorbency and improving dye uniformity. These enzymes are also used for reducing the frequency of streaks and tears in denim’s wear system. Lipase is used along with a-amylase for commercial-scale desizing of denims and other cotton fabrics (Rowe, 2001). Han Huifang et al. (2003) studied the effects of lipases on the dewaxing activity of silk fibers and the effects of lipases and proteases during dewaxing and degumming on silk fibers. Weight loss rate, coloration, wettability, microstructure, gloss, and other properties showed that with proper use and dosage, the use of lipase was superior to no lipase. 8.13.2.4 Flavor development In the food industry, low-molecular-weight esters are widely used for development of flavor. Flavoring agents such as S-methylbutanethioate and S-methyl-3methylbutanethioate are main components of milk flavors, particularly cheese flavors and fruit flavors like banana and strawberry. All of these can be prepared by reactions catalyzed by lipases based on their exceptional specificity, higher reaction rates even at low mole fractions, and activity in organic solvents. Interesterification of tributyrin and hexanol by an immobilized lipase from Rhizomucor miehei (Liposyme IM-77) produced excellent flavor and aroma. Esterification of geraniol and citronellol with shorter chain fatty acids is commonly used for producing beverage (Chang et al., 2003; Shieh and Chang, 2001; Rajendran et al., 2009). 8.13.2.5 Cocoa butter equivalent Lipase-catalyzed transesterification is being used to produce cocoa butter-type triacylglycerols utilizing 1,3-regiospecific microbial lipases (Macrae and Hammond, 1985). Transesterification by regiospecific 1,3-lipases converts inexpensive fats such as palm oil fractions into 1,(3) palmitoyl, 2-oleoyl, 3(1) stearoylglycerol and 1(3) stearoyl, 2oleoyl, 3(1)-stearoylglycerol. It has numerous uses as a confectionery fat. Chocolate contains 30% cocoa butter to give it the necessary crystallization and melting properties. However, cocoa butter is usually very costly. Therefore, alternative sources of fat blends

Industrial applications of thermophilic/hyperthermophilic enzymes

were developed. This required an initial blend of palm medium fraction and stearic acid. Dehydration and lipase reaction follow. Further procedures inclusive of solvent fractionation and distillation are required to shape the favored product. This process is often used to make cocoa butter on a commercial scale (de Castro and Anderson, 1995). 8.13.2.6 Dairy industry Lipase enzymes are used for hydrolyzing the milk fat in the dairy industry. Other applications include accelerated ripening of cheese and lipolysis of fat, cream and butter (Salihu and Zahangir Alam, 2012). Lipases are widely used for modifying fatty acid chain lengths and improve cheese flavor. 8.13.2.7 Baking industry In the bakery industry, (phospho)lipases are used for replacing or supplementing traditional emulsifiers by breaking down wheat lipids to generate emulsifiable lipids in situ (Guerrand, 2017). Lipases during baking also improve the flavor of baked goods by releasing shorter chain fatty acids through esterification. In combination with other commonly used baking enzymes, lipase increases bread volume, improves crumb firmness, extends baked goods shelf life, and improves texture and softness (Robert, 2015). 8.13.2.8 Separation of racemic acid and alcohol Stereospecificity is a unique feature of lipases. It is commonly used to identify mixtures of racemic organic acids in nonmiscible two-phase systems through esterification and transesterification reactions (Klibanov, 1990; Sharma et al., 2001). The properties of both pure and crude lipase enzymes derived from C. rugosa in aqueous and organic solvents was studied by Tsai and Dordick (1996). Although the purified enzyme was found to be less active as compared to the crude enzyme in organic media, the presence of small amounts of water does not affect the activity of the purified enzyme by several times in the esterification of racemic 2-(4-chlorophenoxy) propanoic acid with n-butanol. Lipase enzymes are used to degrade racemic mixtures particularly in enantiomers like nonsteroidal anti-inflammatory drugs that are pharmacologically active especially in the (S)-enantiomeric form. 8.13.2.9 Oleochemical industry Acidolysis, hydrolysis, alcoholysis, and glycerolysis are the general reactions associated with the oleochemical industry. These reactions are energy consuming requiring high temperatures of 240e260
C and high pressures but the use of lipase enzymes can reduce the energy expenditure (Bornscheuer, 2000; Sharma et al., 2001). For example, the use of C. cylindracea lipase in soap production has been reported. Enzymatic methods yielded superior products at lower cost than traditional chemical methods (Saxena et al., 1999). Current trends in the oleochemical industry include using immobilized lipase enzymes for initiating several reactions (hydrolysis, alcoholysis, and glycerolysis) using mixed substrates.

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8.13.2.10 Biodegradation of plastic Biodegradable plastics are frequently employed as a clean technology measure to address environmental problems. But, there are also biodegradable plastics that are used interchangeably, albeit with differences. The major difference between biodegradable and biodestructible plastics is the degree and rate of degradation, with the former requiring further processing as opposed to the latter. The Fermentation Laboratory in Tsukuba, Japan, developed a strategy to make plastic completely destructible using lipase (de Castro and Anderson, 1995). It is based on the ability of lipase to break down polycaprolactones (aliphatic polyesters) and can be blended with plastics to speed up their degradation. 8.13.2.11 Biodiesel production The production of biodiesel as an alternative fuel from natural fats and oils is environmentally friendly as these do not contain sulfur and nitrogen compounds. This process significantly reduces greenhouse effect and fossil fuel air pollution (Li et al., 2009). It is possible to make biodiesel through chemical or enzymatic processes. Oils can be converted to methyl or other alcohol esters having short-chain in a single transesterification reaction with the use of lipase enzymes in organic solvents (Jaeger and Eggert, 2002). Therefore, some issues associated with chemical production, such as the recovery of glycerol and the requirement to use purified fats and oils as the main substrate, can be overcome by using enzymatic transesterification (Kramer, 1995). Residual oil from soybean, canola, and palm oil refinery wastes was methanolyzed by using lipase of R. oryzae in the presence of methanol as well as water recovered by extraction from hexane (Haas and Foglia, 2005). The best conversion to the methyl ester was found in palm oil after 96 h of reaction with about 55% yield. 8.13.2.12 Pharmaceutical industry Enzymes provide many advantages over chemical synthesis in the pharmaceutical industry, which justifies the increasing demand for enzymes. These benefits include enantio- and regioselectivity; milder conditions which avoid isomerization, epimerization, racemization, rearrangement reactions, and overexpression of the enzymes; reuse of the immobilized enzymes; process economics; and mutation of the enzymes for specific reactions. Lipases are able to resolve racemic mixtures by synthesizing single enantiomers. This property is currently exploited in the pharmaceutical industry for drug production. Actually, only one enantiomer of the drug is responsible for the desired therapeutic effect, with milder or fewer side effects seen with the use of the optically pure drug in comparison to those seen with the racemic mixture. Several lipases have been found suitable for use in the synthesis of several enantiomerically pure molecules such as carboxylic acids, amides, alcohols, and esters. These molecules are anti-inflammatory drugs, cancer drugs, antiviral drugs, antihypertensive drugs, anticholesterol drugs, anti-

Industrial applications of thermophilic/hyperthermophilic enzymes

Alzheimer’s drugs, and vitamin A (Houde et al., 2004; Kademi et al., 2004; Bonrath et al., 2002). 8.13.2.6.13 Other applications Lipases are also used in fatty acid unsaturation, paper industry, cosmetics industry, biosensor industry, and environmental protection industry. Lipases can be used for biological antifouling and oily waste treatment. Oil-contaminated soil or beach, fat processing plant or restaurant waste can be treated directly or indirectly with lipases from a variety of sources. In the wastewater treatment, adding a certain amount of lipase can effectively remove fat, reduce the cost of waste water treatment, and reduce secondary pollution. Treating marine oil with lipase and other ingredients breaks down the oil, making it the perfect nutrient for microbes to nourish.

Bibliography Abel, H., Marie, D., Nathalie, R., Danielle, D., Louis, S., Louis, C., 2000. Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit. Enzyme Microb. Technol. 26, 421e430. Aravindan, R., Anbumathi, P., Viruthagiri, T., 2007. Lipase applications in food industry. Indian J. Biotechnol. 6, 141e158. Balcao, V.M., Malcata, F.X., 1998. Lipase catalyzed modification of milk fat. Biotechnol. Adv. 16 (2), 309e341. Bonrath, W., Karge, R., Netscher, T., 2002. Lipase-catalyzed transformations as key-steps in the large-scale preparation of vitamins. J. Mol. Catal. B: Enzym. 19e20, 67e72. Bornscheuer, U.T., 2000. Enzymes in Lipid Modification. Wiley-VCH, Weinheim. Chandra, P., Enespa, S.R., Arora, P.K., 2020. Microbial lipases and their industrial applications: a comprehensive review. Microb. Cell Fact. 19 (1), 169. Chang, S.W., Shaw, J.F., Shieh, C.J., 2003. Optimization of enzymatically prepared Hexyl butyrate by lipozyme IM-77: enzymatic synthesis of Hexyl butyrate. Food Technol. Biotechnol. 41 (3), 237e243. de Castro, H.F., Anderson, W.A., 1995. Fine chemicals by biotransformation using lipases. Quimica Nova 18 (6), 544e554. Dong-Woo, L., You-Seok, K., Ki Jun, K., Byung-Chan, K., Hak-Jong, C., Doo-sik, K., Maggy, T., Yuryang, P., 1999. Isolation and characterisation of thermophilic lipase from Bacillus thermoleovorans ID-1. FEMS Microbiol. Lett. 179, 393e400. Guerrand, D., 2017. Lipases industrial applications: focus on food and agroindustries. OCL 24 (4), D403. Gupta, R., Rathi, P., Bradoo, S., 2003. Lipase mediated upgradation of dietary fats and oils. Crit. Rev. Food Sci. Nutr. 43 (6), 635e644. Haas, M.J., Foglia, T.A., 2005. Alternate feedstocks and technologies for biodiesel production. In: Knothe, G., Gerpen, J.V., Krahl, J. (Eds.), The Biodiesel Handbook. AOCS Press, Champaign, Illinois, pp. 50e69. Haki, G.D., Rakshit, S.K., 2003. Developments in industrially important thermostable enzymes: a review. Bioresour. Technol. 89 (1), 17e34. Han, H., Cui, Y., Cai, L., 2003. Removal method of glial in textile. Guangzhou Ind. Sci. Technol. 75 (19), 102e104. Hasan, F., Shah, A.A., Hameed, A., 2006. Industrial applications of microbial lipases. Enzym. Microb. Technol. 39, 235e251. Houde, A., Kademi, A., Leblanc, D., 2004. Lipases and their industrial applications: an overview. Appl. Biochem. Biotechnol. 118 (1e3), 155e170.

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Jaeger, K., Eggert, T., 2002. Lipases for biotechnology. Curr. Opin. Biotechnol. 13, 390e397. Jaeger, K.E., Dijkstra, B.W., Reetz, M.T., 1999. Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Ann. Rev. Microbiol. 53, 315e351. Kademi, A., Leblanc, D., Houde, A., 2004. In: Pandey, A. (Ed.), Concise Encyclopedia of Bioresource Technology. Haworth Press, Binghamton, NY, pp. 552e560. Khoo, M.L., Ibrahim, C.O., 2003. Development of alkaline lipase for the formulation of detergent. In: Nat. Biol. Conference. 14the16th December, Ipoh, Malaysia. Kim, H., Sung, M., Kim, M., Oh, T., 1994. Occurrence of thermostable lipase in thermophilic Bacillus sp. strain 398. Biosci. Biotech. Biochem. 58, 961e962. Klaus, D., 1998. An enzyme process for the physical refining of seed oils. Chem. Eng. Technol. 21, 278e281. Klibanov, A.M., 1990. Asymmetric transformations catalyzed by enzymes in organic solvents. Acc. Chem. Res. 23, 114e120. Kramer, W., 1995. The potential of biodiesel production. Oils Fats Int 11, 33e34. Lee, S., Rhee, J., 1993. Production and partial purification of a lipase from Pseudomonas putida 3 SK. Enzyme Microbiol. Technol. 15, 617e623. Li, H., Shen, B., Kabalu, J.C., Nchare, M., 2009. Enhancing the production of biofuels from cottonseed oil by fixed-fluidized bed catalytic cracking. Renew. Energy 34, 1033e1039. Luisa, M.R., Schmidt, C., Wahl, S., Sprauer, A., Schmid, R., 1997. Thermoalkalophilic lipase of Bacillus thermocatenulatus. Large scale production, purification and properties: aggregation behavior and its effect on activity. J. Biotechnol. 56, 89e102. Macrae, A.R., Hammond, R.C., 1985. Present and future applications of lipases. Biotech. Gen. Engin. Rev. 3, 193e219. Rajendran, A., Palanisamy, A., Thangavelu, V., 2009. Lipase catalyzed ester synthesis for food processing industries. Brazil. Arch. Biol. Technol. 52 (1), 207e219. Rathi, P., Sapna, B., Sexena, R., Gupta, R., 2000. A hyperthermostable, alkaline lipase from Pseudomonas sp. with the property of thermal activation. Biotechnol. Lett. 22, 495e498. Ray, A., 2012. Application of lipase in industry. Asian J. Pharm. Technol. 2 (2), 33e37. Robert, H., 2015. Lipases in Baking. Lipinov. Adebiotech, Romainville, FR. Rowe, H.D., 2001. Biotechnology in the textile/clothing industry: a review. J. Consum. Stud. Home Econ. 23, 53e61. Salihu, A., Zahangir Alam, M., 2012. Production and applications of microbial lipases: a review. Sci. Rese. Essays 7 (30), 2667e2677. https://doi.org/10.5897/SRE11.2023. Salihu, A., Alam, M.Z., 2014. Thermostable lipases: an overview of production, purification and characterization. Biosci. Biotech. Res. Asia 11 (3), 1095e1107. Saxena, R.K., Ghosh, P.K., Gupta, R., Davidson, W.S., Bradoo, S., Gulati, R., 1999. Microbial lipases: potential biocatalysts for the future industry. Curr. Sci. 77, 101e115. Schmidt, C., Sztajer, H., Stocklein, W., Menge, U., Schimid, R., 1994. Screening, purification and properties of a thermophilic lipase from Bacillus thermocatelnulatus. Biochem. Biophys. Acta 1214, 43e53. Sharma, R., Chisti, Y., Banerjee, U.C., 2001. Production, purification, characterization, and applications of lipases. Biotechnol. Adv. 19, 627e662. Shieh, C.J., Chang, S.W., 2001. Optimized synthesis of lipase-catalyzed hexyl acetate in n-hexane by response surface methodology. J. Agric. Food Chem. 49 (3), 1203e1207. Tsai, S.W., Dordick, J.S., 1996. Extraordinary enantiospecificity of lipase catalysis in organic media induced by purification and catalyst engineering. Biotechnol. Bioengin. 52, 296e300. Wu, X., Jaaskelainen, S., Linko, Y., 1996. An investigation of crude lipase for hydrolysis, esterification, and transesterification. Enzyme Microb. Technol. 19, 226e231. Xiao, F., Li, Z., Pan, L., 2017. Application of microbial lipase and its research progress. Prog. Appl. Microbiol. 8e14. Yan, F., Yong-Goe, J., Kazuhiko, I., Hiroyasu, I., Susumu, A., Tohru, Y., Hiroshi, N., Shugui, C., Ikuo, M., Yoshitsugu, K., 2000. Thermophilic phospholipase A2 in the cytosolic fraction from the archaeon Pyrococcus horikoshii. JAOCS 77, 1075e1084.

Industrial applications of thermophilic/hyperthermophilic enzymes

SUBCHAPTER 8.14

Laccase 8.14.1 Microbial sources and properties Laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) is a multicopper oxidase that catalyzes the oxidation of a wide range of phenolic and nonphenolic aromatic compounds. Because laccase enzymes have potential uses in numerous biotechnological fields, demand for them has increased in the recent years (Bajpai, 2017, 1999; Kunamneni et al., 2008). Laccases are extremely diverse in nature and have a wide variety of specificities. The isoenzyme laccase is primarily found in microbial communities and is encoded by a variety of genes and expressed in different organelles. Yoshida, a Japanese lacquer tree inventor, first described laccase in 1883 using the waste products of Rhus vernicifera. Many aromatic compounds, particularly those having electron-donating groups like phenol and aniline that use molecule oxygen as an electron acceptor, can be oxidized by laccases and polyphenol oxidases (Gianfreda et al., 1999). Wood-degrading fungi of the genus Basidiomycota are the most common and most efficient producers of laccase enzymes (Fernandez-Fernandez et al., 2013; Faria, 2010). The genus Trametes is one of the best studied fungi (Bajpai, 2017). Laccase activity has been found in several fungi (Table 8.14.1) and the enzyme has been purified from many of them. Basidiomycetes, for instance, are white rot fungi that secrete extracellular enzymes, which can break down lignin. One of the enzymes is laccases, and the others are lignin peroxidases and manganese peroxidases (Elisashvili and Kachlishvili, 2009). Laccase is also produced by the saprophyte Ascomycetes (Fernandez-Fernandez et al., 2013). Solid-state fermentation is an interesting technique for production of laccases. This is due to the reason that it imitates the natural environment of white rot fungi but with submerged fermentation also; good results have been also obtained. Microorganism-producing laccase enzymes with high activity in submerged fermentation are Coriolus hirsutus (83,830 U/L), Trametes pubescens (740,000 U/L), Pleurotus ostreatus (3500 U/L), T. versicolor (16,000 U/L, Neurospora crassa (10,000 U/L); Pycnoporus cinnabarinus (10,000 U/L), and T. hirsuta (19,400 U/L) (Font et al., 2003; Koroleva et al., 2002; Lenz and Holker, 2004; Meza et al. al., 2006; Luke and Burton, 2001; RodriguezCouto et al., 2006; Galhaup et al., 2002). Laccase enzymes play a very important role in the biodegradation of lignin and use chemical or natural mediators for oxidizing refractory aromatic compounds at redox potentials exceeding their own (Coll et al., 1993; Xu, 1996; Camarero et al., 2005). Due to their broader substrate specificity as well as broader reactivity, laccases, and laccase

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Table 8.14.1 Laccase-producing fungi.

Pleurotus florida Pleurotus ostreatus P. ostreatus Pleurotus pulmonarius Pleurotus sajor-caju Pleurotus sp. Pleurotus tailandia Trametes hirsuta Trametes versicolor Coriolopsis polyzona Coriolopsis rigida Grifola frondosa Lentinula edodes Phlebia rufa Based on Guimar~aes, L.R.C., Woiciechowski, A.L., Karp, S.G., Coral, J.D., Zandon
a Filho, A., Soccol, C.R., 2017. Chapter 9dLaccases. In: Current Developments in Biotechnology and Bioengineering, Elsevier, pp. 199e216.

mediator systems have great biotechnological potential (Madhavi and Lele, 2009; Marzoorati et al., 2005; Tayhas et al., 1999; Trudeau et al., 1997; Wesenberg et al., 2003; Call, 1994).

