Microbial Enzymes and Metabolites for Health and Well-Being 1032436190, 9781032436197

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Microbial Enzymes and Metabolites for Health and Well-Being This up-to-date reference book discusses the synthesis, production, and application of various microbial enzymes and metabolites for health. Microorganisms like bacteria (lactic acid bacteria, Bacillus species), yeasts, and filamentous fungi have been globally exploited for their biotechnological applications. This book discusses ways to use them commercially. Chapters include the production of fibrinolytic enzymes, microbial lipases, bacteriocin production by lactic acid bacteria, and bioactives produced. It also covers microbial synthesis of alkaloids, terpenoids, and steroids. This book is useful for researchers, academicians, and industry experts in microbiology and biotechnology.

Microbial Enzymes and Metabolites for Health and Well-Being

Edited by

Ranjana Sirohi Amit Kumar Rai Luciana Porto de Souza Vandenberghe Binod Parameswaran

First edition published 2023 by CRC Press 6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487-2742 and by CRC Press 4 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN CRC Press is an imprint of Taylor & Francis Group, LLC © 2023 selection and editorial matter, Ranjana Sirohi, Amit Kumar Rai, Luciana Porto de Souza Vandenberghe, and Binod Parameswaran; individual chapters, the contributors Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, access www.copyright.com or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-7508400. For works that are not available on CCC please contact [email protected] Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identification and explanation without intent to infringe. ISBN: 978-1-032-43619-7 (hbk) ISBN: 978-1-032-43900-6 (pbk) ISBN: 978-1-003-36929-5 (ebk) DOI: 10.1201/9781003369295 Typeset in Times by MPS Limited, Dehradun

Contents Preface..............................................................................................................................................vii Editors ...............................................................................................................................................ix Contributors ......................................................................................................................................xi

SECTION I Microbial Enzymes in Healthcare Chapter 1

Microbial Enzymes and Metabolites in Health Care: An Overview......................... 3 Srichandan Padhi, Puja Sarkar, Md Minhajul Abedin, Ranjana Sirohi, Sudhir P Singh, and Amit Kumar Rai

Chapter 2

Microbial Production of Fibrinolytic Enzymes........................................................25 Kyoungseon Min and Ki-Yeon Kim

Chapter 3

Microbial Enzymes for Synthesis of Chiral Drug Intermediates ............................41 K B Arun, Raveendran Sindhu, Bipin G Nair, Binod Parameswaran, Ashok Pandey, Mukesh Kumar Awasthi, Mohammed Kuddus, and Aravind Madhavan

Chapter 4

Microbial Enzymes in Biomedical Applications......................................................53 Ramzan Ahmed, Manjit Kumar Ray, Debasis Nayak, and Yugal Kishore Mohanta

Chapter 5

Microbial Lipases: Production and Application.......................................................75 T P Sari, Vivek Kumar Gaur, Ayon Tarafdar, Ranjana Sirohi, Raveendran Sindhu, and Amit Kumar Rai

Chapter 6

Health Benefits of Bioactive Compounds Produced Using Microbial Enzymes....91 Swati Sharma, Loreni Chiring Phukon, Rounak Chourasia, Ranjana Sirohi, Binod Parameswaran, Ashok Pandey, Dinabandhu Sahoo, and Amit Kumar Rai

SECTION II Microbial Metabolites in Healthcare Chapter 7

Bacteriocin Production by Lactic Acid Bacteria....................................................111 Luciana Porto de Souza Vandenberghe, Bruna Leal Maske, Gilberto Vinícius de Melo Pereira, Leonardo Wedderhoff Herrmann, Luiz Alberto Junior Letti, and Carlos Ricardo Soccol

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Chapter 8

Contents

Production and Application of Antimicrobial Compounds from Endophytes ......125 Poonam Kumari, Aishwarya Jaiswal, and Akanksha Singh

Chapter 9

Microbial Synthesis and Application of Terpenoids and Steroids ........................ 145 Adriane Bianchi Pedroni Medeiros, Cristine Rodrigues, Denisse Molina-Aulestia, Guilherme Anacleto dos Reis, and Thalita de Oliveira Good

Chapter 10 Microbial Synthesis of Polypeptides and Applications in Healthcare ..................161 T P Sari, Amresh Hindurao Dhamane, Prarabdh C Badgujar, Deepak Kumar, Ayon Tarafdar, Amit Kumar Rai, and Ranjana Sirohi Chapter 11 Microbial Synthesis of Alkaloids and Applications in Healthcare ....................... 173 K B Arun, Bipin G Nair, Raveendran Sindhu, Laya Liz Kuriakose, Binod Parameswaran, Ashok Pandey, Mukesh Kumar Awasthi, and Aravind Madhavan Index..............................................................................................................................................187

Preface The book titled Microbial Enzymes and Metabolites in Healthcare and Well‐Being is published in partnership with Taylor & Francis Group and Biotech Research Society, India (BRSI). In this book, the impact of microbial enzymes and metabolites in the health sector and functional food industry have been discussed, along with the advancement in production-specific metabolites using novel microbial enzymes with unique properties. Microbial enzymes have been applied on different food substrates to produce metabolites with wide range of health benefits, as well as reduction of antinutritional factors. Due to the application of enzymes in functional foods, they are produced using native microorganisms as well as genetically modified candidates with improved properties. Enzymes produced using Bacillus species, lactic acid bacteria, and fungi have been characterised and applied to produce wide range of health benefits metabolites. This book covers a broad range of topic related to microbial enzymes, their sources, and applications in the production of metabolites, which are essential for good health. Chapters in the book have been contributed by global experts in specific area of microbial enzymes. The book is structured into two sections; the first section presents compiled information of microbial enzymes in healthcare. This section contains six chapters. The first chapter provides an introduction to the microbial enzymes and metabolites in healthcare; the second chapter is on microbial production of fibrinolytic enzymes, and it provides a comprehensive understanding of genetic engineering, optimization of fermentation parameters, and downstream process (e.g., separation and purification) to produce industrial fibrinolytic enzymes by various microorganisms. The third chapter is on microbial enzymes for the synthesis of chiral drug intermediates; it covers various biocatalytic microbial platforms that permit the production of the chiral drug intermediates. The fourth chapter is on microbial enzymes in biomedical applicationsl; it focuses on the curative potential of microbial enzymes, including enzybiotics, digestive aids, anti-inflammatory, anticancer, and fibrinolytic agents, as well as potential involvement in sensing applications. The fifth chapter is on the production and application of microbial lipases. This chapter first provides an overview of lipases, followed by their production from bacteria, fungi, and yeast, their purification, and subsequently, their applications in the industrial production of value-added products. The sixth chapter is on the health benefits of bioactive compounds produced using microbial enzymes. This chapter describes different types of nutraceuticals produced using microbial enzymes through hydrolysis and transformation processes. Section two of the book deals with the microbial metabolites used in healthcare and contains five chapters. The seventh chapter is on bacteriocin production by lactic acid bacteria. This chapter provides the classification and production of bacteriocin and presents a comprehensive understanding of recombinant microorganisms for bacteriocins production and mode of action and mechanisms of resistance of bacteriocin. The eighth chapter is on the production and application of antimicrobial compounds from endophytes, which provides an in-depth examination of the antimicrobial compounds generated by endophytes and their applications in the field of medicine and agriculture. The ninth chapter covers examples of terpenoids and steroids with healthcare application obtained by biotransformation, highlighting some microorganisms and the biosynthetic pathways. The tenth chapter is on microbial synthesis of polypeptides and application in healthcare. This chapter discusses the functional properties and characteristics of polypeptides and their application in the health-food industry. The eleventh chapter discusses various genemanipulation strategies to produce alkaloids in microbial hosts and their application in the healthcare industry. We sincerely appreciate the contributions by the authors from different countries who shared their knowledge on microbial enzyme and metabolites in healthcare. We strongly believe that this book provides enriched scientific information that will be useful for researchers, students, vii

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Preface

academicians, and industry experts in food biotechnology and applied microbiology. We are grateful to reviewers for their sincere efforts and acknowledge them for their contribution in the critical review of the chapters. We sincerely acknowledge Prof. Ashok Pandey, Chief Mentor, BRSI, and Managing Editor of the series and the BRSI for providing us the opportunity to prepare this book and guiding us during the editing and publication process for bringing this book to its final form. We thank the team of Taylor & Francis Group comprising Dr Gagandeep Singh, Senior Publisher, Dr. Madhurima Kahali, Editor (Life Sciences), Ms. Neha Bhatt, Editorial Assistant, CRC Press, and the entire team of the CRC Press, Taylor & Francis Group for their consistent support during the publication process. Ranjana Sirohi Amit Kumar Rai Luciana Porto de Souza Vandenberghe Binod Parameswaran

Editors Ranjana Sirohi, PhD, is an Assistant Professor in the University of Petroleum and Energy Studies, Uttarakhand, India. She has served as a post doctorate researcher at Korea University, Seoul, South Korea. She completed her doctorate in Process and Food Engineering from G.B. Pant University of Agriculture and Technology, Pantnagar, India. She has worked as researcher at École Polytechnique Fédérale de Lausanne, Switzerland. Her major research interests are bioprocess technology, food and food waste valorization, waste to wealth, and biofuels. She has more than 120 publications and an h-index of 29 (Google scholar). Her name is listed in the world’s top 2% scientist as per the 2022 study by Stanford University and Elsevier. Amit Kumar Rai, PhD, is Scientist D at the National Agri-Food Biotechnology Institute, Mohali, Punjab, India. He has served as Scientist at Institute of Bioresources and Sustainable Development, regional centre, Sikkim, India from 2012 to 2023. He completed his doctorate from CSIR-Central Food Technological Research Institute, Mysore. His major area of interest is food biotechnology for the production of nutraceuticals and functional foods rich in bioactive peptides and isoflavones using microorganisms. He has developed bioprocess for production of milk and legume based bioactive peptides enriched fermented products that can be beneficial during oxidative stress and hypertension. He has filed 4 patents, 106 publications, including 77 research and review papers, 3 books, and 26 book chapters to his credit. Luciana Porto de Souza Vandenberghe, PhD, is a Full Professor at the Department of Bioprocess Engineering and Biotechnology, Federal University of Paraná, Curitiba, Brazil. She is a member of the Bioprocess Engineering and Biotechnology Graduation Program at UFPR. Dr Vandenberghe earned a PhD in Génie de Procédés Industriels – Biotechnologie at Université de Technologie de Compiègne (2000), France. Her areas of interest include bioprocess engineering and biotechnology and industrial microbiology, with a focus on the valorization of solid and liquid agro-industrial subproducts through submerged and solid-state fermentation for biomolecules production, including industrial enzymes, organic acids, biopolymers and plant growth hormones. She has published 123 papers, 51 book chapters, and 23 deposed patents. Binod Parameswaran, PhD, is a Principal Scientist in the Microbial Processes and Technology Division of Council of Scientific and Industrial Research (CSIR) – National Institute for Interdisciplinary Science and Technology, Trivandrum, India. He earned a PhD in biotechnology at the University of Kerala, Thiruvananthapuram, India. Dr Parameswaran worked as a post-doctoral fellow at the Korea Institute of Energy Research, Daejeon, South Korea, and later joined as a Scientist at CSIR – National Institute for Interdisciplinary Science and Technology, Thiruvananthapuram, India. His research interests include biomass to fuels and chemicals, biopolymers and enzyme technology. He has more than 140 publications and an h-index of 45. His name is listed in the world’s top 2% scientist for his whole career as per the 2020 study by Stanford University and Elsevier.

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Contributors Md Minhajul Abedin Institute of Bioresources and Sustainable Development Regional Centre Tadong, Sikkim, India Ramzan Ahmed Department of Applied Biology University of Science and Technology Meghalaya Ri-Bhoi, Meghalaya, India K B Arun Department of Life Sciences CHRIST (Deemed to be University) Bengaluru, Karnataka, India Mukesh Kumar Awasthi College of Natural Resources and Environment Northwest A&F University Yangling, Shaanxi, China Prarabdh C Badgujar Department of Food Science and Technology National Institute of Food Technology Entrepreneurship and Management Kundli, Sonipat, Haryana, India Rounak Chourasia Institute of Bioresources and Sustainable Development Regional Centre Tadong, Sikkim, India Amresh Hindurao Dhamane Department of Food Science and Technology National Institute of Food Technology Entrepreneurship and Management Kundli, Sonipat, Haryana, India Vivek Kumar Gaur Centre for Energy and Environmental Sustainability Lucknow, Uttar Pradesh, India and School of Energy and Chemical Engineering Ulsan National Institute of Science and Technology (UNIST) Ulsan, Republic of Korea

Thalita de Oliveira Good Department of Bioprocess Engineering and Biotechnology Federal University of Paraná Centro Politécnico Curitiba, Paraná, Brazil Leonardo Wedderhoff Herrmann Department of Bioprocess Engineering and Biotechnology Federal University of Paraná Centro Politécnico Curitiba, Paraná, Brazil Aishwarya Jaiswal Pharmacology Division CSIR – National Botanical Research Institute Lucknow, Uttar Pradesh, India Ki-Yeon Kim Gwangju Bio/Energy R&D Center Korea Institute of Energy Research (KIER) Gwangju, Republic of Korea Mohammed Kuddus Department of Biochemistry University of Hail Kingdom of Saudi Arabia Deepak Kumar Division of Food Technology Department of Nutrition and Dietetics Manav Rachna International Institute of Research and Studies Faridabad, Haryana, India Poonam Kumari Division of Crop Production and Protection CSIR – Central Institute of Medicinal and Aromatic Plants Lucknow, Uttar Pradesh, India and Academy of Scientific and Innovative Research (AcSIR) Ghaziabad, Uttar Pradesh, India Laya Liz Kuriakose Department of Food Technology TKM Institute of Technology Kollam, Kerala, India xi

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Luiz Alberto Junior Letti Department of Bioprocess Engineering and Biotechnology Federal University of Paraná Centro Politécnico Curitiba, Paraná, Brazil Aravind Madhavan School of Biotechnology Amrita Vishwa Vidyapeetham Amritapuri, Kollam, Kerala, India Bruna Leal Maske Department of Bioprocess Engineering and Biotechnology Federal University of Paraná Centro Politécnico Curitiba, Paraná, Brazil Adriane Bianchi Pedroni Medeiros Department of Bioprocess Engineering and Biotechnology Federal University of Paraná Centro Politécnico Curitiba, Paraná, Brazil Kyoungseon Min Gwangju Bio/Energy R&D Center Korea Institute of Energy Research (KIER) Gwangju, Republic of Korea Yugal Kishore Mohanta Department of Applied Biology University of Science and Technology Meghalaya Ri-Bhoi, Meghalaya, India Denisse Molina-Aulestia Department of Bioprocess Engineering and Biotechnology Federal University of Paraná Centro Politécnico Curitiba, Paraná, Brazil Bipin G Nair School of Biotechnology Amrita Vishwa Vidyapeetham Amritapuri, Kollam, Kerala, India Debasis Nayak Department of Wildlife and Biodiversity Conservation Maharaja Sriramchandra Bhanjdeo University Odisha, India

Contributors

Srichandan Padhi Institute of Bioresources and Sustainable Development Regional Centre Tadong, Sikkim, India Ashok Pandey Center for Innovation and Translational Research CSIR – Indian Institute of Toxicology Research (CSIR-IITR) and Centre for Energy and Environmental Sustainability Lucknow, Uttar Pradesh, India Binod Parameswaran Microbial Processes and Technology Division CSIR – National Institute for Interdisciplinary Science and Technology (NIIST) Thiruvananthapuram, Kerala, India Gilberto Vinícius de Melo Pereira Department of Bioprocess Engineering and Biotechnology Federal University of Paraná Centro Politécnico Curitiba, Paraná, Brazil Loreni Chiring Phukon Institute of Bioresources and Sustainable Development Regional Centre Tadong, Sikkim, India Amit Kumar Rai Food and Nutritional Biotechnology Division National Agri-Food Biotechnology Institute Mohali, Punjab, India Manjit Kumar Ray Department of Applied Biology University of Science and Technology Meghalaya Ri-Bhoi, Meghalaya, India Guilherme Anacleto dos Reis Department of Bioprocess Engineering and Biotechnology Federal University of Paraná Centro Politécnico Curitiba, Paraná, Brazil

Contributors

Cristine Rodrigues Department of Bioprocess Engineering and Biotechnology Federal University of Paraná Centro Politécnico Curitiba, Paraná, Brazil Dinabandhu Sahoo Department of Botany University of Delhi New Delhi, India

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Sudhir P Singh Centre of Innovative and Applied Bioprocessing (DBT-CIAB) Mohali, Punjab, India Ranjana Sirohi School of Health Sciences and Technology, University of Petroleum and Energy Studies Dehradun, Uttarakhand, India

T P Sari Department of Food Technology National Institute of Food Technology Entrepreneurship and Management Kundli, Sonipat, Haryana, India

Carlos Ricardo Soccol Department of Bioprocess Engineering and Biotechnology Federal University of Paraná Centro Politécnico Curitiba, Paraná, Brazil

Puja Sarkar Institute of Bioresources and Sustainable Development Regional Centre Tadong, Sikkim, India

Ayon Tarafdar Livestock Production and Management Section ICAR – Indian Veterinary Research Institute Izatnagar, Bareilly, Uttar Pradesh, India

Swati Sharma School of Skill Buildings Shri Ramasamy Memorial (SRM) University Sikkim, Gangtok, India Raveendran Sindhu Department of Food Technology TKM Institute of Technology Kollam, Kerala, India Akanksha Singh Division of Crop Production and Protection CSIR – Central Institute of Medicinal and Aromatic Plants Lucknow, Uttar Pradesh, India and Academy of Scientific and Innovative Research (AcSIR) Ghaziabad, Uttar Pradesh, India

Luciana Porto de Souza Vandenberghe Department of Bioprocess Engineering and Biotechnology Federal University of Paraná Centro Politécnico Curitiba, Paraná, Brazil

Section I Microbial Enzymes in Healthcare

1

Microbial Enzymes and Metabolites in Health Care An Overview Srichandan Padhi, Puja Sarkar, and Md Minhajul Abedin Institute of Bioresources and Sustainable Development, Regional Centre, Tadong, Sikkim, India

Ranjana Sirohi School of Health Sciences and Technology, University of Petroleum and Energy Studies, Dehradun, Uttarakhand, India

Sudhir P Singh Centre of Innovative and Applied Bioprocessing (DBT-CIAB), Mohali, Punjab, India

Amit Kumar Rai Food and Nutritional Biotechnology Division, National Agri-Food Biotechnology Institute, Mohali, Punjab, India

CONTENTS 1.1 1.2

1.3 1.4 1.5 1.6

1.7

1.8 1.9

Introduction.............................................................................................................................4 Microbial Enzymes................................................................................................................. 4 1.2.1 Microbial Enzymes in Disease Diagnosis .................................................................5 1.2.2 Determination of Serum Triglycerides ......................................................................5 Therapeutic Drug Monitoring ................................................................................................6 Drug Assay Systems ..............................................................................................................6 Antibody-Antigen Reaction ...................................................................................................7 Microbial Enzymes in Treatment of Diseases ......................................................................7 1.6.1 As Anti-Inflammatory Agents....................................................................................7 1.6.2 As Wound Healers .....................................................................................................9 1.6.3 As Antibacterials ........................................................................................................9 1.6.4 As Antithrombolytic Agents ....................................................................................10 1.6.5 As Digestive Aids ....................................................................................................10 1.6.6 As Anticancer Chemotherapeutics........................................................................... 10 Microbial Enzymes in Drug Manufacturing .......................................................................11 1.7.1 In Synthesis of Antimicrobials ................................................................................11 1.7.2 In Dynamic Kinetic Resolution (DKR) of Drugs ...................................................12 1.7.3 In Synthesis of L-Tert-Leucines ..............................................................................12 1.7.4 In Synthesis of Statin Intermediates........................................................................12 Other Applications ...............................................................................................................12 Microbial Metabolites .......................................................................................................... 13

DOI: 10.1201/9781003369295-2

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Microbial Enzymes and Metabolites for Health and Well-Being

1.9.1 As Antibiotics...........................................................................................................13 1.9.2 As Antitumor Agents ............................................................................................... 15 1.9.3 As Immunosuppressant Drugs .................................................................................16 1.9.4 As Enzyme Inhibitors...............................................................................................16 1.10 Conclusion ............................................................................................................................17 References........................................................................................................................................ 17

1.1 INTRODUCTION The demand and necessity of microbial diagnostic and healthcare products is growing and projected to rise in coming years. Individuals, whether living in underdeveloped or developed countries, have extended their use of microbials both in primary (for e.g. immunization) and secondary (for e.g. chemotherapy) healthcare systems. The global market size of the microbial products was estimated at 189 billion USD in 2021 and is expected to increase up to 302 billion USD by 2030, with a compound annual growth rate (CAGR) of 5.35% from 2022 to (“Microbial Products Market Size US$ 302 Billion by 2030”, 2022). Microorganisms such as bacteria, filamentous fungi, and yeasts have been used since ancient times for biosynthesis or production of various value-added products (Chourasia et al., 2020a). One of the best-known industrial processes, production of alcohol from malt or fruit extracts using yeast, is still accepted in beverage making. Since then, the use of microorganisms has become enormous in the field of agriculture, food and beverages, energy and medicine. The useful microbial products include macromolecules like proteins, peptides enzymes, polymers, and other chemical substances of biological significance, such as antimicrobial, immunosuppressant, agro­ chemicals, enzyme inhibitors, and many others (Gurung et al., 2013; Chourasia et al., 2020b; Abedin et al., 2022a; Abedin et al., 2022b). Microbial enzymes have drawn particular interest for their prevalent use in industrial applications because of their special characteristics, including maximal catalytic activity, stability, and ease of production and optimization compared to those of their plant and animal counterparts (Sarkar et al., 2022). Among other features, tolerance to a wide range of pH and temperatures make them ideal for many bio-pharmaceutical and -medical productions (Singh et al., 2016). Similarly, microbes produce secondary metabolites to compete with one another and the environment. Their synthesis can be finely accustomed by growth conditions, nutritive sources, and enzyme induction or inactivation. These metabolites are very unusual and diverse, and they hold significant potential for use in healthcare systems (Sharrar et al., 2020; Chourasia et al., 2022a). This chapter describes the use and significance of microbial secondary metabolites and secreted enzymes in the healthcare applications.

1.2 MICROBIAL ENZYMES Enzymes are proteinaceous in nature (except ribozymes, which are catalytic RNAs) and popularly known as biocatalysts; they support and carry out almost all chemical reactions occuring in our body. They are highly specific and speed up the rate of a particular reaction, lowering the acti­ vation energy, and do not undergo any permanent changes in themselves. Compositionally, en­ zymes are made up of amino acids that hold one another with amide bonds. The catalytic site, which determines the specificity of the enzyme of one substrate over the others, forms the basis of enzyme classification. The Enzyme Commission (EC) set by the International Union of Biochemistry and Molecular Biology (IUBMB) and International Union for Pure and Applied Chemistry (IUPAC) classified enzymes into six main classes; these are based on the type of reactions they catalyze (oxidoreductases, transferases, hydrolases, lyases, ligases, and isomerases). Microorganisms are preferred sources of enzymes for various industrial applications for many reasons: (i) ease of availability, (ii) faster growth rate, (iii) economical feasibility, (iv) grease of product modification and optimization, (v) high yields, and (vi) greater catalytic activity and stability. In addition, the enzyme production can be enhanced with the implementation of

Microbial Enzymes and Metabolites in Health Care

5

FIGURE 1.1 The applications of microbial enzymes in different health sectors.

recombinant DNA technology with little or no difficulty. The use of microbial enzymes in industry over the conventional chemical catalysts has witnessed a rapid growth. Their use is associated with less harm to the environment, greater efficiency, better product quality, and, more importantly, the ability to work in varied physical and chemical conditions (Illanes et al., 2012; Sharma et al., 2022). The applications of microbial enzymes in different health sectors is presented in Figure 1.1. The following sections briefly discuss the importance and use of different enzymes of microbial origin in healthcare setups and applications.

1.2.1 MICROBIAL ENZYMES

IN

DISEASE DIAGNOSIS

Several enzymes isolated from different sources, including plant, animal, and microorganisms have been used as diagnostic reagents. Estimation of particular metabolites in body fluids and tissues (for example, blood or urine, biopsies), such as glucose, triglycerides, alcohol, and cholesterol, routinely employ enzyme systems or kits. However, there has been a swift expansion in the use of specific microbial enzymes in clinical chemistry as microorganisms can be a more practical source than those purified from animal tissues. Such enzymes can be used to determine and quantify body metabolites. For example, microbial cholesterol oxidase can be used for determining and quan­ tifying cholesterol in blood. Some of the microbial enzymes used, or having potential to be used in disease diagnosis, are discussed below.

1.2.2 DETERMINATION

OF

SERUM TRIGLYCERIDES

Triglycerides, if found concentrated beyond the normal range, have important clinical implication in the diagnosis of arteriosclerosis and lipid disorders. Glycerol kinase (GK, EC 2.7.1.30) has been

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Microbial Enzymes and Metabolites for Health and Well-Being

widely used in the diagnostic kits for determining the level of triglycerides in human serum. GK catalyzes the ATP-dependent phosphorylation of glycerol released from serum triglycerides (either by alkaline hydrolysis or by lipase). Glycerol levels are determined spectrophotometrically by coupling the GK reaction producing α-glycerol-3-phosphate. In microorganisms, GK makes the possible utilization of glycerol as an important source of carbon. GK has been commercialized from microorganisms like Escherichia coli and Candida mycoderma (Hayashi and Lin, 1967). To date, isolation of GKs from various microbial sources is known. Such microorganisms include few bacterial species, like Bacillus stearothermophillus (Atkinson, 1983) and Thermus flavus (Huang et al., 1998), Thermus thermophilus, Enterococcus casseliflavus (Yeh et al., 2009), a hyper­ thermophilic archaeon Thermococcus kodakarensis (Koga et al., 2008), and a thermophilic fungus Chaetomium thermophilum (Wilk et al., 2020).

1.3 THERAPEUTIC DRUG MONITORING TDM facilitates individualization in the drug treatment to optimize its clinical benefit and mini­ mize the side effects. Patients with critically ill septic shock conditions pose major setbacks for antimicrobial therapy due to the pathophysiological impact of sepsis on the drug pharmacokinetics (Varghese, Roberts, and Lipman, 2011). Determination of the level of antibiotics in blood or urine is particularly important in case the antibiotics are reported to cause any toxicity (for example, gentamycin, which is being used to treat urinary tract infection, bone infection, endocarditis, and many others; even at 10 µg/mL, serum concentrations can display toxic side effects). Many microbial enzymes, which can modify or degrade or inactivate the antibiotics, have found wide applications in clinical chemistry. β-lactamases (EC 3.5.2.6) are widely being used in determi­ nation of penicilins and cephalosporins in clinical plasma and urine samples. Their uses have also been implicated in sterility testing of sensitive antibiotics, as well as testing of new antibiotics for enzymatic degradation susceptibility (Atkinson, 1983). β-lactamases are commonly produced by bacterial species such as E. coli, Bacillus licheniformis, B. cereus, Staphylococcus aureus, Pseudomonas aeruginosa, P. cepacia, Entrococcus cloacae, and many others (Ferreira et al., 2011). In addition, genes encoding β-lactamases in fungal species such as Fusarium spp., have been speculated; however, more research to validate the findings is warranted (Gao et al., 2017). Other enzymes such as gentamycin acetyl transferase (EC 2.3.1.60) and chloramphenicol acetyl transferase (EC 2.3.1.28), which modify the antibiotics gentamycin and chloramphenicol respec­ tively, have been used in certain instances to determine antibiotic levels. Most of the gram-negative bacteria (in particular, E. coli and S. aureus) and Mycobacterium tuberculosis are frequent pro­ ducers of these enzymes (Belaynehe et al., 2017). Nevertheless, their use in clinical chemistry is not seen so far.

1.4 DRUG ASSAY SYSTEMS The monitoring of specific drug molecules in body fluid, such as serum, provides important information about the effective balance between useful dose and its toxic concentrations. In such circumstances, an enzyme inhibition assay to find out the serum concentrations of that drug can be applied. A chemotherapeutic drug methotrexate, which is being used as folic acid antagonist in cancer therapy, challenges the treatment in patients having weak renal clearance mechanisms (Strang and Pullar, 2017). An enzymatic assay applying the inhibition of dihydrofolate reductase (EC 1.5.1.3, isolated from Lactobacillus casei) by serum methotrexate has been developed. In addition, similar enzymes were also purified from E. coli (Bolin et al., 1982). Paracetamol (N-acetyl p-aminophenol), a major component of various analgesic preparations in severe over­ doses can cause extensive liver damage, and therefore, knowledge of an accurate serum level of paracetamol is important. Aryl acylamidase (EC 3.5.1.13), which breaks down paracetamol to

Microbial Enzymes and Metabolites in Health Care

7

acetate and p-aminophenol, has found its application in clinical diagnosis. This enzyme is widely distributed in bacteria and fungi. Bacterial species like Pseudomonas fluorescens, Bacillus sphaericus, and Rhodococcus erythropolis, and an ascomycete Nocardia farcinica, are some of the common producers of aryl acylamidase (Heumann et al., 2009; Ko et al., 2010).

1.5 ANTIBODY-ANTIGEN REACTION Antibodies, which are small proteins often created by the immune system itself, are considered protectors and help prevent infections. Therefore, assessing antibody formation and determi­ nation of its level is important in suspect patients. Protein A, a surface protein initially isolated from S. aureus, is being used as a universal probe in the determination and quantification of antibody levels through antigen-antibody reaction. Protein A has been validated to bind a variety of human IgG (subclass 1, 2 and 4, 2 moles/mole). In addition, its reaction with other antibodies that are IgA and IgM has also been detected (Kronvall and Williams, 1969; Hjelm, Sjödahl, and Sjöquist, 1975). Specific microbial enzymes like alkaline phosphatases (EC 3.1.3.1) hold a great potential to be used as an alternate; they are superior to that of the calf alkaline phosphatase used in the enzyme-linked immunosorbent assay (ELISA) test kits (“Alkaline Phosphatase from Microorganism - TOYOBO USA”, 2022). ELISA test uses col­ orimetric detection of current or past infections (antigen or antibody) in the blood mediated through an antigen-antibody reaction.

1.6 MICROBIAL ENZYMES IN TREATMENT OF DISEASES Characterization and application of therapeutic agents (enzymes and molecules) for combating human diseases and disorders is the prime focus of research (Chourasia et al., 2022b; Chourasia et al., 2022c). Many pathological circumstances are known to be linked with increased or diminished enzymatic activity and also with surplus or deficiency of the same. On the other hand, enzymes of microbial origin are potential therapeutic agents that are also cost effective, consistent, and expedient in isolation and genetic modification. Microbial enzymes commercialized and used in healthcare are presented in Table 1.1. The diverse applications of microbial enzymes as anticancer, anti-inflammatory and fibrinolytic agents, enzybiotics, and digestive aids have widely been documented and are discussed below.

1.6.1 AS ANTI-INFLAMMATORY AGENTS Serratiopeptidase (EC 3.4. 24.40) is a protease that belong to the trypsin family and has been widely used in therapeutic applications (Metkar et al., 2020). It was first isolated from Serratia marcescens, which inhabits the intestine of silkworm, Bombyx mori. Later on, its production has been detected in other members of Serratia. In addition, other bacterial species such as Bacillus licheniformis and Streptomyces hydrogenans have been reported to produce this enzyme (Wagdarikar, Joshi, and Shaikh, 2015; Nageswara, Guntuku, and Yakkali, 2019). Serratiopeptidase possesses a special ability as a potential anti-inflammatory candidate to break up the damaged and dead tissue without affecting living tissue (Kotb, 2013). It has also shown to have a substantial effect on immune cell movement. At the site of inflammation, the enzyme modulates the entry of Proinflammatory mediator molecules (PMN) and other lymphocytes (Mouneshkumar Chappi et al., 2015). Further, it has demonstrated significant antiedemic and analgesic properties in various fields, including gynecology, otorhinolaryngology, orthopae­ dics, dentistry, and surgery (Jadhav et al., 2020). According to studies, it has been observed that it reduces capillary permeability caused by histamine, bradykinin, and serotonin; breaks down aberrant exudates and proteins; and improves the absorption of degraded products via blood and lymphatics (Tiwari, 2017).

8

Microbial Enzymes and Metabolites for Health and Well-Being

TABLE 1.1 Common Microbial Enzymes Commercialized and Used in Health Care Enzyme Arginine deiminase

Bafibrinase

Source Organism Enterococcus spp. Halobactericum salinarum Lactococcus lactis Mycoplasma arginine Mycoplasma hominis Pseudomonas aeruginosa P. putida Bacillus spp

Application/Treatment Anti-tumor Anti-cancer

References Han et al., 2016

Anti-thrombotic

Mukherjee et al., 2012

Collagenases

Clostridium histolyticum Actinomycetes Aspergillus oryzae Streptomyces parvulus

Wound healing Eliminates dead cells

Klasen, 2000; Beygmoradi and Homaei, 2017

L-asparaginase

Erwinia chrysanthemi Escherichia coli

Anti-cancer

Dhankhar et al., 2020

L-Glutaminase

Aeromonas veroni Alcaligenes faecalis Bacillus cereus B. subtilis Brevundimonas diminuta Vibrio azureus Kosakonia radicincitans LG8 Pseudomonas spp. Streptomyces Burkholderia Chromobacterium Pseudomonas Rhizopus Arthobacter crystalopoites Bacillus subtilis B. thuringiensis Enterococcus hirae Staphylococcus aureus Streptomyces griseus

Anticancer effects especially in acute lymphocytic leukemia

Gomaa, 2022; Spiers and Wade, 1976; Nandakumar et al., 2003

Fat digestion Overcome exocrine pancreatic enzyme deficiency Anticancer Anti-inflammatory Immunomodulatory Antiviral

Blonk et al., 2021; Basso and Serban, 2020; Phukon et al., 2022

Lipase

Lysozymes

Masschalck and Michiels, 2008; Ercan and Demirci, 2015

Methioninase

Aeromonas sp. Brevibacterium linens Citrobacter freundii Clostridium sporogenes Entamoeba histolytica Pseudomonas putida Streptomyces avermitilis

Anti-cancer

Cavuoto and Fenech, 2012; Kato, Inagaki, and Oikawa, 2019; Hoffman et al., 2019; B. Sharma, Singh, and Kanwar, 2014

Nattokinase

B. subtilis var. natto

Anti-thrombolytic

Weng et al., 2017

Sacrosidase

Saccharomyces cerevisiae

Serratiopeptidase

Serratia marcescens Bacillus licheniformis Streptomyces hydrogenans

Congenital sucrase-isomaltase deficiency Anti-inflammatory antiedemic Analgesic

Treem et al., 1999; Puntis and Zamvar, 2015 Wagdarikar, Joshi, and Shaikh, 2015; Nageswara, Guntuku, and Yakkali, 2019; Kotb 2013

Streptokinase

Streptococcus hemolyticus

Anti-thrombolytic

Banerjee, Chisti, and Banerjee, 2004

Microbial Enzymes and Metabolites in Health Care

9

TABLE 1.1 (Continued) Common Microbial Enzymes Commercialized and Used in Health Care Enzyme α- amylases

β-galactosidase

Source Organism Bacillus amyloliquefaciens B. licheniformis B. stearothermophilus Bifidobacterium spp. Lactobacillus spp Aspergillus spp. Kluyveromyces spp.

Application/Treatment Digestive disorder Improve fibre digestibility Malnutrition Lactose intolerance

References de Souza and e Magalhães, 2010 Saqib et al., 2017

1.6.2 AS WOUND HEALERS Collagenases (EC 3.4.24.3) are a group of metalloproteases that can specifically hydrolyze natural collagen and appear to be cheap and convenient medications for burns, wound healing, and other related diseases. Collagenases produced from the microorganisms have attracted attention in biological and industrial applications. Clostridium histolyticum produces collagenase, which is used in the treatment of burn scars because it promotes the healing of wounds by producing collagen-derived peptides, resulting in the increase of macrophage chemotaxis and cytokine release (Klasen, 2000). Collagenase enzyme eliminates necrotic dead tissues in a precise and painless manner without harming the cell membrane. It also helps break down the tough cords in Dupuytren and is widely used in human cell isolation, a use which has been approved by the Food and Drug Administration (FDA) (Miller et al., 2015). Production and isolation of collagenases have been reported from a variety of microbial sources, including Actinomycetes, Aspergillus oryzae, Streptomyces parvulus, and many other members (Beygmoradi and Homaei, 2017).

1.6.3 AS ANTIBACTERIALS Enzymes purified from the microorganisms have displayed antibacterial activity. Microbial lysins, which are peptidoglycan-degrading enzymes, have been found to be beneficial in treating Grampositive bacterial infections (Górski et al., 2020). During the lytic cycle of the phage, lysins are spontaneously produced in phage-infected bacterial cells and spread through the peptidoglycan layer via membrane lesions released by holins. They subsequently break the peptidoglycan layer, causing host lysis and allowing progeny phages to be released. Some lysins can also kill bacteria without utilizing enzymes by disrupting the bacterial plasma membrane with peptides with properties similar to cationic antimicrobial peptides (CAPs), such as hydrophobicity, positive charge, and amphipathic secondary structure, which have been identified in T4 phage lysins en­ coded by Φ KZ and phage D3 in Pseudomonas aeruginosa (Rotem et al., 2006). Another example is the lys1521 (bacteriophage secondary lysine) synthesized in Bacillus amyloliquefaciens, whose positively charged C-terminal sequences increased the permeability of P. aeruginosa’s outer membrane and allowing the enzyme’s N-terminal domain access to the peptidoglycan layer (Orito et al., 2004). Lysozymes which can be obtained from a variety of sources including animals, plants, and mi­ croorganisms, have demonstrated immunomodulatory, anticancer, anti-inflammatory, and antiviral properties in addition to antibacterial properties. Lysozymes can lyse bacteria via non-enzymatic processes. In addition, they can cause plasma membrane destabilization by eliminating divalent ions from the membrane surface (Masschalck and Michiels, 2008). Several bacterial species have been reported to produce lysozymes such as Arthobacter crystalopoites, B. thuringiensis, Streptomyces

10

Microbial Enzymes and Metabolites for Health and Well-Being

griseus, Bacillus subtilis, Enterococcus hirae, and Staphylococcus aureus (Ercan and Demirci, 2015). Further, various recombinant fungal species have also been reported to produce recombinant human lysozymes such as Aspergillus niger, Escherichia coli, Pichia pastoris, Kluyveromyces lactis, and Saccharomyces cerevisiae (MacKenzie et al., 1994; Masuda, Ide, and Kitabatake, 2005; Lamppa, Tanyos, and Griswold, 2013; Ercan and Demirci, 2015).

1.6.4 AS ANTITHROMBOLYTIC AGENTS Fibrinolytic enzymes are specialized proteases having the ability to degrade the fibrin mesh of blood clots. Microbial fibrinolytic enzymes, on the other hand, have attracted enormous attention not only in blood clot removal but also in blood pressure regulation (Sharma, Osmolovskiy, and Singh, 2021). Microorganisms such as actinomycetes, microalgae, and filamentous fungi are the major sources of fibrinolytic enzymes. Bacillus spp., isolated from the traditional fermented food, have demonstrated the ability to produce fibrinolytic enzymes that have been used as a food additive to treat and/or prevent thrombosis (Peng, Yang, and Zhang, 2005). Further, fermented food products such as Korean Chungkook-Jang, Japanese Natto, Chinese Douchi, and Indonesian Tempe have produced a variety of effective fibrinolytic enzymes that have been isolated and characterized (Stephani et al., 2017). Notably, fibrinolytic enzymes, such as streptokinase (from Streptococcus hemolyticus), nattokinase (from B. subtilis var. natto), and bafibrinase (from Bacillus spp.) are some of the potential candidates for use in biopharmaceuticals (Banerjee, Chisti, and Banerjee, 2004; Mukherjee et al., 2012; Taneja et al., 2017; Weng et al., 2017).

1.6.5 AS DIGESTIVE AIDS Microbial enzymes such as α-amylases (EC 3.2.1.1), β-galactosidase (EC 3.2.1.23), sacrosidases (EC 3.2.1.26), and triacylglycerol lipases (EC 3.1.1.3) have been used to hydrolyze complex biomolecules such as carbohydrates and lipids into simpler forms. These enzymes help treat digestive disorders and also help overcome malnutrition and discomfort. Amylases break down starch into simple sugars, such as dextrins, glucose, maltose, and maltotriose, and improve the digestibility of fibre. Bacillus amyloliquefaciens, B. licheniformis, and B. stearothermophilus have been reported to produce amylases (de Souza and e Magalhães, 2010). β-galactosidases, which can hydrolyze lactose into simple sugars, such as galactose and glucose, and aid in the treatment of lactose intolerance, have reportedly been produced by a variety of bacteria (Bifidobacterium spp. and Lactobacillus spp.) and fungi (Aspergillus spp., Kluyveromyces spp.) (Saqib et al., 2017; Thakur, Rai, and Singh, 2022). The role of lipases in fat digestion and fulfilling exocrine pancreatic enzyme deficiency is well known (Blonk et al., 2021). Species of Burkholderia, Chromobacterium, Pseudomonas, and Rhizopus have been documented as producers of lipases (Basso and Serban, 2020; Phukon et al., 2022). Sacrosidase, a hydrolase enzyme, helps digest and absorb sucrose from food and has been reported to prevent the symptoms of gas and bloating, abdominal cramps, and diarrhea in patients suffering from congenital sucrase-isomaltase deficiency (CSID) consuming a normal sucrose rich and carbohydrate-containing diet. This enzyme has originally been produced by the yeast Saccharomyces cerevisiae (Treem et al., 1999; Puntis and Zamvar, 2015).

1.6.6 AS ANTICANCER CHEMOTHERAPEUTICS Use of enzymes sourced from different microorganisms in the treatment of cancer has been re­ ported. L-asparaginase is a chemotherapeutic drug being used to treat acute lymphoblastic leu­ kemia, a cancer form of the blood and bone marrow (Muneer et al., 2020; Wang et al., 2021), and extensively produced from Erwinia chrysanthemi and Escherichia coli. The enzyme depletes asparagines (necessary for biosynthesis of proteins) in tumor cells; as a result, it inhibits the mammalian target of rapamycin and its downstream targets, such as 4E-binding protein-1

Microbial Enzymes and Metabolites in Health Care

11

(4E-BP1) and serine threonine kinase (p70s6k). This leads to inhibition of translation, causing tumor cell apoptosis (Dhankhar et al., 2020). Another arginine degrading enzyme arginine dei­ minase (ADI) is known to be useful in the treatment of human melanomas, renal cell carcinomas, and hepatocellular carcinomas. These cancers are reported to be auxotrophic for arginine (lacking argininosuccinate synthetase that catalyzes synthesis of argininosuccinate from citrulline and aspartate). Several arginine deiminase enzymes have been isolated from Enterococcus spp., Halobactericum salinarum, Lactococcus lactis, Mycoplasma arginini, Mycoplasma hominis, Pseudomonas aeruginosa and P. putida having potential for use in antitumor and anticancer therapy (Han et al., 2016). Methionine dependence is considered as the only known metabolic defect in cancer. Methioninase, which utilizes methionine as a substrate, has demonstrated therapeutic potential in cancers such as acute lymphoblastic leukemia, carcinoma, glioblastoma, glioma, osteosarcoma, primary ductal melanoma, and non-small lung cancer (Cavuoto and Fenech, 2012). Aeromonas sp., Brevibacterium linens, Citrobacter freundii, Clostridium sporogenes, Entamoeba histolytica, Pseudomonas putida, and Streptomyces avermitilis have been reported to secrete the methioninase (Sharma, Singh, and Kanwar, 2014; Hoffman et al., 2019; Kato, Inagaki, and Oikawa, 2019). L-Glutaminase, an amide enzyme known for its contributory role in the cellular nitrogen metabolism in living cells, has shown significant anticancer effects especially in acute lymphocytic leukemia (Spiers and Wade, 1976; Gomaa, 2022). Glutamine metabolism appears to be deregulated in cancer cells and its deprivation suppresses their growth and even persuades cell death. L-glutaminase is reported to be a major enzyme that regulates the delectable taste of several fer­ mented foods such as soy sauce (Nandakumar et al., 2003). Many bacteria have been reported to synthesize both extracellular and intracellular L-glutaminases, which include Aeromonas veroni, Alcaligenes faecalis, Bacillus cereus, B. subtilis, Brevundimonas diminuta, Vibrio azureus, Kosakonia radicincitans LG8, and species of Pseudomonas and Streptomyces (Barzkar et al., 2021). L-glutaminase isolated from Alcaligenes faecalis and B. cereus inhibited the proliferation of HepG2 cell lines of hepatocellular cancer and HeLa cell lines of cervical cancer (Singh and Banik, 2013; Dhankhar et al., 2020). Similarly, the same from Bacillus sp. DV2–37 inhibited human breast (MCF-7), hepatocellular (HepG-2), and colon (HCT-116) carcinoma cell lines (Gomaa, 2022).

1.7 MICROBIAL ENZYMES IN DRUG MANUFACTURING Besides their potential application in disease diagnosis and treatment, microbial enzymes are also being utilized in the drug-manufacturing industries. In their immobilized forms, enzymes are used for commercial production of many drugs and antibiotics. During the drug-development processes, pro-drugs (less active or inactive) are converted to pharmacologically active drugs by the action of enzymes. In addition, microbial enzymes are also found to be applicable in converting plant steroids for the synthesis of steroidal drugs.

1.7.1 IN SYNTHESIS

OF

ANTIMICROBIALS

Penicillin acylases, which include penicillin G acylases, penicillin V acylases, and ampicillin acylases, are a group of enzymes that have been used in the synthesis of 6-amino penicillanic acid (6-APA), an intermediate used in the synthesis of β-lactam antibiotics. These enzymes break the acyl chain of penicillins and convert them to 6-APA and corresponding organic acids. Penicillin acylases have also been used in the synthesis of semisynthetic penicillins, which demonstrated better stability, easier absorption, and lesser side effects. Over the past decade, microorganisms have been screened for production of these enzymes for industrial applications. Penicillin acylases sourced from E. coli, Bacillus megaterium and Alcaligenes faecalis have found application in drug manufacturing (Illanes et al., 1994; Rajendhran, Krishnakumar, and Gunasekaran, 2003). Recently,

12

Microbial Enzymes and Metabolites for Health and Well-Being

(Velasco-Bucheli et al., 2020) has reported a penicilin acylase from Streptomyces lavendulae, which is yet to find its application.

1.7.2 IN DYNAMIC KINETIC RESOLUTION (DKR)

OF

DRUGS

Because of their high chemo-selectivity, some enzymes are able to perform enantio-selective reactions by selectively acting on one enantiomer and transforming it to the corresponding product. As such, the product can easily be separated from the other enantiomer. Optically pure α-amino acids production from α-aminonitriles using DKR has been reported (Yasukawa, Hasemi, and Asano, 2011). Sequentially, the enzyme nitrile hydratase catalyzed the nonselective hydrolysis of racemic α-aminonitrile resulting into an amide. This was followed by hydrolysis of the amide by a stereoselective amino acid amide hydrolase. The last step involved was racemization of the α-amino acid amide by α-amino-ε-caprolactam racemase. In a similar investigation, a lipase iso­ lated from the bacterium Burkholderia cepacia was used to catalyze the DKR of racemic α-aminonitrile to produce acetylated amine with substantial yield (Vongvilai et al., 2011).