8.14.2 Applications of laccases Laccase is now considered by many to be the ideal green catalyst. There is growing interest in using laccase as an alternative to traditional chemical processes in the forestry, textile, and pharmaceutical industries. Laccases also show potential applications in many other fields, like food, paints, cosmetics, organic synthesis and bioremediation (Bajpai, 2017). Laccase has also been utilized for the production of ethanol from lignocelluloses. In fact, research is ongoing in the potential use of laccases for commercial applications. 8.14.2.1 Forestry The forest products sector includes companies that grow, harvest, and/or process wood and wood fibers, manufacture pulp and paper and board products, and process traditional wood products. In the pulp and paper industry, efforts are being made to develop environment friendly bleaching technologies, reduction in energy consumption in pulping, bleaching and paper making processes, efficient recycling of fiber, reduction of wastewater generation, and detoxification to conserve freshwater and protect natural water bodies. Producers of particle board, medium-density fiberboard, structural panels, and solid wood composites reduce the cost of manufacturing and harmful release of

Industrial applications of thermophilic/hyperthermophilic enzymes

formaldehyde from adhesives, while improving the reusability of product (Maloney, 1996; Sellers, 2001). In order to meet these challenges, innovative approaches are required for reducing the quantity of binder while maintaining the same product quality. One more issue associated with wood products is the need to improve surface as well as bulk properties for improving permanence, range, and compatibility with other materials for use in the hybrid products like wood plastic composites (Sellers, 2001). The cost of pulping fiberboard can also be an issue, as the manufacturing process for fiberboard involves pulping. Due to its diversity, laccases are one of the most significant enzymes in the forest industry. Laccase enzymes can be used almost throughout the papermaking chain because of its high redox potential (Leonowicz et al., 2001; Yaropolov et al., 1994; Mayer and Staples, 2002). In the forest industry, laccases are being investigated for the functionalization of cellulose fibers. Additionally, a new durable and stable lignocellulose material is engineered using a laccase-catalyzed grafting process with phenolic compounds. Laccase has been investigated to improve the compressibility of engineered wood panels through in situ enzymaticelignin coupling without the use of formaldehyde-containing toxic adhesives. In order to overcome the aforementioned difficulties, the forest products industry is more and more relying on enzyme technology (Bajpai et al., 1999, 2012; Borch et al., 2003; Ragauskas, 2002; Viikari, 2002). 8.14.2.2 Textile industry In the textile industry, the use of laccases is rapidly increasing. Laccases are used for decolorizing textile effluents, bleaching textiles and even synthesizing dyes (Bajpai, 2017; Abadulla et al., 2000; Rodríguez Couto et al., 2005; 2006; Rodriguez Couto, 2012; Setti et al., 1999; Upadhyay et al., 2016; Blanquez et al., 2004; Hou et al., 2004; Madhavi and Lele, 2009). 8.14.2.3 Food industry Laccase enzymes show great potential for use in the food industry. They are used as food additives in food processing (Mate and Alclade, 2016; Bajpai, 2017; Brijwani et al., 2010; Osma et al., 2010). In the food industry, laccases are used to facilitate homopolymerization and heteropolymerization reactions. Laccase is widely used to produce inexpensive and healthy foods. Laccase has the potential for making food processing more cost effective and environment friendly. 8.14.2.4 Personal care and medical applications The potential personal care and medical applications of laccase have attracted vigorous research activity because of its specificity and bio-based nature. Some products produced by laccase enzymes are detoxifying, antibacterial, or active personal care products. Laccase enzymes can be used for synthesing complex pharmaceuticals such as anti-inflammatory drugs, anesthetics, tranquilizers, and antibiotics (Bajpai, 2017).

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8.14.2.5 Bioremediation The use of laccase enzyme with and without redox mediators is of great interest. Laccase or laccase mediator systems oxidize xenobiotic compounds to release highly bioavailable, lesser toxic products, which can be more easily removed by physical and or mechanical means (Chandra and Chowdhury, 2015; Bajpai, 2017). 8.14.2.6 Organic synthesis Laccase has emerged as an exciting catalyst in the field of synthetic organic chemistry over the last decades. In organic synthesis, the usefulness of laccases is reflected by the variety of chemical transformations they produce (Piscitelli et al., 2013; Nakamura, 1960; Kunamneni et al., 2008; Nicotra et al., 2004a,b; Mogharabi and Faramarzi, 2014). 8.14.2.7 Biofuel Laccases play an important role in lignin degradation and has been widely investigated for potential applications in biomass processing. Laccases can be used for detoxification of lignocelluloses after thermochemical pretreatment and the production of biofuels and value-added products from biomass (Kudanga and Le Roes-Hill, 2014; Mate and Alclede, 2016). 8.14.2.8 Nanobiotechnology Nanotechnology research has expanded significantly in recent years. Laccases are being researched for use in biosensors and biofuel cells because they are able to catalyze electron transfer reactions and do not need additional cofactors. Therefore, laccase can be utilized as a biosensor or bioreporter. Applications of bioreporters are highly sought-after in the field of extremely sensitive diagnostics. Biosensors based on laccases have been created for electroimmunoassay and for detecting morphine and codeine, catecholamines, and plant flavonoids (D’Souza, 2001; Ferry and Leech, 2005; Franzoi et al., 2009; JaroszWilkołazka et al., 2004; Kunamneni et al., 2008; Rodríguez-Delgado et al., 2015; Rodrıguez Couto and Toca Herrera, 2006).

Bibliography Abadulla, E., Tzanov, T., Kosta, S., Robra, K.H., Cavaco-Paulo, A., Gubitz, G., 2000. Decolourization and detoxification of textile dyes with a laccase from Trametes hirsute. Appl. Environ. Microbiol. 66, 3357e3362. Bajpai, P., 1999. Application of enzymes in the pulp and paper industry. Biotechnol. Prog. 15, 147e157. Bajpai, P., 2012. Biotechnology in Pulp and Paper Processing. Springer, New York, 412 pp. Bajpai, P., 2017. Laccases and Their Applications, first ed. Bookboon. 169 pp. Baldrian, P., 2006. Fungal laccases: occurrence and properties. FEMS Microbiol. Rev. 30, 215e42. Bl
anquez, P., Casas, N., Font, X., Gabarrell, M., Sarr
a, M., Caminal, G., 2004. Mechanism of textile metal dye biotransformation by Trametes versicolor. Water Res. 38, 2166e2172. Borch, K., Franks, N., Lund, H., Xu, H., Luo, J., 2003. Oxidizing enzymes in the manufacturing of paper materials. Patent (USA) US 2003/0124710 A1. Brijwani, K., Rigdon, A., Vadlani, P.V., 2010. Fungal laccases: production, function, and applications in food processing. Enzyme Res. 2010, 149748.

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Call HP (1994). WO 9429510. Camarero, S., Ibarra, D., Martinez, M.J., Martinez, A.T., 2005. Lignin-derived compounds as efficient laccase mediators for decolorization of different types of recalcitrant dyes. Appl. Environ. Microbiol. 71, 1775e1784. Chandra, R., Chowdhary, P., 2015. Properties of bacterial laccases and their application in bioremediation of industrial wastes. Environ. Sci. Process Impacts 17, 326e342. Coll, P.M., Abalos, J.M.F., Villanueva, J.R., Santamaria, R., Perez, P., 1993. Purification and characterization phenoloxidase (Laccase) from the lignin degrading basidiomycete PM1. Appl. Environ. Microbiol. 59, 2607e2613. D’Souza, S.F., 2001. Microbial biosensors. Biosens. Bioelectr. 16, 337e353. Elisashvili, V., Kachlishvili, E., 2009. Physiological regulation of laccase and manganese peroxidase production by white-rot basidiomycetes. J. Biotechnol. 144, 37e42. Faria, R.A., 2010. Estudo da producxaeo de enzimas lignolı’ticas por Ceriporiopsis subvermispora. USP, Sao Paulo, 102 pp.
Moldes, D., 2013. Recent developments and applications of Fern
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an, M.A., immobilized laccase. Biotechnol. Adv. 31 (8), 1808e1825. Ferry, Y., Leech, D., 2005. Amperometric detection of catecholamine neurotransmitters using electrocatalytic substrate recycling at a laccase electrode. Electro Anal. 17, 2113e2119. Font, X., Caminal, G., Gabarrell, X., Romero, S., Vicent, M.T., 2003. Black liquor detoxification by laccase of Trametes versicolor pellets. J. Chem. Technol. Biotechnol. 78, 548e54. Franzoi, A.C., Peralta, R.A., Neves, A., Vieira, I.C., 2009. Biomimetic sensor based on Mn (III) and Mn (II) complex as manganese peroxidise mimetic for determination of rutin. Talanta 78, 221e226. Galhaup, C., Wagner, G., Hinterstoisser, B., Haltrich, D., 2002. Increased production of laccase by the wood degrading basidiomycete Trametes pubescens. Enzyme Microb. Technol. 30, 529e36. Gianfreda, L., Xu, F., Bollag, J.M., 1999. Laccases: a useful group of oxidoreductive enzymes. Biorem. J. 3 (1), 1e25. Guimar~aes, L.R.C., Woiciechowski, A.L., Karp, S.G., Coral, J.D., Zandon
a Filho, A., Soccol, C.R., 2017. Chapter 9dLaccases. In: Current Developments in Biotechnology and Bioengineering. Elsevier, pp. 199e216. Hou, H., Zhou, J., Wang, J., Du, C., Yan, B., 2004. Enhancement of laccase production by Pleurotus ostreatus and its use for the decolourization of anthraquinone dye. Process Biochem. 39, 1415e1419. Jarosz-Wilkołazka, A., Ruzgas, T., Gorton, L., 2004. Use of laccase-modified electrode for amperometric detection of plant flavonoids. Enzyme Microb. Technol. 35, 238e241. Kierulff, J.V., 1997. Denim bleaching. Text. Horiz. 17, 33e36. Koroleva, O.V., Gavrilova, V.P., Stepanova, E.V., Lebedeva, V.I., Sverdlova, N.I., Landesman, E.O., Yavmetdinov, I.S., Yaropolov, A.I., 2002. Production of lignin modifying enzymes by co-cultivated white-rot fungi Cerrena maxima and Coriolus hirsutus and characterization of laccase from Cerrena maxima. Enzyme Microb. Technol. 30, 573e580. Kudanga, T., Le Roes-Hill, M., 2014. Laccase applications in biofuels production: current status and future prospects. Appl. Microbiol. Biotechnol. 98, 6525e6542. Kunamneni, A., Plou, F.J., Antonio Ballesteros, A., Alcalde, M., 2008a. Laccases and their applications: a patent review. Recent Patents Biotechnol. 15, 10e24. Kunamneni, A., Camarero, S., García-Burgos, C., Plou, F.J., Ballesteros, A., Alcalde, M., 2008b. Engineering and applications of fungal laccases for organic synthesis. Microb. Cell Fact. 7, 1e17. Lenz, J., Holker, U., 2004. Trickle-film processing: an alternative for producing fungal enzymes. BioFor. Eur. 6, 55e7. Leonowicz, A., Cho, N.S., Luterek, J., Wilkolazka, A., Wojtas-Wasilewska, M., Matuszewska, A., 2001. Fungal laccase: properties and activity on lignin. J. Basic Microbiol. 41, 185e227. Li, K., Xu, F., Eriksson, K.-H.L., 1999. Comparison of fungal laccases and redox mediators in oxidation of a nonphenolic lignin model compound. Appl. Environ. Microbiol. 65, 2654e2660. Luke, A.K., Burton, S.G., 2001. A novel application for Neurospora crassa: progress from batch culture to a membrane bioreactor for the bioremediation of phenols. Enzyme Microb. Technol. 29, 348e56. Madhavi, V., Lele, S.S., 2009. Laccase: properties and applications. BioResources 4 (4), 1694e1717. Maloney, T.M., 1996. The family of wood composite materials. For. Prod. J. 46, 19e26. Marzoorati, M., Danieli, B., Haltrich, D., Riva, S., 2005. Selective laccase-mediated oxidation of sugars derivatives. Green Chem. 7, 310e315.

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Mate, M.D., Alclade, M., 2016. Laccase: a multi-purpose biocatalyst at the forefront of biotechnology. Microb. Biotechnol. 2016. https://doi.org/10.1111/1751-7915.12422. Mayer, A.M., Staples, R.C., 2002. Laccase: new functions for an old enzyme. Phytochemistry 60, 551e565. Meza, J.C., Sigoillot, J.C., Lomascolo, A., Navarro, D., Auria, R., 2006. New process for fungal delignification of sugar-cane bagasse and simultaneous production of laccase in a vapor phase bioreactor. J. Agric. Food Chem. 54, 3852e8. Minussi, R.C., Pastore, G.M., Duran, N., 2002. Potential applications of laccase in the food industry. Trends Food Sci. Technol. 13, 205e216. Mogharabi, M., Faramarzi, M.A., 2014. Laccase and laccase-mediated systems in the synthesis of organic compounds. Adv. Synth. Catal. 356, 897e927. Nakamura, T., 1960. On the process of enzymatic oxidation of hydroquinone. Biochem. Biophys. Res. Commun. 2, 111e113. Nicotra, S., Cramarossa, M.R., Mucci, A., Pagnoni, U.M., Riva, S., Forti, L., 2004a. Biotransformation of resveratrol: synthesis of trans-dehydrodimers catalyzed by laccases from Myceliophtora thermophyla and from Trametes pubescens. Tetrahedron 60, 595e600. Nicotra, S., Intra, A., Ottolina, G., Riva, S., Danieli, B., 2004b. Laccase-mediated oxidation of the steroid hormone 17b-estradiol in organic solvents. Tetrahedron-Asymmetr 15, 2927e2931. Osma, J.F., Toca-Herrera, J.L., Rodríguez-Couto, S., 2010. Uses of laccases in the food industry. Enzyme Res. 2010, 918761. Palonen, H., Viikari, L., 2004. Role of oxidative enzymatic treatments on enzymatic hydrolysis of softwood. Biotechnol. Bioeng. 86, 550e557. Piscitelli, A., Pezzella, C., Lettera, V., Giardina, P., Faraco, V., Sannia, G., 2013. Fungal laccases: structure, function and applications. In: Polizeli, M.T.M., Rai, M. (Eds.), Fungal Enzymes. CRC Press, Boca Raton, FL, pp. 113e151. Ragauskas, A., 2002. Biotechnology in the pulp and paper biotechnology. A challenge for change. Prog. Biotechnol. 21, 7e12. Rodríguez-Couto, S., Rodríguez, A., Paterson, R.R.M., Lima, N., Teixeira, J.A., 2006. High laccase activity in a 6 l airlift bioreactor by free cells of Trametes hirsuta. Lett. Appl. Microbiol. 42, 612e6. Rodríguez Couto, S., L
opez, E.R., Sanrom
an, M.A., 2006. Utilisation of grape seeds for laccase production in solid-state fermentors. J. Food Eng. 74, 263e267. Rodríguez Couto, S., Sanrom
an, M.A., G€ ubitz, G.M., 2005. Influence of redox mediators and metal ions on synthetic acid dye decolourization by crude laccase from Trametes hirsuta. Chemosphere 58, 417e422. Rodriguez Couto, S., Toca Herrera, J.L., 2006. Industrial and biotechnological applications of laccases: a review. Biotechnol. Adv. 24 (5), 500e513. Rodríguez-Couto, S., 2012. Laccases for denim bleaching: an eco-friendly alternative. Open Text. J. 5, 1e7. Rodríguez-Delgado, M.M., Alem
an-Nava, G.S., Rodríguez-Delgado, J.M., Dieck-Assad, G., MartínezChapa, S.O., Barcel
o, D., Parra, R., 2015. Laccase-based biosensors for detection of phenolic compounds. Trend Anal. Chem. 74, 21e45. Sellers, T., 2001. Wood adhesive innovations and applications in North America. For. Prod. J. 51, 12e22. Setti, L., Giuliani, S., Spinozzi, G., Pifferi, P.G., 1999. Laccase catalyzed oxidative coupling of 3-methyl 2benzothiazolinone hydrazone and methoxyphenols. Enzyme Microb. Technol. 25, 285e289. https:// doi.org/10.1016/S0141-0229(99)00059-9. Tayhas, G., Palmore, R., Kim, H.-H., 1999. Electro-enzymatic reduction of dioxygen to water in the cathode compartment of a biofuel cell. J. Electroanal. Chem. 565, 110e117. Trudeau, F., Diagle, F., Leech, D., 1997. Reagent less mediated laccase electrode for the detection of enzyme modulators. Anal. Chem. 69, 882e886. Upadhyay, P., Shrivastava, R., Agrawal, P.K., 2016. Bioprospecting and biotechnological applications of fungal laccase. 3 Biotech 6, 1e12. Viikari, L., 2002. Trends in pulp and paper biotechnology. Prog. Biotechnol. 21, 1e5. Wesenberg, D., Kyriakides, I., Agathos, S.N., 2003. White-rot fungi and their enzymes for the treatment of industrial dye effluents. Biotechnol. Adv. 22, 161e187. Xu, F., 1996. Oxidation of phenols, anilines, and benzenethiols by fungal laccases: correlation between activity and redox potentials as well as halide inhibition. Biochem 35, 7608e7614. Yaropolov, A.I., Skorobogatko, O.V., Vartanov, S.S., Varfolomeyev, S.D., 1994. Laccase: properties, catalytic mechanism and applicability. Appl. Biochem. Biotechnol. 49, 257e280.

Industrial applications of thermophilic/hyperthermophilic enzymes

SUBCHAPTER 8.15

Phytase 8.15.1 Microbial sources and properties Phytase (myoinositol hexakisphosphate phosphohydrolase, EC 3.1.3.8) catalyzes the hydrolysis of phytate (myoinositol (1,2,3,4,5,6)-hexakisphosphate) to inorganic phosphate and less phosphorylated myo-inositol derivatives (Zhu et al., 2011). The global phytase market size is expected to reach over US$1 billion by 2025 and is expected to grow at a CAGR of around 6.3% in terms of revenue over the next 5 years (https://www. acumenresearchandconsulting.com/phytasemarketplace). Phytase is a phosphatase enzyme. It catalyzes the hydrolysis of phytic acid. It breaks down phytic acid and improves the availability and digestibility of calcium, zinc, magnesium, sodium, and amino acids in broilers and pigs. Recently, thermostable phytases have received a lot of attention due to the reduction in phytase activity seen upon exposure to heat during feed pelleting. Phytase is approved as a feed additive in commercial poultry diets for improving digestibility of phosphorus. Many studies have shown that dietary phytase supplementation may also improve energy and amino acid utilization efficiency (Selle et al., 2000; Amerah et al., 2014). But, the use of phytase in feed is limited because higher temperatures during pelleting can lead to phytase inactivation. Apart from this thermostability issue, there is controversy about the effects of industrial phytases on protein and amino acid availability (Zhang et al., 1999; Adeola and Sands, 2003; Snow et al., 2003). Phytases, or phytases, have attracted much research interest ecologically and industrially due to their roles in pollution control and diverse applications. Phytases constitute the major phytate phosphatase enzymes advised for addition to the diet of nonruminant animals such as swine, poultry, and fish (Singh and Satyanarayana, 2014). In addition to enhancing an animal’s intake of phosphate, calcium, magnesium, zinc, and iron, phytase supplementation enhances nitrogen retention capacity through effective use of proteins and amino acids (Lei and Stahl, 2000). Specifically, adding phytase to the diet reduces global warming, acidification, and eutrophication by 17, 110, and 700 times compared to the use of monocalcium phosphate in the diet (Nielsen and Wenzel, 2007). The utilization of phytase-induced plant growth promotion has also been practiced in agriculture, mostly due to improved exploitation of inorganic phosphates by plants (Memenza et al., 2016; Li et al., 2007). The commercial importance of phytase is illustrated by its role in the food industry, production of biofuel, and detoxification of industrial waste. The occurrence of phytase in bread-making processes in the food industry like steeping, malting, germination, fermentation, and vegetable protein production has called this enzyme a boom in the food industry (Fredrikson et al., 2001; Mittal et al.