1.7.3 IN SYNTHESIS

OF

L-TERT-LEUCINES

L-Tert-leucines are industrially used as a building block to many antiviral drugs acting as protease inhibitors, namely, atazanavir (HIV protease), boceprevir (hepatitis C virus genotype 1 protease), and telaprivir (hepatitis C NS3–4A serine protease). The recombinant expression of leucine dehydrogenase (LeuDH, from Exiguobacterium sibiricum) and glucose dehydrogenase (from Bacillus megaterium) in E. coli to catalyze the synthesis of L-Tert-leucines from trimethylpyruvic acid was found to be successful (Li et al., 2014). Similarly, use of a recombinant LeuDH of Thermoactinomyces intermedius and a recombinant format dehydrogenase of Pichia pastoris has been shown to synthesize L-tert-leucine from the same substrate (Patel, 2018).

1.7.4 IN SYNTHESIS

OF

STATIN INTERMEDIATES

Statins are being used as lipid-lowering medications for patients who are at high risks of cardio­ vascular diseases. They act as specific inhibitors to the metabolic enzyme hydroxylmethylglutaryl coenzyme A (HMG-CoA) reductase, which converts HMG-CoA to mevalonate, a rate-limiting point in synthesis of cholesterol. Use of microbial enzymes in synthesis of intermediates to these drugs has been reported (Patel, 2009). Alcohol dehydrogenase (KleADH) of Klebsiella oxytoca has been used industrial synthesis of t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate. Similarly, ke­ toreductase of Acinetobacter calcoaceticus has been used in synthesis of (3S,5R)-dihydroxy-6(benzyloxy) hexanoic acid, ethyl ester by enantioselective reduction of the corresponding diketoe­ sters such as 3,5-dioxo-6-(benzyloxy) hexanoic acid, ethyl or tert-butyl esters. Both the intermediates are being used in the synthesis of the cardiovascular drugs, such as atorvastatin and rosuvastatin.

1.8 OTHER APPLICATIONS Superoxide dismutase (EC 1.15.1.1) and peroxidases such as catalase (EC 1.11.1.6), lactoperoxidases (EC 1.11.1.7) and glutathione peroxidases (EC 1.11.1.9) have been used as skin exfoliate and serve as scavengers of free radicals, protecting the skin from the detrimental effects of UV light. Such en­ zymes from Marinomonas sp., Sulfolobus acidocaldarius, and Thermusthermophilus have found applications in skin care (Bafana et al., 2011). Microbial proteases like alkaline aspartic proteases (EC 3.4.23) have been used to treat various skin disorders, including xeroderma (dry skin), ich­ thyoses (thick and scaly skin), and psoriasis (skin rash with itchy and scaly patches). Fungal species like Aspergillus, Penicillium, Rhizopus, and Neurospora are common producers of aspartic proteases (Mandujano-González et al., 2016). Kerratinases (EC 3.4.21), which are produced by wide source of

Microbial Enzymes and Metabolites in Health Care

13

microorganisms (species of Thermosipho, Thermococcus, Microbacterium, Xanthomonas, Vibrio, and many others) are being used in the treatment of stretch marks, scar tissues, and, more importantly, in epithelial cell regeneration (a part of skin-healing process) (Gopinath et al., 2015). Lactoperoxidase, along with glucose oxidase (EC 1.1.3.4), have been used in toothpaste for oral care. Microorganisms, especially fungi, have been widely investigated for the production of glucose oxidases. The filamentous fungi Aspergillus and Penicillium serve as largest producers of glucose oxidase on an industrial scale (Dubey et al., 2017).

1.9 MICROBIAL METABOLITES Microbial metabolites are the multi-potential low-molecular weight organic compounds mi­ croorganisms produce in response to competition and hardship. These metabolites over the past decades have gained enormous economic importance and established a foundation for a plethora of crucial products in agriculture, biotechnology, and medicine as well. Besides antibiotics, which alone share a major piece of the commercial market, a significant number of metabolites, such as anticancer, immunosuppressive, enzyme-inhibiting agents, and many others, have shown remarkable market potential. Production of secondary metabolites involves biosynthetic path­ ways governed through biosynthetic gene clusters (BGCs), which remain inactive under normal environmental conditions. Moreover, these biosynthetic pathways are related to a network of primary metabolism applying the same intermediates and regulatory mechanisms, and they are formed by pathways deviated from primary metabolic pathways at a few points (Ranghar, Agrawal, and Agrawal, 2019). Microbial metabolites can be classified based on their chemical structure and biosynthetic pathways into (1) nitrogen-containing compounds originated from shikimic acid pathway (alkaloids, glucosinolates, and cyanogenic glycosides), (2) terpenes originated from mevalonic acid and MEP pathway (volatiles, sterols, carotenoids, saponins, and glycosides), and (3) phenolic compounds originated from malonic acid pathway (phenolic acids, flavonoids, lignin, stilbenes, lignans, coumarins, and tannins) (Agostini-Costa et al., 2012; Kumar et al., 2020; Sarkar et al., 2022). Microbial metabolite commercialized and used in healthcare applications is presented in Table 1.2.

1.9.1 AS ANTIBIOTICS Antibiotics are antimicrobial peptides produced from microorganisms to serve a survival function in nature. Fungi, bacteria (gram positive & gram negative), and archaea all are capable of releasing antimicrobial peptides during their late exponential phase or late stationary phase as a secondary metabolite. Antimicrobial products are found to be effective against bacteria, fungi, parasites, viruses and biofilms (a naturally resistant structure comprising microbial cells. Plenty of micro­ organisms were searched for metabolite-producing abilities; they include antibacterial (penicillin, cephalosporin, bacitracin, polymixin B, erythromycin, rifamycin, gentamicin), antifungal (amphotericin B, aspergillic acid, asperfumin), and antiviral (emerimidine A, emerimidine B, emeriphenolicin A, emeriphenolicin D, cytonic acids A & B). Thousands of antibiotics were isolated from bacteria and fungi, from which only hundreds are getting used to treat against dis­ eases related to humans, animals, and plants. Penicillium crysogenum produces penicillin as a secondary metabolite in the stationary phase. Cephalopsorins are developed from cephalosporin C, a natural product produced by Cepahlosporium acreminium. Various members of the Bacillus genus do counts for their ability to produce different antimicrobial peptides. Cephamycins are the drugs pro­ duced from Streptomyces. Gliomastix, a endophytic fungi from Parispolyphylla var. yunnanensis (medicinal plant), are also being reported to produce secondary metabolites such as ergosta-5,7, 22trien-3-ol, 2,3–dihydro-5-hydroxy-α,α-dimethyl-2-benzofuranmethanol, and 4-hydroxymellein. Lactic acid bacteria are a heterogeneous group of gram-positive bacteria classified according to their glucose fermentation metabolisms, growth temperature, cell morphology, utilization of sugar, and

Streptomyces nodosus

Acremonium spp. Streptomyces tsukubaenis

Streptomyces verticillus

Tolypocladium inflatum Streptomyces pervulus

Streptomyces hygroscopicus

Amphotericin B

Cephalosporins Tacrolimus

Bleomycin

Cyclosporin-A Actinomycin-D/Dactinomycin

Sirolimus

Cholramphenicol

Penicillium spp.

Amycolatopsis mediterranei Streptomyces venezuelae

Penicillin

Source Organism

Rifamycin

Microbial Metabolites/ Generic Name of Drugs

RX

Siromus

PSORID 50 Dactinoget

TM

Bleomycin

TAXIM-O Pangraph RX

FUNGIZONE

Paraxin

AEMCOLO

BISTREPIN

Brand Name

Zydus Biogen

Biocon GLS Pharma Ltd.

Salius pharma

ALKEM Panacea Biotech Ltd.

Abbott

RedHill Biopharma Ltd Abbott

ALEMBIC

Manufacturer

TABLE 1.2 Microbial Metabolites Commercialized and Used in Healthcare Applications

Immunosuppressive

Immunosuppressive Antitumor

Antitumor

Antimicrobial/Antibacterial Immunosuppressive

Antimicrobial/Antifungal

Antimicrobial/Antibacterial

Antimicrobial/Antibacterial

Antimicrobial/Antibacterial

Healthcare Application

Kino et al., 1987

Borel et al., 1976 Williams and Katz, 1977

Sugiyama et al., 2002

Adinarayana et al., 2003 Kino et al., 1987

Zhang et al., 2020

Ahmed and Vining, 1983

August et al., 1998

Agostini-Costa et al., 2012

References

14 Microbial Enzymes and Metabolites for Health and Well-Being

Microbial Enzymes and Metabolites in Health Care

15

optimized growing temperature range. The bacteriocins produced from LAB (examples are genera such as Lactobacillus, Lactococcus, Pediococcus, Leuconostoc, Streptococcus) have various positive aspects (Mokoena, 2017). Bacteriocins are peptides composed of 20–60 amino acid residues that are cationic and hydrophobic, ribosomally synthesized and transcribed from the genes located in operons in chromosomes, plasmids, or other mobile genetic elements (Kumariya et al., 2019; Zimina et al., 2020). The important criteria of bacteriocin in comparison to the conventionally used antibiotics is that it acts against pathogens, opportunistic bacteria such as multidrug resistant strains, unless any discrimination exists between antibiotic resistant and anti­ biotic susceptible strain.

1.9.2 AS ANTITUMOR AGENTS In the 21st century, cancer has become a major concern to human health because of its worldwide rate of morbidity and mortality. Remarkable treatment and diagnostic approaches are available to treat the disease, but due to the acquiring resistance against the conventionally used drugs with their side effects and toxicity, there is a need of developing novel anticancer agents. Thus, developing anticancer agents with an advanced mode of action has become one of the major concerns in research and development. Recently, bacteria and bioactive compounds produced from them, such as antibiotics, bacteriocin, toxins, non-ribosomal peptides, and polyketides, have shown significant results as alternative cancer therapeutic agents; they show different mechanisms on regulating essential signaling pathways, necrosis, reducing angiogenesis, apoptosis, and through translational and splicing inhibition. On an addition in distinctive cancer therapy, it has been found that live bacteria work as an antitumor like a standby method (Baindara and Mandal, 2020). Significantly, bacteria can be used in antitumor therapy as it can solely target the anoxic region inside of a solid tumor as, for example, the Clostridium sp., which is an obligate anaerobe (Felgner et al., 2016). William Coley first used the supernatant from Streptococcus pyogenes and Serretia marcescens to treat tumor patients. Later, it is known as Coley’s toxins (McCarthy, 2006). S. pyogenes OK-432 was prepared, which is now successfully used in the treatment of lym­ phangioma tumors (Olivieri et al., 2016). The engineered strain of Salmonella enteric Serovar typhimurium has been shown to induce the antitumor activity (Loeffler et al., 2007). ActinomycinD from Streptomyces antibioticus is one of the primarily used antitumor agents (Amanzadeh and Amanzadeh, 2021). Bacteria ribosomally synthesized proteins and peptides, which act as toxins and bacteriocins. These are capable with antimicrobial properties, and some of them are also reported to have antic­ ancer activities through different studies, among which few are on clinical trials and others have already filed for patent due to their significant anticancer properties. Clostridium perfringens en­ terotoxins (CPE) from C. perfringens, Botulinum toxin from C. botulinum, Diptheria toxin from C. diptheriae, Interlukin-4 Pseudomonas exotoxin (IL-4PE) from P. aerugenosa (Falnes and Sandvig, 2000; Kominsky et al., 2004; Ansiaux and Gallez, 2007; Puri et al., 2009). Listeriolysin O from Listeria monocytogenes (Elingarami and Zeng, 2011) and Burkholderia lethal factor 1 (BLF1) from Burkholderia pseudomallei (Cruz-Migoni et al., 2011) all are reported to have an antitumor effect as a toxin in various aspects with different mechanisms of action. Other than these, there are bacteriocins like bovicin from Streptococcus bovis (Mantovani et al., 2002), Colicin from Escherichia coli (Šmarda et al., 1978), Laterosporulin 10 from B. laterosporus SKDU10 (Baindara et al., 2016), Microcin E492 from Klebsiella pneumoniae RYC492 (Hetz et al., 2002), Nisin from Lactococcus lactis (Baindara, Korpole, and Grover, 2018), Pediocins from the genus of Pediococcus (Villarante et al., 2011), Plantaricin A from Lactobacillus plantarum C11 (Sand et al., 2010). Pyocins from Pseudomonas aeruginosa (Michel-Briand and Baysse, 2002) having antitumor potentiality has been reported. Podophyllotoxin, kaempferol and guanacastane diterpenoids are few of the antitumor agents produced from endophytic fungus Mucor fragilis and Cercospora sp. Another antitumor agent, fusidienol from Fusarium griseum, is being reported to act against tumors causing enzyme

16

Microbial Enzymes and Metabolites for Health and Well-Being

farnesyl transferase. Balanol is a protein kinase-C inhibitor from Verticillium balanoides. The conventionally used antitumor drugs such as epirubicin, pentostatin, and peplomycin were sourced from microorganisms (Kulanthaivel et al., 1993). Rhinocladiella sp., which is an endophyte on Tripterygium wilfordii, produces an antitumour alkaloid known as 22-oxa-12-cytochalasin. Some of the microbial metabolites have been reported to act as antitumor agents, less toxic side effects; hence, they can be considered better than standardized drugs and used as a superior alternate to the con­ ventionally prescribed drugs. However, there is a need of more research in the therapeutic approach and clinical trials on these metabolites.

1.9.3 AS IMMUNOSUPPRESSANT DRUGS Immunosuppressant drugs are used to suppress the immune system by acting on the function of lymphocytes. These drugs are useful during the transplantation of organs and tissues to suppress the action of the immune system and inhibit the process of allograft rejection (Rainsford, 2007). The immunosuppressants are being extensively used for the restriction of autoimmune disorders (insulindependent diabetes, rheumatoid arthritis) and other non-autoimmune inflammatory conditions (Li et al., 2017). Different types of immunosuppressive agents have been isolated as metabolites from microorganisms, such as cyclosporine A (Tolyplocladium inflatum) acting in T cell regulation without exhibiting excessive toxicity; Tacrolimus (Streptomyces tsukubaenis) used in controlling allogenic organ transplantation is a macrolides class of drug, and Sirolimus and ripamycin (Streptomyces hygroscopicus), mycophenolate mofetil (Penicillium stoloniferum), and gliotoxin (Aspergillus and Trichoderma) all are reported for their activity as immunosuppressive and in organ transplantation (Vézina and Kudelski, 1975; Borel, 1976; Kino et al., 1987; Demain, 1992).

1.9.4 AS ENZYME INHIBITORS Enzyme inhibitors are increasing in popularity as effective tools for exploring enzyme structures and reaction pathways, as well as prospective applications in medicine and agriculture. Several enzyme inhibitors have been isolated from microorganisms, which are being employed to target amylases, glucosidases, lipases, proteases, and xanthine oxidase (Baria et al., 2020). Amylase inhibitors prevent in the absorption of dietary carbohydrates, and thus are helpful in the treatment and prevention of diseases, such as diabetes, hyperlipidemia, obesity, and rumen acidosis (Li, Fan, and Zhao, 2022). Microbial α-amylase inhibitor, such as tau aggregation inhibitors and paim has been isolated from Streptomyces calvus TM-521 and Streptomyces corchorushii respectively (Singh et al., 2017). Similarly, acarbose isolated from Actinoplanes utahensis consists of aminocyclitol moiety and va­ lienamine, inhibits intestinal α-glucosidase and sucrase, resulting in low starch metabolism in the intestine and helps in the treatment of type 2 mellitus diabetes (Demain and Sanchez, 2009). Further, lipstatin produced by Streptomyces toxytricini acts as lipase inhibitor, which decreases the fat absorption in the intestine and helps in the treatment of diabetes and obesity. Orlistat is a saturated derivative of lipstatin (Weibel et al., 1987; Rodgers, Tschöp, and Wilding, 2012). Protease inhibitors produced by Streptomyces spp., such as antipain, chymostatin, and leu­ peptin, have been employed to treat certain diseases by targeting proteases in the pathogenic pathways of diseases, such as AIDS, arthritis, cancer, emphysema, and pancreatitis (Butler et al., 2008). Xanthine oxidase inhibitor, hydroxyakalone isolated from Agrobacterium aurantiacum, helps in the reduction of uric acid level in the blood and therefore helps in the prevention of gout (Borges, Fernandes, and Roleira, 2012). Monostatin and marinostatin isolated from the Alteromonas spp. are the cysteine and serine inhibitors, respectively. Monostatin exhibit activity against bacterial pathogens such as Vibrio anguillarum and Aeromonas hydrophilia, whereas marinostatin helps reduce in the pancreatitis pathogenesis (Ruocco et al., 2017). Enzyme inhibitors from fungus have been also used for the treatment of several diseases, such as Alzheimer’s disease, cancer, and diabetes (Paterson, 2008; Sharma et al., 2020).

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1.10 CONCLUSION Due to the emerging incidences of infectious and deadly diseases, the demand for pharmaceutically important products seems to be growing. However, in comparison to the earlier chemical or synthetic methods, microbial synthesis of such products using latest biotechnological interventions has reduced the production cost and side effects, and provided better catalytic properties and stability. The continued developments on microbial processes and technologies have launched patented production of several enzymes and other chemical substances, implicating their prevalent applications in pharmaceutical industries and healthcare. Nonetheless, only a fraction of microorganisms of what existed on this planet has been explored, and a larger part remains untapped exploring, which may lead to discovery and development of many novel enzymes and products for the benefits of mankind.

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2

Microbial Production of Fibrinolytic Enzymes Kyoungseon Min and Ki-Yeon Kim Gwangju Bio/Energy R&D Center, Korea Institute of Energy Research (KIER), Gwangju, Republic of Korea

CONTENTS 2.1 2.2 2.3

Introduction.............................................................................................................................25 Microbial Sources of Fibrinolytic Enzymes..........................................................................27 Recombinant Fibrinolytic Enzymes.......................................................................................28 2.3.1 General Genetic Engineering Approach to Produce Fibrinolytic Enzyme by Microorganisms ......................................................................................29 2.3.2 Recombinant Fibrinolytic Enzymes Produced by Microorganisms .........................30 2.4 Optimization of Culture Conditions for Producing Recombinant Fibrinolytic Enzyme .....31 2.5 Recovery of Microbial Fibrinolytic Enzymes .......................................................................32 2.6 Conclusions............................................................................................................................. 34 References........................................................................................................................................ 34

2.1 INTRODUCTION According to the World Health Organization (WHO), 17.9 million people worldwide die from cardiovascular diseases (CVDs) each year (Sharma et al., 2021). A thrombus, colloquially called a blood clot, is a major causative agent of CVDs. Since fibrin is a primary component of the thrombus, fibrinolytic enzymes that can hydrolyze the thrombus have been important in thrombosis therapy. Several fibrinolytic enzymes not only can successfully dissolve the thrombus and perform homogeneous blood flow maintenance, but they also are applicable for antibacterial agents (Jadhav et al., 2020), detergent additives for removing blood stains from fibers (Guleria et al., 2016), and functional food additives for preventing and treating CVDs (Weng et al., 2017). Fibrinolytic en­ zymes are currently classified into serine protease, metalloprotease, and serine metalloprotease, as shown in Table 2.1. For the economically feasible production of fibrinolytic enzymes, pharmaceutical industries have aimed to screen novel producers with certain advantages over what is already known. Insects, plants, snakes, and earthworms as potential sources of fibrinolytic enzymes have been studied, and their characteristics, production, and purification have also been reported. However, it is very expensive to directly produce fibrinolytic enzymes from them, and undesirable side effects (e.g. a short half-life, low specificity for fibrin, and fatal complications) have been reported (Kumar and Sabu, 2019; Sharma et al., 2021). Accordingly, there are increasing demands for alternative sources of fibrinolytic enzymes and cost-effective production. Various microorganisms including bacteria, fungi, and algae can also produce fibrinolytic enzymes (Kumar and Sabu, 2019). Comprehensive biochemical diversity, easy genetic modification, and scale-up feasibility make microorganisms a promising source in industrial-scale production. With an aim of commercial production of fibrinolytic enzymes, various microbial producers, such as recombinants, random mutants, and metagenomic libraries, have been developed (Cai et al., 2016; Faraji et al., 2017; Liu et al., 2019). In addition, statistical optimization of culture DOI: 10.1201/9781003369295-3

25

Serine proteases are endopeptidases to cleave peptide bonds in proteins, where serine is the nucleophilic amino acid at the enzyme active site.

Metalloproteases are any exo- and endo-peptidases whose catalytic mechanism involves a metal. In these enzymes, a divalent cation usually activates the water molecule. Therefore, fibrinolytic enzymes belonging to the metalloproteases mostly require divalent metal ions (e.g. Zn2+, Mg2+, Ca2+, Hg2+ or Co2+) for their activity.

Serine metalloproteases have properties of both serine proteases and metalloproteases.

Metalloprotease

Serine metalloprotease

Definition

Serine protease

Fibrinolytic Enzyme

TABLE 2.1 Classification of Fibrinolytic Enzymes

Direct fibrinolytic activity

M179, CFR15, AprE176

CMase, PoFE, FVP-I, AMMP, Bacillokinase II

Tissue plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), xylarinase, starase, subtilisin

Direct fibrinolytic activity

Plasmin, trypsin, nattokinase, brinase

Direct fibrinolytic activity.

Example of Fibrinolytic Enzyme

Indirect fibrinolytic activity.

Function

( e Silva et al., 2018; Moula Ali and Bavisetty, 2020)

( Moula Ali and Bavisetty, 2020; Rawlings and Barrett, 1995)

( Hedstrom, 2002; Moula Ali and Bavisetty, 2020; Sekar and Hageman, 1979)

Reference

26 Microbial Enzymes and Metabolites for Health and Well-Being

Microbial Production of Fibrinolytic Enzymes

27

media (e.g. carbon, nitrogen, substrate, mineral) and physicochemical parameters (e.g. tempera­ ture, pH, agitation speed, inoculum size) have been intensively researched (Biji et al., 2016; Moula Ali and Bavisetty, 2020). To improve economic feasibility, agricultural wastes and residues have been used as an inexpensive and sustainable feedstock for the microbial production of fibrinolytic enzymes (Meshram et al., 2017). Subsequently, commercial fibrinolytic enzymes should be required complex separation and purification steps, including precipitation, membrane filtration, dialysis, ion exchange, hydrophobic interaction, gel permeation, and affinity chromatography (Sharma et al., 2021). The multistage separation and purification processes are often timeconsuming, causing low product quality membrane fouling and protein aggregation. However, a few effective alternatives have recently been developed for the concentration and purification of various industrially important enzymes, thereby being applicable for the microbial production of fibrinolytic enzymes (Ahn et al., 2015; da Silva et al., 2019; Nascimento et al., 2016). Along with the development of biotechnology, there are great advances in the field of microbial production of fibrinolytic enzymes. This chapter deals with the recent progress on microbial producers, genetic engineering, optimization of the fermentable parameters, and downstream process for producing fibrinolytic enzymes.

2.2 MICROBIAL SOURCES OF FIBRINOLYTIC ENZYMES Microorganisms are excellent repositories of fibrinolytic enzymes. Bacteria, fungi, and algae are well-known microbial sources of fibrinolytic enzymes (Diwan et al., 2021). Given that i) bac­ terial sources usually secrete their fibrinolytic enzymes in a few days of submerged fermentation and ii) secreted enzymes can be purified without cell disruption, the main advantage of bacterial sources is the easy and economically feasible in large-scale production (Liu et al., 2013). Like bacterial sources, fungi also have an excellent ability to secrete various hydrolytic enzymes. Since fungi can grow on insoluble agricultural wastes, their endogenous fibrinolytic enzymes can be produced through solid-state fermentation (Meshram et al., 2017; Tao et al., 1998), as well as submerged fermentation (Tharwat, 2006). Solid-state fermentation is considered more cost-effective because it consumes less water than submerged fermentation (Meshram et al., 2017; Tao et al., 1998). Additionally, algae have been researched to photo-autotrophically produce fibrinolytic enzymes since algae can produce complex organic compounds with min­ imum nutritional requirements (i.e. carbon dioxide and water) (e Silva et al., 2018; Páblo et al., 2017). Since the above merits of microbial sources increase the feasibility of large quantitative production of fibrinolytic enzymes at a low cost through process engineering and/or genetic engineering, efforts are ongoing to secure information (i.e. the enzymatic properties and the gene sequences) on fibrinolytic enzymes from various microorganisms. Among microbial fibrinolytic enzymes, nattokinase (also known as subtilisin NAT) is a directacting serine protease with few side effects in the human body (Kurosawa et al., 2015; Lampe and English, 2016; Nagata et al., 2017). This enzyme was first reported from B. subtilis var. natto, which is used as a starter in the production of Natto, a fermented soybean food (Sumi et al., 1987). This gram-positive bacterium synthesizes immature nattokinase containing the signal peptide in the cytoplasm and then excretes functional mature polypeptides out of the cell through the wellestablished secretory system (Nakamura et al., 1992). Thus, commercial nattokinase production is conducted simply through the downstream extraction and purification process from the fermented Natto (Weng et al., 2017). Nattokinase produced in this way is currently available as a food supplement in many countries (Weng et al., 2017), but it is not currently used as a therapeutic medicine for thrombosis, due to impurities. Accordingly, new microbial sources along with genetic-engineering approaches (Cai et al., 2016; Che et al., 2020; Liang et al., 2007; Liu et al., 2019) have been explored to increase the production amount and purity of the nattokinase available as a therapeutic purpose. In addition, numerous microbial resources producing direct-acting serine protease have been secured in efforts to find better fibrinolytic enzymes, resulting from bacteria

28

Microbial Enzymes and Metabolites for Health and Well-Being

(e.g. B. amyloliquefaciens (Agrebi et al., 2010; Jo et al., 2011; Kim et al., 2009), B. cereus (Narasimhan et al., 2018), B. licheniformis (Hwang et al., 2007), B. subtilis (Hu et al., 2019; Jeong et al., 2001), Paenibacillus polymyxa (Lu et al., 2010), Streptomyces megaspores (Chitte and Dey, 2000), Streptomyces omiyaensis (Uesugi et al., 2011), from fungi (e.g. Aspergillus oryzae (Shirasaka et al., 2012), Cordyceps sinensis (Li et al., 2007), Fomitella fraxinea (Lee et al., 2006), Lyophyllum shimeji (Moon et al., 2014), Paecilomyces tenuipes (Kim et al., 2011), Pleurotus eryngii (Cha et al., 2010)) and algae (e.g. C. diaricatum (Matsubara et al., 2000), C. intricatum (Matsubara et al., 1998), and C. latum (Matsubara et al., 1999). Serrapeptase (also known as serratiopeptidase or serralysin), a metalloprotease containing zinc ion essential for its activity (Miyata et al., 1971), is also a well-known direct-acting fibrinolytic enzyme. Since it was first purified from the culture broth of Serratiamarcescens E-15(Miyata et al., 1970), this extracellular enzyme has been widely used as an anti-inflammatory agent (Jadhav et al., 2020). To enhance the production of this enzyme, heterologous overexpression was conducted in E. coli (Srivastava et al., 2019), and several homologies were also identified from various species of Pseudomonas (Louis et al., 1998), Serratia (Bhargavi and Prakasham, 2013; Wu et al., 2016), and Xenorhabdus (Massaoud et al., 2011). Additionally, direct-acting metalloproteases have also been reported from microorganisms; in particular, fungi are the richest source of metalloprotease (e.g. Fusarium pallidoroseum (El-Aassar, 1995), Rhizopus chinensis (Xiao-lan et al., 2005), Ganoderma lucidum (Choi and Sa, 2000), Perenniporia fraxinea (Kim et al., 2008), Flammulinavelutipes (Park et al., 2007), Pleurotus ostreatus (Choi and Shin, 1998), Schizophyllum commune (Park et al., 2010), Tricholoma saponaceum (Kim and Kim, 2001), Armillaria mellea (Lee et al., 2005), Cordyceps militaris (Cui et al., 2008), Fomitella fraxinea (Lee et al., 2006)). Besides direct-acting fibrinolytic enzymes, indirect fibrinolytic enzymes (i.e. plasminogen ac­ tivators) have been also produced from microorganisms. Plasminogen activators, which convert plasminogen into a direct fibrinolytic enzyme (i.e. plasmin), can be broadly divided into two categories: eucaryotic and bacterial (Kunamneni et al., 2007). Bacterial plasminogen activators include streptokinase and staphylokinase. Streptokinase is the first clinically applied bacterial plasminogen activator and mainly from Streptococcus dysgalactiae subsp. equisimilis H46A. This strain excreted the most active streptokinase among more than a hundred variants isolated from human sources and did not produce an erythrogenic toxin (Christensen, 1945). Thus, the com­ mercial streptokinase is easily prepared by simply growing this strain in a semi-defined medium (Banerjee et al., 2004). Given that the entire nucleotide sequence of the streptokinase gene was reported (Malke et al., 1985; Malke and Ferretti, 1984), overexpression has been attempted in several types of microorganisms, for example, Escherichia coli (Ko et al., 1995), S. equisimilis H46A (Müller and Malke, 1990), B. subtilis (Wong et al., 1994), and Pichia pastoris (Hagenson et al., 1989; Pratap et al., 2000). Several homologies were identified from Streptococcus species (Babu and Subathra Devi, 2015; Bhardwaj and Angayarkanni, 2015; Johnsen et al., 1999; Rajendran and Selvan, 2011). Staphylokinase is an extracellular protein produced by Staphylococcus aureus. Because sta­ phylokinase is a monomer without any disulfide bond and its molecular weight is much smaller than that of streptokinase (Bokarewa et al., 2006), the functional expression has easily been achieved in Escherichia coli (Sako, 1985), Bacillus subtilis (Wong et al., 1994), and yeast Hansenula polymorpha (Moussa et al., 2012). Compared to recombinant streptokinase, the recombinant staphylokinase exhibited more fibrinolysis and higher therapeutic efficiency and safety (i.e. t-PA) (Collen and Van de Werf, 1993). Nonetheless, this plasminogen activator is not still cost-effective to produce and has several side effects.

2.3 RECOMBINANT FIBRINOLYTIC ENZYMES Numerous recombinant fibrinolytic enzymes have been expressed in several types of micro­ organisms thus far. From an economic viewpoint, production rate and yield are significantly

Microbial Production of Fibrinolytic Enzymes

29

considered in the production of recombinant enzymes. These factors can be guaranteed when adopting the appropriate microbial expression system (i.e. expression vectors and microbial hosts), but functional expression might be hampered by the physiological effects of host cells. Hence, genetic modifications are often required, and this chapter summarizes the recent genetic en­ gineering strategies for the high-level expression of fibrinolytic enzymes in microbial hosts.

2.3.1 GENERAL GENETIC ENGINEERING APPROACH ENZYME BY MICROORGANISMS

TO

PRODUCE FIBRINOLYTIC

Two complementary approaches have been commonly applied to express recombinant fibrino­ lytic enzymes: i) activation of the recombinant DNA in a heterologous host with the wellestablished genetic technologies, or ii) expression of the recombinant DNA in the native producer that may require the establishment of new genetic engineering tools. Although the manipulation of gene expression is generally easier to leverage the heterologous host with wellestablished genetic tools, the expression is often hampered by the host’s physiological effects, such as the codon usage. On the contrary, if the native producers of fibrinolytic enzymes can use established genetic tools, they can be applied to recombinant DNA expression. In this way, it is important to understand the protein expression characteristics of the host when producing recombinant proteins. For industrial and pharmaceutical applications, a few fibrinolytic enzymes have been produced simply by growing wild-type strains in nutrient media and further purified from the extracellular broth (Banerjee et al., 2004; Jadhav et al., 2020; Weng et al., 2017). However, various native sources of fibrinolytic enzymes are associated with pathogenicity and infections. For instance, Serratia marcescens, the wild microbial producer of serrapeptase, is an opportunistic pathogen and is associated with infections (Mahlen, 2011), e.g. pneumonia, endocarditis, bacteremia, urinary tract infection, and meningitis. Nevertheless, the accidental bulk release of these pathogenic mi­ croorganisms is common in the large-scale production of fibrinolytic enzymes. These accidents often expose the people involved in industrial operations and cause the risk of potential infections. In addition, the multi-drug resistant strains among the released pathogenic biomass are associated with clinical outbreaks (Gastmeier, 2014). Therefore, the recombinant expression of fibrinolytic enzymes in non-pathogenic microorganisms is a viable option. Various recombinant fibrinolytic enzymes derived from pathogens have been produced in Escherichia coli, Bacilli, lactic acid bacteria, and yeasts which are generally regarded as safe (GRAS) strains (Tang et al., 2013), and then can be used in the food and drug industries. Another criterion for selecting an expression host is whether the host organism synthesizes the recombinant protein as the original structure with activity. For example, given that protein N-glycosylation is the most common post-translational modification in eukaryotes, the glycosyl­ ated fibrinolytic enzyme of eukaryotic origin has been heterologously expressed in eukaryotic yeast systems (Hu et al., 2005; Sugimoto and Nakajima, 2001; Vu et al., 2015). However, it is worth noting that glycosylation patterns vary depending on the type of eukaryotes. Streptokinase is a non-glycosylated fibrinolytic enzyme, natively produced by a pathogenic bacterium Streptococcus dysgalactiae. When expressed in yeast Pichia pastoris, the recombinant strepto­ kinase was found to be heavily glycosylated (Pratap et al., 2000). On the contrary, yeast Schizosaccharomyces pombe produced recombinant streptokinase without glycosylation (Kumar and Singh, 2004). Although researchers successfully engineered an E. coli strain and obtained N-glycosylation of a recombinant protein (Valderrama-Rincon et al., 2012), no successful gly­ cosylated fibrinolytic enzymes have been reported from bacteria to date. The expression host is often a significant factor to determine the production yield. E. coli is the most common host strain for the production of fibrinolytic enzymes. Bacilli, lactic acid bacteria, and yeast have also been chosen as the expression hosts for the high-yield production of recom­ binant fibrinolytic enzymes, due to the ability to grow rapidly and at high density in inexpensive

30

Microbial Enzymes and Metabolites for Health and Well-Being

substrates, their well-characterized genetics, and the availability of functional expression vectors and efficient mutant strains. In particular, they can directly secrete proteins into the extracellular medium. The secreted enzymes are properly folded, soluble, and biologically active. Although fungi also have common advantages (e.g. rapid growth, extracellular secretion of fibrinolytic enzymes, the possibility of solid-state fermentation), the information on the production of recombinant fibrinolytic enzymes is not enough up to date (Liu et al., 2013). Some of these genetically engineered strains have also been developed as hosts that can further improve production yield. Given that host-specific intracellular and extracellular proteases often degrade the recombinant protein and negatively affect the production yield, E. coli BL21, two deleted main proteases, is the preferred host for the expression of recombinant fibrinolytic enzymes (Ghasemi et al., 2012). Extracellular protease-deleted Bacilli, such as B. subtilis WB800 and B. licheniformis BL10 were also used to increase the production yield of recombinant fibrinolytic enzymes (Liu et al., 2019). In the case of yeast, protease deficient strains have been developed (Liu et al., 2013), but there is a lack of information on the production of recombinant fibrinolytic enzymes. Codon usage bias of the expression hosts often negatively affects to the expression level of heterologous protein. For instance, the arginine codons AGA and AGG, which are particularly rare in E. coli, often cause lower protein expression. Hence, the heterologous expression of fibrinolytic enzymes can be improved by the use of codon-optimized synthetic genes (Hu et al., 2005). The secretory expression strategy is an effective way to improve the yield of fibrin-degrading enzymes because it allows the acquisition of soluble active proteins by simpler purification methods (Liu et al., 2013). Bacilli, lactic acid bacteria, and yeasts essentially secrete recombinant fibrinolytic enzymes. However, the production of fibrinolytic enzymes in E. coli was mostly produced as intracellular insoluble aggregates, and thus renaturation or purification from the inclusion bodies should be required to obtain active fibrinolytic enzymes. To prevent insoluble inclusion bodies, the fusion of signal peptides is a commonly used strategy. For example, to increase the nattokinase yield, signal peptides for extracellular proteins in B. licheniformis were screened, resulting in the overexpression of the signal peptidase I SipVled to a 4.2-fold improved the nattokinase yield in this optimized strain (Cai et al., 2016). In addition to the expression host, characteristics of the expression vector (e.g. promoter and copy number) are also significant to determine the expression level. When using an expression vector with a high copy number, the amount of expressed protein might be increased, but other elements of the expression vector (e.g. antibiotic resistance gene) often cause a metabolic burden. Therefore, medium-copy expression vectors with a strong promoter have been preferred for the expression of the recombinant fibrinolytic enzymes.

2.3.2 RECOMBINANT FIBRINOLYTIC ENZYMES PRODUCED

BY

MICROORGANISMS

Recombinant fibrinolytic enzymes are produced through the individual or combinatorial use of the already mentioned methodology. To increase yields of fibrinolytic enzymes and simplify the downstream purification process, the fibrinolytic enzyme genes have been cloned and expressed in various microbial host systems, including Escherichia coli, Bacilli, and yeasts. A representative list of the recombinant fibrinolytic enzymes produced by microbial hosts is presented in Table 2.2. E. coli has been extensively investigated as the easiest and cheapest host strain to produce recombinant direct-acting fibrinolytic enzymes. Although direct-acting fibrinolytic enzymes can be expressed in E. coli, a large amount of cytoplasmic recombinant protein often forms insoluble and inactive inclusion bodies, thereby resulting that most of the aggregated proteins are lost their catalytic activities during solubilization and refolding processes (Chiang et al., 2005; Liang et al., 2007; Ni et al., 2016). To overcome this limitation, Liang et al. (2007) expressed a fused natto­ kinase gene with a periplasmic secretion signal sequence (i.e. pelB leader sequence). As a result, active nattokinase was secreted from the E. coli, but the activity was quite lower than native nattokinase (Liang et al., 2007).

Microbial Production of Fibrinolytic Enzymes

31

TABLE 2.2 Genetic Engineering Strategy for Producing Recombinant Fibrinolytic Enzymes Fibrinolytic Enzyme Category Direct

Indirect

Expression Host

Genetic Strategy

Expression Level (mg/L)

Fold Change

Reference

Escherichia coli Periplasmic secretion

49.3

Bacillus subtilis Strong artificial promoter

643

No activity without ( Liang et al., 2007) secretion 2.36 ( Wu et al., 2011)

Bacillus subtilis Use of protease deficient strain

600

1.80

Escherichia coli Periplasmic secretion

70

Pichia pastoris

310 (Glycosylated enzyme)

No activity without ( Schlott et al., 1994) secretion – ( Faraji et al., 2017)

Codon optimization

( Nguyen et al., 2013)

646 (After deglycosylation)

Given that Bacillus species can produce secretory proteins, they are mainly preferred for the homologous expression of direct-acting recombinant fibrinolytic enzymes. The intrinsic serine protease promoter has been substituted witha strong promoter to improve the serine protease expression in B. subtilis. As shown in Table 2.2, the productions of the serine protease in the control strain using the intrinsic promoter and in the engineering strain with a strong artificial promoter were of 272 and 643 mg/L, respectively (Wu et al., 2011). B. subtilis itself, however, produces a substantial number and quantity of native extracellular proteases, and thus, an extra­ cellular protease deficient strain has been used to produce recombinant nattokinase. For example, the nattokinase gene was expressed under the control of the acoA promoter in B. subtilis WB800, a strain that lacks eight extracellular proteases. As a result, the expression level of nattokinase was increased to 1.8-foldcompared to that of the previously most expressed strain, as shown in Table 2.2 (Nguyen et al., 2013; Ye et al., 1999). The genes encoding indirect fibrinolytic enzymes have also been expressed to varying levels in different expression systems. As shown in Table 2.2, a fibrinolytic enzyme from Staphylococcus aureus was expressed in thermal inducible E. coli strain K12 and IPTG inducible E. coli TGI, respectively. However, the expression levels were low with20 and 70mg/L, respectively, because of inclusion body formation (Sako, 1985; Schlott et al., 1994). In accordance with Faraji et al. (2017), indirect fibrinolytic enzymes expression in Pichia strain was reported in the 100% gly­ cosylated form. An indirect fibrinolytic enzyme from Staphylococcus aureusafter codon optimi­ zation was expressed in P. pastoris. As shown in Table 2.2, the expression maximum reached up to 310 mg/L of the culture medium with 3% methanol. Further, deglycosylation enhanced the activity up to 646 mg/L (Faraji et al., 2017).

2.4 OPTIMIZATION OF CULTURE CONDITIONS FOR PRODUCING RECOMBINANT FIBRINOLYTIC ENZYME High production cost often hinders microbial fibrinolytic enzymes to commercial applications. In addition to recombinant technology and genetic engineering, fermentation process has also been optimized for reducing production costs. Given that microorganisms have diverse physiological and nutritional needs, optimization of the nutritional and physical conditions is mandatory for

32

Microbial Enzymes and Metabolites for Health and Well-Being

producing fibrinolytic enzymes. Since microbial sources of fibrinolytic enzymes are equipped with proteases, complex nitrogen sources are usually preferred rather than simple inorganic nitrogen (Wu et al., 2019). In the case of metalloprotease-type fibrinolytic enzymes, they in evitably require metal ions as cofactors for growth and metabolism (Xin et al., 2018). Accordingly, nitrogen source and metal ion are significant factors for optimizing nutrients in culture media. Temperature, pH, and agitation are also important variables. Most microbial sources of fibrino­ lytic enzymes used for fermentation have an optimum temperature from 30 °C to 40 °C (Avhad and Rathod, 2015; Pan et al., 2019), but a few thermophilic bacteria exhibit higher optimal temperatures, for example, S. megasporus at 55 °C (Chitte et al., 2011). The pH of culture media might be affected to enzyme yield by improper structure formation, cleavage of disulfide bonds, protein aggregation, and deamination as a result of improper homeostasis (Yon, 1996), and most fibrinolytic enzymes are usually produced in pH range from neutral to slightly alkaline (Taneja et al., 2017). Fibrinolytic enzyme-producing bacteria grown in agitated conditions show increased enzyme production, due to the homogenous distribution of nutrients and oxygen (Chitte et al., 2011). Above fermentable parameters have traditionally been optimized by changing one variable (e.g. cultivation time, temperature, nutrition concentration, pH, inoculum level) at a time while keeping all the others constant (Moula Ali and Bavisetty, 2020). Traditional methods are not only timeconsuming due to a large number of experiments but can also lead to incorrect results. To over­ come this complexity, statistically optimized conditions are commonly used to design experiments for desirable responses using the minimum number of experiments. Table 2.3 summarized sta­ tistically optimized parameters for fibrinolytic enzymes.

2.5 RECOVERY OF MICROBIAL FIBRINOLYTIC ENZYMES Most of the fibrinolytic enzymes produced from bacteria (except E. coli) and fungi have been produced in the form of extracellular proteases at the end of the exponential growth phase (Choi et al., 2009; Wang et al., 2006). As shown in Figure 2.1, centrifuges and filtration are generally used for the exclusion of contaminants such as microbial cell debris and colloids from the culture broth and the crud extract. The crude fibrinolytic enzymes and the cell-free extracts were concentrated and fractionated either through precipitation using ammonium sul­ fate, ethanol, and acetone, dialysis, ultrafiltration, or ultracentrifugation. Such methodologies are used either individually or in combination, accompanied by chromatographic techniques (e.g. affinity chromatography, gel filtration chromatography, ion-exchange chromatography, and hydrophobic interaction chromatography) for further purification (Choi et al., 2009; Hu et al., 2019; Kim et al., 2011). For instance, a novel fibrinolytic enzyme from the culture broth of Streptomyces radiopugnans was purified using ammonium sulfate precipitation accompanied by dialysis and concentrated with a syringe filter-ultrafiltration membrane. The enzyme was further purified using gel-filtration and ion-exchange chromatography. A 22.36-fold increase in specific activity (3891 U/mg) of the purified enzyme was observed with a yield of 35% relative to crude enzymes (Dhamodharan, 2019). Most recombinant fibrinolytic enzymes from E. coli and all of the algal fibrinolytic enzymes were produced in the form of intracellular enzymes. Therefore, cell destruction is inevitably required to purify the intracellular fibrinolytic enzymes. As already mentioned, most recom­ binant fibrinolytic enzymes produced by E. coli already aggregate to an insoluble inclusion body in the cytoplasm. The aggregated fibrinolytic enzymes preclude the application of con­ ventional purification methods (e.g. gel-filtration, ion-exchange, or affinity chromatography) for their isolation. In these situations, it is crucial to develop a suitable and optimized puri­ fication protocol for each protein and these protocols include denaturation and refolding processes. Additionally, complex downstream purification processing leads to several disadvantages and thus other strategies (e.g. aqueous two-phase systems, reverse micelles systems, and three-phase

TLFD, CCD, RSM

OFAT

SmF

SmF

SSF

SmF

SSF

SmF

Streptococcus agalactiae EBL-31 (Recombinant)

Serratia marcescens RSPB11

Pseudoalteromonas sp. IND11

Bacillus subtilis (Recombinant)

Bacillus cereus IND1 Serratia rubidaea KUAS001

PBD, BBD, RSM

TLFD, CCD, RSM

PBD, CCD, RSM

RSM, CCRD

TLFD, OFAT, RSM, CCD

SSF

Shewanella sp. IND20

Optimization Methods

Fermentation Modes

Producers

Optimized Parameters

Moisture (%): 100 Medium composition (%): Beef extract (0.3), NaH2PO4 (0.05) pH: 6.0 Temperature (°C): 30 Medium compositions (%): Maltose (1.5), Yeast extract (1.5), NaCl (1.5), Skimmed milk powder (0.15)

Medium compositions (%): Soybean hydrolysate (6.1), KH2PO4 (0.415), CaCl2 (0.015)

pH: 7.0 Moisture: 121.4% Medium composition (%): maltose (1.0), NaH2PO4 (0.1)

pH:7.0 Temperature (°C): 37.5 Medium compositions (%): Dextrose (0.35), MgSO4 (0.1), KH2PO4 (0.08), NaCl (0.1), CaCl2 (0.005) Aeration: 150 rpm pH:7.0 Temperature (°C): 30 Medium compositions (%): Dextrose (0.35), MgSO4 (0.1), KH2PO4 (0.08), NaCl (0.1), CaCl2 (0.005)

pH: 10.0 Moisture (%): 120.8 Medium composition (%): Trehalose (1.21)

TABLE 2.3 Statistical Optimization for Producing Fibrinolytic Enzymes

394.9

3,699

77,400

1,573

23,910

147.08

2,751

Enzyme Production (U/mL)

2.57

4.0

2.0

3.0

1.518

2.01

2.5

Fold Change

( Vijayaraghavan and Prakash Vincent, 2014a) ( Anusree et al., 2020)

( Chen et al., 2007)

( Vijayaraghavan and Vincent, 2014b)

( Bhargavi and Prakasham, 2016)

( Vijayaraghavan and Prakash Vincent, 2015)

Reference

Microbial Production of Fibrinolytic Enzymes 33

34

Microbial Enzymes and Metabolites for Health and Well-Being

FIGURE 2.1 Flow chart of purification steps of microbial fibrinolytic enzymes.

partitioning) have been developed (Avhad et al., 2014; Liu et al., 2004). For example, an aqueous two-phase system was used for the purification of fibrinolytic enzymes from B. subtilis and Mucorsubtilissimus. The aqueous two-phase system is formed by two mutually immiscible phases that are generated by mixing phase-forming components above a threshold concentration (Ashipala and He, 2008; Nascimento et al., 2016). An aqueous two-phase system is also suitable for largescale production because polyethylene glycol, which is often used in one of its phases, has favorable physical and chemical properties (da Silva et al., 2019); Polyethylene glycol is a polydispersed polymer that can react with a variety of functional groups of proteins, and polyethylene glycol binding to proteins improves their solubility in water and organic solvents, increases its biocompatibility, and makes the scale-up easier (Hutanu et al., 2014).