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al. 2013). Incorporating phytase into corn-based biofuel production also reduces waste disposal constraints and contributes to savings in ethanol yield (Shetty et al., 2008). Phytases are ubiquitous in nature as they are found in microorganisms, plants, and animals. For instance, enzyme-degrading phytate were reported in the blood of calves, birds, reptiles, and fishes and also in plants like maize, soybean wheat, and rice (Hamada, 1996; McCollum and Hart, 1908; Nagai and Funahashi, 1962; Nakano et al., 1999; Rapoport et al., 1941; Huebel and Beck, 1996; Hayakawa et al., 1989; Maugenest et al., 1999). Microbial phytases are the most promising candidate to produce phytases for biotechnological applications. The majority of these enzymes is members of the histidine acid phosphatase and alkaline phosphatase subfamilies and exhibit a variety of biochemical characteristics, stereospecificity, and kinetics. In contrast to bacteria, yeasts and molds have extracellular phytate-degrading enzymes, whereas Bacillus and Enterobacter have these enzymes primarily associated with the cell. These enzymes are periplasmic proteins in Escherichia coli, whereas in Selenomonas ruminantium and Mitsuokella multiacidus, phytatedegrading activity was connected to the outer membrane (D’Silva et al., 2000; Kim et al., 1998; Greiner et al., 1993). The histidine acid phosphatase family includes glycosylated phytoses that are derived from yeast and fungi (Wyss et al., 1999). Nakamura et al. (2000) reported phytase activity in 35 yeast species and demonstrated that these yeast species varied in terms of pH and thermal characteristics. Phytases from Basidiomycetes (belonging to 3-phytases) and Ascomycetes (belonging to 6-phytases) have different amino acid sequences, which cause them to fall into different clades in the tree according to the analysis of phylogenetic relationships between yeast and fungal phytases. The Ascomycetes fungi and yeasts share a significant amount of amino acid sequence similarity because they both belong to the phylum Ascomycota. Therefore, yeast phytases are grouped with those from the fungi Neosartorya and Monascus (Gontia-Mishra and Tiwari, 2013). Due to several properties like substrate specificity, tolerance to proteolysis, and catalytic effectiveness, bacterial phytases have proven to be true alternatives to fungal enzyme (Konietzny and Greiner, 2002). Some strains of bacteria like Pseudomonas, Bacillus species, and Klebsiella produces phytase enzyme (Sasirekha et al., 2012; Reddy et al., 2015; Tambe et al., 1994; Mittal et al., 2012; Richardson and Hadobas, 1997). Periplasmic phytases from E. coli are more efficient than commercial phytases from Aspergillus niger in releasing phosphorus phytate in swine and poultry diets (Augspurger et al., 2003; Greiner et al., 1993).

8.15.2 Applications of phytase enzymes Phytase enzymes have wide applications in industry, agriculture, and biotechnology because of their ability to break down keratin.

Industrial applications of thermophilic/hyperthermophilic enzymes

8.15.2.1 Dietary supplement Phytases are of paramount importance for biotechnological applications in animal and human nutrition processing and manufacturing due to their ability to enhance phosphorus efficiency and minimize phytic acid concentrations in livestock and feed. Monogastric animals, for instance, pigs, poultry, and fish have low or no gastrointestinal phytase levels and cannot utilize phytic acid and phosphorus in food and feed. Consequently, inorganic phosphate supplementation is needed for meeting nutritional and growth requirements, increasing feed costs and phosphorus load. Phytase enzymes are important in the feed sector as they improve the phosphorus digestion process (infinitabiotech.com/ blog/applications-of-phytase-enzyme/). 8.15.2.2 Food additive Phytic acid is abundant in grains and whole grains in many breads and doughs, so phytase is used as a food and feed additive in the fermentation processes and various bread-making applications. For example, the phytase enzyme from A. ficuum has been used for dephosphorylation of legumes. Up to 78% of phytic acid was removed after 15 h incubation of soybean meal with fungal phytase (infinitabiotech.com/blog/applications-of-phytaseenzyme/). 8.15.2.3 Application for promoting plant growth Phosphorus is an important and necessary component of cells, playing a role in energy metabolism, acid production, and biosynthesis of cell membrane. It is also an essential macronutrient for plant growth and development processes. Soil deficiency of phosphorus is a main problem for farmers all over the world. Most soils have substantial amounts of total soil phosphorus. It can be found in inorganic as well as organic forms. Phytic acid is the main source of organic phosphorus in the soil. This accounts for 10%e50% of available organic phosphorus. Therefore, the enzyme phytase is necessary for digesting it. 8.15.2.4 Therapeutic applications In several parts of the world, people use plant-based foods as their primary nutritional source of raw materials as these are rich in nutrients (carbohydrates, protein, fiber, vitamins) and nonnutrients. Phytic acid is the main phosphorus storage molecule in seeds and grains and account for up to 90% of total seed phosphorus. It causes mineral deficiencies in humans by forming complexes with food elements like, magnesium, calcium, iron, zinc, copper, and manganese. 8.15.2.5 Other commercially available phytase enzyme products Phytase is an enzyme that is beneficial in animal nutrition. It boasts the top sales share in the industry at 83.6%. Phytase is added to approximately 70% of monogastric feeds. A. niger provided the first industrial phytase classified as a 3-phytase. Later, Peniophora lycii

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was used to produce an industrial product, 6-phytase. Many products from fungal phytases have been manufactured and commercialized by various companies over the years. Phytase enzymes are produced on a commercial scale using either phytate-producing bacteria or recombinant DNA technology. Fig. 8.15.1 shows significance of phytases (Rebello et al., 2017).

Figure 8.15.1 Significance of phytase. (A) Phytase in preventing environmental pollution. (B) Phytase in preventing antinutritional properties of phytate in body. (From Rebello, S., Jose, L., Sindhu, R., Aneesh, E.M., 2017. Molecular advancements in the development of thermostable phytases. Appl. Microbiol. Biotechnol. 101 (7), 2677e2689. Reproduced with permission.)

Industrial applications of thermophilic/hyperthermophilic enzymes

Bibliography Adeola, O., Sands, J.S., 2003. Does supplemental dietary microbial phytase improve amino acid utilization? A perspective that it does not. J. Anim. Sci. 81 (E.Suppl. 2), E78eE85. https://doi.org/10.2527/ 2003.8114_suppl_2E78x. Amerah, A.M., Plumstead, P.W., Barnard, L.P., Kumar, A., 2014. Effect of calcium level and phytase addition on ileal phytate degradation and amino acid availability of broilers fed corn-based diets. Poultry Sci. 93 (4), 906e915. Augspurger, N.R., Webel, D.M., Lei, X.G., Baker, D.H., 2003. Efficacy of an E. coli phytase expressed in yeast for releasing phytate-bound phosphorus in young chicks and pigs. J. Anim. Sci. 81, 474e483. D’Silva, C.G., Bae, H.D., Yanke, L.J., Cheng, K.J., Selinger, L.B., 2000. Localization of phytase in Selenomonas ruminantium and Mitsuokella multiacidus by transmission electron microscopy. Can. J. Microbiol. 46, 391e395. Fredrikson, M., Biot, P., Alminger, M.L., Carlsson, N.-G., Sandberg, A.-S., 2001. Production process for high-quality pea-protein isolate with low content of oligosaccharides and phytate. J. Agric. Food Chem. 49, 1208e1212. Gibson, D., 1987. Production of extracellular phytase from Aspergillus ficuum on starch media. Biotechnol. Lett. 9, 305e310. Gontia-Mishra, I., Tiwari, S., 2013. Molecular characterization and comparative phylogenetic analysis of phytases from fungi with their prospective applications. Food Technol. Biotechnol. 51, 313e326. Greiner, R., Konietzny, U., Jany, K.D., 1993. Purification and characterization of two phytases from Escherichia coli. Arch. Biochem. Biophys. 303, 107e113. Hamada, J.S., 1996. Isolation and identification of the multiple forms of soybean phytases. J. Am. Oil Chem. Soc. 73, 1143e1151. Hayakawa, T., Toma, Y., Igaue, I., 1989. Purification and characterization of acid phosphatases with or without phytase activity from rice bran. Agric. Biol. Chem. 53, 1475e1483. Huebel, F., Beck, E., 1996. Maize root phytase. Purification, characterization, and localization of enzyme activity and its putative substrate. Plant Physiol. 112, 1429e1436. Kim, Y.O., Kim, H.K., Bae, K.S., Yu, J.H., Oh, T.K., 1998. Purification and properties of a thermostable phytase from Bacillus sp. DS11. Enzyme Microb. Technol. 22, 2e7. Konietzny, U., Greiner, R., 2002. Molecular and catalytic properties of phytate-degrading enzymes (phytases). Int. J. Food Sci. Technol. 37, 791e812. Lei, X.G., Stahl, C.H., 2000. Nutritional benefits of phytase and dietary determinants of its efficacy. J. Appl. Anim. Res. 17, 97e112. Lei, X., Stahl, C., 2001. Biotechnological development of effective phytases for mineral nutrition and environmental protection. Appl. Microbiol. Biotechnol. 57, 474e481. Li, X., Wu, Z., LiW, Y.R., Li, L., Li, J., Li, Y., LiM, 2007. Growth promoting effect of a transgenic Bacillus mucilaginosus on tobacco planting. Appl. Microbiol. Biotechnol. 74, 1120e1125. Maugenest, S., Martinez, I., Godin, B., Perez, P., Lescure, A.M., 1999. Structure of two maize phytase genes and their spatio-temporal expression during seedling development. Plant Mol. Biol. 39, 503e514. McCollum, E.V., Hart, E.B., 1908. On the occurrence of a phytin splitting enzyme in animal tissue. J. Biol. Chem. 4, 497e500. Memenza, M., Mostacero, E., Camarena, F., Zuniga, D., 2016. Disease control and plant growth promotion (PGP) of selected bacterial strains in Phaseolus vulgaris. In: Biological Nitrogen Fixation and Beneficial Plant-Microbe Interaction. Springer, pp. 237e245. Mittal, A., Gupta, V., Singh, G., Yadav, A., Aggarwal, N.K., 2013. Phytase: a boom in food industry. Octa J. Biosci. 1 (2), 158e169. Mittal, A., Singh, G., Goyal, V., Yadav, A., Aggarwal, N.K., 2012. Production of phytase by acidothermophilic strain of Klebsiella sp.DB-3FJ711774.1 using orange peel flour under submerged fermentation. Innov. Roman. Food Biotechnol. 10, 1827. Nagai, Y., Funahashi, S., 1962. Phytase (myo-inositol hexaphosphate phosphohydrolase) from wheat bran. Agric. Biol. Chem. 26, 794e803. Nakamura, Y., Fukuhara, H., Sano, K., 2000. Secreted phytase activities of yeast. Biosci. Biotechnol. Biochem. 64, 841e844. Nakano, T., Joh, T., Tokumoto, E., Hayakawa, T., 1999. Purification and characterization of phytase from bran of Triticum aestivum L. Cv. Nourin #61. Food Sci. Technol. Res. 5, 18e23.

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Nielsen, P.H., Wenzel, H., 2007. Environmental assessment of RonozymeÒ P5000 CT phytase as an alternative to inorganic phosphate supplementation to pig feed used in intensive pig production. Int. J. LCA 12, 514e520. Rapoport, S., Leva, E., Guest, G.M., 1941. Phytase in plasma and erythrocytes of vertebrates. J. Biol. Chem. 139, 621e632. Rebello, S., Jose, L., Sindhu, R., Aneesh, E.M., 2017. Molecular advancements in the development of thermostable phytases. Appl. Microbiol. Biotechnol. 101 (7), 2677e2689. Reddy, C.S., Achary, V.M., Manna, M., Singh, J., Kaul, T., Reddy, M.K., 2015. Isolation and molecular characterization of thermostable phytase from Bacillus subtilis (BSPhyARRMK33). Appl. Biochem. Biotechnol. 175, 3058e3067. Richardson, A.E., Hadobas, P.A., 1997. Soil isolates of Pseudomonas spp. that utilize inositol phosphates. Can. J. Microbiol. 43, 509e516. Sasirekha, B., Bedashree, T., Champa, K.L., 2012. Statistical optimization of medium components for improved phytase production by Pseudomonas aeruginosa. Int. J. ChemTech Res. 4, 891e895. Selle, P.H., Ravindran, V., Caldwell, A., Bryden, W.L., 2000. Phytate and phytase: consequences for protein utilisation. Nutr. Res. Rev. 13 (2), 255e278. Shetty, J.K., Paulson, B., Pepsin, M., Chotani, G., Dean, B., Hruby, M., 2008. Phytase in fuel ethanol production offers economical and environmental benefits. Int. Sugar. J. 110 (1311), 160e174. Singh, B., Satyanarayana, T., 2014. Fungal phytases: characteristics and amelioration of nutritional quality and growth of non-ruminants. J. Anim. Physiol. Anim. Nutr. 99, 646e660. Snow, J.L., Douglas, M.W., Parsons, C.M., 2003. Phytase effects on amino acid digestibility in molted laying hens. Poultry Sci. 82 (3), 474e477. Tambe, S.M., Kaklij, G.S., Keklar, S.M., Parekh, L.J., 1994. Two distinct molecular forms of phytase from Klebsiella aerogenes: evidence for unusually small active enzyme peptide. J. Ferment. Bioeng. 77, 23e27. Wyss, M., Brugger, R., Kronenberger, A., R
emy, R., Fimbel, R., Oesterhelt, G., Lehmann, M., van Loon, A.P., 1999. Biochemical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): catalytic properties. Appl. Environ. Microbiol. 65 (2), 367e373. Zhang, X., Roland, D., Mcdaniel, G., Rao, S., 1999. Effect of Natuphos phytase supplementation to feed on performance and ileal digestibility of protein and amino acids of broilers. Poultry Sci. 78 (11), 1567. Zhu, D., Wu, Q., Wang N, N., 2011. 3.02 industrial enzymes. In: Comprehensive Biotechnology. Elsevier.

Relevant websites infinitabiotech.com/blog/applications-of-phytase-enzyme/. https://www.acumenresearchandconsultingcom/phytasemarketplace.

Industrial applications of thermophilic/hyperthermophilic enzymes

SUBCHAPTER 8.16

Alcohal dehydrogenases 8.16.1 Microbial sources and properties Alcohol dehydrogenase (ADH) is part of a family of oxidoreductases that are ubiquitous within the three domains of life. This cofactor-dependent enzyme catalyzes alcohol oxidation in the presence of NADþ or NADPþ (Reid and Fewson, 1994). ADH is involved in interconversions between alcohols, aldehydes, and ketones. They are important in biotechnological processes such as the production of optically active alcohols for fragrances, pharmaceuticals, pesticides, foods, and fine chemicals. These enzymes are also useful in bioelectrocatalysis for the production of biosensors, biofuels, optically active compounds, or fermented spirits (Eichler, 2001; Kroutil et al., 2004; Yakushi and Matsushita, 2010; Reyes-Valadez et al., 2016; Yang et al., 2012; Nie et al., 2007; De los RiosDeras et al., 2015). Hence, industry demand for active and stable ADHs having the improved ability to withstand high temperatures, organic solvents, or different pH values has increased. Extremophiles contain enzymes that can withstand harsh conditions in order to survive (Tsigos et al., 1998). The production of enantiomerically and diastereomerically pure diols is mainly desirable as these are essential chemical building blocks. Thermoplasma acidophilus is an archaea that possesses enzymes with robust properties to survive in extreme conditions and has many applications in biotechnology (Ruepp et al., 2000; Darland et al., 1970). ADHs are classified into following types based upon their cofactor requirements (Radianingtyas and Wright, 2003). ➢ ADH that is dependent on nicotinamide adenine dinucleotide (NAD) ➢ ADH that is dependent on pyrroloquinoline quinine (PQQ) ➢ ADH that is dependent on either heme or cofactor F420 or flavine adenine dinucleotide (FAD) NAD-dependent ADH is further divided into following types depending on the alcohol metabolized (Thatcher, 1980; Eklund et al., 1976; Neale et al., 1986; Conway et al., 1987). ➢ The Drosophila ADH (type I) is an example of a short-chain ADH. ➢ The horse liver ADH is an example of a medium-chain or zinc ADH (type II or ZnADH). ➢ Zymomonas ADH2 is an example of long-chain or iron ADHs (type III or Fe-ADH). These three families have little in common and diverged independently all through evolution, indicating that these ADHs have unique structures and reaction mechanisms.

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Since a type III ADH was first reported from Zymomonas mobilis, numerous type III ADHs, have been biochemically characterized. These are Bacillus methanolicus methanol dehydrogenase, Escherichia coli L-1,2-propanediol oxidoreductase, Saccharomyces cerevisiae ADH4, Clostridium acetobutylicum ADH1, C. acetobutylicum butanol dehydrogenases, and a human iron-activated ADH (Williamson and Paquin, 1987; Conway and Ingram, 1989; Youngleson et al., 1989; Walter et al., 1992; de Vries et al., 1992; Deng et al., 2002). Crystal structure and high thermostability of bacterial type III ADH show high thermostability, which is different from other mesophilic ADHs. Though many ADHs have been found from HA, but thermostable ADHs are still in demand because of their exceptional characteristics, showing different substrate specificities and physiological functions. So far, 19 hyperthermophilic ADHs have been characterized. Several ADHs from (ultra)thermophilic organisms have attracted substantial interest regarding their use (Radianingtyas and Wright, 2003). Thermoanaerobium brockii’s NADP-dependent ADH is the most extensively researched (TbADH). This enzyme exhibits very good stability in the presence of organic solvents and displays a broader substrate spectrum (Miroliaei and Nemat-Gorgani, 2002; Keinan et al., 1986). TbADH’s reliance on the pricey cofactor NADP(H), which is about five times more costly than its unphosphorylated form, is a significant disadvantage, though (Wichmann and Vasic-Racki, 2005). Therefore, using ADHs that require NAD is advantageous for preparative applications (H). The thermophile Thermus species ATN1 (alkanedegrading) was initially cultured contained one such enzyme (Höllrigl et al., 2008).