2.6 CONCLUSIONS Given that fibrinolytic enzyme can be a major causative agent in cardiovascular diseases, it might be a significant protein applicable for pharmaceutical and clinical area. Although fibrinolytic enzymes are widely distributed in nature, their applications have been limited thus far, due to high production costs, low enzyme stability, and therapeutic side effects. This chapter provides a comprehensive understanding of genetic engineering, optimization of fermentation parameters, and downstream process (e.g. separation and purification) for massive production of fibrinolytic enzymes in microorganisms. The results discussed in this chapter would give viable opportunity novel development of fibrinolytic enzyme-based clinical applications.

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Sharma, C., Osmolovskiy, A., and Singh, R. 2021. Microbial fibrinolytic enzymes as anti-thrombotics: Production, characterisation and prodigious biopharmaceutical applications. Pharmaceutics 13:1880. Shirasaka, N., Naitou, M., Okamura, K., Fukuta, Y., Terashita, T., and Kusuda, M. 2012. Purification and characterization of a fibrinolytic protease from Aspergillus oryzae KSK-3. Mycoscience 53: 354–364. Srivastava, V., Mishra, S., and Chaudhuri, T.K. 2019. Enhanced production of recombinant serratiopeptidase in Escherichia coli and its characterization as a potential biosimilar to native biotherapeutic counterpart. Microbial Cell Factories 18:1–15. Sugimoto, M., and Nakajima, N. 2001. Molecular cloning, sequencing, and expression of cDNA encoding serine protease with fibrinolytic activity from earthworm. Bioscience, Biotechnology, and Biochemistry 65:1575–1580. Sumi, H., Hamada, H., Tsushima, H., Mihara, H., and Muraki, H. 1987. A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet. Experientia 43:1110–1111. Taneja, K., Bajaj, B.K., Kumar, S., and Dilbaghi, N. 2017. Production, purification and characterization of fibrinolytic enzyme from Serratia sp. KG-2-1 using optimized media. 3 Biotech 7:1–15. Tang, Y., Xiao, J., Chen, Y., Yu, Y., Xiao, X., Yu, Y., and Wu, H. 2013. Secretory expression and char­ acterization of a novel peroxiredoxin for zearalenone detoxification in Saccharomyces cerevisiae. Microbiological Research 168:6–11. Tao, S., Peng, L., Beihui, L., Deming, L., and Zuohu, L. 1998. Successive cultivation of Fusarium oxy­ sporum on rice chaff for economic production of fibrinolytic enzyme. Bioprocess Engineering 18: 379–381. Tharwat, N.A. 2006. Purification and biochemical characterization of fibrinolytic enzyme produced by thermophilic fungus Oidiodendron flavum. Biotechnology 5:160–165. Uesugi, Y., Usuki, H., Iwabuchi, M., and Hatanaka, T. 2011. Highly potent fibrinolytic serine protease from Streptomyces. Enzyme and Microbial Technology 48:7–12. Valderrama-Rincon, J.D., Fisher, A.C., Merritt, J.H., Fan, Y.Y., Reading, C.A., Chhiba, K., Heiss, C., Azadi, P., Aebi, M., and DeLisa, M.P. 2012. An engineered eukaryotic protein glycosylation pathway in Escherichia coli. Nature Chemical Biology 8:434–436. Vijayaraghavan, P., and Prakash Vincent, S.G. 2014a. Statistical optimization of fibrinolytic enzyme pro­ duction using agroresidues by Bacillus cereus IND1 and its thrombolytic activity in vitro. BioMed Research International 2014:1–11. Vijayaraghavan, P., and Prakash Vincent, S.G. 2015. A low cost fermentation medium for potential fibrinolytic enzyme production by a newly isolated marine bacterium, Shewanella sp. IND20. Biotechnology Reports 7:135–142. Vijayaraghavan, P., and Vincent, S.G.P. 2014b. Statistical optimization of fibrinolytic enzyme production by Pseudoalteromonas sp. IND11 using cow dung substrate by response surface methodology. SpringerPlus 3:1–10. Vu, T.B.N., Do, T.T., Le, T.H., Nguyen, T.T., and Nguyen, S.L.T. 2015. Enhance production of recombinant lumbrokinase by optimizing gene codon usage for expression in Pichia pastoris and its properties. Journal of Biotech Research 6:96–106. Wang, C.T., Ji, B.P., Li, B., Nout, R., Li, P.L., Ji, H., and Chen, L.F. 2006. Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi. Journal of Industrial Microbiology and Biotechnology 33:750–758. Weng, Y., Yao, J., Sparks, S., and Wang, K.Y. 2017. Nattokinase: An oral antithrombotic agent for the prevention of cardiovascular disease. International Journal of Molecular Sciences 18:523. Wong, S.L., Ye, R., and Nathoo, S. 1994. Engineering and production of streptokinase in a Bacillus subtilis expression-secretion system. Applied and Environmental Microbiology 60:517–523. Wu, D., Li, P., Zhou, J., Gao, M., Lou, X., Ran, T., Wu, S., Wang, W., and Xu, D. 2016. Identification of a toxic serralysin family protease with unique thermostable property from S. marcescens FS14. International Journal of Biological Macromolecules 93:98–106. Wu, R., Chen, G., Pan, S., Zeng, J., and Liang, Z. 2019. Cost-effective fibrinolytic enzyme production by Bacillus subtilis WR350 using medium supplemented with corn steep powder and sucrose. Scientific Reports 9:1–10. Wu, S.M., Feng, C., Zhong, J., and Huan, L.D. 2011. Enhanced production of recombinant nattokinase in Bacillus subtilis by promoter optimization. World Journal of Microbiology and Biotechnology 27: 99–106.

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Xiao-lan, L., Lian-xiang, D., Fu-ping, L., Xi-qun, Z., and Jing, X. 2005. Purification and characterization of a novel fibrinolytic enzyme from Rhizopus chinensis 12. Applied Microbiology and Biotechnology 67:209–214. Xin, X., Ambati, R.R., Cai, Z., and Lei, B. 2018. Purification and characterization of fibrinolytic enzyme from a bacterium isolated from soil. 3 Biotech 8:1–8. Ye, R., Kim, J.H., Kim, B.G., Szarka, S., Sihota, E., and Wong, S.L. 1999. High-level secretory production of intact, biologically active staphylokinase from Bacillus subtilis. Biotechnology and Bioengineering 62:87–96. Yon, J.M. 1996. The specificity of protein aggregation. Nature Biotechnology 14:1231–1231.

3

Microbial Enzymes for Synthesis of Chiral Drug Intermediates K B Arun Department of Life Sciences, CHRIST (Deemed to be University), Bengaluru, Karnataka, India

Raveendran Sindhu Department of Food Technology, TKM Institute of Technology, Kollam, Kerala, India

Bipin G Nair School of Biotechnology, Amrita Vishwa Vidyapeetham, Amritapuri, Kollam, Kerala, India

Binod Parameswaran Microbial Processes and Technology Division, CSIR-National Institute for Interdisciplinary Science and Technology (NIIST), Thiruvananthapuram, Kerala, India

Ashok Pandey Center for Innovation and Translational Research, CSIR-Indian Institute of Toxicology Research (CSIR-IITR) and Centre for Energy and Environmental Sustainability, Lucknow, Uttar Pradesh, India

Mukesh Kumar Awasthi College of Natural Resources and Environment, Northwest A&F University, Yangling, Shaanxi, China

Mohammed Kuddus Department of Biochemistry, University of Hail, Kingdom of Saudi Arabia

Aravind Madhavan School of Biotechnology, Amrita Vishwa Vidyapeetham, Amritapuri, Kollam, Kerala, India

CONTENTS 3.1 3.2

Introduction.............................................................................................................................42 Key Microbial Enzymes for Chiral Drug Intermediates.......................................................42 3.2.1 Lipases ........................................................................................................................43 3.2.2 Aldolases.....................................................................................................................44 3.2.3 Lyases and Hydrolases............................................................................................... 45

DOI: 10.1201/9781003369295-4

41

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Microbial Enzymes and Metabolites for Health and Well-Being

3.2.4 Oxidoreductases.......................................................................................................... 46 3.2.5 Transaminases.............................................................................................................47 3.3 Engineered Enzymes ..............................................................................................................47 3.4 Conclusions............................................................................................................................. 48 References........................................................................................................................................ 48

3.1 INTRODUCTION The synthesis of single enantiomers by biocatalysis or chemical route has become progressively significant for expanding and using pharmaceutic drugs and other chemicals (Federsel, 2005). Biological catalysis is nowadays attaining promising developments and momentum. It is attracting drug chemists as an essential drug intermediate technology, and it possesses a similar role to chromatographic techniques and chemical catalysis (Pollard and Woodley, 2007). Whole cell-based and extracted enzyme-based biocatalytic platforms are progressively applied as an effective way to synthesize complex drug intermediate compounds and other chemicals (Garzón-Posse et al., 2018). Additionally, the biotransformation area has attained more attention for producing chiral drug intermediates similar to bio-active compounds (de Carvalho, 2011), providing the progress and engagement of a more eco-friendly and cost-effective production process. Compared to the chemical route of production, the biocatalytic synthesis demonstrates the advantage of decreased ecological pollution and the large regio-, chemo-, and enantio-selectivity. Biocatalytic reactions can be conducted at standard pressure and temperature to circumvent harsh situations, which frequently could cause difficulties with racemization, isomerization, re-arrangement of the compound, and epimerization. Thus, biological catalysis provides several attractive features in green chemical synthesis, especially for producing chiral compounds used to synthesize food ingredients or enantiopure drugs (de María et al., 2019). And further progress and application of biocatalysis for large-production processes and sustainable chemistry can also be anticipated (Pollard and Woodley, 2007). Microbial enzymes are widely used for chiral synthesis. Enzymes isolated from microbes can be immobilized and reused for many generations. Microbes can be engineered to produce desired biocatalysts to transform chemical compounds to synthesize drugs. This chapter outlines the microbial enzymes involved in producing chiral drug compounds.

3.2 KEY MICROBIAL ENZYMES FOR CHIRAL DRUG INTERMEDIATES The molecule’s chirality mainly determines the potential and safeness of drugs. A molecule is said to be chiral if that specific molecule cannot be overlaid on its mirror image through any rotations and translations. The biomolecules showed stringent chirality as the sugar and DNA are D-chiral and amino acids are L-chiral. Remarkably, most of the drug intermediates exhibit chirality that significantly affects the pharmacological potential of the compound. Conventional drug synthesis usually ends up with paired chiral products having different toxicity and efficiency because one of these chiral products may be inactive or highly lethal. The active chiral molecule is called eutomer, and the inactive form is mentioned as distomer. For example, (S)-ibuprofen inhibits cyclooxygenase I 100 times better than (R)-ibuprofen, and when comparing the anaesthetic effect, S(-)-secobarbital is more effective than R(+)-secobarbital (Nguyen et al., 2006). Pharmaceutical sectors greatly depend on chiral intermediates to synthesize drugs (Patel, 2011, 2000), and chemical and biological methods can make these intermediates. The chirality of drug intermediates is often complex, and efforts are always made to develop new methods to synthesize the chiral intermediate of our interest. Conventional techniques are always expensive and have several disadvantages. Studies have shown that microorganisms and microbial enzymes have immense capacity to synthesize selective chemo, regio, and patio chemicals (Patel, 2013). However, advancements in molecular biotechnology have enhanced the synthesis of these chiral intermediates using engineered microbes and recombinant microbial enzymes.

Microbial Enzymes for Synthesis of Chiral Drug Intermediates

43

TABLE 3.1 Microbes for Chiral Drug Intermediates Microbe A. niger P. putida

Enzyme Mutant D11 monoamine oxidase Dioxygenases and monooxygenases

Yield

Product

>90%

solifenacin and levocetirizine Various biologically active compounds

86%

Reference ( Ghislieri et al., 2013) ( Wong et al., 2002)

Streptomyces sp.

cytochrome P-450sca-2

70%

Pravastatin

( Park et al., 2003)

Arthrobacter sp

x-transaminase

99%

( Richter et al., 2014b) ( Hussain and Ward, 2003)

Streptomyces griseolus

Recombinant reductase

80%

Agrochemicals & pharmaceuticals pharmaceuticals

B. megaterium

Transaminase

42%

Antagonists

( Hanson et al., 2008)

C. macedoniensis

Enoate reductase

87%

Levodione

( Kataoka et al., 2004)

Microbial synthesis has advantages over the chemical synthesis of desired chiral products. The engineered microbes and recombinant enzymes are economically feasible and can act efficiently in harsh industrial conditions such as pH, temperature, and pressure. The recombinant enzymes and the engineered microbes are integral for biocatalysis that execute discerning and precise chemical reactions to synthesize chiral products with reliable homochirality. Unlike the chemical method, biocatalysis can often be performed in a wide range of reaction conditions in fewer steps with less energy utilization and less harmful by-product formation, thus lowering the entire process steps and expense. The pharmaceutical industries depend significantly on custom-made enzymes for the critical execution of biocatalysis. Identifying enzymes or enzymes engineered with characteristic enantioselectivity properties is significant for synthesizing desired products. Earlier, the pharmaceutical industry mainly relies on hydrolases, especially lipases, catalyzing ester hydrolysis. Enzymes can be engineered to enhance their catalytic activity, constancy, and precision. These engineered enzymes can be overexpressed to meet industrial needs. Advancements in enzyme engineering have allowed choosing an unlimited number of enzymes, facilitating the designing the best pathways for synthesizing chiral intermediates. Microorganisms have tremendous potential to produce enzymes that aid in synthesizing compounds with better chemo-, regio- and enantioselectivity. Lipases, aldolases, transaminases, oxidoreductases, lyases, and hydrolases from microbial sources are critical enzymes used to synthesize chiral intermediates. Microbes and their enzyme participating in chiral drug synthesis are listed in Table 3.1.

3.2.1 LIPASES Lipases (triacylglycerol hydrolases E.C. 3.1.1.3) catalyze the hydrolysis reaction, converting triacylglycerols to glycerol and fatty acids. The water-soluble lipases usually hydrolyze water-insoluble ester substrates. Lipases are generally involved in activating esterification, interesterification, transesterification, aminolysis, and alcoholysis reactions. Monoacylglycerols, diacylglycerols, triacylglycerols, cholesteryl esters, and wax esters are the most common substrates of lipases. Lipases have wide applications in the food, chemical, and, most notably, pharmaceutical industries. For industrial applications, lipases are mainly produced from Achromobacter sp., Bacillus subtilis, Aspergillus sp., Fusarium sp., Staphylococcus aureus, Alcaligenes sp., Pseudomonas aeruginosa, Arthrobacter sp., and Serratia marcescens (Chandra et al., 2020; Singh and Kumar, 2019). The market value of microbial lipases in 2021 is 535 million USD and is projected to reach approximately

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990 million USD by 2031. Lipases are among the most regularly utilized enzymes for synthesizing chiral intermediates and drugs. Lipases do not require cofactors, and the regio-, chemo-, and enantioselectivity in the resolution of racemic mixtures is the fundamental reason for their employment. Lipases have significant role in the production of acids, alcohols, amines, and esters (GotorFernández et al., 2006). Lipases are widely used in pharmaceutical sectors for stereospecific hydrolysis to resolve the racemic mixtures and produce chiral intermediates. Burgess and Jennings have reported the enantioselective esterification of unsaturated alcohols using the lipases isolated from Pseudomonas sp (Burgess and Jennings, 1991). Profens are one of the primary nonsteroidal anti-inflammatory medicines, and the potential is mainly obliged to S-enantiomer. Lipases from Candida antarctica and Candida rugosa are used to produce enantiomerically pure profens (Sikora et al., 2014). Lipase from Candida rugosa have more enantioselectivity to the S-flurbiprofen. Meanwhile, immobilized lipase from Candida antarctica showed high enantioselectivity to R-flurbiprofen. Arylazetidione class of chiral intermediates is synthesized to produce Paclitaxel analogues, an anticancer drug. The lipase from Arthrobacter sp. has better potential to resolve the racemic mixtures of arylazetidinones, the intermediates for synthesizing amino acid side chains of paclitaxel and its analogues (Anand et al., 2007). Immobilized lipase from Candida cylindracea is used to resolve the isomers of baclofen, a muscle relaxant (Muralidhar et al., 2001). The lipase B from Candida antarctica (CalB) immobilized on cashew apple bagasse was found to resolve the racemic mixtures of rac-indanyl acetate (de Souza et al., 2020). The immobilized CalB is also reported to synthesize (S)-γfluoroleucine ethyl ester, the specific chiral intermediate, from 2-(3-butenyl) azlactone for the production of the cathepsin K inhibitor drug - Odanacatib (Laquièvre et al., 2012; Okeke and Frankenberger, 2003). Lipase from Geotricumcandidum can work in an organic solvent medium to synthesize the chiral molecule S-(1-(acetoxyl)-4-(3-phenyl) butyl) phosphonic acid that inhibits squalene synthase (Patel et al., 1997). S-(-)-acetate is the primary chiral intermediate for the drug named (-)-15-deoxyspergualin. S-(-)-acetate was synthesized by the stereospecific acetylation of racemic 7-[N,N′-bis-(benzyloxy-carbonyl)N-(guanidinoheptanoyl)]-alpha hydroxy-glycine, mediated by lipase (Chandra et al., 2020). Lipase isolated from Rhizopus oryzae hydrolyzes selectively the racemic mixture of 3-acylthio2-methylpropanoic acid to synthesize pharmacologically active (S)-captopril, the antihypertensive drug (Patel et al., 1992). (S)-2-Pentanol is one of the active chiral intermediates for synthesizing various anti-Alzheimer’s drugs, and CalB was used to synthesize (S)-2-pentanol from the racemic mixture of 2-pentanol (Patel et al., 2000). Immobilized lipase from Burkholderiacepacia efficiently resolves the racemic mixture of 1-phenylbut-3-yn-1-ol (Borowiecki and Dranka, 2019). Immobilized CalB on acrylic resin helps the lysis of chlorohydrin-synthon acetate mixture, which was then transformed to active pharmaceutical ingredients (R)-(-)-diprophylline and (S)(+)-xanthinol nicotinate (Borowiecki et al., 2021). The lipase from Pseudomonas fluorescens (PFL) was used for the synthesis of chiral intermediates, such as atenolol (Agustian et al., 2016), derivatives of N‐substituted benzimidazole (Łukowska-Chojnacka et al., 2016), propranolol (Wang et al., 2012), and 3‐hydroxymethyl‐1‐tetralone tosylates (Caro et al., 2003). PFL is also reported to resolve the racemic mixtures of β‐aryloxyalcohols – (RS)‐1‐chloro‐3‐(2,5‐dichlorophenoxy) propan‐2‐ol, and (RS)‐3‐(4‐chlorophenoxy)propane‐1,2‐diol (Soni et al., 2018). Lipase from Arthrobacter species is utilized to resolve the racemic mixture of methyl-1,4benzodioxan-2-carboxylate, which is used to synthesize the antihypertension drug doxazosin. The same chiral intermediate is used to produce the active (S) enantiomers of α-adrenergic receptor antagonists such as dibozane, prosimpal, and piperoxan (Rouf et al., 2012).

3.2.2 ALDOLASES C-C bond formation is one of the prominent methods for building complex molecules. Enzymes can create stereoselective C-C bonds, as their chemo-, regio-, and enantioselectivity is very high.

Microbial Enzymes for Synthesis of Chiral Drug Intermediates

45

Aldolases (E.C.4.1.2) are one of the enzymes that catalyze the enantioselective construction of C-C bonds, typically by creating two chiral centres (Wagner et al., 1995; Windle et al., 2014). Aldolases catalyze aldol condensation reactions with a broad range of substrates. Based on the donor’s characteristics, aldolases are classified as dihydroxyacetone phosphate-dependent, pyruvate-dependent, acetaldehyde-dependent, and glycine-dependent aldolases (Liu et al., 2021). The aldol condensation end-products such as monoalcohol, polyalcohol, and amino alcohol are vital components for the production of chiral drugs. The chiral molecule 2,4-dideoxyhexose and its derivatives are used as the precursor molecule for producing the HMG CoA reductase inhibitors such as lovastatin and mevastatin. 2,4dideoxyhexose was synthesized using the 2-deoxyribose-5-phosphate aldolase (DERA) enzyme (Gijsen and Wong, 1994; Greenberg et al., 2004). DERA can accept two substrates and is widely used to synthesize drugs, including pravastatin and atorvastatin. The aldehydes present in the media inhibit the undesirable DERA activity. The DERA gene from Pectobacteriumatrosepticum was engineered and expressed in E. coli. This recombinant protein was tolerant to aldehydes and showed better activity at alkaline pH (Haridas et al., 2020). β-Hydroxy-α-amino acids and the equivalent alcohols are one of the critical components used for synthesizing drugs. They also form the building units of glycopeptides, chloramphenicol, mycestericins, sphingofungins, callipeltin E13, polyoxypeptin A, cyclomarins and lactacystin. The synthesis of β-Hydroxy-α-amino acids from glycine and various aldehydes has been catalyzed by threonine aldolase, phenylserine aldolase, and hydroxyaspartate aldolase. Steinreiber et al. have produced β-hydroxy-α-amino acids by overexpressing D-threonine aldolase from Alcaligenes xylosoxidans and L-threonine aldolase from Pseudomonas putida in E. coli (Steinreiber et al., 2007). The sialic acid, N-Acetyl-d-neuraminic acid, is the key precursor for synthesizing the antiflu drug zanamivir. The production of N-Acetyl-d-neuraminic acid is a two-step enzymatic reaction involving the epimerization of N-acetyl-d-glucosamine (catalyzed by N-acetyl-d-glucosamine epimerase) and the condensation of the epimer with the pyruvate (catalyzed by aldolase) (Gao et al., 2019). The N-acetyl-Dneuraminic acid aldolase from E. coli (Aisaka and Uwajima, 1986), Haemophilus influenzae (Lilley et al., 1998), Clostridium perfringens (Traving et al., 1997) and Trichomonas vaginalis (Meysick et al., 1996) were employed to synthesize N-acetyl-D-neuraminic acid. The aldolase from Lactobacillus plantarum also showed better condensation activity (Sá nchez-Carró n et al., 2011). Fused epimerase and aldolase expressed in immobilized and soluble forms of E. coli actively catalyze the synthesis of N-Acetyl-d-neuraminic acid and N-acetyl-Dglucosamine (used for the treatment of osteoarthritis) (Jain et al., 2016; Wang et al., 2009). Microbes producing lipase and aldolase are listed in Table 3.2.

3.2.3 LYASES

AND

HYDROLASES

Lyases are applied to synthesize chiral drug compounds. They can transform their substrate compounds to synthesize newer double bonds by eliminating the substrate’s functional groups by techniques other than oxidation and hydrolysis. The γ lactamase from Bradyrhizobium japonicum was isolated and recombinantly expressed in E. coli (Zhu et al., 2012). The recombinant lactamase was used in the production of γ lactam, a crucial drug intermediate for abacavir and carbovir (Gao et al., 2015). Another intermediate compound for abacavir is γ lactam 2-azabicyclo-hept-5en-3-one, an inhibitor for reverse transcriptase of HIV and hepatitis B viruses. The compound was synthesized using the γ-lactamase from Rhodococcus and Pseudomonas solanacearum (Evans et al., 1992). Tyrosine phenol lyase synthesized from Erwinia herbicola was applied to produce the dihydroxyphenylalanine, a medication for Parkinson’s disease (Patel, 2008). To synthesize industrially important lactulose, cellobiose 2-epimerase from Caldicellulosiruptorsaccharolyticus was recombinantly expressed in E. coli (Wang et al., 2015). The G. kaustophilus derived N succinylamino acid racemase and G. stearothermophilus derived L-carbamoylase was immobilized to synthesize a series of L amino acids (Soriano-Maldonado et al., 2015).

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Microbial Enzymes and Metabolites for Health and Well-Being

TABLE 3.2 Lipase and Aldolase for Chiral Drug Synthesis Enzyme Lipase

Microbe Pseudomonas sp Arthrobacter sp

Aldolase

Chiral Intermediate Esterification of unsaturated alcohols Methyl-1,4-benzodioxan2-carboxylate

Reference ( Burgess and Jennings, 1991) ( Rouf et al., 2012)

Candida antarctica

R-flurbiprofen

Candida rugosa Candida antarctica

S-flurbiprofen (S)-γ-fluoroleucine ethyl ester

( Laquièvre et al., 2012)

Candida antarctica

(S)-2-pentanol

( Patel et al., 2000)

Candida antarctica Candida antarctica

Chlorohydrin-synthon acetate Rac-indanyl acetate

( Borowiecki et al., 2021) ( de Souza et al., 2020)

Candida antarctica

Enantiopure precursors and derivatives for β-blockers practolol, pindolol and carteolol

( Gundersen et al., 2021)

Geotricumcandidum

( Patel et al., 1997)

Pseudomonas cepacia

S-(1-(acetoxyl)-4-(3-phenyl) butyl) phosphonic acid (S)-Practolol

Pseudomonas fluorescens

Atenolol

( Agustian et al., 2016)

Candida cylindracea Rhizopus oryzae

Baclofen 3-acylthio-2-methylpropanoic acid

( Muralidhar et al., 2001) ( Patel et al., 1992)

Pseudomonas fluorescens

β‐aryloxyalcohols

( Soni et al., 2018)

E. coli Haemophilus influenzae Trichomonas vaginalis Lactobacillus plantarum Clostridium perfringens

N-Acetyl-d-neuraminic acid

( Aisaka and Uwajima, 1986; Lilley et al., 1998; Meysick et al., 1996; Sá nchez-Carró n et al., 2011; Traving et al., 1997)

Alcaligenes xylosoxidans (D-threonine aldolase) Pseudomonas putida (L-threonine aldolase) Alcaligenes (Achromobacter) xylosoxidans IFO 12699 and Arthrobacter sp. DK-38 (D-threonine aldolases)

β-Hydroxy-α-amino acids

( Steinreiber et al., 2007)

(2R,3S)-2-amino-3-hydroxy-3(pyridin-4-yl)-propanoic acid

( Goldberg et al., 2008)

Shewanellahalifaxensis (2-deoxyribose 5-phosphate aldolase)

Islatravir

Lactobacillus brevis (2-deoxyribose 5-phosphate aldolase)

Statin precursor-(3R,5S)-6-chloro2,4,6-trideoxyhexapyranoside

( Jiao 2015)

Pectobacterium atrosepticum (2-deoxyribose-5-phosphate aldolase

2,4-dideoxyhexose

( Haridas et al., 2020)

( Mulik et al., 2016)

3.2.4 OXIDOREDUCTASES Oxidation and reduction reactions have been widely used in the pharmaceutical industry. The monoamine oxidase mutant from A. niger was developed by directed evolution, and highthroughput screening was applied to produce the critical pharmaceutical ingredients for

Microbial Enzymes for Synthesis of Chiral Drug Intermediates

47

synthesizing solifenacin and levocetirizine (Ghislieri et al., 2013). A novel carbonyl reductase from Kluyveromyces lactis was identified and applied to reduce t-butyl 6-cyano-(5R)-hydroxy-3oxohexanoate to t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate, the essential chiral precursor for the synthesis of the atorvastatin calcium (Luo et al., 2015). The genetically engineered ketone reductases Microbacteriumcampoquemadoensis, which was obtained by directed evolution and showed higher activity than the wild type enzyme, were used in the synthesis of (S, E)-methyl 2-(3-(3-(2-(7-chloroquinolin-2-yl)-vinyl)phenyl)-3hydroxypropyl)benzoate, anti-asthma drug montelukast, with >90% yield (Liang et al., 2010).

3.2.5 TRANSAMINASES Transaminases are extensively used in chiral amines or chiral amino acid production. They are the essential building materials for several biologically active pharmaceutic drugs and natural compounds like atazanavir and sitagliptin (Paul et al., 2014). A transaminase mutant from Arthrobacter was isolated for synthesizing amino steroids, resulting in >80% product yield (Richter et al., 2014a). Ruegeria sp. transaminases were isolated from synthesizing several significant bulk chiral amines, and they displayed large stereoselectivity and >8000-fold high enzymatic activity than the wild counterpart (Pavlidis et al., 2016). Bacillus megaterium transaminase has been applied to produce R-sec-butylamine and R-1-cyclopropylethylamine, key intermediates for corticotropin release factor antagonist used for the medication of depression. Arthrobacter transaminase mutants are engineered by site-directed mutagenesis. They have been used in the asymmetric production of the suvorexant, an orexin receptor antagonist (Mangion et al., 2012), silodosin, a prodrug for prostatic hypertrophy (Simon et al., 2014), and mexiletine (Koszelewski et al., 2009). Rational engineering was used to enhance the Vibrio fluvialis amine transaminase substrate specificity, and the optimized mutant showed >1700 times improved rate of reaction (Dourado et al., 2016). ω-Transaminases from Pseudomonas jessenii was engineered using computational design enhanced the substrate scope to produce enantiopure bulky amines.

3.3 ENGINEERED ENZYMES The NADPH-dependent carbonyl reductase isolated from Yarrowialipolytica was heterologously expressed in E. coli cells. It was used for the biotransformation of ethyl 4-chloro-3oxobutanoate to ethyl (S)-4-chloro-3- hydroxybutanoate, a crucial intermediate for synthesizing statins (He et al., 2014). Another NADPH-dependent carbonyl reductase and glucose dehydrogenase isolated from Rhodococcuspyridinivorans was applied to synthesize ethyl (S)-4chloro-3-hydroxybutanoate with >90% yield (Xu et al., 2016). The engineered E. coli possesses a Mycobacterium sp. formate dehydrogenase and Pichia finlandica carbonyl reductase used to synthesize chiral intermediates for anti-cholesterol drugs. The engineered E. coli, which contains an enoate reductase from Candida macedoniensis, was applied for the C=C bond reduction to produce 6R-levodione, a key intermediate in the synthesis of chemical flavones and carotenoids (Kataoka et al., 2004). Using engineered whole cells as biocatalysts, an engineered form of phenylalanine dehydrogenase from Thermoactinomyces intermedius and recombinant FDH expressed in E. coli were used for the synthesis of the (S)-3hydroxyadamantylglycine, an important chiral intermediate for the production of saxagliptin, an antidiabetic drug (Salis et al., 2009). A ketoreductase isolated from Acinetobacter was identified and expressed in the E. coli expression system to produce (3S,5R)-dihydroxy-6(benzyloxy) hexanoic acid ethyl ester. This reaction was performed using the modified E. coli’s whole cells or lysate with >90% yield (Goldberg et al., 2008). Using engineered E. coli to produce LeuDH from Thermoactinomyces intermedius and FDH from the yeast P. pastoris, the L-Tle yielded >90 (Kragl et al., 1996).

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3.4 CONCLUSIONS The chiral intermediates are critical players in the pharmaceutical sector for synthesizing drugs. There are numerous chemical reactions to synthesize the chiral intermediates. Even though the chemical catalysis methods are significant, these strategies are often confined due to the high cost of precursor molecules and inadequate stereoselectivity. Hence, biocatalysis for the synthesis of chiral products is gaining much attention due to its advantages such as low cost, work in mild conditions, reduced reaction steps, and exhibits higher regio-, chemo-, and enantioselectivity in resolving racemic mixtures. A wide range of enzymes such as lipases, aldolases, transaminases, oxidoreductases, proteases, decarboxylases, and many more are employed to produce a diverse group of desired chiral molecules. Protein biochemistry, cloning, mutagenesis, directed evolution, and fermentation knowledge have progressed over the years, allowing for endless provision to a vast array of enzymes and microorganisms as biocatalytic devices in synthesizing chiral molecules. Drug resistance and the emergence of new pathogens will demand the development of new drugs. Hence, an interdisciplinary approach is required to identify new chiral intermediates or novel chemical modifications of available chiral molecules for better pharmaceutical potential, along with identifying new enzymes and enzyme sources for better biocatalysis.

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Microbial Enzymes in Biomedical Applications Ramzan Ahmed and Manjit Kumar Ray Department of Applied Biology, University of Science and Technology Meghalaya, Ri-Bhoi, Meghalaya, India

Debasis Nayak Department of Wild Life and Biodiversity Conservation, Maharaja Sriramchandra Bhanjdeo University, Odisha, India

Yugal Kishore Mohanta Department of Applied Biology, University of Science and Technology Meghalaya, Ri-Bhoi, Meghalaya, India

CONTENTS 4.1 4.2

Introduction.............................................................................................................................53 Applications of Microbial Enzymes in Biomedical Sectors.................................................55 4.2.1 Analytical/Diagnostic Enzymes .................................................................................55 4.2.2 Biosensors................................................................................................................... 55 4.2.3 Therapeutics Enzymes................................................................................................59 4.2.3.1 Asparginase..................................................................................................61 4.2.3.2 Chitinase ......................................................................................................61 4.2.3.3 Collagenase ..................................................................................................62 4.2.3.4 Lipase...........................................................................................................62 4.2.3.5 Nattokinase ..................................................................................................62 4.2.3.6 Serratiopeptidase..........................................................................................62 4.2.4 Thrombolytic Drugs ...................................................................................................62 4.2.4.1 Streptokinase................................................................................................ 63 4.2.4.2 Staphylokinase .............................................................................................63 4.2.4.3 Superoxide Dismutase .................................................................................63 4.3 Nutraceutical Enzymes...........................................................................................................63 4.4 Microbial Enzymes in Pharmaceutical Industry Applications..............................................64 4.5 Problems and Safety Concerns ..............................................................................................65 4.6 Future Prospects of Microbial Enzymes in Biomedicine .....................................................65 4.7 Conclusions............................................................................................................................. 66 References........................................................................................................................................ 66

4.1 INTRODUCTION Enzymes are functional proteins or nucleic acids (ribozymes) that aid in the execution of bio­ chemical reactions at rates that are appropriate for the regular functioning, growth, and prolifer­ ation of any biological system, including unicellular and multicellular plants and animals (Brasil et al., 2017; Mitchell, 2017; Nelson and Cox, 2017). Enzymes’ ability to remain viable and execute catalytic activities outside of their source organism, or in in situ conditions (Copley, 2017), enables DOI: 10.1201/9781003369295-5

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them to be used in a variety of industrial processes involving chemical transformations of sub­ strates to their corresponding products. Enzyme-catalyzed reactions are very efficient, meaning they take place under normal environmental conditions, such as pH, temperature, and pressure (the conditions depend on the physiological conditions of the source organisms and its environmental conditions) (Kumari et al., 2021). An enzyme derived from a mesophilic bacterium that lives in a neutral environment, for example, can be effective at mild temperatures, atmospheric pressure, and neutral pH. Enzymes can be employed in both isolated and immobilised forms (Cao et al., 2016; Miller, Akbar, and Morris, 2015). Enzymes are used in a variety of industrial applications, including the synthesis of pharmaceuticals such as drugs, the leavening of dough for bread pro­ duction, the processing of grain juices into lager and wine, the production of agrochemicals, biopolymers, nutraceuticals, artificial flavours, waste remediation, and many others (Chourasia et al., 2022; Hathwar et al., 2011; Newton et al., 2018). Because enzymes have successfully replaced several chemical catalyst-based pharmaceutical manufacturing methods, they have a bright future in this industry. Enzymes have recently been used to treat cancer and infectious disorders where antibiotics are no longer effective (due to appearance of resistance in bacteria against the antibiotics). Proteases, lipases, and amylases, for example, are utilised as digestive aids and have been used alone or in cocktails to treat digestive issues. Despite recent improvements, enzymes still have a lot of potential in the pharmaceutical industry. Other than as digestion aids, enzymes were mostly overlooked as medications. Crude proteo­ lytic enzymes, such as pepsin for dyspepsia, were only employed to treat digestive problems in the late 19th century. Later, researchers discovered that a Bacillus pyocyaneus extracellular secretion, nuclease (an enzyme that denatures nucleic acid), kills anthrax germs and protects mice from other lethal bacterium. This marked a turning point in the application of parental enzymes in the treatment of various diseases, malignancies, and, eventually, a wide range of diseases. Enzyme supplements come in pill, capsule, and powder form, and are frequently made up of several en­ zymes (Gonzalez and Isaacs, 1999). Enzymes have chiral selectivity, which is used to make medications that are enantiomerically pure (Underkofler et al., 1958). While it is evident that enzymes catalyse practically all biological events, it is equally crucial to note that enzymatic reactions are rarely, if ever, carried out in isolation. Enzymes and enzyme-rich foods are recom­ mended to consume on a regular basis to maintain good health, prevent disease, and slow down the aging process. Enzymes are required for the biochemical operations of each cell in our body, and lack of these enzymes will hasten the aging process. Enzymes have a variety of roles in the body, including regulating growth from a single cell to a mature organism, converting food into energy to meet the body’s demands, and breaking down or building up particular compounds within the cell (Kaur and Sekhon, 2012). A small number of fungi, yeast, and bacteria provide the majority of medically essential enzymes (Teal and Wymer, 1991; Yang et al., 2016). Out of the 4,000 known microbial enzymes, only about 200 are currently used commercially (X. Liu and Kokare, 2017). In comparison to industrially important enzymes, medically relevant enzymes are required in much smaller quantities. They should, however, be extremely pure and specific. Because these enzymes have a low Michaelis-Menten constant (Km) andmaximum velocity (Vmax), they have highest productivity even at minimal enzyme and substrate concentrations. The sources of such enzymes should be identified with great attention and consideration in order to avoid any unwanted con­ tamination by unsuitable substances and to allow for easy purification. The majority of medically significant enzymes are sold as lyophilized pure formulations with biocompatible buffering salts and mannitol solvent. These enzymes are expensive, although not as expensive as or similar to medicinal drugs or treatments (Gurung et al., 2013). As a result, enzymes have a wide range of applications in a variety of industries, including food, textiles, medicine, dairy, and others. We may now add or modify the competence of the genes that are crucial for us to make these novel enzymes, thanks to advances in modern biotechnology and protein engineering. This chapter’s goal is to highlight the current role of microbial enzymes in biomedical applications.

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55

4.2 APPLICATIONS OF MICROBIAL ENZYMES IN BIOMEDICAL SECTORS 4.2.1 ANALYTICAL/DIAGNOSTIC ENZYMES The presence of particular enzymes in the blood has been used to diagnose cellular damage, which leads to the release of intracellular components into the bloodstream. As a result, when a physician informs a patient that neurological enzymes assay is required, the goal is to determine whether or not brain damage or other diseases has occurred. In human and veterinary medicine, many additional enzymes are used in the clinical diagnosis of various disorders. Because of their high specificity and efficiency, enzymes have proven to be excellent analytical reagents. They can be used to determine the concentration of a substrate in a quantitative manner. The quantity of tissue damage is determined by the level of diagnostic enzymes. One of the most useful enzymes for bioanalytical applications is alcohol dehydrogenase. There are other additional enzymes that can be utilised to diagnose diseases (Table 4.1). Enzymes are also used in clinical diagnostics for the detection and quantification of diabetes and other health disorders, such as glucose oxidase (EC 1.1.3.4) extracted from Aspergillus niger for glucose (Hossain and Park, 2016), urease (EC 3.5.1.5) and glutamate dehydrogenase (EC 1.4.1.2) for urea, lipase (from Bacillus sp., Aspergillus sp., Penicillium sp.), carboxyl esterase, and glycerol kinase for triglycerides (Basso and Serban, 2020; Stevens et al., 2018) and urate oxidase (EC 1.7.3.3) for uric acid; creatinase (EC 3.5.3.3) and sarcosine oxidases (EC 1.5.3.1) for creatinine (Dordick, 2013; Le Roes-Hill and Prins, 2016). Cholesterol oxidase (EC 1.1.3.6) extracted from Alcaligenessp. has also been discovered to be effective in biotechnological applications such as cholesterol detection and conversion (Aggarwal et al., 2016; Lata, Dhull, and Hooda, 2016; Shukla, Turner, and Tiwari, 2015). Putrescine oxidase (EC 1.4.3.10) is an enzyme that detects biogenic amines like putrescine, a food spoilage marker (Le Roes-Hill and Prins, 2016). Enzymes are essential in nucleic acid manipulation for genetic engineering research and development, such as restriction endonucleases for site-specific frag­ mentation of DNA for molecular cloning (Newman, Strzelecka, and Dorner, 1995) and DNA polymerases for polymerase chain reaction (PCR) DNA amplification. The following enzymes aid in the quick identification of a variety of diseases (Table 4.1) and few imprortant diagnostics microbial enzymes are depicted in Figure 4.1 with its sources of microbial origin.

4.2.2 BIOSENSORS The 21st century has provided various scientific innovations and advantages. Each and every appliance or machinery we use, from mobile phones to household appliances like televisions, refrigerators, air conditioners, microwave ovens, and computers, are embedded with sensors (Chambers et al., 2008; Goode, Rushworth, and Millner, 2015). The concept of sensors is not new; from time immemorial, nature has deeply embedded the sensing ability, starting with the amoeba, which moves according to the environment it is suited for. Similarly, certain species of algae have the capability to sense the nearby presence of toxins. The olfactory sensing capability of predators is quite important for their survival. Therefore, we can define a sensor as a device that helps in identifying and noticing any physio-chemical changes such as pressure, temperature, moisture, movement, force, and an electrical quantity like current, and thus, transforms those to signals that can be detected and analyzed (Llandro et al., 2010). Similarly, for biological samples, the term “biosensors” can be suitably adopted, which can be defined accordingly to the International Union of Pure and Applied Chemistry (IUPAC), “a device that uses specific biochemical reactions mediated by isolated enzymes, immune systems, tissues, organelles, or whole cells to detect chemical compounds usually by electrical, thermal, or optical signals” (Ziegler and Göpel, 1998). These biosensors have evolved as an excellent tool in diagnostic, medical, and healthcare along with food-quality measurement and national security and defense establishments (Karim and Fakhruddin, 2012).

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Microbial Enzymes and Metabolites for Health and Well-Being

TABLE 4.1 Microbial Enzymes Commonly Employed in Clinical Diagnosis Diagnostic Enzymes

Function/Disease

Microbial Sources

References

Acid phosphatase (ACP) Alanine aminotransferase

Prostate carcinoma, malaria Hepatocellular damage (hepatitis B and C)

Aspergillus niger, A. terreus

Devi et al., 2017; Iqbal, 1998

Bacillus fordii, Pyrococcus furiosus

Huang et al., 2006; Corathers, 2006

Alkaline phosphatase (ALP)

Liver (carcinoma) or bone disease (rickets), chronic kidney disease

Aspergillus sp. Meyerozyma caribbica

Freethi et al., 2016

Alanine transaminase (ALT) Amylase, lipase (LPS)

Hepatitis (liver damage)

Escherichia coli B

Ishiguro et al., 2009

Pancreatitis, Skin disorders

Bacillus sp., Geobacillus sp.

Horseradish peroxidase

Ab or Ag detection (ELISA)

Bacillus sp.

Sivaramakrishnan et al., 2006; Kumar et al., 2000; Singh et al., 1995; Esmaili et al., 2017 Wang et al., 2010

Aspartate aminotransferase (AST)

Myocardial, Hepatic parenchymal and muscle diseases

Pseudomonas striata, Escherichia coli K-12, Bacillus stearothermophilus

Kamma, Nakou, and Persson, 2001

Cholinesterase

Muscular dystrophy, chronic renal disease, pancreatitis, hepatobiliary diseases. Hepatic parenchymal disease, Clostridium difficile infections

Pseudomonas aeruginosa

Mabrouk et al., 2011

Pyrococcus furiosus, Thermococcus sp.

Cheng et al., 2015

Glutamate dehydrogenase (GLDH) Gamma glutamyltransferase

Cardiovascular mortality

K. pneumonia, E. faecalis

Junna Wang et al., 2017

Lactate dehydrogenase (LDH),

Heart attack or liver diseaseBreast cancer, Necrosis

Leuconostoc mesenteroides, Lactobacillus sp.

Rayamajhi, Zhang, and Miao, 2013; Brown et al., 2012

Gelatinase B

Gastric cancer, Rheumatoid arthritis For L-arginine levels in plasma and urine/ Antitumor

Bacillus sp.

Dragutinović et al., 2006

Bacillus subtilis & E. coli

Kaur and Sekhon, 2012

Cholesterol esterase

For serum cholesterol levels

Actinomycetes, Pseudomonas fluorescens, Acinetobacter sp.

Takayuki and Osamu, 1976

Uricase

For uric acid, gout

A. flavus

Acid phosphatase

Malaria

Morganella morganii, Aspergillus sp.

Sherman, Saifer, and PerezRuiz, 2008; Terkeltaub, 2009 Kirschenbaum et al., 2011

Cathepsin D

Rheumatoid arthritis, breast cancer

Candida albicans

Arginase

Vasudev and Banks, 2011; Rossman, Maida, and Douglas, 1990

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FIGURE 4.1 Few important microbial diagnostic enzymes and their sources.

The first biosensors were developed in the 1960s by Clark and Lyons for detecting glucose by using an enzyme-linked electrode; therefore, they are known as the fathers of biosensors. From there, the biosensors have been modified gradually. The first-generation biosensors also known as mediator-less amperometric biosensors, measured the content of the analytes and products of the bioreceptor reactions, which diffuse to the surface of the transducer and produce an electric response (Purohit et al., 2020). In 1975, the second-generation biosensors were conceptualized by Lubbers and Opitz, who used auxiliary enzymes and co-reactants into the biosensors for enhancing analytical efficacy. Thus, these biosensors were termed as mediator amperometric biosensors (Turner, 1989). The third-generation biosensors were able to detect the biomolecule without the requirement of intermediate stages. In 1987, researchers from Cambridge, USA, measured the blood glucose level directly using a pen-size detector. Thus paving the way for low cost and high feasibility of repeated measurement of the sample directly by the patient without the intervention of any mediators (Luong, Male, and Glennon, 2008). Currently, various biosensors such as proteinbased biosensors, aptamer-based Biosensors, cell-based biosensors, antibody-based biosensors, enzyme-based biosensors, and nanoparticle-based biosensors are available based on their bior­ ecognition principle (Kirsch et al., 2013; Sin et al., 2014). Enzyme-based biosensors play an crucial role in the health application in early detecting and diagnosing diseases (Srivastava and Joshi, 2012). Several applications of biosensors have been established, like pregnancy test kits where hCG protein is detected in the urine. Commercially, glucose-based biosensors have captured about 80% of the global market (Table 4.2). Bloodglucose-level monitoring in diabetes patients using commercial biosensors has been established namely Accu-Chek, One Touch, Glucocard, Freestyle, etc. (Heller and Feldman, 2008). Advanced techniques, such as enzyme-linked immunosorbent assay, fluorimetric, and immune-affinity column assay, have been developed for cardiovascular disease detection. Biosensors are also being employed based on bio-molecule recognition with respective selectivity for a particular biomarker of interest (Marchi et al., 2010). For example, in the detection of cancer biomarkers, DNA, peroxides, etc., different biosensors are available for the early diagnosis of cancer based on detection of tumor-associated antigen and its corresponding antibodies. A biochip is available for quick and accurate detection of multiple cancer markers. In detection of bacteria in blood platelets, a real-time biosensor is used for the detection of bacteria (Escherichia coli, Bacillus cereus,

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Microbial Enzymes and Metabolites for Health and Well-Being

TABLE 4.2 Examples of Oxidase and Dehydrogenase Enzymes Used in Amperometric Biosensors Enzymes

Substrate

Sources

References

Glucose oxidase (E.C. 1.1.3.4) Glutamate oxidase (EC 1.4.3.11)

β-D-Glucose

Aspergillus niger

Hossain and Park, 2016

L-Glutamate

Streptomyces sp.

Soldatkin et al., 2015

Alcohol oxidase (EC 1.1.3.13)

Ethanol

Pichia pastoris Hansenula polymorpha

Secchi et al., 2013

Lactate oxidase (EC 1.1.3.2)

L-Lactate

Pediococcus sp. Aerococcus viridians

Ascorbate oxidase (EC 1.10.3.3)

L-Ascorbic acid

Cucurbita sp.