8.16.2 Applications of alcohal dehydrogenases 8.16.2.1 Ethanol production ADH is an enzyme commonly used in alcohol production, especially in industrial ethanol production. Appropriate substrates are required for the fermentation of microbes for producing ethanol. Plant-based raw materials are commonly used and have a high sugar content, which is required for ethanol production. It starts with converting sugars, i.e., sucrose or glucose, by glycolysis process (i.e., converts glucose to pyruvate). Pyruvate gets converted to ethanol via a two or three-step pathway, under anaerobic conditions. ADH is a main enzyme in both the pathways (Eram and Ma, 2013). In this conversion, actually pyruvate gets first converted to the intermediate product acetaldehyde, which is then converted to ethanol with the release of carbon dioxide as a byproduct. A main hindrance to diverting more popularity to bioethanol production is the difficulty in finding suitable microbes, which can convert biomass into biofuel (Dien et al., 2003). This sparked research interest in finding appropriate microorganisms that could have genetically engineered pathways for selectively producing ethanol. Z. mobilis can ferment glucose and fructose to ethanol (Dien et al., 2003). While most eukaryotic and prokaryotic microorganisms use the EmbdenMeyerhof-Parnas (EMP) pathway for glycolysis, Z. mobilis can use the Entner-

Industrial applications of thermophilic/hyperthermophilic enzymes

Doudoroff (ED) pathway (Kang and Lee, 2015). An advantage of the ED pathway is that more carbon is available for producing bioethanol as only one ATP molecule is generated from one glucose molecule as compared to the EMP pathway where two ATP are generated from one glucose molecule (Kang and Lee, 2015). E. coli is generally used industrial microorganism because of its simpler growth conditions and capability of fermenting different types of sugars (Dien et al., 2003). Attempts have been made to maximize ethanol yield. It has been reported that methods can be devised for redirecting the flow of sugar (Kang and Lee, 2015). Rerouting the flux of sugars from biomass to ethanol production has been found to enhance the ethanol production when examined in S. cerevisiae (Kang and Lee, 2015). Similar to mesophiles, hyperthermophiles initiate fermentation by converting glucose to the intermediary pyruvate. Mesophilic and a few moderately thermophilic microorganisms convert pyruvate to ethanol using both two-step and three-step pathways. Some bacteria, including S. cerevisiae and Z. mobilis, go through a two-step process that involves pyruvate decarboxylase (PDC) nonoxidative decarboxylation of pyruvate to acetaldehyde and ADH’s subsequent reduction to ethanol (Eram and Ma, 2013). Traditional, bioethanol production is conducted at temperatures in the range of 25
C and 37
C to ensure optimal growth for mesophiles producing ethanol. Hightemperature fermentation is reported to have several benefits, including substantial savings in cost. According to Abdel-Banat et al. (2010), raising the fermentation temperature by 5
C, saves a 30,000 kL ethanol plant about $800,000 per year in terms of cooling, pollution reduction and simultaneous saccharification and fermentation. Thermostable ethanol producers can be used in place of mesophiles like S. cerevisiae and Z. mobilis when using high fermentation temperatures. In contrast to mesophiles producing ethanol several hyper/thermophilic microorganisms are known to use pentoses from lignocelluloses, which is available in abundance. Use of this substrate makes bioethanol production more economical (Barnard et al., 2010; Olson et al., 2015). 8.16.2.2 Chiral pharmaceutical intermediates ADH is an antidepressant, anxiolytic, antiasthmatic, anticholesterol, antihypertensive, antithrombotic, antiepileptic, b-lactam antibiotic, nonsteroidal anti-inflammatory, and anticancer drug. It is also used in the production of chiral pharmaceutical intermediates such as emetics, anti-AIDS drugs, and adrenaline receptor agonist (Zheng et al., 2017). 8.16.2.3 Fine chemicals ADH is also used for producing fine chemicals such as g-valerolactone, (2S,3S)-2,3butanediol and phenylethanol (Zheng et al., 2017). 8.16.2.4 Other applications ADH is also used in the production of biosensors (Park et al., 1999; Cai et al., 1997; Malinauskas et al., 2000; Mullor et al., 1996; Spures et al., 1996; Boujitita et al., 1996). It is widely used as a bioselective compound for ethanol biosensors. A new biosensor based on the co-immobilization of TBO, NADH, and ADH on glassy carbon electrodes coated

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with cellulose acetate was developed for the measurement of ethanol (Alpat and Telefoncu, 2010). ADH is widely used as a biocatalyst for the dynamic kinetic resolution of racemic substrates and for preparing enantiomerically pure chemicals.

Bibliography Abdel-Banat, B.M.A., Hoshida, H., Ano, A., Nonklang, S., Akada, R., 2010. High temperature fermentation: how can processes for ethanol production at high temperatures become superior to the traditional process using mesophilic yeast? Appl. Microbiol. Biotech. 85, 861e867. Alpat, S., Telefoncu, A., 2010. Development of an alcohol dehydrogenase biosensor for ethanol determination with toluidine blue O covalently attached to a cellulose acetate modified electrode. Sensors (Basel, Switzerland) 10 (1), 748e764. Barnard, D., Casanueva, A., Tuffin, M., Cowan, D., 2010. Extremophiles in biofuel synthesis. Environ. Technol. 31, 871e888. Boujitita, M., Chapleau, M., Murr, N.E., 1996. Biosensors for analysis of ethanol in food: effect of the pasting liquid. Anal. Chim. Acta 319, 91e96. Cai, C.X., Xue, K.H., Zhou, Y.M., Yang, H., 1997. Amperometric biosensor for ethanol based on immobilization of alcohol dehyrogenase on a nickel hexacyanoferrate modified microband gold electrode. Talanta 44, 339e347. Conway, T., Ingram, L.O., 1989. Similarity of Escherichia coli propanediol oxidoreductase (fucO product) and an unusual alcohol dehydrogenase from Zymomonas mobilis and Saccharomyces cerevisiae. J. Bacteriol. 171, 3754e3759. Conway, T., Sewell, G.W., Osman, Y.A., Ingram, L.O., 1987. Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis. J. Bacteriol. 169 (6), 2591e2597. Thatcher, D.R., 1980. The complete amino acid sequence of three alcohol dehydrogenase alleloenzymes (Adh-N11, Adh-s and Adh-UF) from the fruitfly Drosophila melanogaster. Biochem. J. 187, 875e886. Darland, G., Brock, T.D., Samsono, W., Conti, S.F., 1970. A thermophilic, acidophilic mycoplasma isolated from a coal refuse pile. Science 170, 1416e1418. de Vries, G.E., Arfman, N., Terpstra, P., Dijkhuizen, L., 1992. Cloning, expression, and sequence analysis of the Bacillus methanolicus C1 methanol dehydrogenase gene. J. Bacteriol. 174 (16), 5346e5353. Deng, Y., Wang, Z., Gu, S., Ji, C., Ying, K., Xie, Y., Mao, Y., 2002. Cloning and characterization of a novel human alcohol dehydrogenase gene (ADHFe1). DNA Seq. 13 (5), 301e306. Dien, B., Cotta, M., Jeffries, T., 2003. Bacteria engineered for fuel ethanol production: current status. Appl. Microbiol. Biotechnol. 63, 258e266. Eichler, J., 2001. Biotechnological uses of archaeal extremozymes. Biotechnol. Adv. 19, 261e278. Eklund, H., Nordström, B., Zeppezauer, E., Söderlund, G., Ohlsson, I., Boiwe, T., Söderberg, B.O., Tapia, O., Br€and
en, C.I., Akeson, A., 1976. Three-dimensional structure of horse liver alcohol dehydrogenase at 2-4 A resolution. J. Mol. Biol. 102 (1), 27e59. Eram, M.S., Ma, K., 2013. Decarboxylation of pyruvate to acetaldehyde for ethanol production by hyperthermophiles. Biomolecules 3, 578e596. Höllrigl, V., Hollmann, F., Kleeb, A.C., Buehler, K., Schmid, A., 2008. TADH, the thermostable alcohol dehydrogenase from Thermus sp. ATN1: a versatile new biocatalyst for organic synthesis. Appl. Microbiol. Biotechnol. 81 (2), 263e273. Kang, A., Lee, T.S., 2015. Converting sugars to biofuels: ethanol and beyond. Bioeng 2, 184e203. Keinan, E., Hafeli, E.K., Seth, K.K., Lamed, R., 1986. Thermostable enzymes in organic synthesis. 2. asymmetric reduction of ketones with alcohol dehydrogenase from Thermoanaerobium brockii. J. Am. Chem. Soc. 108, 162e169. Kroutil, W., Mang, H., Edegger, K. y, Faber, K., 2004. Recent advances in the biocatalytic reduction of ketones and oxidation of sec-alcohols. Curr. Opin. Chem. Biol. 8, 120e126. Littlechild, J.A., Guy, J.E., Isupov, M.N., 2004. Hyperthermophilic dehydrogenase enzymes. Biochem. Soc. Trans. 32 (Pt 2), 255e258. Malinauskas, A., Ruzgas, T., Gorton, L., Kubota, L.T., 2000. A reagentless amperometric carbon paste based sensor for NADH. Electroanalysis 12, 194e198.

Industrial applications of thermophilic/hyperthermophilic enzymes

Miroliaei, M., Nemat-Gorgani, M., 2002. Effect of organic solvents on stability and activity of two related alcohol dehydrogenases: a comparative study. Int. J. Biochem. Cell Biol. 34, 169e175. Mullor, S.G., Cabezudo, M.S., Ordieres, A.J.M., Ruiz, B.L., 1996. Alcohol biosensor based on alcohol dehydrogenase and Meldola Blue immobilized into a carbon paste electrode. Talanta 43, 779e784. Neale, A.D., Scopes, R.K., Kelly, J.M., Wettenhall, R.E., 1986. The two alcohol dehydrogenases of Zymomonas mobilis. Purification by differential dye ligand chromatography, molecular characterisation and physiological roles. Eur. J. Biochem. 154 (1), 119e124. Nie, Y., Xu, Y., Mu, X.Q., Wang, H.Y., Yang, M., Xiao, R., 2007. Purification, characterization, gene cloning, and expression of a novel alcohol dehydrogenase with anti-prelog stereospecificity from Candida parapsilosis. Appl. Environ. Microbiol. 73, 3759e3764. Olson, D.G., Sparling, R., Lynd, L.R., 2015. Ethanol production by engineered thermophiles. Curr. Opin. Biotechnol. 33, 130e141. Park, J.K., Yee, H.J., Lee, K.S., Lee, W.Y., Shin, M.C., Kim, T.H., 1999. Determination of breath alcohol using a differential-type amperometric biosensor based on alcohol dehydrogenase. Anal. Chim. Acta 390, 83e91. Radianingtyas, H., Wright, P.C., 2003. Alcohol dehydrogenases from thermophilic and hyperthermophilic archaea and bacteria. FEMS Microbiol. Rev. 27, 593e616. Reid, M.F. y, Fewson, C.A., 1994. Molecular characterization of microbial alcohol dehydrogenases. Crit. Rev. Microbiol. 20, 13e56. Reyes-Valadez, J.N., Quintana-Hernandez, P.A., Coronado-Velasco, C., Castro-Montoya, A.J., 2016. Simulation of the bioethanol production process from a mixture of glucose/xylose including temperature, pH and sugar concentration effects. Revista Mexicana de Ingenierıa Quımica 15, 1e9. Rios-Deras, G.C., Rutiaga-Qui~ nones, O.M., L
opez-Miranda, J., P
aez-Lerma, J.B., L
opez, M., SotoCruz, N.O., 2015. Improving Agave duranguensis must for enhanced fermentation: C/N ratio effects on mezcal composition and sensory properties. Revista Mexicana de Ingeniería Química 14 (2), 363e371. Ruepp, A., Graml, W., Santos-Martinez, M.L., Koretke, K.K., Volker, C., Mewes, H.W., Frishman, D., Stocker, S., Lupas, A.N. y B., W., 2000. The genome sequence of the thermoacidophilic scavenger Thermoplasma acidophilum. Nature 407, 508e513. Spures, S.D., Hartely, I.C., Wedge, R., Hart, J.P., Pittson, R., 1996. A disposable reagentless screen-printed amperometric biosensor for the measurement of alcohol in beverages. Anal. Chim. Acta 329, 215e221. Tsigos, I., Velonia, K., Smonou, I., Bouriotis, V., 1998. Purification and characterization of an alcohol dehydrogenase from the Antarctic psychrophile Moraxella sp. Tae123. Eur. J. Biochem. 254, 356e362. Walter, K.A., Bennett, G.N., Papoutsakis, E.T., 1992. Molecular characterization of two Clostridium acetobutylicum ATCC 824 butanol dehydrogenase isozyme genes. J. Bacteriol. 174 (22), 7149e7158. Wichmann, R., Vasic-Racki, D., 2005. Cofactor regeneration at the lab scale. In: Kragl, U. (Ed.), Technology Transfer in Biotechnology, vol 92. Springer, Berlin, pp. 225e260. Williamson, V.M., Paquin, C.E., 1987. Homology of Saccharomyces cerevisiae ADH4 to an iron-activated alcohol dehydrogenase from Zymomonas mobilis. Mol. Gen. Genet. 209 (2), 374e381. Yakushi, T., Matsushita, K., 2010. Alcohol dehydrogenase of acetic acid bacteria: structure, mode of action, and applications in biotechnology. Appl. Microbiol. Biotechnol. 86, 1257e1265. Yang, C., Ying, X., Yu, M., Zhang, Y., Xiong, B., Song, Q., Wang, Z., 2012. Towards the discovery of alcohol dehydrogenases: NAD(P)H fluorescence-based screening and characterization of the newly isolated Rhodococcus erythropolis WZ010 in the preparation of chiral aryl secondary alcohols. J. Ind. Microbiol. Biotechnol. 39, 1431e1443. Youngleson, J.S., Jones, W.A., Jones, D.T., Woods, D.R., 1989. Molecular analysis and nucleotide sequence of the adh1 gene encoding an NADPH-dependent butanol dehydrogenase in the Gram-positive anaerobe Clostridium acetobutylicum. Gene 78 (2), 355e364. Zheng, Y.G., Yin, H.H., Yu, D.F., Chen, X., Tang, X.L., Zhang, X.J., Xue, Y.P., Wang, Y.J., Liu, Z.Q., 2017. Recent advances in biotechnological applications of alcohol dehydrogenases. Appl. Microbiol. Biotechnol. 101 (3), 987e1001.

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Developments and Applications of Enzymes From Thermophilic Microorganisms

SUBCHAPTER 8.17

Esterases 8.17.1 Microbial sources and properties Esterases belong to the lipolytic hydrolase family and catalyze hydrolysis and formation of ester bond (Kulkarni et al., 2013). Esterases catalyze esterification, transesterification, and interesterification reactions (Kawamoto et al., 1987). These enzymes influence the rate of reversible reactions according to their thermodynamics. Thus, in the organic phase, esterases promote the formation of ester and in the aqueous phase hydrolyze the ester (Dodrick, 1989). Esterases are different from lipases primarily on the basis of substrate specificity and surface activation (Long, 1971). Esterases hydrolyze shorter chain carboxylic acids (lesser than C-12) and lipases hydrolyze insoluble longer chain (higher than C12) triglycerides (Meghji et al., 1990; Kim et al., 2004; Faiz et al., 2007). They are attractive biocatalysts because the cofactors are not required (Godinho et al., 2011). The Enzyme Commission number assigned to esterases is E.C 3.1.1. x, where x is substrate dependent (Bornscheuer, 2002). Esterases play an important role in breaking down natural and industrial pollutants such as grain waste, plastics, and other toxic chemicals. Hydrolases such as esterases and lipases share not only some common features but also certain unique properties (Table 8.17.1) (Sharma et al., 2017). Table 8.17.2 shows the classification of esterases by mechanism of action (Sharma et al., 2017). Table 8.17.1 Characteristics of esterases and lipases. Property

Esterases

Lipases

Substrate preference

Ester, triglycerides

Enantio-selectivity Chain length of triglycerides Interfacial activation pH optima Substrate hydrophobicity Solvent stability Water solubility MichaeliseMenten kinetics

Intermediate Below C6

Triglycerides, alcohol Usually high Above C6

No 6.0 High to low

Yes 8.0e9.5 High

High to low Soluble Obey

Prominent Insoluble Does not obey

Based on Sharma, T., Sharma, A., Sharma, S., Kanwar, S.S., 2017. An overview on esterases: structure, classification, sources and their application. In: Rai, V. (Ed.), Recent Advances in Biotechnology, vol. 2, Shree Publishers & Distributors, New Delhi.

Industrial applications of thermophilic/hyperthermophilic enzymes

Table 8.17.2 Classification of esterases on the basis of their mechanism of action.

Arlyesterases (EC 3.1.1.2) Act on aromatic esters. Resistant to organophosphates but are inhibited by sulfydryl regeants such as chloromercuribenzoate Acetylesteres (EC 3.1.1.6) Act on aromatic esters; not inhibited by organophosphates, serine or sulfydryl reagents. Carboxylesterases (EC 3.1.1.1) Catalyze the hydrolysis of aliphatic esters and are inhibited by organophosphates but not by serine. Cholinesterase hydrolases (EC 3.1.1.7; 3.1.1.8) Catalyze the hydrolysis of the neurotransmitter acetylcholine into choline and acetic acid; repressed by organophosphates as well as serine. Based on Sharma, T., Sharma, A., Sharma, S., Kanwar, S.S., 2017. An overview on esterases: structure, classification, sources and their application. In: Rai, V. (Ed.), Recent Advances in Biotechnology, vol. 2, Shree Publishers & Distributors, New Delhi.