Giménez-Gómez et al., 2016; Hernández-Ibáñez et al., 2016; Andrus et al., 2015 Wen et al., 2012; M. Liu et al., 2011

Cholesterol oxidase (EC 1.1.3.6)

Cholesterol

Streptomyces sp porcine pancreas

Lata, Dhull, and Hooda, 2016; Aggarwal et al., 2016; Shukla, Turner, and Tiwari, 2015

Choline Oxidase Choline (EC 1.1.3.17)

Acetylcholine

Alcaligenessp

Rahman and Asiria, 2015; Tunç, Koyuncu, and Arslan, 2015

Laccase (EC 1.1.3.4)

Polyphenols

Tyrosinase (EC 1.14.18.1)

Monophenols Dihydroxyphen­ ols Bisphenol A

Trametespubescens, Paraconiothyrium variable Trametes versicolor Mushroom

Gonzalez-Rivera and Osma, 2015; Sadeghi, Fooladi, and Malekaneh, 2015; Casero et al., 2013 Campanhã Vicentini et al., 2016; Kochana et al., 2015; Njagi et al., 2010

Alcohol dehydrogenase (E.C. 1.1.1.1)

Ethanol

Saccharomyces cerevisiae

Gómez-Anquela et al., 2015; Wu and Zaman, 2015; Li, Lu, and Deng, 2013

Glucose dehydrogenase (EC 1.1.1.47)

Glucose

Pseudomonas sp. Escherichia coli

Guo et al., 2015; Y. Lin et al., 2014; Liang et al., 2013

Pseudomonas aeruginosa, etc.) in platelet concentrates. Real-time in vivo detection of dopamine with the immobilization of tyrosine onto the implantable microelectrode surface is by an enzymebased carbon fiber micro biosensor (Njagi et al., 2010). Enzyme immobilization is a most widely used and promising technology in the field of medical diagnostics, pharmaceutical, and many more to come. There is a huge demand for specific, rapid, highly sensitive, low-cost, handy, and home-use compatibility sensors having a reliable, efficient, and systematic approach. Many methods are being employed for enzyme immobilization in various large-scale processes. Biosensors are also developed for detection of different metabolites, nucleic acid, and proteins using affinity-based biosensors. For example, based on electrochemical tech­ nology, glucose meters are now highly used. New technology of lab-on-chip, microfluidic devices, and nano-sensors offer a new generation biosensors. Today, this process is going through matu­ ration, and with many years to come, through modifications and by overcoming challenges. Moreover, to make the microbial enzyme-based biosensors more efficient, the nanotechnology has been incorporated to make faster and more efficient results within few minutes of sampling. The details of different sensing activity of microbial enzymes, along with nanotechnology is depicted in Figure 4.2.

Microbial Enzymes in Biomedical Applications

59

FIGURE 4.2 Schematic diagram represents (a) micro/nanotechnolgies enhancing the performance of microbial biosensors, (b) limitations of conventional microbial biosensors, and (c) general features of bio­ sensors ( Lim et al., 2015).

4.2.3 THERAPEUTICS ENZYMES Enzymes derived from microorganisms are regarded as the most potent therapeutic agents of the 21st century. They are advantageous due to their cost efficiency, predictability with controllable con­ centration, and reliable raw material sources. Microbial enzymes offer a wide range of applications in medication and pharmacology, and their significance in this sector has just recently been recognized. Various companies, including enzyme producers, are currently conducting extensive research in the field of enzymology to discover novel and better ways to use enzymes for industrial as well as medical applications (Table 4.3). Therapeutic enzymes have a large and expanding market. Therapeutic enzymes are currently accessible in various forms like tablets, powders, capsules, and as food additives. Studies are being carried out to make use of the diverse microbial resources available, which include both terrestrial and marine microbial flora. Antimicrobials, anti-inflammatories, an­ ticoagulants, fibrinolytics, oncolytics, thrombolytics, mucolytics, and digestive reliefs are all ex­ amples of medically essential enzymes produced by microbes (Gurung et al., 2013). Microbial enzymes can also be used to treat diseases that have resurfaced after developing antibody resistance. Combinations of enzymes and medications can also produce synergistic ef­ fects, which can be used to treat a variety of diseases by reducing their negative effects. Therapeutic enzymes produced by microorganisms are used to remove cytotoxic compounds from the bloodstream, to treat life-threatening illnesses as oncolytics, thrombolytics, and anticoagulants,

60

Microbial Enzymes and Metabolites for Health and Well-Being

TABLE 4.3 Microbial Enzymes Used in Therapeutics Enzymes

Microorganisms

Biomedical Applications

References

L-Asparaginase, L-glutaminase,

Escherichia coli, Bacillus cereus, Alcaligenes faecalis, Aeromonas veroni

Antitumor, Acute Lymphoblastic Leukaemia

Dinndorf et al., 2007 P. Singh and Banik, 2013; Dhankhar et al., 2020; Aravinth Vijay Jesuraj et al., 2017

β-galactosidase

Beauveria bassiana, Acinetobacter Aspergillus sp., Kluyveromyces

Lactose intolerance

Saqib et al., 2017; Kochana et al., 2015

L- tyrosinase

Pseudomonas acidovorans

Parkinson’s disease, myocardium neurogenic injury

Zaidi et al., 2014

Streptokinase, urokinase

Streptococci sp., Bacillus subtilis

Anticoagulants, cardiovascular disorders

Penicillin oxidase, rifamycin B oxidase

Penicillium sp.

Antibiotic synthesis

Aneja et al., 2013; Sikri and Bardia, 2007; Zaitsev et al., 2010 Erickson and Bennett, 1965

Superoxide dismutases,

Lactobacillus plantarum, Corynebacterium glutamicum

Antioxidants, myocardial ischemia, Peyronie’s disease, colitis, multiple sclerosis, and breast cancer

Emerit, Samuel, and Pavio, 2006; C. Riedl et al., 2005; Ishihara et al., 2009; Griess et al., 2017

Glutathione peroxidases, Catalase

Corynebacterium glutamicum Penicillium notatum, Aspergillus niger Clostridium perfringens, Clostridium histolyticum

Anti-iflammatory, antioxidant

Sabir et al., 2007

Skin ulcers, Dupuytren’s disease

( Klasen, 2000; HartigAndreasen, Schroll, and Lange, 2019) Gonzalez-Rivera and Osma, 2015; Casero et al., 2013

Collagenase

Laccase, Rhodanese

Pseudomonas aeruginosa, Polyporus versicolor, Microbial consortium

Detoxification

β-Lactamase

Klebsiella pneumonia, Citrobacter freundii, Serratia marcescens

Antibiotic resistance

Gupta et al., 2012

Ribonuclease

Saccharomyces cerevisiae and Bacteriophages Serratia marcescens

Antiviral

Scherbik et al., 2006; Lin et al., 2013 Gupte and Luthra, 2017; Jadhav et al., 2020

Uricase

Aspergillus flavus, Bacillus subtilis, Pseudomonas putida

Gout and Hyperuricemia

Nelapati and PonnanEttiyappan, 2019

α-Amylase, Lipase

Bacillus spp., Candida lipolytica, A. oryzae

Digestive disorders

Sivaramakrishnan et al., 2006; J. Kumar et al., 2000; D. Singh, Dahiya, and Nigam, 1995; Esmaili et al., 2017

Serrapeptase

Anti‐Inflammatory

Microbial Enzymes in Biomedical Applications

61

FIGURE 4.3 Therapeutic role of various therapeutic microbial enzymes.

and to compensate for metabolic deficits such as cystic fibrosis (Kaur and Sekhon, 2012). Pro-drug activator enzymes and antineoplastic enzymes are used to treat cancer as well as other disorders, such as Fabry, cystic fibrosis (CF), Gaucher, Mucopolysaccharidosis type I (MPS I), Pompe, Mucopolysaccharidosis type VI (MPS VI) also known as Maroteaux-Lamy syndrome, and severe combined immunodeficiency (SCID) and few infectious diseases caused by varieties of micro­ organisms like bacteria, fungi or protozoa. Moreover, in lactose intolerant people, some of the microbes help in digestion by providing some important enzymes like amylases, proteases, and lipases, etc. Therapeutic enzymes, i.e. both the metabolic and digestive enzymes can be used singly or in amalgamation with other treatments to safely treat a wide range of disorders. The following sections go over various microbial therapeutic enzymes and few important therapeutics importance of the microbial enzymes are illustrated in Figure 4.3. 4.2.3.1 Asparginase The enzyme asparginase is used to treat severe lymphocytic leukaemia. It has a little effect on normal cells that can produce L-asparagine for their individual needs, but it does induce a decrease in the free extracellular concentration leading tumour cells to starve to death. Asperginase can be applied intravenously and is effective only when the asparagine level in the blood stream are severely low (Gurung et al., 2013). Clinically available asparaginase is generated from two sources: Escherichia coli and Erwinia chrysanthemi, according to investigations conducted in the 1960s employing bacteria to seek alternate sources of asparaginase. E. coli enzymes are native and derivatized by adding monomethoxypolyethylene glycol. This enzyme showed a good potentiality against acute lymphoblastic leukemia (ALL) (Dinndorf et al., 2007). 4.2.3.2 Chitinase The cell wall of many pathogenic microbes contain chitin as a component. It was also reported as a good antibacterial target (Fusetti et al., 2002). A lytic enzyme generated from bacteriophage was used to attack the cell walls of Bacillus anthracis, Clostridium perfringens, and Streptococcus

62

Microbial Enzymes and Metabolites for Health and Well-Being

pneumonia (Zimmer et al., 2002). It was observed that the enzyme produced from bacteriophage showed good result against new drug-resistant bacterial strains and can be utilised to treat a variety of ailments. They have anti-inflammatory properties. A large number of these bacterial proteolytic enzymes can also be used to remove shedding skins from scalding (Gurung et al., 2013). 4.2.3.3 Collagenase It aids in the healing of burns and ulcers on the skin. It aids in the repair procedure by breaking up and removing dead skin and tissue. As a result, antibiotics work better, and the natural healing process of an individual’s body is accelerated (Ostlie et al., 2012). It is a one-of-a-kind protease capable of hydrolyzing native collagen in a specific manner. Bacterial collagenases are metallo­ proteases that may breakdown extracellular matrix, making them essential virulence agents. It’s administered topically to burns or ulcers on the skin to speed up healing and get rid of dead tissue (Alipour et al., 2016). 4.2.3.4 Lipase It is utilized to help with digestion. Because they have the ability to activate tumour necrosis factor, they are also utilised in the treatment of malignant tumours. In the past, lipases were used to treat dyspepsia, gastrointestinal problems, cutaneous symptoms of digestive allergies, and a variety of other infections. Alcresta Therapeutics, in collaboration with Chiral Vision, developed a single-use cartridge containing immobilised lipases from Chromobacteriumviscosum, Pseudomonas fluor­ escens, Burkholderiacepacia, and Rhizopus oryzae, capable of hydrolyzing up to 90% of the fats in the enteral preparation. This product was marketed as Relisorb® and is still widely used today (Basso and Serban, 2020; Stevens et al., 2018). 4.2.3.5 Nattokinase It’s a serine proteinase that Bacillus subtilis produces. Nattokinase is fibrinolytic, considered as third-generation enzyme isolated and developed from a fermented soybean food product of Japan, Bacillus natto (Metkar et al., 2017). It can lower blood coagulation and cholesterol levels. Huge volumes of enzymes may be manufactured quickly and effectively since they are produced by microbes. 4.2.3.6 Serratiopeptidase Serratiopeptidase alone or in combination is considered as a powerful anti-inflammatory agent, and it has potentiality against various diseases and disorders like atherosclerosis, amyloidosis, bron­ chitis, sinusitis, rheumatoid arthritis, carpal tunnel syndrome, etc. (Bhagat, Agarwal, and Roy, 2013; Gupte and Luthra, 2017; Jadhav et al., 2020; Malshe, 1998; Metkar et al., 2017; Murugesan, Sreekumar, and Sabapathy, 2012; Sannino et al., 2013; Tiwari, 2017). This enzyme also has application for the treatment of periodontal inflammatory disorders caused due to dental implants (Tamimi et al., 2021). Serratiopeptidase is one of the most important proteolytic enzymes derived from nonpathogenic enterobacteria Serratia marcescens sp. E15. found in silkworms. Bacteria like E. coli have recently been genetically modified to overexpress recombinant serratiopeptidase (Rouhani et al., 2020). Serratiopeptidase is blooming all around the world as a supplement in the diet or as medicinal agent. However, the regulatory status of the enzyme varies by country to country (Jadhav et al., 2020).

4.2.4 THROMBOLYTIC DRUGS These drugs lyse thrombi or clots and restore normal blood flow to blocked blood arteries. They are curative rather than preventive. The activation of the natural fibrinolytic system, and hence, the activation of plasminogen is the mechanism by which they work. Streptokinase, urokinase, and other plasminogen activators are examples. The enzymatic breakdown of fibrin clots is known as

Microbial Enzymes in Biomedical Applications

63

thrombolysis or fibrinolysis. Proteases are microbial fibrinolytic enzymes that have been shown to break blood clots effectively without any side effects maintaining regular blood stream in the arteries. Proteases are simple to manufacture on a big scale and at a low cost. These fibrinolytic enzymes are produced by most common genera like Bacillus, Staphylococcus, Streptomyces, Serratia, and Pseudomonas etc. (Mukherjee et al., 2012; Peng, Yang, and Zhang, 2005; Taneja et al., 2017; Weng et al., 2017). 4.2.4.1 Streptokinase Streptokinase is regarded as a first-generation activator plasminogen produced by a variety of hemolytic Streptococci bacterial strains. Streptokinase is found as a lyophilized powder, which can be administered intravenously or intracoronally for treatment. As compared to other plasminogen activators, it is inexpensive, due to which utilization of streptokinase is highest in developing nations (Adivitiya and Khasa, 2017). 4.2.4.2 Staphylokinase Staphylokinase is a possible thrombolytic drug and a third-generation fibrinolytic enzyme (Nedaeinia et al., 2020; Vakili et al., 2018). Staphylococcal fibrinolysin, also known as Müller’s factor, is an extracellular protein produced by Staphylococcus aureus. It has thrombolytic action similar to Streptokinase. 4.2.4.3 Superoxide Dismutase SOD is a metalloenzyme that is found all over the body. SODs prevent disease progression by destructing the inflammatory phenomenon by cleaning harmful free radicals. Drugs for critical coronary artery destruction, penile fibrosis, breast cancer, colitis, and multiple sclerosis contain superoxide dismutase (Emerit et al., 2006; Griess et al., 2017; Ishihara et al., 2009; Riedl et al., 2005). These enzymes are are also used in cosmetic compositions for reducing damage of the skin caused by free radicals. Microbes are proving to be a cost-effective source for the manufacture of various SODs. For e.g. Corynebacterium glutamicum (El Shafey et al., 2008).

4.3 NUTRACEUTICAL ENZYMES Nutrition and health are the two parameters for healthy lifestyle. The importance of naturally derived phyto-constituents and dietary supplements from plant and animal products have been known from time immemorial (Galanakis, 2021). Hippocrates, who is considered the father of modern medicine, established the importance of food and its effect on health way back in 460 BC. India, the land of Ayurveda, is the storehouse of indigenous medicinal system derived from nature (J. Singh and Sinha, 2012; Tapal and Tiku, 2019). Nutrition has a direct relationship with various ailments, proper diet, and intake of food has been proved to have beneficial role in optimizing the progression of various diseases. The word “nutraceutical” is a combination of two words “nutri­ tion” and “pharmaceutical.” Hence, it can be said that the food supplement has a vital role in regulating the normal metabolism of various cells and thereby thwarts the development of any disease in the body (Kamran and Reddy, 2018; Weng et al., 2021). Nutraceuticals are basically a concoction of locally available herbs and foods that can stimulate and nourish our body to maintain active rate of metabolism. These nutraceuticals can provide an alternative to the commercially available antibiotics in our daily life, as regular uptake of anti­ biotics may develop short-term and long-term tolerance in our body, thereby increasing the emergence of multiple drug-resistant microorganisms (Kuddus, 2019). Regular use of nutraceu­ ticals in the form of herbs, spices, and plant-derived natural phytochemicals has exhibited the capacity to normalize the body metabolism along with immune-boosting and defending for pro­ phylaxis of various disease conditions (Chanda et al., 2019). Nutraceuticals can be broadly clas­ sified as chemical constituents (basically the primary metabolites such as amino acids, vitamins,

64

Microbial Enzymes and Metabolites for Health and Well-Being

fatty acids, plant- and animal-derived components); herbals (basically, the natural products, such as flavonoids, terpenoids, tannins, curcumin, carotenoids, and phenolic acids present as secondary metabolites in various shurbs and herbs used in our daily life); probiotics microorganisms (basi­ cally they belong to a class of microorganism that boost our metabolism and help in the absorption and assimilation of food particles in the intestine thereby replacing the toxic micro flora of the intestine with good intestine friendly bacterial strains) and nutraceuticals enzymes (these enzymes act as biocatalyst, which helps in fastening the metabolic rate and helps in various conditions such as constipation, diarrhea, and ulcerative colitis) (Galanakis, 2021; Jagtap and Manohar, 2021). Various nutraceutical enzymes, such as Xylanase (Trichoderma sp.), Neutral Protease (Bacillus sp.), Lipase (Rhizophussp.), Lactase (Aspergillus sp.), Invertase (Saccharomyces certevisiae); Hemicellulase and Cellulase (Trichoderma sp.), Glucoamylase (Aspergillus sp.) Beta glucanase, Amylase (Aspergillus sp.), Alpha Amylase (Bacillus sp.), Alpha Galactosidase (Aspergillus sp.), and Acid Protease NE (Aspergillus sp.) are available commercially for direct intake by patients (Charoensiddhi et al., 2017; Fatima and Khare, 2018; Jian Wang et al., 2016). These nutraceuticals enzymes are basically derived from microorganism, which are supplemented to eliminate the symptoms for individuals who are suffering from various medical disorders such as hypertension, diabetes, gastrointestinal tract problems, malnutrition, and obesity (Alagawany et al., 2021; Kumar and Kumar, 2015).

4.4 MICROBIAL ENZYMES IN PHARMACEUTICAL INDUSTRY APPLICATIONS The enzyme industry is one of the world’s most important sectors, and there is a large market for enzymes overall. The pharmaceutical business is becoming more well-known as a major buyer of commercial enzymes. Enzymes are in high demand as therapeutic treatments for a variety of disorders. Accelerated and comprehensive research into the immense microbial resources—both terrestrial and marine—as a source of new therapeutic enzymes is critical. Microbial enzymes have the potential to treat a variety of serious diseases that have become resistant to antibiotics. In the pharmaceutical and diagnostic sectors, enzymes play a variety of important and critical tasks. These are widely utilised as therapeutic medications for enzymatic deficiency and digestive diseases, as well as in diagnostic techniques like ELISA and diabetes testing kits (Mane and Tale, 2015). Enzyme uses in medicine are as diverse as they are in industry, and they are continually expanding. Proteolytic enzymes, which remove dead skin and burns, and fibrinolytic enzymes, which break up clots, are currently the most common medicinal applications of microbial enzymes. Nattokinase (EC 3.4.21.62) is a powerful fibrinolytic enzyme that could be used to treat thrombosis (Cho et al., 2010; Sumi et al., 1987). Acid protease, dextranase (EC 2.4.1.2), and rhodanase (EC 2.8.1.1) are enzymes that can be used to treat gastrointestinal dyspepsia, tooth decay, and cyanide poisoning (Okafor, 2007). Lipases (EC 3.1.1.3) are the most common enzymes employed in organic synthesis, and they are utilised to make optically active alcohols, acids, esters, and lactones (Cambou and Klibanov, 1984; Saxena et al., 1999). The synthesis of (2R, 3S)-3-(4methoxyphenyl) methyl glycidate (an intermediary for diltiazem) and 3, 4-dihydroxylphenyl ala­ nine (DOPA, for Parkinson’s disease treatment) is aided by microbial lipases and polyphenol oxidases (EC 1.10.3.2). Tyrosinase (EC 1.14.18.1) is a key oxidase enzyme involved in melano­ genesis and the synthesis of L-Dihydroxyphenylalanine (L-Dihydroxyphenylalanine) (L-DOPA). L-DOPA is a precursor for the creation of dopamine, a powerful medicine for treating Parkinson’s disease and preventing myocardial neurogenic injury (Babar et al., 2015; Ikram-ul-Haq, Ali, and Qadeer, 2002; Zaidi et al., 2014). Chitosanase (EC 3.2.1.132) catalyses the hydrolysis of chitosan to biologically active chitosan oligosaccharides (COS), which are used as antimicrobials, anti­ oxidants, blood cholesterol and blood pressure lowering, arthritis control, infection protection, and antitumor properties (Kim and Rajapakse, 2005; Ming et al., 2006; Thadathil and Velappan, 2014; Zhang, Sang, and Zhang, 2012). Table 4.3 shows how microbial enzymes can be used to treat a variety of health issues (Kaur and Sekhon, 2012; Mane and Tale, 2015; Vellard, 2003). Enzymes

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are widely employed for scientific and analytical applications, such as estimating substrate con­ centrations and determining the catalytic activity of enzymes present in biological materials. In diverse immunoassays, that are used to determine a range of proteins and hormones, advances in enzyme technology have removed or minimised the usage of dangerous radioactive elements (Palmer and Bonner, 2007).

4.5 PROBLEMS AND SAFETY CONCERNS Before introducing a microbial enzyme into the market as food and feed, it is of utmost importance to carry out a full safety study. Global regulators, along with European regulators, also require review on toxicological study of the particular enzyme from microbial origin before registration. On the basis of intended applications as food, feed, or any other technological uses, different countries have different control regulations. For example, according to European Commission and European Food Safety Authority, a sequence of toxicological studies is required in-vitro and in-vivo for inclusion of food enzymes in the community positive list. Likewise, in other countries also like China and Brazil, these investigations are also necessary for the same. Again, the enzyme used animal feed also needs an elaborate studies on toxicogenic and dermal sensitization studies in European Union (Regulation (EC) No 105 429/2008). Moreover various alternative assays in animals in vitro have been estab­ lished, authenticated, and accepted by various regulatory bodies for conducting such examinations (Jain et al., 2018). According to the weight of evidences, enzymes do not produce acute toxicity, genotoxicity, repeated dose oral toxicity, or cutaneous sensitization. Some of the proteases, on the other hand, have been demonstrated to induce skin and ocular irritation, and there is a welldocumented danger of enzyme sensitization at work. Significantly, occupational safety can be achieved by establishing suitable industrial hygiene standards and observing the air quality, as well as health monitoring and imposing strict limits on enzyme air exposure (60 ng active enzyme protein/m3). Demonstrating the safety of the production strain is the most crucial aspect of determining the safety of an enzyme preparation, according to sources (Sewalt et al., 2016). The two basic approaches of confirming the host strain is safe are: first, the host strain should be ensured for non-pathogenicity and non-toxigenicity against non-genetically produced strains, and second, to establish an Squalene Synthase-like (SSL) gene for expression of desired enzyme sequences using genetic-engineering techniques (Pariza and Johnson, 2001). An SSL can be created by regularly assessing members of the lineage using the decision tree, which comprises considerable antibacterial activity and harmful microbial metabolites along with oral toxicity assessment for repeated dosing for determining the marginal safety for extensive use. After the establishment of SSL, any strains in the lineage that are upstream from the production strain are allowed by the decision tree. Formerly, these strains were evaluated toxicologically, which will support as safe production host for other enzymes, and the new enzyme produced in the cascade will be considered safe (Pariza and Johnson, 2001). A number of regulatory agencies have been effectively exposed to the concept by the enzyme industry. (Sewalt et al., 2016).