Esterases are extensively distributed in animals, plants, and microorganisms. Microbial sources are believed to be important for production of esterases. These include bacteria, yeasts, actinomycetes, and fungi. Samples like the surface of cheese, oil-contaminated areas of municipal waste, or squid have been used for producing microbial esterases (Gandolfi et al., 2000; Ranjitha et al., 2009). Various sources provide different esterases such as carboxylesterases, cholinesterases, acetylxylanesterases, arylesterases, phosphotriesterases, phenolesterases, porcine liver esterases, acetylcholinesterases, and tannin esterases (Panda and Gowrishankar, 2005). In particular, esterases of extremophilic origin are the most robust biocatalysts, as their inherent thermostability and tolerance to organic solvents allow them to function under the severe conditions of industrial processes, which combined with their high chemo-, regio-, and enantioselectivity make them an attractive biocatalyst for various applications in industry (Lopez-Lopez et al., 2014). These substrates typically include the necessary inducer. Contrarily, thermostable intracellular esterases from Bacillus sp., phosphotriesterases from Pseudomonas montellii that are controlled by phosphate and carbon sources, and tributyrin-induced esterases from Lactobacillus casei are distinct from the enzymes produced by Aspergillus sp. or Sporotrichum sp. (Fenster and Parkin, 2003; Choi and Lee, 2001). Esterases are produced in a variety of ways and often do not require high purity (Peterson et al., 2001). However, by developing suitable production strategies, the wide range of applications and growing requirement for esterases can be met (Table 8.17.1). Highly thermostable esterases have been isolated from wild-type strains like Thermus thermophilus HB27’s E34Tt, which has been reported to have a half-life of 135 min at 85
C, a temperature optimum of more than 80
C, and an optimum pH of 8.1. They have also been expressed in heterologous hosts, as in the case of an esterase gene from Aeropyrum pernix K1 that has been expressed in Escherichia coli displaying an optimal

269

270

Developments and Applications of Enzymes From Thermophilic Microorganisms

temperature of 90
C and the half-life of more than 160 h at 90
C (Fuci~ nos et al., 2011; Gao et al., 2003). Ikeda and Clark (1998) reported the cloning of esterases from Pyrococcus furiosus. The resultant recombinant enzyme was found to be the most thermostable esterase reported so far. It showed a temperature optimum of 100
C and a half-life of 50 min at 126
C. Many lipase and esterases producing (hyper)thermophiles have been studied (Atomi and Imanaka, 2004; Bruins et al., 2001; Levisson et al., 2009; Salameh and Wiegel, 2007; Egorova and Antranikian, 2005). Table 8.17.3 shows the properties of esterases from (hyper)thermophilic microorganisms (Fuci~ nos et al., 2012). To date, three esterases with known amino acid sequences of thermophilic archaeal origin belonging to family IV have been biochemically characterized. In addition to these enzymes, esterases from Sulfolobus shibatae, Sulfolobus acidocaldarius, and Pyrococcus furiosus have been purified and characterized, but their associated genes have not been described (Hotta et al., 2002; Huddleston et al., 1995; Ikeda and Clark, 1998; Manco et al., 2000; Morana et al., 2002; Sobek and Gorisch, 1989, 1988).

8.17.2 Application of esterases Esterases have several commercial uses in the production of food (including dairy), animal feed, detergents, paper, leather, pharmaceuticals, and cosmetics (Coughian Laura et al., 2015; Panda and Gowrishankar, 2005; Bornscheuer, 2002; Kohli and Gupta, 2016; Lai et al., 2019). Esterase enzymes are the most common novel enzymes found in soil metagenomes and are important biocatalysts for biotechnological applications (Lee and Lee, 2013). 8.17.2.1 Food and dairy, beverages, and perfume Esterases are used in the dairy industry to hydrolyze milk fat for improving the flavor in the production of cheese products. Esterases are also used in the production of wine, fruit juices, alcohol, and beer. Both esterases and lipases are used as transesterification catalysts to convert inferior fats and oils into more valuable ones. It has been widely used to hydrolyze milk fat for this purpose. Fruity strawberry flavor with esterase and lipase obtained from Pseudomonas fragi has been developed (Choi and Lee, 2001; Kermasha et al., 2000). Fakuda et al. (1998) have reported that an acetylesterase derived from Saccharomyces cerevisiae plays an important role in the production of isoamyl acetate, which determines the flavor of sake during fermentation. A large amount of high fructose corn syrup sweeteners are made from corn starch with the use of hydrolytic enzymes. Yeast esterases also play an important role in determining the final ester content of products like membrane-filtered and bottle-fermented beers (Dufour and Bing, 2001). In addition to acetyltransferases, esterases from S. cerevisiae play an important role in producing isoamyl acetate, which contributes significantly to determining the taste of sake (Fakuda et al., 1998).

Table 8.17.3 Characteristics of esterases/lipases from (hyper)thermophilic microorganisms. Enzyme properties Enzyme

MW (kDa)

Aeropyrum pernix K1

Esterase

63

90

8

p-Nitrophenyl C8

A. fulgidus

Esterase (AFEST)

35.5

80

6.5e7.5

p-Nitrophenyl C6

Alicyclobacillus (formerly Bacillus) acidocaldarius

Esterase (EST2)

34

70

7.1

p-Nitrophenyl C6

Picrophilus torridus

Esterase (Est A)

66 (trimer)

70

6.5

Pyrobaculum calidifontis VA1

Esterase (Est)

34

90

7.0

p-Nitrophenyl C2 p-Nitrophenyl C6

Half-life of 21 h at 90
C Half-life of 56 min at 110
C

Pyrococcus furiosus

Esterase

NA

100

7.6

Sulfolobus solfataricus P1

Esterase

34

85

4-Methylumbelliferyl C2 p-Nitrophenyl C6

Thermotoga maritima

Esterase 267 (hexamer) Esterase (E34Tt)

267 (hexamer)

>95

8.5

34

>80

8.1

Esterase (SsoPEst)

58.4

80

5.5

Half-life of 50 min at 126
C 41% of remaining activity after 5 days at 80
C Half-life of 1.5 h at 100
C Half-life of 135 min at 85
C Half-life of 1 h at 80
C

Thermus thermophilus HB27 Sulfolobus solfataricus P2

Opt. temp.

Opt. pH

Best substrate

Microorganism

8

p-Nitrophenyl C8 p-Nitrophenyl C10 p-Nitrophenyl C8

Stability Half-life over 160 h at 90
C Half-life of 26 min at 95
C Half-life of 24 min at 85
C

Remarks

References Gao et al. (2003)

Maintain 80% activity at 110
C Active in water immiscible solvents

Maintain high activity levels at ambient temperature (16% at 30
C). Active and stable in the presence of 80% watermiscible organic solvents

Manco et al. (2000) Manco et al. (1998) Manco et al. (2000) Hess (2008) Hotta et al. (2002)

Ikeda and Clark (1998) Park and Choi (2006) Sehgal et al. (2002) Levisson et al. (2009) Fuci~ nos et al. (2011) Shang et al. (2010)

Reproduced with Permission from Fuci~ nos, P., Gonzalez, R., Atanes, E., Sestelo, A.B., Perez-Guerra, N., Pastrana, L., Rua, M.L., 2012. Lipases and esterases from extremophiles: overview and case example of the production and purification of an esterase from Thermus thermophilus HB27. Methods Mol. Biol. 861, 239e266.

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Developments and Applications of Enzymes From Thermophilic Microorganisms

Ferulic acid, sinapic acid, caffeic acid, and coumaric acid are extensively used in the food, beverage, and perfume industries. Their esters are found in cereals, agricultural industry residues, and biomass (Chaabouni et al., 1996; Asther et al., 2002). Feruloyl and cinnamoyl esterase enzymes from Aspergillus niger, along with pectinase, cellulase, and xylanase enzymes, can liberate such hydroxycinnamic acids from rice bran, wheat bran, sugar cane, bamboo, sugar beet pulp, and others. Esterases from Fusarium oxysporum play an important role in the production of fragrance and flavoring compounds from geraniol and fatty acids (Chaabouni et al., 1996; Christakopoulos et al., 1998). Pentylferulate esters are flavor precursors for food processing as well as cosmetics and are products of feruloyl esterases using water-in-oil microemulsions (Giuliani et al., 2001). Studies are now being conducted on improving the taste of fermented meat products. Ostdal et al. (1996) used a lipase-esterase enzyme from Pedicoccus pentosauces SV6 for improving the flavor of fermented sausages (Ostdal et al., 1996). 8.17.2.2 Degradation of synthetic materials Cholesterol esterases and polyuretenases are commonly used for degrading man-made contaminants such as plastics, polyurethanes, polyesters and polyethylene glycol adipates (Jahangir et al., 2003). Polyurethane degrading enzymes from Pseudomonas aeruginosa and P. chlororophis are extensively used for polyester degradation and are analogous to carboxylesterases and acetylcholinesterases (Howard et al., 2001). 8.17.2.3 Agriculture Phosphotriesters, e.g., synthetic organophosphorus compounds, like coumaphos and its oxone derivative coroxon, have several applications as nematicides and insecticides. Residues of these compounds are toxic to the environment and can end up in food. Phosphotriesterases from Alteromonas sp and Brevundimonas diminuta are extensively used to detoxify/degrade these organophosphorus compounds (Horne et al., 2002). 8.17.2.4 Chemical industries Esterases are widely used in other industries like pulp and paper, leather, textile, and bakery. Ophiostoma piceae sterol esterase is used in papermaking because it effectively hydrolyzes both sterol esters as well as triglycerides. In addition, cholesteryl esterases and steryl esterases from Pseudomonas species, Candida rugosa, and Chromobacterium viscosum also play an important role in the reduction of pitch problems during papermaking. During softwood and hardwood paper pulp production, resin deposits are formed that adversely affect paper machine runnability and paper quality (Kontkanen et al., 2004).

Bibliography Asther, M., Haon, M., Roussos, S., Record, E., Delattre, M., Lesage-Meessen, L., Labat, M., Asther, M., 2002. Feruloyl esterase from Aspergillus niger a comparison of the production in solid state and submerged fermentation. Process Biochem. 38, 685e691.

Industrial applications of thermophilic/hyperthermophilic enzymes

Atomi, H., Imanaka, T., 2004. Thermostable carboxylesterases from hyperthermophiles. Tetrahedron Asymmetry 15, 2729e2735. Bornscheuer, U.T., 2002. Microbial carboxyl esterases: classification, properties and application in biocatalysis. FEMS Microbiol. Rev. 26 (1), 73e81. Bruins, M.E., Janssen, A.E.M., Boom, R.M., 2001. Thermozymes and their applications. Appl. Biochem. Biotechnol. 90, 155e186. https://doi.org/10.1385/ABAB:90:2:155. Chaabouni, M.K., Pulvin, S., Touraud, D., Thomas, D., 1996. Enzymatic synthesis of geraniol esters in a solvent free system by lipases. Biotechnol. Lett. 18, 1083e1088. Choi, Y.J., Lee, B.H., 2001. Culture conditions for the production of esterase from Lactobacillus casei CL 96. Bioprocess Biosyst. Eng. 24, 59e63. Christakopoulos, P., Tzalas, B., Mamma, D., Stamatis, H., Liadakis, G.N., Tzia, C., Kekos, D., Kolisis, F.N., Macris, B.J., 1998. Production of esterases from Fusarium oxysporum catalyzing transesterification reactions in organic solvents. Process Biochem. 33, 729e733. Coughian Laura, M., Cotter Paul, D., Will, C., Alvarez-Ordonez, A., 2015. Biotechnological applications of functional metagenomice in the food and pharmaceutical industries. Front. Microbiol. 6, 672e678. Dodrick, J.S., 1989. Enzymatic catalysis in monophasic solvent. Enzyme Microbiol. Technol. 11, 194e211. Dufour, J.P., Bing, Y., 2001. Influence of yeast strain and fermentation conditions on yeast esterase activities. Brew Dig. 76, 44. Faiz, O., Colak, A., Saglam, N., Canakci, S., Belduz, A.O., 2007. Determination and characterization of thermostable esterolytic activity from a novel thermophilic bacterium Anoxybacillus gonensis A4. J. Biochem. Mol. Biol. 40, 588e594. Fakuda, K., Yamamoto, N., Kiyokawa, Y., Yanagiuchi, T., Wakai, Y., Kitamoto, K., Inoue, Y., Kimura, A., 1998. Balance of activities of alcohol acetyltransferase and esterase in Saccharomyces cerevisiae is important for production of isoamyl acetate. Appl. Environ. Microbiol. 64, 4076e4078. Fenster, K.M., Parkin, K.L., 2003. Intracellular esterase from Lactobacillus casei LILA: nucleotide sequencing, purification, and characterization. J. Dairy Sci. 86, 1118e1129. Fuci~ nos, P., Gonz
alez, R., Atanes, E., Sestelo, A.B., P
erez-Guerra, N., Pastrana, L., R
ua, M.L., 2012. Lipases and esterases from extremophiles: overview and case example of the production and purification of an esterase from Thermus thermophilus HB27. Methods Mol. Biol. 861, 239e266. Fuci~ nos, P., Pastrana, L., Sanrom
an, A., Longo, M.A., Hermoso, J.A., R
ua, M.L., 2011. An esterase from Thermus thermophilus HB27 with hyperthermoalkalophilic properties: purification, characterisation and structural modelling. J. Mol. Catal. B Enzym. 70, 127e137. Egorova, K., Antranikian, G., 2005. Industrial relevance of thermophilic Archaea. Curr. Opin. Microbiol. 8 (6), 649e655. https://doi.org/10.1016/j.mib.2005.10.015. Gandolfi, R., Gaspari, F., Franzetti, L., Molinari, F., 2000. Hydrolytic and synthetic activities of esterases and lipases of non-starter bacteria isolated from cheese surface. Ann. Microbiol. 50, 183e189. Gao, R., Feng, Y., Ishikawa, K., et al., 2003. Cloning, purification and properties of a hyperthermophilic esterase from archaeon Aeropyrum pernix K1. J. Mol. Catal. B Enzym. 24e25, 1e8. Gao, R.J., Feng, Y., Ishikawa, K., Ishida, H., Ando, S., Kosugi, Y., Cao, S.G., 2003b. Cloning, purification and properties of a hyperthermophilic esterase from archaeon Aeropyrum pernix K1. J. Mol. Catal. B Enzym. 24e25, 1e8. Giuliani, S., Piana, C., Setti, L., Hochkoeppler, A., Pifferi, P.G., Williamson, G., Faulds, C.B., 2001. Synthesis of pentylferulate by a feruloyl esterase from Aspergillus niger using water-in-oil microemulsions. Biotechnol. Lett. 23, 325e330. Godinho, L.F., Reis, C.R., Tepper, P.G., Poelarends, G.J., Quax, W.J., 2011. Discovery of an Escherichia coli esterase with high activity and enantio-selectivity towards 1, 2-O-Isopropylideneglycerol esters. Appl. Environ. Microbiol. 77, 6094e6099. Hess, M., 2008. Thermoacidophilic proteins for biofuel production. Trends Microbiol. 16, 414e419. Hess, M., Katzer, M., Antranikian, G., 2008. Extremely thermostable esterases from the thermoacidophilic euryarchae on Picrophilus torridus. Extremophiles 12 (3), 351e364. Horne, I., Harcourt, R.L., Tara, D., Sutherland, R.R.J., Oakeshott, J.G., 2002. Isolation of Pseudomonas monteilli strain with a novel phosphotriesterase. FEMS Microbiol. Lett. 206, 51e55. Hotta, Y., Ezaki, S., Atomi, H., Imanaka, T., 2002. Extremely stable and versatile carboxylesterase from a hyperthermophilic archaeon. Appl. Environ. Microbiol. 68 (8), 3925e3931. Howard, G.T., Crother, B., Vicknair, J., 2001. Cloning, nucleotide, sequencing and characterization of a polyurethanase gene (pueB) from Pseudomonas chlororaphis. Int. Biodeterior. Biodegrad. 47, 141e149.

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Huddleston, S., Yallop, C.A., Charalambous, B.M., 1995. The identification and partial characterisation of a novel inducible extracellular thermostable esterase from the archaeon Sulfolobus shibatae. Biochem. Biophys. Res. Commun. 216, 495e500. Ikeda, M., Clark, D.S., 1998. Molecular cloning of extremely thermostable esterase gene from hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli. Biotechnol. Bioeng. 57, 624e629. Jahangir, R., Mc Closkey, C.B., Mc Clung, W.G., Labow, R.S., Brash, J.L., Santerre, J.P., 2003. The influence of protein adsorption and surface modifying macromolecules on the hydrolytic degradation of a poly (ether-urethane) by cholesterol esterase. Biomaterials 24, 121e130. Kawamoto, T., Sonomoto, K., Tanaka, A., 1987. Esterification in organic solvents: selection of hydrolases and effects of reaction conditions. Biocatalysis 1, 137e145. Kermasha, S., Bisakowski, B., Ismail, S., Morin, A., 2000. The effect of physical and chemical treatments on the esterase activity from Pseudomonas fragi CRDA 037. Food Res. Int. 33, 767e774. Kim, H.K., Na, H.S., Park, M.S., Oh, T.K., Lee, T.S., 2004. Occurrence of ofloxacin ester hydrolyzing esterase from Bacillus niacin EM001. J. Mol. Catal. B: Enzym. 27, 237e241. Kohli, P., Gupta, R., 2016. Medical aspects of esterases: a mini review. Int. J. Pharm. Pharm. Sci. 8 (8), 21e26. Kontkanen, H., Tenkanen, M., Fagerstrom, R., Reinikainen, T., 2004. Characterisation of steryl esterase activities. J. Biotechnol. 108, 51e59. Kulkarni, S., Patil, S., Satpute, S., 2013. Microbial esterases: an overview. Int. J. Curr. Microbiol. Appl. Sci. 2, 135e146. Lai, O.M., Lee, Y.Y., Phuah, E.T., Akoh, C.C., 2019. Lipase/esterase: properties and industrial applications. In: Melton, L., Shahidi, F., Varelis, P. (Eds.), Encyclopedia of Food Chemistry, vol. 2. Elsevier, pp. 158e167. Lee, M.H., Lee, S.W., 2013. Bioprospecting potential of the soil metagenome: novel enzymes and bioactivities. Genom. Inform. 11, 114e120. Levisson, M., Sun, L., Hendriks, S., Swinkels, P., Akveld, T., Bultema, J.B., Barendregt, A., van den Heuvel, R.H., Dijkstra, B.W., van der Oost, J., Kengen, S.W., 2009. Crystal structure and biochemical properties of a novel thermostable esterase containing an immunoglobulin-like domain. J. Mol. Biol. 385 (3), 949e962. Long, C., 1971. Biochemists Handbook. Redwood London, pp. 273e274. Lopez-Lopez, O., Cerdan, M.E., Maria, I., Gonzalez, S., 2014. New extremophilic lipases and esterases from metagenomics. Curr. Protein Pept. Sci. 15, 445e455. Manco, G., Febbraio, F., Rossi, M., 1998. Thermophilic esterases and the amino acid “traffic rule” in the hormone sensitive lipase subfamily. Prog. Biotechnol. 15, 325e330. Manco, G., Giosue, E., D’Auria, S., Herman, P., Carrea, G., Rossi, M., 2000. Cloning, overexpression, and properties of a new thermophilic and thermostable esterase with sequence similarity to hormonesensitive lipase subfamily from the archaeon Archaeoglobus fulgidus. Arch. Biochem. Biophys. 373, 182e192. Meghji, K., Ward, O.P., Araujo, A., 1990. Production, purification and properties of extracellular carboxyl esterases from Bacillus subtilis NRRL 365. Appl. Environ. Microbiol. 56, 3735e3740. Morana, A., Di Prizito, N., Aurilia, V., Rossi, M., Cannio, R., 2002. A carboxylesterase from the hyperthermophilic archaeon Sulfolobus solfataricus: cloning of the gene, characterization of the protein. Gene 283 (1e2), 107e115. Ostdal, H., Baron, C.P., Blom, H., Andersen, H.J., 1996. Production, isolation and partial characterization of lipase-esterase from Pediococcus pentosaceus SV61. Lebensm Wiss Technol. 29, 542e546. Panda, T., Gowrishankar, B.S., 2005. Production and applications of esterases. Appl. Microbiol. Biotechnol. 67 (2), 160e169. Park, Y., Choi, S.Y., Lee, H., 2006. A carboxylesterase from the thermoacidophilic archaeon Sulfolobus solfataricus P1; purification, characterization, and expression. Biochim. Biophys. Acta 1760, 820e828. Peterson, E.I., Valinger, G., Solkner, B., Stubenrauch, G., Schwab, H., 2001. A novel esterase from Burkholderia gladioli which shows high deacetylation activity on cephalosporins is related to b-lactamases and DD-peptidases. J. Biotechnol. 89, 11e25. Ranjitha, P., Karthy, E.S., Mohankumar, A., 2009. Purification and partial characterization of esterase from marine Vibrio fischeri. Mod. Appl. Sci. 3, 73e82. Salameh, M., Wiegel, J., 2007. Lipases from extremophiles and potential for industrial applications. Adv. Appl. Microbiol. 61, 253e283. https://doi.org/10.1016/S0065-2164(06)61007-1.

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Sehgal, A.C., Tompson, R., Cavanagh, J., Kelly, R.M., 2002. Structural and catalytic response to temperature and cosolvents of carboxylesterase EST1 from the extremely thermoacidophilic archaeon Sulfolobus solfataricus P1. Biotechnol. Bioeng. 80 (7), 784e793. Shang, Y.S., Zhang, X.E., Wang, X.D., Guo, Y.C., Zhang, Z.P., Zhou, Y.F., 2010. Biochemical characterization and mutational improvement of a thermophilic esterase from Sulfolobus solfataricus P2. Biotechnol. Lett. 32 (8), 1151e1157. Sharma, T., Sharma, A., Sharma, S., Kanwar, S.S., 2017. An overview on esterases: structure, classification, sources and their application. In: Rai, V. (Ed.), Recent Advances in Biotechnology, vol. 2. Shree Publishers & Distributors, New Delhi. Sobek, H., Gorisch, H., 1988. Purification and characterization of a heat-stable esterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Biochem. J. 250, 453e458. Sobek, H., Gorisch, H., 1989. Further kinetic and molecular characterization of an extremely heat-stable carboxylesterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Biochem. J. 261, 993e998.

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Developments and Applications of Enzymes From Thermophilic Microorganisms

SUBCHAPTER 8.18

DNA polymerase 8.18.1 Microbial sources and properties DNA polymerases (EC 2.7.7.7) are a specific class of enzymes found in all organisms. It replicates DNA and helps to repair and maintain it (Ishino and Ishino, 2013). In 1956, Escherichia coli DNA polymerase I, the first identified enzyme involved in the replication process was discovered (Kornberg, 1957). Thermostable DNA polymerases are of particular importance in essential genetic engineering techniques, polymerase chain reaction (PCR), and LDR/LCR. DNA polymerases are grouped into different families (A, B, C, D, X, and Y) according to how similar their primary amino acid sequences are (Burgers et al., 2001). Enzymes are essential in the transmittance of genetic information from generation to generation. Since their identification, DNA polymerases have opened up new avenues for research into the replication and transcription of DNA. They were also crucial in the development of PCR and DNA sequencing, which are the foundations of much of contemporary biotechnology. Polymerases are now essential tools for DNA amplification, sequencing, and labeling. Additionally, DNA polymerases are crucial for creating molecular diagnostics for individualized medicine. For instance, we are at the forefront of techniques for identifying genomic changes that can result in illnesses like cancer or drug side effects in patients (Drouin et al., 2007). The major role of a DNA polymerase is to duplicate DNA using one of its strands as a template and a small DNA or RNA fragment as a primer for extension from the 50 end to the 30 -OH end. Additionally, DNA polymerases are involved in replication and repair of DNA, homologous recombination, cell cycle checkpoints, sister chromatid joining, and maintenance of genome integrity during immune system development. Dysfunction of DNA polymerase is associated with diseases such as extraocular paralysis, xeroderma pigmentosum, and tumorigenesis (Kannoche and Stary, 2003; Loeb, 2001; Ponamarev et al., 2002; Copeland and Longley, 2003). Polymerases do not create new DNA strands from scratch. Instead, it produces a new DNA strand based on templates from two existing DNA strands. This is done with the aid of one more enzyme called helicase. This frees the double helical structure of the DNA molecule into two single DNA strands. Additionally to the template strand, a primer is required for the polymerase to function. This is a nucleic acid fragment, which is the starting point for replicating DNA. A primer, usually a short strand of RNA, must be complementary to the template. DNA polymerases work by sliding along a singlestranded DNA template, reading its nucleotide bases, and introducing new complementary nucleotides into the primer to produce a sequence complementary to the template. It can replicate 749 nucleotides per second.

Industrial applications of thermophilic/hyperthermophilic enzymes

Two new DNA molecules are created, at the end of the replication process. These are identical to the parent molecule. Such precise replication is aided by the fact that DNA polymerases have built-in capabilities to detect and correct errors that occur during the replication process. Many families of DNA polymerases have now been recognized, and new ones continue to be discovered. Some of the most important polymerases for biotechnology fall into the families labeled A and B. These are single-subunit polymerases. Genetic engineering is also expanding its repertoire to include tailor-made polymerases. These genetically engineered DNA polymerases have helped to improve the speed and accurateness of PCR, making it possible to perform PCR directly from tissue. Also the development of next-generation sequencing tools and development of whole-genome amplification is possible. An overview of the structure of DNA polymerase is shown in Fig. 8.18.1 (Ishino and Ishino, 2013). The common three-dimensional core structure of polynucleotide polymerases bear resemblance to the human right hand and contains three different domains: palm, finger, and thumb (Rothwell and Waksman, 2005). Polymerases usually used for PCR are obtained from several thermophilic microorganisms: Pyrococcus furiosus (Pfu polymerase), Thermus aquaticus (Taq), Thermus Thermophile (Tth polymerase), and Thermococcus litoralis (Wind or Tli polymerase or Vent polymerase).

family A Taq DNA polymerase Finger

family B Pfu DNA polymerase 3'-5' exo

Palm

Thumb

family Y Sso DinB Finger

Thumb

Thumb Finger

Palm 5'-3' exo

Little finger (Wrist)

Palm

Figure 8.18.1 Structural overview of DNA polymerases. Ribbon diagrams of the three DNA polymerases from family A, B, and Y are shown. Taq DNA polymerase (PDB code 1TAQ), Pfu DNA polymerase (PDB code 3A2F), and Sso DinB DNA polymerase (PDB code 1K1S) represented from each family. The three distinct domains of the DNA polymerases are shown in different colors. A unique C-terminal domain in Sso DinB is called the “little finger” or “wrist” domain. (From Ishino, S., Ishino, Y., 2013. DNA polymerases and DNA ligases. In: Satyanarayana, T., Littlechild, J., Kawarabayasi, Y. (Eds.), Thermophilic Microbes Environ Ind Biotechnol Biotechnol Thermophiles, second ed. Springer Netherlands, Dordrecht, pp. 429e458. Reproduced with permission.)

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The use of a particular polymerase depends upon the type of PCR being performed and the type and size of template. The mixture of polymerases has been developed to solve the problems associated with using individual polymerases in specific types of PCR. PCR method is currently used now a days in biological and medical research laboratories for detection of genetic diseases, diagnosis of infectious diseases by gene cloning, paternity testing, identification of genetic fingerprints, and detecting bacterial or viral infections (particularly AIDS) (Drouin et al., 2007). The first thermostable DNA polymerase applied to PCR was Taq polymerase. It lacks 0 3 -50 proofreading exonuclease activity, making it unable to excise mismatches (Cabrera and Blamey, 2018). Therefore, archaeal DNA polymerases are the best choice when higher fidelity is required for reducing the error rates. These DNA polymerases possess 30 -50 proofreading activity, have 10-fold lesser error rates as compared to Taq polymerase, and are more thermostable but sluggish. DNA polymerases most commonly used are from archaea bacteria of the genera Pyrococcus (Pfu, Pwo, Deep Vent, Platinum Pfx) and Thermococcus (KOD1, Tli, 9
N-7). DNA polymerases create complementary strands of DNA according to template DNA in living cells. Several enzymes were identified from each organism and the common functions of these enzymes were studied. Besides their fundamental role in maintaining genome integrity during replication and repair, DNA polymerases are extensively used for DNA manipulation in vitro. Factors to consider before choosing a DNA polymerase are thermostability, processivity, speed or elongation rate, specificity, precision, robustness, and efficiency.

8.18.2 Applications DNA polymerases are playing a central role in modern molecular biology and biotechnology and are used for DNA cloning and sequencing, PCR, whole genome amplification and single nucleotide polymorphism detection. It enables technologies such as synthetic biology and molecular diagnostics, biodiversity assessment, and in vitro mutagenesis. Each of these applications relies on the ability of polymerases to replicate DNA, creating products that precisely represent the original input. One of the best-known and earliest DNA polymerase-based biotechnology applications is PCR (Gardner and Kelman, 2014). PCR has become one of the most important methods presently used in bioscience, forensic science, and diagnostics. PCR has been a fundamental tool for amplification and detection of specific alleles (Erlich et al., 1991). Developments in DNA polymerase accuracy, speed, and throughput continue for improving PCR workflows for genetic analysis, cloning, and diagnostics. Elshawadfy et al. (2014) have shown that the speed and throughput of PCR can be increased by combining advantageous protein domains from various DNA polymerases into a single engineered chimeric enzyme. Similar to this, Yamagami et al. (2014) created new DNA polymerases by swapping the domains of DNA polymerases discovered in hot springs for the selection of specific hybrid polymerases with acceptable PCR characteristics.

Industrial applications of thermophilic/hyperthermophilic enzymes

Castillo-Lizardo et al. (2014) analyzed replication slippage during PCR of repetitive sequences and found that the DNA polymerase processivity clamp, proliferating cell nuclear antigen (PCNA) reduces slippage and promotes error-free replication of repeat sequences (Indiani and O’Donnell, 2006). In addition to PCR, DNA polymerases play an important role in DNA sequencing technology. For sequencing the first draft of the human genome in 2001, Sanger DNA sequencing was used. This is the standard and widely used method for determining DNA sequences (Venter et al., 2001; Lander et al., 2001). The latest DNA sequencing technologies are collectively called next-generation sequencing. There are various next-generation sequencing methods that use different techniques. But, most share common features which distinguish them from Sanger sequences.

Bibliography Burgers, P.M., Koonin, E.V., Bruford, E., Blanco, L., Burtis, K.C., Christman, M.F., et al., 2001. Eukaryotic DNA polymerases: proposal for a revised nomenclature. J. Biol. Chem. 276, 43487e43490.
Blamey, J.M., 2018b. Biotechnological applications of archaeal enzymes from extreme Cabrera, M.A., environments. Biol. Res. 51, 37. Castillo-Lizardo, M., Henneke, G., Viguera, E., 2014. Replication slippage of the thermophilic DNA polymerases B and D from the Euryarchaeota Pyrococcus abyssi. Front. Microbiol. 5, 403. Cline, J., Braman, J., Hogrefe, H., 1996. PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases. Nucl. Acids Res. 24, 3546e3551. Copeland, W.C., Longley, M.J., 2003. DNA polymerase gamma in mitochondrial DNA replication and repair. Sci. World J. 3, 34e44. Drouin, R., Dridi, W., Samassekou, O., 2007. DNA polymerases for PCR applications. In: Polaina, J., MacCabe, A.P. (Eds.), Industrial Enzymes. Springer, Dordrecht. Elshawadfy, A.M., Keith, B.J., Ee Ooi, H., Kinsman, T., Heslop, P., Connolly, B.A., 2014. DNA polymerase hybrids derived from the family-B enzymes of Pyrococcus furiosus and Thermococcus kodakarensis: improving performance in the polymerase chain reaction. Front. Microbiol. 5, 224. Erlich, H.A., Gelfand, D., Sninsky, J.J., 1991. Recent advances in the polymerase chain reaction. Science 252, 1643e1651. Gardner, A.F., Kelman, Z., 2014. DNA polymerases in biotechnology. Front. Microbiol. 5, 659. Indiani, C., O’Donnell, M., 2006. The replication clamp-loading machine at work in the three domains of life. Nat. Rev. Mol. Cell Biol. 7, 751e761. Ishino, S., Ishino, Y., 2013. DNA polymerases and DNA ligases. In: Satyanarayana, T., Littlechild, J., Kawarabayasi, Y. (Eds.), Thermophilic Microbes Environ Ind Biotechnol Biotechnol Thermophiles, second ed. Springer Netherlands, Dordrecht, pp. 429e458. Kannouche, P., Stary, A., 2003. Xeroderma pigmentosum variant and error-prone DNA polymerases. Biochimie 85, 1123e1132. Kornberg, A., 1957. Enzymatic synthesis of deoxyribonucleic acid. Harvey Lect. 53, 83e112. Lander, E.S., Linton, L.M., Birren, B., Nusbaum, C., Zody, M.C., Baldwin, J., et al., 2001. Initial sequencing and analysis of the human genome. Nature 409, 860e921. Loeb, L.A., 2001. A mutator phenotype in cancer. Cancer Res. 61, 3230e3239. Ponamarev, M.V., Longley, M.J., Nguyen, D., Kunkel, T.A., Copeland, W.C., 2002. Active site mutation in DNA polymerase gamma associated with progressive external ophthalmoplegia causes error-prone DNA synthesis. J. Biol. Chem. 277, 15225e15228. Rothwell, P.J., Waksman, G., 2005. Structure and mechanism of DNA polymerases. Adv. Protein Chem. 71, 401e440. Venter, J.C., Adams, M.D., Myers, E.W., Li, P.W., Mural, R.J., Sutton, G.G., et al., 2001. The sequence of the human genome. Science 291, 1304e1351. Yamagami, T., Ishino, S., Kawarabayasi, Y., Ishino, Y., 2014. Mutant Taq DNA polymerases with improved elongation ability as a useful reagent for genetic engineering. Front. Microbiol. 5, 461.

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SUBCHAPTER 8.19

DNA ligases 8.19.1 Microbial sources and properties DNA ligases are one of the most important enzymes in modern biotechnology. In 1967, DNA ligase was discovered independently by the Gellert, Lehman, Richardson, and Hurwitz Institute. Since its discovery, DNA ligase has become an important tool in molecular biology. This enzyme plays a significant role in DNA replication and repair. It acts like a glue, which can bind different pieces of DNA together. Scientists use this molecular glue for joining DNA fragments into entirely new sequences. But, people are using it as a tool without knowing the history and mechanism of DNA ligase. DNA ligases are important in maintaining genomic integrity by bridging the cleavage of the phosphodiester backbone of DNA. This break occurs during replication and recombination and as a result of DNA damage and its repair. Since the discovery of the enzyme in 1968, DNA ligases from several organisms have been investigated for their biochemical potential and physiological importance. The identification of DNA ligases from thermophiles is also of fundamental interest. Such thermostable DNA ligases retain catalytic activity in nick-binding reactions during temperature cycling, including thermal (90e100
C) denaturation steps of double-stranded DNA. Thermostable DNA ligases are important as ligase detection reaction (LDR)/ligase chain reaction (LCR) enzymes. LDR/LCR is a method for detection of singlenucleotide mutations in DNA strands and is useful in diagnosing genetic disorder (Barany, 1991; Zirvi et al., 1999). Yang et al. (2010) reported that this method has also been used to detect microRNAs that play key regulatory roles in fundamental cellular processes through the formation of RNA-induced silencing complexes (RISCs) with target mRNAs. LCR using thermostable DNA ligase also enabled background removal caused by target-independent ligation. DNA modifications of LCR have been conducted for improving sensitivity and utility, including gapped PCR and realtime quantitative LCR (Abravaya et al., 1995; Psifidi et al., 2011). Hyperthermophilic DNA ligases are particularly suitable for LCR. DNA ligase from P. furiosus and T. aquaticus, T. filiformis, T. scotodactus (eubacteria), T. Thermophilus, and Thermococcus 9
N (archaea) are currently commercially available. Table 8.19.1 shows thermostable DNA ligases from archaea and bacteria. A lack of any DNA ligase gene results in some developmental and immunological deficiencies even though DNA ligases are functionally linked to DNA replication and repair. DNA repair goes wrong when there is no DNA ligase. The lack of ligase frequently results in Bloom syndrome, LIG4 syndrome, Ataxia-telangiectasia, and Fanconi anemia (https://geneticeducation.co.in/).

Table 8.19.1 Thermostable DNA ligases for archaea and bacteria characterized to date and their cofactor utilities. Species

Cofactor

References

Archaea

ATP ATP ATP ATP ATP

Sulfophobococcus zilligii Sulfolobus acidocaldarius Sulfolobus shibatae Thermococcus fumicolans Thermococcus kodakarensis Thermococcus sp. Thermococcus sp. 1519 Thermoplasma acidophilum

ATP ATP ATP ATP ATP ATP ATP ATP

ATP ATP ATP ATP

ADP NADþ

NAD*

ADP GTP ADP NAD. NAD* NAD* NAD*

Ferrer et al. (2008) Jeon and Ishikawa (2003) Jackson et al. (2007) Ferrer et al. (2008) Sriskanda et al. (2000) Ferrer et al. (2008) Keppetipola and Shuman (2005) Kiyonari et al. (2006) Seo et al. (2007) Sun et al. (2008) Ferrer et al. (2008) Lai et al. (2002) Rolland et al. (2004) Nakatani et al. (2000) Kim et al. (2006) Bezsudnova et al. (2009) Ferrer et al. (2008)

Bacteria

Aquifex aeolicus VF5 Aquifex pyrophilus Bacillus stearothermophilus

NAD* NAD. NAD.

Rhodothermus marinus Thermus filiformis Thermus scotoductus (Ts) Thermus species AKI6D Thermus thermophilus HB8 Zymomonas mobilis

NAD* NAD* NAD. NAD. NAD* NAD*

Tong et al. (2000) Lim et al. (2001) Singleton et al. (1999) and Timson and Wigley (1999) Thorbjarnard
ottir et al. (1995) Lee et al. (2000) Thorbjarnard
ottir et al. (1995) Tong et al. (1999) Takahashi et al. (1984) Shark and Conway (1992)

281

From Ishino, S., Ishino, Y., 2013. DNA polymerases and DNA ligases. In: Satyanarayana, T., Littlechild, J., Kawarabayasi, Y. (Eds.), Thermophilic Microbes Environ Ind Biotechnol Biotechnol Thermophiles, second ed., Springer Netherlands, Dordrecht, pp. 429e458. Reproduced with permission.

Industrial applications of thermophilic/hyperthermophilic enzymes

Acidithiobacillus ferrooxidans Aeropyrum pet-nix Ferroplasma acidarmanus Ferroplasma acidophilutn Methanothermobacterium thermoautrophicum Picrophilus torridus Pyrococcus horikoshii Pyrococcus furiosus Staphylothermus marinus

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Developments and Applications of Enzymes From Thermophilic Microorganisms

T4 DNA ligase is the name given to the DNA ligase that is most frequently used in biotechnological experiments because it was discovered in bacteriophage T4. T4 DNA ligase uses ATP as energy, in contrast to the enzyme present in E. coli. Additionally, T4 DNA ligase can bind to DNA, RNA, or DNA/RNA hybrids and can ligate both sticky and blunt ends.

8.19.2 Application DNA ligase has been extensively utilized in a variety of molecular biology and biotechnology applications since its discovery. In recombinant DNA experiments, DNA ligases and restriction enzymes are frequently combined. The enzyme has been used in various ways to sequence DNA. It is advantageous for this enzyme that DNA ligase does not require dNTPs as substrates for its activity because excessive dNTP concentrations in solution, a common occurrence in DNA polymerase reactions, can interfere with accurate measurement. Assays for protein-protein interactions also use DNA ligase (Landegren et al., 1988). These methods are widely used for rapid and effective in situ studies of protein-protein interactions (Söderberg et al., 2006; Tanabe et al., 2015). Research in microbiology, immunology, molecular genetics, and oncology frequently uses in vitro DNA manipulation, and it also has useful applications in clinical assays. DNA-related enzymes found through research into the molecular processes of DNA replication and repair were used to create this technology. DNA primase is the enzyme that starts DNA replication. A small RNA primer is created by DNA primase and extended by a DNA polymerase. DNA polymerase can be used in the PCR due to its primer extension activity. In a short in vitro reaction, it amplifies a single copy of DNA by several orders of magnitude (Foy and Parkes, 2001; Conaway and Lehman, 1982; Erlich, 1989; Levin et al., 1997). Additionally, DNA ligase helps in joining Okazaki fragments during lagging strand maturation. DNA ligases have contributed to recombinant DNA techniques such as DNA cloning based on their ability to join two DNA strands in vitro (Copeland et al., 2001). In addition, DNA ligases are extensively used in ligase-mediated mutation detection methods and DNA sequencing. DNA ligase is used in genetic engineering to create recombinant DNA. First, two DNA fragments, presumably plasmid and genomic DNA, are cleaved using the same restriction enzyme. Some restriction enzymes cut DNA unevenly, creating single-stranded regions known as sticky ends. Ligase then joins two complementary sticky ends of plasmid and genomic DNA to create a new double-stranded DNA sequence. Utilizing DNA ligase, next-generation DNA sequencing has been researched. Genomic and genetic research is being revolutionized by next-generation sequencing (NGS) technologies such as Roche’s 454 FLX Pyrosequencer, Illumina’s Illumina Genome Analyzer, and Life Technologies’ Sequence by Oligonucleotide Ligation and Detection (SOLiD) Sequencer (Tanabe et al., 2015; Bentley, 2006; Mardis, 2008; Margulies et al., 2005). Although these platforms differ significantly in terms of sequence biochemistry and sequence recognition, the workflows are conceptually analogus. Table 8.19.2 summarzes application of DNA ligase.

Industrial applications of thermophilic/hyperthermophilic enzymes

Table 8.19.2 Application of DNA ligase.

Ligates both the cohesive blunt ends and sticky ends. Ligate single-stranded and double-stranded DNA Used in a ligase chain reaction Used during the DNA repair mechanism and replication Used to insert a gene in the plasmid Also useful in nanochemistry and DNA origami experiments https://geneticeducation.co.in/.

Bibliography Abravaya, K., Carrino, J.J., Muldoon, S., Lee, H.H., 1995. Detection of point mutations with a modified ligase chain reaction (Gap-LCR). Nucleic Acids Res. 23 (4), 675e682. Barany, F., 1991. The ligase chain reaction in a PCR world. PCR Methods Appl. 1, 5e16. Bentley, D.R., 2006. Whole-genome re-sequencing. Curr. Opin. Genet. Dev. 16 (6), 545e552. Bezsudnova, E.Y., Kovalchuk, M.V., Mardanov, A.V., Poliakov, K.M., Popov, V.O., Ravin, N.V., Skryabin, K.G., Smagin, V.A., Stekhanova, T.N., Tikhonova, T.V., 2009. Overexpression, purification and crystallization of a thermostable DNA ligase from the archaeon Thermococcus sp. 1519. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 65 (Pt 4), 368e371. Conaway, R.C., Lehman, I.R., 1982. A DNA primase activity associated with DNA polymerase alpha from Drosophila melanogaster embryos. Proc. Natl. Acad. Sci. U. S. A. 79 (8), 2523e2527. Copeland, N.G., Jenkins, N.A., Court, D.L., 2001. Recombineering: a powerful new tool for mouse functional genomics. Nat. Rev. Genet. 2 (10), 769e779. Erlich, H.A., 1989. Polymerase chain reaction. J. Clin. Immunol. 9 (6), 437e447. Ferrer, E., Golyshina, O.V., Beloqui, A., Bottger, L.H., Andreu, J.M., Polaina, J., De Lacey, A.L., Trautwein, A.X., Timmis, K.N., Golyshin, P.N., 2008. A purple acidophilic di-ferric DNA ligase from Ferroplasma. Proc. Natl. Acad. Sci. U. S. A. 105, 8878e8883. Foy, C.A., Parkes, H.C., 2001. Emerging homogeneous DNA-based technologies in the clinical laboratory. Clinical Chemistry 47 (6), 990e1000. Ishino, S., Ishino, Y., 2013. DNA polymerases and DNA ligases. In: Satyanarayana, T., Littlechild, J., Kawarabayasi, Y. (Eds.), Thermophilic Microbes Environ Ind Biotechnol Biotechnol Thermophiles, second ed. Springer Netherlands, Dordrecht, pp. 429e458. Jackson, B.R., Noble, C., Lavesa-Curto, M., Bond, P.L., Bowater, R.P., 2007. Characterization of an ATPdependent DNA ligase from the acidophilic archaeon “Ferroplasma acidarmanus” Fer1. Extremophiles 11 (2), 315e327. Jeon, S.J., Ishikawa, K., 2003. A novel ADP-dependent DNA ligase from Aeropyrum pernix K1. FEBS Lett. 550 (1e3), 69e73. Keppetipola, N., Shuman, S., 2005. Characterization of a thermophilic ATP-dependent DNA ligase from the euryarchaeon Pyrococcus horikoshii. J. Bacteriol. 187 (20), 6902e6908. Kim, Y.J., Lee, H.S., Bae, S.S., Jeon, J.H., Yang, S.H., Lim, J.K., Kang, S.G., Kwon, S.T., Lee, J.H., 2006. Cloning, expression, and characterization of a DNA ligase from a hyperthermophilic archaeon Thermococcus sp. Biotechnol. Lett. 28 (6), 401e407. Kiyonari, S., Takayama, K., Nishida, H., Ishino, Y., 2006. Identification of a novel binding motif in Pyrococcus furiosus DNA ligase for the functional interaction with proliferating cell nuclear antigen. J. Biol. Chem. 281 (38), 28023e28032. Lai, X., Shao, H., Hao, F., Huang, L., 2002. Biochemical characterization of an ATP-dependent DNA ligase from the hyperthermophilic crenarchaeon Sulfolobus shibatae. Extremophiles 6 (6), 469e477. Landegren, U., Kaiser, R., Sanders, J., Hood, L., 1988. A ligase-mediated gene detection technique. Science 241 (4869), 1077e1080. Lee, J.Y., Chang, C., Song, H.K., Moon, J., Yang, J.K., Kim, H.K., Kwon, S.T., Suh, S.W., 2000. Crystal structure of NAD (þ)-dependent DNA ligase: modular architecture and functional implications. EMBO J. 19 (5), 1119e1129. Levin, D.S., Bai, W., Yao, N., O’Donnell, M., Tomkinson, A.E., 1997. An interaction between DNA ligase I and proliferating cell nuclear antigen: implications for Okazaki fragment synthesis and joining. Proc. Natl. Acad. Sci. U. S. A. 94 (24), 12863e12868.

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Lim, J.H., Choi, J., Han, S.J., Kim, S.H., Hwang, H.Z., Jin, D.K., Ahn, B.Y., Han, Y.S., 2001. Molecular cloning and characterization of thermostable DNA ligase from Aquifex pyrophilus, a hyperthermophilic bacterium. Extremophiles 5 (3), 161e168. Mardis, E.R., 2008. Next-generation DNA sequencing methods. Ann. Rev. Genom. Hum. Genet. 9, 387e402. Margulies, M., Egholm, M., Altman, W., et al., 2005. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376e380. Nakatani, M., Ezaki, S., Atomi, H., Imanaka, T., 2000. A DNA ligase from a hyperthermophilic archaeon with unique cofactor specificity. J. Bacteriol. 182 (22), 6424e6433. Psifidi, A., Dovas, C., Banos, G., 2011. Novel quantitative real-time LCR for the sensitive detection of SNP frequencies in pooled DNA: method development, evaluation and application. PLoS ONE 6 (1), e14560. Rolland, J.L., Gueguen, Y., Persillon, C., Masson, J.M., Dietrich, J., 2004. Characterization of a thermophilic DNA ligase from the archaeon Thermococcus fumicolans. FEMS Microbiol. Lett. 236 (2), 267e273. Seo, M.S., Kim, Y.J., Choi, J.J., Lee, M.S., Kim, J.H., Lee, J.H., Kwon, S.T., 2007. Cloning and expression of a DNA ligase from the hyperthermophilic archaeon Staphylothermus marinus and properties of the enzyme. J. Biotechnol. 128 (3), 519e530. Shark, K.B., Conway, T., 1992. Cloning and molecular characterization of the DNA ligase gene (lig) from Zymomonas mobilis. FEMS Microbiol. Lett. 75, 19e26. Singleton, M.R., Håkansson, K., Timson, D.J., Wigley, D.B., 1999. Structure of the adenylation domain of an NADþ-dependent DNA ligase. Structure 7 (1), 35e42. Söderberg, O., Gullberg, M., Jarvius, M., Ridderstråle, K.K., Leuchowius, K., Jarvius, J., Wester, K., Hydbring, P., Bahram, F., Larsson, L., Landegren, U., 2006. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat. Methods 3, 995e1000. Sriskanda, V., Kelman, Z., Hurwitz, J., Shuman, S., 2000. Characterization of an ATP-dependent DNA ligase from the thermophilic archaeon Methanobacterium thermoautotrophicum. Nucleic Acids Res. 28 (11), 2221e2228. Sun, Y., Seo, M.S., Kim, J.H., Kim, Y.J., Kim, G.A., Lee, J.I., Lee, J.H., Kwon, S.T., 2008. Novel DNA ligase with broad nucleotide cofactor specificity from the hyperthermophilic crenarchaeon Sulfophobococcus zilligii: influence of ancestral DNA ligase on cofactor utilization. Environ. Microbiol. 10 (12), 3212e3224. Takahashi, M., Yamaguchi, E., Uchida, T., 1984. Thermophilic DNA ligase. J. Biol. Chem. 259, 10041e10047. Tanabe, M., Ishino, Y., Nishida, H., 2015. From structure-function analyses to protein engineering for practical applications of DNA ligase. Archaea 2015. Article ID 267570, 20 pages. Thorbjarnard
ottir, S.H., J
onsson, Z.O., Andr
esson, O.S., Kristj
ansson, J.K., Eggertsson, G., Palsdottir, A., 1995. Cloning and sequence analysis of the DNA ligase-encoding gene of Rhodothermus marinus, and overproduction, purification and characterization of two thermophilic DNA ligases. Gene 161 (1), 1e6. Timson, D.J., Wigley, D.B., 1999. Functional domains of an NADþ-dependent DNA ligase. J. Mol. Biol. 285 (1), 73e83. Tong, J., Barany, F., Cao, W., 2000. Ligation reaction specificities of an NAD(þ)-dependent DNA ligase from the hyperthermophile Aquifex aeolicus. Nucleic Acids Res. 28 (6), 1447e1454. Tong, J., Cao, W., Barany, F., 1999. Biochemical properties of a high fidelity DNA ligase from Thermus species AK16D. Nucleic Acids Res. 27 (3), 788e794. Yang, J., Li, Z., Liu, C., Cheng, Y., 2010. Simple and sensitive detection of microRNAs with ligase chain reaction. Chem. Commun. 46, 2432e2434. Zirvi, M., Bergstrom, D.E., Saurage, A.S., Hammer, R.P., Barany, F., 1999. Improved fidelity of thermostable ligases for detection of microsatellite repeat sequences using nucleoside analogs. Nucleic Acids Res. 27 (24), e41.

Relevant websites https://www.edvotek.com/. https://geneticeducation.co.in/.

CHAPTER 9

Future perspectives Hyperthermophilic enzymes have developed into model systems for research into the evolution of enzymes, their stability and mechanisms of action, the relationships between protein structure and function, and biocatalysis in harsh environments. These applications result from the discovery that the cloning and expression of genes from hyperthermophiles in mesophilic hosts facilitate biochemical and molecular biology studies, such as protein purification and characterization. A wide variety of enzymes from the remarkable variety of archaeal and bacterial hyperthermophiles can be chosen to create new biotechnological applications. This field has a fascinating and endless future (Vieille and Zeikus, 2001). Microorganisms that are thermophilic have been isolated and identified. About this group’s physiology, little is known. Thermophilic microorganisms produce extracellular enzymes that can withstand high temperatures. The fact that mesophilic organisms can also produce extracellular enzymes with high thermostability shows how important it is to understand the genetic and structural details of these enzymes. Thermostable enzymes are used in several industries, for example, food, textile, pulp and paper, and biofuels, but there is still a lot of room for finding new applications. This is why there is a growing market for the production of natural or genetically altered enzymes (Gomes et al., 2016). In particular, thermophilic enzymes show great potential for applications in biotechnology because of their higher tolerance to temperature extremes, chemicals, organic solvents, and pH values (Dumorne et al., 2017). There is a great requirement for new thermostable enzymes with new functionalities with improved performance that can save time, money, and energy in the production of high value chemicals and other “white” biotechnology applications. Thermophilic/hyperthermophilic microorganisms are a more accessible and sustainable source in several biotechnology fields for developing biobased economies. These enzymes have had a major impact from a commercial and biotechnological point of view and are already being used in many industrial applications, but to a limited extent in biofuels, but still have the potential to be discovered (Secades et al., 2003). For biomass conversion and biofuel production, hyperthermophilic enzymes are attracting attention in the field of metabolic engineering. Improving the productivity and activity of hyperthermophilic enzymes is an important step for low-cost and effective utilization of lignocellulosic biomass (Ebaid et al., 2019). The applicability of thermostable enzymes as biocatalysts for the depolymerization of lignocellulosic feedstocks in the production of biofuels has attracted broad Developments and Applications of Enzymes From Thermophilic Microorganisms ISBN 978-0-443-19197-8, https://doi.org/10.1016/B978-0-443-19197-8.00004-9

© 2023 Elsevier Inc. All rights reserved.

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industrial and biotechnology interest. Their robust thermal stability makes them better suited to the harsh processing conditions required to efficiently break down lignocellulose into fermentable products. Various chemical methods have been used to improve the performance of enzymes and have shown encouraging results. However, they have several negative environmental impacts. In recent years, new gene expression systems have been developed for the recombinant production of hyperthermophilic enzymes, offering a wide range of options for engineering hyperthermophilic cells. A complete financial evaluation of biofuel manufacturing from lignocellulosic waste using hyperthermophilic enzymes requires exhaustive studies that directly or indirectly strengthen the biofuel market. Combining functional genomics and metabolic engineering with other omics techniques can also help in developing robust strains for a broad range of applications (Ebaid et al., 2019). In the genomics period, many hyperthermophilic enzymes with novel properties are being found through comprehensive comparative genomic proteomics analysis with high-throughput structural and functional characterization. The genomes of many hyperthermophiles have been sequenced and more are in preparation (http://www.genomesonline.org/). Hyperthermophiles are hosts for many genes, several of which are able to encode proteins with unknown functions. A broad range of thermostable and biologically novel enzymes for a variety of possible applications is anticipated to become available merely by investigating the ever-increasing database of (meta)genome sequences. For the pharmaceutical and chemical industry, the characterization of these novel proteins has great potential as they are used to synthesize compounds that are presently not easy to synthesize by the use of conventional synthetic methods. Moreover, these natural enzymes form the basis for further protein engineering by computational and/or combinatorial laboratory techniques described and will certainly lead to a new step in high-temperature enzymes (Unsworth et al., 2007). Thermophiles at the moment are advanced enough to start developing metabolically engineered strains for producing industrially applicable chemicals but so far there have been no major breakthroughs in this area. Only P. furiosus has made progress. Research on thermophiles has historically focused on answering fundamental questions about how life functions at higher temperatures: how transcription and translation occur, how the protein molecules fold, how metabolism occurs through thermolabile intermediates, and whether early life might have evolved at higher temperatures? But, the emerging industrial biotechnology sector is focusing more on producing chemicals and fuels by exploiting thermophilicity (Zeldes et al., 2015). Extreme thermophiles are well positioned to meet the demand for mass production of chemicals from renewable feedstocks due to their extraordinary benefits and the recent expansion of genetic systems enabling metabolic engineering. They can withstand higher temperatures which results from the heat produced in huge bioreactors, and operating at these temperatures makes them less

Future perspectives

prone to be contaminated by microbes and phages from the environment. It also exhibits exceptional metabolic properties as a product of extreme environments. More work is still needed to realize the promise of producing large amounts of renewable fuels and chemicals using thermophilic hosts, but genetic tools now exist to enable this work (Zeldes et al., 2015).

References Dumorne, K., C ordova, D.C., Astorga-El o, M., Renganathan, P., 2017. Extremozymes: a potential source for industrial applications. J. Microbiol. Biotechnol. 27 (4), 649e659. Ebaid, R., Wang, H., Sha, C., Abomohra, A.E., Shao, W., 2019. Recent trends in hyperthermophilic enzymes production and future perspectives for biofuel industry: a critical review. J. Clean. Prod. 238 (20), 117925. Gomes, E., de Souza, A.R., Orjuela, G.L., Da Silva, R., de Oliveira, T.B., Rodrigues, A., 2016. Applications and benefits of thermophilic microorganisms and their enzymes for industrial biotechnology. In: Schmoll, M., Dattenböck, C. (Eds.), Gene Expression Systems in Fungi: Advancements and Applications. Fungal Biology. Springer, Cham. Secades, P., Alvarez, B., Guijarro, J.A., 2003. Purification and properties of a new psychrophilic metalloprotease (Fpp2) in the fish pathogen Flavobacterium psychrophilum. FEMS Microbiol. Lett. 226, 273e279. Unsworth, L.D., van der Oost, J., Koutsopoulos, S., 2007. Hyperthermophilic enzymes-stability, activity and implementation strategies for high temperature applications. FEBS J. 274 (16), 4044e4056. Vieille, C., Zeikus, G.J., 2001. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65 (1), 1e43. Zeldes, B.M., Keller, M.W., Loder, A.J., Straub, C.T., Adams, M.W.W., Kelly, R.M., 2015. Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals. Front. Microbiol. 6, 1209.

Relevant websites http://www.genomesonline.org/. https://worldwidescience.org/.

287

Index ‘Note: Page numbers followed by “f ” indicates figures and “t” indicates tables.’

A Agriculture esterases, 272 pectinase, 215e216 Alcohal dehydrogenases applications, 264e266 chiral pharmaceutical intermediates, 265 ethanol production, 264e265 fine chemicals, 265 microbial sources and properties, 263e264 types, 263 Alkaliphilic microorganisms, 23 Amylase, discovery, 1 Amylases, 82te83t a-amylase amino acid residues, 108 applications, 111e123, 111t Bacillus amyloliquefaciens, 109f bacterial and archaeal, 112te114t bioremediation, 122 clinical and pharmaceutical industry, 122 commercially available bacterial, 116t detergent industry, 118 food industry, 119e120 fuel alcohol production, 118e119 fungal, 115t mode of action, 109t paper industry, 120e122 starch conversion, 116e118 starch hydrolysis, 106 textile industry, 120 three-dimensional structures, 108f two-dimensional structure, 107e108 b-amylase, 106, 108f categories, 106 g-amylase, 107 microbial sources and properties, 106e111 polypeptide chain, 108 starch hydrolysis, 107f Amylopullulanases baking industry, 168e169 brewing and alcohol industries, 169 food industry, 168 hyperthermophilic archaeal, 166t

industrial applications, 166e169 microbial sources, 164e166 properties, 164e166 thermophilic bacterial, 165t Antistaling agent, pullulanase, 159 Aquifex aeolicus, 18e19 Aquifex pyrophilus, 18e19, 32 Aquificales, 33te36t, 37 Archaeoglobales, 33te36t Arrhenius plots, 51e52 Aspergillus niger glucoamylase, 134f

B Bacillus thermoproteolyticus, 232e233 Baking industry amylopullulanases, 168e169 lipase, 247 b-amylase, 106 Bent Arrhenius plots, 51e52 b-glucosidase biofuel production, 146 cassava detoxification, 147 cellulolytic microbes, 141 classification, 142 cloning, 143 deficiency, 141 flavor industry, 147 fungal, 143 industrial application, 144e148 intracellular, 143 microbial sources and properties, 141e144 microorganisms producing, 144t recombinant, 145t thermal stability, 143 vitamins, 148 waste paper deinking, 148 Bioblasting, cellulases, 187 Biocatalyst, 47e48 chemical modification and immobilization, 47 chemical reactions, 47 metagenomic genetic discovery, 47 Biodiesel production, lipase, 248 Biodiversity hyperthermophilic microorganisms, 33te36t

289

290

Index

Biodiversity (Continued) Aquificales and Thermotogale, 37 deep-sea hyperthermophiles, 32e37 environmental 16S rRNA sequences analysis, 29 enzyme model system, 31 Euryarchaeota and Crenarchaeota, 37 higher sulfur content, 31 hot natural and industrial environments, 29 hot natural environments, 32 single hot spring, Yellowstone National Park, 32 Sulfolobales, 37e38 thermal springs, Vulcano Island, 38t thermophilic microorganisms archea and bacteria, 31 culture-dependent approach, 29e31 culture-independent approach, 29e31 hot springs, 31e32 optimum growth temperature, 29 thermal springs, Vulcano Island, 38t Biofuel industry b-glucosidase, 146e147 cellulases, 191e192 laccase, 254 xylanases, 206 Biomats and sediments, 29e31, 30f Biorefineries Clostridium thermocellum, 70e71 ethanol biorefineries, 67 Kluyveromyces marxianus, 67, 70 Saccharomyces cerevisiae, 67 second-generation, 66, 67t Thermoanaerobacterium saccharolyticum, 71 Bioremediation a-amylase, 122 laccase, 254 Bio-stonewashing, 187 Biphasic Arrhenius plots, 51e52 Bright orange thermophiles, 24f

C Caldicellulosiruptor bescii, 182e183 Caldolysin, 95 Cassava detoxification, b-glucosidase, 147 Cell-free dehydrogenase, 65 Cellulases, 82te83t animal feed industry, 188e189 applications, 186e192 biofuels, 191e192

Caldicellulosiruptor bescii, 182e183 commercial applications, 182 food industry, 189e191, 190f microbial sources, 182e186 pulp and paper industry, 187e188 textile and detergent industry, 187 thermostable, 182e186, 184te185t Chaperonins, 50 Chemical industry esterases, 272 proteases, 236 xylanases, 205e206 Chemical modifications advantages, 93f caldolysin, 95 circular dichroism studies, 92 cross-linked enzyme aggregates (CLEAs), 95 D-hydantoinase cross-linking, 95 enzyme catalysis, 93 vs. genetic modification, 90e95 intramolecular cross-linking, 94 site-specific, 90e91, 92f Chitinases actinomycetes and bacterial species, 223, 224t action of, 223f applications, 225e228 biocontrol agents, 227 classification, 222e223 fungal, 224e225, 225t microbial sources, 222e225 pharmaceutical and medical uses, 227e228 properties, 222e225 waste management, 226e227 Chitobiosidase, 223 Circular dichroism, 48, 92 Clostridium thermocellum, 70e71 Commercially available hyperthermophilic enzymes, 82te83t Confocal laser scanning microscopy (CLSM) analyses, a-amylase, 123 Conformational changes, 48 Crenarchaeota, 21, 37, 38t Culture-dependent strategies, 17 Culture-independent strategies, 17 Cuprophan membranes, 84e85 Cyclodextrin glycosyl transferase (CGTase) application, 176e178 chemical structure, 174f cyclodextrin isolation, 174e176, 175f

Index

3D structure, 176 microbial sources, 173e176 properties, 173e176 sources and optimum growth conditions, 175t Cyclodextrins production, pullulanase, 159

D Deep-sea hyperthermophiles, 32e37 Desulfurococcales, 33te36t Detergent industry a-amylase, 118 cellulases, 187 lipase, 245e246 proteases, 235e236 pullulanase, 158e159 xylanases, 204e205 Dextrose equivalent (DE), 157 Diastase, discovery, 1 Dictyoglomus turgidum, 182e183 Directed evolution, 105 DNA ligases application, 282, 283t archaea and bacteria, 281t ligase chain reaction (LCR), 280 ligase detection reaction (LDR), 280 microbial sources and properties, 280e282 recombinant DNA techniques, 282 DNA polymerase applications, 278e279 genetic engineering, 277 microbial sources and properties, 276e278 PCR method, 278 structural overview, 277, 277f

E Eichhornia crassipes, 90 Endochitinases, 222 Endoglucanase, 89e90 Enzyme production animal origin, 78 commercial enzymes, 78 hyperthermophilic enzyme production biomass yield, 84e85 cellulase and hemicellulase, 84e85 commercial, 79, 82te83t dialysis fermentation, 84e85 extremophilic microbes, 78 micro filtration reactor, 85 novozymes, 78

paper industry, 84 solid-state fermentation (SSF), 85 xylanases, 79 plant origin, 78 steps involved, 77t thermophiles biomass production, 63 biorefineries, 66e71 commercial fermentation, 63e64 genetic engineering methods, 64 optimum growth temperature, 63 pretreatment methods, 65e66 vectors, 64, 64t whole cells/isolated enzymes, 65e66 Enzymes as biocatalyst, 47e48 commercial prospects, 1e2 historical background, 1e2 industrial markets, 2 Esterases, 82te83t agriculture, 272 application, 270e272 beverages and perfume, 270e272 characteristics, 268t chemical industries, 272 classification, 269t food and dairy application, 270e272 highly thermostable, 269e270 microbial sources and properties, 268e270 synthetic materials degradation, 272 (hyper)thermophilic microorganisms, 271t Ethanol production alcohal dehydrogenases, 264e265 a-amylase, 118e119 glucoamylases, 135e136 glucose (xylose) isomerase, 241 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), 94 Euryarchaeota, 37, 38t Exochitinases, 223 Extreme thermophiles, 5, 286e287 Extremophiles, 48 Extremozymes, 8

F Facultative aerobes, 31 Facultative thermophiles, 5

291

292

Index

Feed industry proteases, 234 xylanases, 204 Flavor industry b-glucosidase, 147 lipase, 246 Food industry esterases, 270e272 laccase, 253 lipase, 245, 245t pectinase, 214e215 proteases, 234 xylanases, 203 Food industry, a-amylase, 119e120 Food-protein-derived peptides, 234f Forestry, laccase, 252e253 Fuel alcohol production, a-amylase, 118e119 Fuelzyme, 79

G Gene expression systems, 286 Generally recognized as safe (GRAS), 78 Genetic engineering, 95 arabinose addition, 98e99 cI gene, bacteriophages, 99 E. coli, 96t expression vectors, 97 pHsh vector, 99e100 expression plasmid, 97f genetic mutagenesis, 100 glutamate dehydrogenase (gdhA) gene, 98 heterologous expression, 95e96 hyperthermostable enzyme, 96 isopropyl-b-D-thiogalactopyranoside (IPTG), 99 LacS gene, 98 maltose/dextrin addition, 98e99 molecular engineering, 98 multiple transformation techniques, 98 promoter, 97 shuttle vector-based transformation system, 98 transient DNA expression, 95e96 Geobacillus thermoleovorans NP33 amylopullulanase, 164 Geothermal hot springs, 31e32 Glucoamylases applications, 132e137 Aspergillus niger, 134f ethanol production, 135e136 fungal, 131

glycosylation, 132 manufacturers, 137, 137t microbial sources and properties, 131e132 microorganisms producing, 133t modular arrangements, 133f sugar production, 134e135 yeast, 132 Glucose (xylose) isomerase ethanol production, 241 high fructose corn syrup, 241 microbial sources, 239e240 microorganisms, 240t properties, 239e240 Thermobifida fusca WSH03-11 (TfuGI), 240 Thermotoga maritime (TmaGI), 240 Thermus oshimai (TosGI), 240 Glucose (xylose) isomerases, 82te83t Glucose oxidase, 82te83t Glutamate dehydrogenase, 82te83t Glutamate dehydrogenase (gdhA) gene, 98

H Hemicellulases, 82te83t High-amylose starch, 158 High fructose corn syrup (HFCS), 239, 241 Hot springs, 29e31, 30f Hyperthermophiles directed evolution, 8e9 extremozymes, 8 growth temperature, 4e5 heat-stable enzymes, 9 protein stability, 8e9 research, 11f thermostability, 7e8 Hyperthermophilic enzymes, 53te55t, 285

I Industrial applications alcohal dehydrogenases, 263e264 amylases, 106e111 amylopullulanases, 164e166 cellulases, 182e186 chitinases, 222e225 cyclodextrin glycosyl transferase (CGTase), 173e176 DNA ligases, 280e282 DNA polymerase, 276e278 esterases, 268e270 glucoamylases, 131e132

Index

glucose (xylose) isomerase, 239e240 laccase, 251e252 lipase, 251e252 phytase, 257e258 proteases, 232e233 thermostable enzyme, 105 xylanases, 198e200 Industrially relevant feedstock Isolated enzymes, thermophilic enzyme production, 65 Isopropyl-b-D-thiogalactopyranoside (IPTG), 99

K Kinetic stability, 51e52 Kluyveromyces marxianus, 67, 70

L Laccase applications, 252e254 biofuel, 254 bioremediation, 254 food industry, 253 forestry, 252e253 fungi, 251, 252t microbial sources and properties, 251e252 nanobiotechnology, 254 organic synthesis, 254 personal care and medical applications, 253 textile industry, 253 Laccases, 82te83t Ligase chain reaction (LCR), 280 Lipases, 82te83t applications, 244e249 baking industry, 247 biocatalyzed reactions, 243e244 biodiesel production, 248 characteristics, 268t cocoa butter equivalent, 246e247 dairy industry, 247 detergents, 245e246 flavor development, 246 food industry, 245 microbial sources, 243e244 microbial sources and properties, 251e252 oleochemical industry, 247 pharmaceutical industry, 248e249 plastic biodegradation, 248 properties, 243e244 racemic acid and alcohol separation, 247

textile industry, 246 thermostable, 244t

M Maltooligosaccharide preparation, pullulanase, 159e160 Maltotriose syrup, 167e168 Manmade thermal habitats, 17 Mesophilic enzymes, 51e52 Methanobacteriales, 33te36t Methanococcales, 33te36t Methanopyrales, 33te36t Moderate thermophiles, 5 Multifunctional polymers, 94

N Nanobiotechnology, laccase, 254 Natural thermal habitats, 17 Novamyl, 79 Novozym 435, 94 Novozymes, 84, 114

O Obligate thermophiles, 5 Oleochemical industry, lipase, 247

P Paper industry a-amylase, 120e122 boil-outs, 121e122 chemical modification, 121 deinking, 121 enzymatic modification, 120 cellulases, 187e188 pectinase, 217 xylanases, 202e203 Pectinase agriculture, 215e216 applications, 214e218, 218f bacterial source, 213e214 classification, 212e213 food industry, 214e215 paper and pulp industry, 217 plant development, 212 polygalacturonases, 212 prebiotics/functional foods, 218 waste water treatment, 217e218 wine industry, 216e217

293

294

Index

Pectin-degrading enzymes microbial sources, 212e214 pectinase. See Pectinase plant development, 212 properties, 212e214 Pectin-derived oligosaccharides (PDOs), 218 Pharmaceutical industry a-amylase, 122 lipase, 248e249 xylanases, 205e206 Photographic industry, proteases, 236 Physical modification, 93 Physiological and morphological aspects alkaliphilic microorganisms, 23 anaerobic bacteria, environmental stress, 20 archaeal genera, 21 Bright orange thermophiles, 24f crenarchaeal genera, 21 fermentation products, 18 heterotrophic bacteria, 18 hyperthermophilic Aquifex Aquifex pyrophilus and Aquifex aeolicus, 18e20 metabolic process, 18 obligate autotrophy, 20 phylogenetic analysis, 19e20 Thermoplasma species, 22e23 Thermotoga, 18 Phytase applications, 258e260 bacteria source, 258 commercially available products, 259e260 dietary supplement, 259 food additive, 259 fungi and yeasts, 258 microbial, 258 microbial sources and properties, 257e258 plant growth promotion, 259 signifince, 260f therapeutic applications, 259 Polyethersulphonic membrane, 84e85 Prebiotics/functional foods, pectinase, 218 Production/activity enhancement, 89e104 chemical modifications, 90e95 Eichhornia crassipes, 90 endoglucanase, 89e90 genetic engineering, 95e100 growth conditions, 89e90 medium composition, 89e90

Proteases applications, 233e237 Bacillus species, 233 categories, 232 chemical industry, 236 detergent industry, 235e236 food and feed industry, 234 leather industry, 235 medical area, 236e237 microbial sources and properties, 232e233 photographic industry, 236 silk degumming, 236 thermolysin, 232e233 thermophiles, 232e233 waste management, 235 Protein engineering studies, 49 Protein stability, 48e49 Pullulanase amylopullulanases action of, 156f hyperthermophilic archaeal, 166t thermophilic bacterial, 165t antistaling agent, 159 cyclodextrins production, 159 detergents, 158e159 enzymes degrading pullulan, 153 high-amylose starch, 158 industrial application, 157e160 maltooligosaccharide preparation, 159e160 mesophilic bacteria, 155 microbial sources and properties, 153e155 purification and characterization, 155 reaction specificities, 154t starch processing industry, 157e158 Pulp and paper industry cellulases, 187e188 xylanases, 202e203 Pyrobaculum islandicum, 21 Pyrococcus furiosus, 84e85 Pyrodictium abyssi, 21e22 Pyrodictium brockii, 21 Pyrodictium occultum, 21 Pyrolobus fumarii, 32

R Resistant starch, 168 Rhizomucor pusillus, 200

Index

S Saccharification, 65, 134e135 Saccharomyces cerevisiae, 67 Shuttle vector-based transformation system, 98 Silk degumming, proteases, 236 Simultaneous saccharification and fermentation (SSF) process, 66e67 Solid-state fermentation (SSF), 77, 85 Sporotrichum thermophile, 186 Starch conversion, a-amylase, 116e118 Starch hydrolysis, amylase, 107f Stereospecificity, lipase, 247 Submerged fermentation (SmF), 77 Sugar production, glucoamylases, 134e135 Sulfolobales, 33te36t, 37e38 Sulfolobus shibatae, 84e85, 182e183 Sulfolobus solfataricus, 85

T Textile industry a-amylase, 120 cellulases, 187 laccase, 253 lipase, 246 xylanases, 205 Thermoanaerobacterium saccharolyticum, 71 Thermoanaerobacter mathranii, 65e66 Thermobifida fusca WSH03-11 (TfuGI), 240 Thermococcales, 33te36t Thermocrinis ruber, 32 Thermophiles, 286e287 applications, 7t definition, 2 enzymes and roles, 6t extremozymes, 8 facultative, 5 heat-stable enzymes, 9 heat tolerance, 9 microorganisms, 2, 3t obligate, 5 research, 11f temperature-dependent growth, 4e5, 4f Thermophilic/hyperthermophilic enzymes vs. mesophilic enzymes, 52 properties, 48e52 Arrhenius plots, 51e52 chaperonins, 50 conformational changes, 48e49

direct sequence comparisons, 50 kinetic stability, 51e52 molecular mechanisms, 49 optimal operation temperature, 53te55t optimum growth temperature, 48 protein crystal structure, 51 protein engineering studies, 49 protein stability, 48e49 site-directed mutagenesis methods, 51 structural data, 51 thermostability, 49e50 Thermoproteales, 33te36t Thermostability, 49 hyperthermophilic enzymes, 49 kinetic stability, 51e52 mesophilic organisms, 49 Thermotogales, 33te36t, 37 Thermotoga maritima, 32, 37 Thermotoga maritima (TmaGI), 240 Thermotoga species, 18, 19t Thermus oshimai (TosGI), 240 Theroascus aurantiacus, 85 Thielavia terrestris, 89 Thielavia terrestris Co3Bag1, 199e200 Trichoderma reesei, 182

V Vectors, thermophilic expression system, 64t

W Waste management chitinases, 226e227 pectinase, 217e218 proteases, 235 Whole-cell biocatalytic processes, 65 Wine industry esterases, 270e272 pectinase, 216e217

X Xylanases, 79, 82te83t Achaetomium sp. Xz-8, 200 application, 200e207 biofuel industry, 206 detergents, 204e205 feed industry, 204 food industry, 203 Geobacillus sp. Strain WSUCF1, 198e199 microbial sources, 198e200

295

296

Index

Xylanases (Continued) pharmaceuticals and chemicals, 205e206 plant defense mechanisms, 206 properties, 198e200 pulp and paper industry, 202e203 Rhizomucor pusillus, 200

textiles, 205 Thermoascus aurantiacus M-2, 200 thermostable, 199, 201t Thielavia terrestris Co3Bag1, 199e200 transcriptomics and proteome profiling, 198 xynBCA, 199