4.6 FUTURE PROSPECTS OF MICROBIAL ENZYMES IN BIOMEDICINE Microorganism are the most diverse organisms that inhabitate the planet earth owing to their diversity in geographical, genomic, and physiological characteristics. Hence, to sustain life, they have evolved to employ greater diversity in producing various types of enzymes. These microbial enzymes have now opened great opportunities in the field of medical diagnostic and healthcare sector (Elegbede and Lateef, 2021; Gurung et al., 2013). The microbial enzymes are immensely used as alternative therapeutics against various disease conditions. Owing to their economical, cost effective, high-yield capacity and greater convenience for isolation, the microbial enzymes are produced for use in various food, beverage, healthcare, and biofuel industries (Singh et al., 2016). Enzymes being proteinacious in nature are highly labile to heat and other environmental conditions, their targeted delivery to their

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specific substrates provides a challenge for their large-scale productions. The emergence of PEGlyation and nano-encapsulation and conjugation with various proteins and nanoparticles have open new avenues for their standardized therapeutic applications (Nigam, 2013; Thapa et al., 2019). With advancement in science and innovation microbial enzymes can be explored for their application as anticancer agents, antioxidants, antimicrobial agents, anti-inflammatory agents, and can aid in boosting immune response of individuals.

4.7 CONCLUSIONS In the 21st century, the potential for industrial usage of microbial enzymes has risen considerably and is continuing to expand, as enzymes hold immense promises for many sectors in meeting the demands of a rapidly growing population while also coping with natural resource exhaustion. Enzymes have been shown to have great promise in a wide range of industries, including medi­ cines, food-feed and beverages, detergents, processing of leather, paper & pulp, management of waste, and sustainable environmental development. Furthermore, microbial enzymes are quite significant and have a lot of medicinal potential. Bacterial production of therapeutic enzymes is both cost-effective and environmentally friendly. The need of the hour is to test new enzymes as well as improve and upgrade existing ones. The isolation, regulation, overproduction, and appli­ cations of these microbial enzyme biopharmaceuticals should be researched to their maximum potential to reap the benefits of these microbial enzyme biopharmaceuticals over chemical ther­ apeutic agents and in the treatment of many human diseases. For the creation of medicines, the ability to transfer enzymes into the body is critical. Catalytically active enzymes must be in their original states to bind to their prospective substrates. Because of their fragile structure, their development as therapies is impeded. PEGylation, nanocarriers and encapsulation of nanoparticles, and creation of mutant varieties by recombinant DNA technologies have all emerged as com­ plementary ways to overcoming some of the hurdles connected with enzyme structure and func­ tion. Research is being in progress to find the best nanocarrier for providing these enzymatic medications. Recent biotechnology breakthroughs have ushered in a new era of biopharmaceutical research, in which biomolecules like enzymes can be linked to a variety of inorganic and organic materials to satisfy specific therapeutic targeting requirements.

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Microbial Lipases Production and Application T P Sari Department of Food Technology, National Institute of Food Technology Entrepreneurship and Management, Kundli, Sonipat, Haryana, India

Vivek Kumar Gaur Centre for Energy and Environmental Sustainability, Lucknow, Uttar Pradesh, India School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Republic of Korea

Ayon Tarafdar Livestock Production and Management Section, ICAR – Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India

Ranjana Sirohi Department of Food Technology, School of Health Sciences and Technology, University of Petroleum and Energy Studies, Dehradun, Uttarakhand, India

Raveendran Sindhu Department of Food Technology, TKM Institute of Technology, Kollam, Kerala, India

Amit Kumar Rai Food and Nutritional Biotechnology Division, National Agri-Food Biotechnology Institute, Mohali, Punjab, India

CONTENTS 5.1 5.2 5.3 5.4

5.5 5.6

Introduction.............................................................................................................................76 5.1.1 Historical Overview ...................................................................................................76 Chemical Characterization of Lipases ...................................................................................77 Sources and Classification of Lipase..................................................................................... 78 Microbial Lipases ................................................................................................................... 79 5.4.1 Bacterial Lipases ........................................................................................................81 5.4.2 Fungal Lipases............................................................................................................81 5.4.3 Yeast Lipases..............................................................................................................82 Production and Purification of Lipases .................................................................................82 5.5.1 Screening for Lipolytic Activity................................................................................83 Applications............................................................................................................................83 5.6.1 Application in Food and Nutraceutical Industry.......................................................83

DOI: 10.1201/9781003369295-6

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5.6.1.1 Food Products ..............................................................................................83 5.6.1.2 Pharmaceuticals and Nutraceuticals............................................................83 5.6.2 Application of Lipase in Textile Industry ................................................................. 84 5.6.3 Cosmetics.................................................................................................................... 85 5.6.4 Biodiesel Production ..................................................................................................85 5.7 Conclusion .............................................................................................................................. 86 References........................................................................................................................................ 86

5.1 INTRODUCTION Enzymes are proteins that act as natural catalysts and possess the ability to catalyze various chemical and biochemical reactions (Wu et al., 2021). Enzymatic biocatalytic reactions are attaining research focus due to their wide industrial applications and green, safe, and sustainable tag (Kumari et al., 2021). Lipolysis embraces the dissociation of ester bonds in triglycerides, leading to the formation of fatty acids and glycerol. Lipases are important industrial enzymes after protease and amylase (Ülker et al., 2011). Lipolytic enzymes include esterases (carboxylesterases; EC. 3.1.1.1) and lipases (triacylglycerol lipases; EC 3.1.1.3); the former acts on triglycerides with short-chain, water-soluble fatty acids; lipases catalyze the hydrolysis of triglycerides into fatty acids and glycerol (Gilham and Lehner, 2005). Lipases are ubiquitous and widely distributed among plants, animals, insects, and microbes. However, lipases from microbes hold the leading position as industrial biocatalysts due to their high throughput, ease of isolation, and regio- and enantio-selectivity properties (He et al., 2010; Sangeetha et al., 2011). Also, the possible genetic manipulation makes the microbial enzymes superior to other sources (Hong Tan et al., 2017; Thakur et al., 2022). The recent advancement in recombinant DNA technology and other bioprocessing resulted in well-characterized preparations of lipases on a large scale, which finds application in many areas, including food, beverage, detergents, paper and pulp, textiles, pharmaceutical and nutraceuticals, cosmetics, biodiesel production, biosensors, etc. as a viable alternative to harmful chemicals (Sarmah et al., 2018). Specificity, improved efficiency, and environment compatibility are the major reasons enzymes are more attractive for industrial applications than chemicals. Immobilization, bioim­ printing techniques, chemical modification, and genetic engineering to improve the catalytic efficacy of enzymes also bolstered the demand on a large scale (Sánchez et al., 2021). The enzyme industry is growing in size, complexity, and diversity. According to an industrial enzyme market analysis report by Grand View Research, the world market size value for enzymes in 2020 was USD 5.93 billion, which is forecasted to USD 9.14 billion in 2027 with a CAGR of 6.4% from 2020 to 2027 (detailed report can be accessed through https://www.grandviewresearch. com/industry-analysis/industrial-enzymes-market). The significant product divisions among en­ zymes include saccharases, proteases, and lipases. The report also headlights the significance of micro-organism segment, which accounts for more than 80% share of the global revenue in 2019.

5.1.1 HISTORICAL OVERVIEW Enzymes have long history of human use. They have been used in different applications like aging, brewing, curdling, etc. involving the use of added preparations such as calf’s rumen, fruit purees, or homemade cultures. Commercial exploitation of enzymes gained importance when Danish chemist Christian Hansen extracted rennet from calves’ stomach, which opened up extensive research on enzymes and their versatile applications in many industries (Hua et al., 2018). Lipase was first identified by French physiologist Claude Bernard from pancreatic juice in 1856 (Bernard, 1856), which we now call pancreatic lipase. The isolated ‘fat ferment’ could dissolve oil droplets in an aqueous medium; however, the fundamental aspects of lipolysis in vertebrate physiology were discovered later by Whitehead (Whitehead, 1909). It took more than a century for isolation and characterization of the main fat-hydrolyzing ferments, which have been called lipases since

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1900 (Zechner et al., 2012). The first bacterial lipases were successfully extracted from Serratia marescens and Pseudomonas aeruginosa in the beginning of 20th century (Hasan et al., 2006). The very first commercial lipase used as detergent additive was isolated from Thermomyces lanugi­ nosus (Al-Ghanayem and Joseph, 2020); however, currently, traditional lipases have been replaced by recombinant lipases for application in commercial use.

5.2 CHEMICAL CHARACTERIZATION OF LIPASES Lipases are monomeric proteins with a molecular weight ranging from 19–60 kDa and exhibit pH and temperature dependent activity. The optimum pH and temperature of activity for most of lipases falls under 30 °C–55 °C and 5 °C–11 °C respectively (Takó et al., 2017). Lipases from Chromobacterium viscosum, A. niger and Rhizophus sp., exhibit optimum activity at acidic pH, whereas lipase from P. nitroaeducens are active at pH 11.0 (Abreu Silveira et al., 2017). Presence of calcium is reported to stimulate the catalytic activity of lipases; whereas Co, Ni2+, Hg2+ and Sn2+suppressed the activity (Melani et al., 2020) In addition to hydrolysis of ester bonds, lipases can also catalyze esterification, transester­ ification, interesterification, acidolysis, alcoholysis, and aminolysis (Borchert et al., 2017) (Table 5.1). In the absence of water, lipases catalyze esterification and interesterification by re­ versing the reaction (Rmili et al., 2019). In hydrolysis, ester bonds are cleaved in the presence of water. Esterification happens in the presence of anhydrous solvents and supercritical fluids, where the absence of water eliminates the chance of hydrolysis and the kinetics of enzyme action can be controlled by varying the reaction medium (Stergiou et al., 2013). The exceptional biocatalytic activity of lipase is contributed by a catalytic triad, which is found in the active site of lipase, given as Glycine-Histidine-Serine- Glutamic/ Aspartic acid- Glycine motif (Vaquero et al., 2016). This catalytic triad, commonly called ‘lid,’ is buried inside the protein and covered by two nearly parallel amphiphathic helices. This key structural component of lipase, known as mobile lid domain and located over the active site, is essential for exposing the active site in the presence of substrate. It maintains equilibria between active and inactive enzyme conformations by opening and closing the active site according to the hydrophobicity of sur­ rounding media (Hou and Shimada, 2009). The lid is mostly closed in aqueous media, whereas partially opened in presence of hydrophilic layer (Khan et al., 2017). Conformational changes to the mobile lid domain leads to interfacial activation, a unique property of lipases facilitating hydrolysis of insoluble esters. Determination of this lid domain is an important criterion in pre­ dicting the substrate preference, thermostability, and interfacial properties of lipases, which are important in protein designing and engineering (Sarmah et al., 2018). Interfacial activation is strongly influenced by lid, as reported by Cheng et al. (2012); wherein the deletion of lid2, which is

TABLE 5.1 Lipase Catalyzed Reactions Reaction

Mechanism

Hydrolysis

R1COOR2 + H2O → R1COOH + R2OH

Esterification Inter esterification

R1COOH + R2OH → R1COOR2 + H2O R1COOR2 + R3COOR4 → R3COOR2 + R1COOR4

Acidolysis

R1COOR2 + R3COOH → R3COOR2 + R1COOH

Alcoholysis Aminolysis

R1COOR2 + R3OH → R1COOR3 + R2OH R1COOR2 + R3NH2 → R1CONHR3 + R2OH

78

Microbial Enzymes and Metabolites for Health and Well-Being

unique to the family I.3 lipase from Pseudomonas sp. MIS38, failed to undergo interfacial acti­ vation, proposing the importance of lid2 (Cheng et al., 2012).

5.3 SOURCES AND CLASSIFICATION OF LIPASE Owing to their industrial worth as biocatalysts and versatile biotechnological tools in many aqueous, non-aqueous, and micro-aqueous environments, diverse research on microbial lipases, their isolation, purification, and characterization from different sources, has attained global focus. Various sources for microbial lipases, both from bacterial, fungal, and yeast, were identified by researchers with high production rates intended for large-scale applications. Lipases can be classified based on the source of origin and specificity (Figure 5.1). Based on origin, lipases can be classified as animal sourced, plant sourced, insect sourced, and microbial sourced. Lipases obtained from animal sources like pig and human pancreas were extensively studied for their ability in catalyzing fat digestion, adsorption, and lipoprotein metabolism. Plants, seeds, and energy reserve tissues are the major sources of lipases (Cavalcanti et al., 2007). In insects, lipases are found majorly in plasma, salivary glands, muscles, and adipose tissues (Santana et al., 2017). However, considering the production time, yield, environmental compatibility, and ease of genetic manipulation, microorganisms are the preferred choice for industrialists in gen­ erating lipases. Lipases from microbial sources can be produced by bacteria, fungi, and yeasts by providing a suitable environment and energy source for growth. Though many microbial strains have been reported by researchers as potent lipase producers, the genera Candida, Rhizopus, Bacillus and Pseudomonas are mostly used for industrial applications (Patel et al., 2019). The most important criteria for the selection of lipase for industrial application is the specificity of action. Lipases can be classified based on specificity of action into different categories; these include substrate specific lipases, regio-selective lipases (1, 3-specific lipases and fatty-acid

FIGURE 5.1 Classification of lipases ( Nagarajan, S. 2012).

Microbial Lipases

79

specific lipases) and stereo-specific lipases (Khan et al., 2017). Substrate specific lipases act on specific substrate in a mixture allowing the synthesis of desired material as in biofuel production (Ribeiro et al., 2011). Nonspecific lipases (produced by Candida rugosa, Staphylococcus aureus, and Pseudomonas sp.) act randomly and produce mono- and diacyl glycerol, as well as free fatty acids and glycerol. 1, 3-specific lipases (produced by Aspergillus niger, Mucor javanicus, Rhizopus delemar, Rhizopus oryzae, Yarrowialipolytica, Rhizopus niveus, and Penicillium roquefortii) act on 1 and 3 position of triglyceride. Fatty acid specific lipases act faster on long-chain saturated fatty acids as compared to unsaturated ones (Ribeiro et al., 2011). Stereospecific lipases (isolated from Burkholderiacepacia, Pseudozyma antarctica, Candida rugosa, and Rhizopus delemar) finds application in pharmaceutical and agrochemical industries where preferential hydrolysis of one isomer happens over another (Khan et al., 2017). Lipases can be classified based on unique properties, such as thermostable lipases, cold active lipases, acid stable, alkali stable, and organic solvent tolerant lipases (Nagarajan, 2012). Thermostable lipases are produced primarily by thermophiles and operate well at a temperature above 60 °C (Li et al., 2005), e.g. Lipase from Bacillus thermoleovorans, Geobacillus thermo­ denitrificans, and R. oryzaeoperate well at 60 °C–70 °C. The activity of lipase from Geobacillus thermodenitrificans was highest at 65 °C at pH 7.0 and changing the temperature of medium to 80 °C caused reduction in activity by 80% (Balan et al., 2012). The structural features contributing the thermostability of lipases were analyzed by Chakravorty et al. (2011). The researcher found that an increase in surface-charged residues and decrease in beta-branched residues at helices was found to be correlated with improved thermostability. Also, increase in titratable amino acids in the serine residue of active site also contributes to thermostability (Chakravorty et al., 2011). In contrary to thermostable lipases, cold active lipases function well at low temperatures. The well-defined bacterial genera for production of cold active lipase include Aeromonas, Pseudoalteromonas, Moraxella, Acinetobacter, Staphylococcus, Serratia, Psychrobacter, and Pseudomonas sp. Among fungal strains, Candida spp, Aspergillus nidulans, and Penicillium roqueforti were found to produce cold active lipases (Joseph et al., 2008; Phukon et al., 2020). Thermotolerant acid stable lipases are generally used in food and flavour industries for the production ofaromatic esters like isoamyl acetate (Hasan et al., 2006). The optimum activity of lipase produced from Pseudomonas gessardii was evaluated as pH of 3.5 by Ramani et al. (2010) indicating its application in pharma­ ceutical industry as a substitute for pancreatic lipases for enzyme therapy. Thermostable alkaline lipases better found its application as additives in detergent industries. Their ability in producing biodiesel is also an area of interest among researchers (El-Ghonemy et al., 2021). Lipases tolerant in organic solvents are an interesting area for researchers because many industrially important reactions like esterification, transesterification, and biodiesel production perform well as increased solubility of non-polar compounds in the presence of organic solvents. Initially, scientists used many methods to improve lipolytic activity of enzymes in the presence of organic solvents; later, the research on screening the microbial strain, which is solvent-tolerant, gained importance as the former adds high cost of production (Kumar et al., 2016). Microbial lipases exhibit high stability toward a number of organic solvents (Gupta et al., 2004).

5.4 MICROBIAL LIPASES Lipases from microbial sources are witnessing significant growth today and expected in near future owing to their broad-spectrum applications (Table 5.2). Many microorganisms, including bacteria, fungi, and yeasts, are found to be a potential source for lipase production. Microbial lipases can be majorly classified based on their origin as bacterial, fungal, and yeast lipases. Based on site of production inside the cells, the lipases can be categorized as intracellular, extracellular, and membrane-bound lipase (Figure 5.2). The most widely used ones are extracellular lipases, which need further purification by suitable techniques depending on the source and composition. Intracellular and cell-bound lipases do not need extensive purification steps and can be directly

C.cylindracea

Rhizopus nodosus

NovoCor AD

Lactobacillus casei, Lactobacillus paracasei,

Lipases

Lipases

Bacillus subtilis Candela lipolytica

Lipases Lipases

Pseudomonas aeruginosa

Thermomyceslanoginosus

Lipase type II

Lipases

Synthesis of esters

Aspergillus niger Pseudomonas fluorescens

Amano Lipase A Amano Lipase AK

P. fluorescens

Candida cylindracea

Lipases

P. alcaligenes S. marcescens

Improvement of flavor

Immobilized P. cepacia Rhizomucormeihei

Lipases

Synthesis of lovastin drug

Candida rugosa

Lipases

Lipases Lipases

Alkaline lipases Lipases

Acts as a biocatalysts

Candida utilis Immobilized Rhizomucormeihei

Lipases Lipases

Hydrolysis of triglyceride to glycerol and free fatty acids

---

Synthesis of fluoxetine intermediates

---Synthesis of diltiazem hydrochloride

Desizing

Surface modification

Transesterification of crude soybean oils Enhanced the level of polyunsaturated fatty acid

Flavour ingredients synthesis (methyl ketones,lactonesflavour esters, and beta-keto acids)

A. niger and A. oryzae

Lipases

Enzyme Properties

Alkaline Lipases

Source Organism

Mucormiehei and Candida Transesterification of fatty acid Antarctica Acinetobacter radioresistens -------

Lipases

Name of Enzyme

TABLE 5.2 Application of Different Microbial Lipases and Their Properties

( Xiao et al., 2017)

( Chandra et al., 2020) ( Andualema and Gessesse, 2012)

( Rajmohan et al., 2002)

( Chandra et al., 2020)

( Sen et al., 2021)

( Kumar et al., 2021)

( Hasan et al., 2006)

( Hsu et al., 2002) ( Stevenson et al., 1993)

( Matsumae et al., 1993)

( Hasan et al., 2006)

( Chen et al., 1998)

( Akoh, 1993)

References

Production of personal care products in cosmetics ( Andualema and industry Gessesse, 2012) Degreasing of fatty raw materials in leather industry ( Hasan et al., 2006)

pharmaceutical industry Helps to treat depression, and obsessive-compulsive disorder (OCD)

Removal of fatty stains Helps in vasodialation of coronary in medical

Food flavouring

Cheese Industry

Denims and other fabrics Denims in textile industry

Surface modification of polyester fabrics

Polyethylenetere-phthalate fibers Polyethylenetere-phthalate fibers

Production of soap

Biodiesel production Tea processing industry

Lowering of serum cholesterol level

Production of flavoured alcoholic beverages Cosmetics industry

Cheese manufacturing industry

Detergent industry

Fat and oleochemical industry

Application Area

80 Microbial Enzymes and Metabolites for Health and Well-Being

Microbial Lipases

81

FIGURE 5.2 Classification of microbial lipases.

taken for immobilization; contributing to reduced cost of production as compared to extracellular ones (Vishnoi et al., 2020). However, considering the conversion rate, extracellular lipases out­ weigh intracellular ones (Mehmood et al., 2021). Easiness in packaging, handling, end use and stability, the powdered form of lipase is most convenient choice for many industrial applications (Bano et al., 2017).

5.4.1 BACTERIAL LIPASES The genera Bacillus sp, Pseudomonas sp., Burkholderia sp. and Staphylococcus sp. are considered as the most potent, industrially dominant lipase-producing bacteria (Bharathi et al., 2019; Phukon et al., 2022). They have been isolated and characterized from different natural environments, including oil industrial wastes, fat-processing factories, milk-processing plants, paper industries, and oil-contaminated soils. Bacterial lipases are mostly glycoproteins, and some extracellular li­ poproteins also possess catalytic activity (Vishnoi et al., 2020). They are mostly nonspecific in their substrate selectivity (Tembhurkar et al., 2012). Most lipases work in a wide range of pH; however, alkaline bacterial lipases are more common. Some exceptions to the optimal temperature and pH are also reported in literature as the lipase isolated from P. fluorescens SIK W1 exhibited optimum activity at acidic pH of 4.8 (Andersson et al., 1979). Some bacterial lipases perform well over a wide range of pH from 3–12. Most of bacterial lipases works over a wide range of tem­ perature between 30 °C–60°C. Addition of stabilizers like ethylene glycol, sorbitol, and glycerol proved to improve the thermal stability of bacterial lipases. In comparison with fungal-sourced lipases, bacterial lipases exhibit optimum activities in neutral or alkaline pH and also offer higher thermostability (Chandra et al., 2020).

5.4.2 FUNGAL LIPASES The chief industrial sources of fungal lipases are from the genera Candida, Rhizopus, Penicillium, Aspergillus, and Rhizomucor (Ribeiro et al., 2011). The ease of extraction, purification, and processing steps makes filamentous fungi a suitable source as compared to other fungal sources for lipase production at industrial scale. Fungal lipases are preferred for some applications as they are

82

Microbial Enzymes and Metabolites for Health and Well-Being

extracellularly produced, which makes the purification easier contributing to reduced cost of production (Vishnoi et al., 2020). Lipase-producing fungi were isolated from diverse sources, including industrial wastes, composts, oil-contaminated soils, etc.; commercial fungal lipases are majorly produced by A. niger, C. rugosa, H. lanuginosa, M. miehei, R. arrhizus, R. delemar, R. japonicus, R. niveus and R. oryzae (Chandra et al., 2020).

5.4.3 YEAST LIPASES Commercially dominant genus of yeast for isolation of lipase falls under Candida; especially C. rugosa and C. antarctica are well recognized for immobilized extracellular lipases. Lipase from C. rugosa is well known for aroma and flavour enhancer, also used in pharmaceutical industries and as a biosensor (Vanleeuw et al., 2019). C. antarctica produces two different lipases, lipases A and B; lipase B is the most commonly used one in biocatalysis (de María et al., 2005). C. antarctica B is also used for synthesis of cyclic esters (Kundys et al., 2018). Apart from above-mentioned genus, Yarrowia spp, Rhodotorula sp., Pichia sp., Saccharomycopsis sp, Trichosporon sp, and Geotrichum candidum has also been explored for potential industrial applications (Vishnoi et al., 2020).

5.5 PRODUCTION AND PURIFICATION OF LIPASES The methods for production of lipases falls under submerged culture (SF) and solid-state fer­ mentation (SSF); in few cases, immobilized cell culture methods were also used. As the name indicates, SSF involves culturing of microbes on a substrate with low moisture content, whereas SF involves a liquid medium for culturing. Lipases are secreted into the medium they grow (extra­ cellular), and some are intracellular and also found bound to the cell wall (Nagarajan (2012). The rate of production depends on the characteristics of media, including concentration of carbon, nitrogen, and dissolved oxygen, pH, and growth temperature. Age of culture, concentration of inoculum, and source of lipid also plays an important role in the rate of production (Deive et al., 2009). A variety of studies aimed to optimize the medium composition and operational variables for enhancing the rate of production and ease of purification of lipases (Colla et al., 2015). Purification of microbial lipases aims to remove contaminants, which also aids in further improvement in their activity, stability, and shelf life. High level of purity is necessary, especially for therapeutic applications where homogeneity of enzyme is a key factor (Silva et al., 2019). Extracellular lipases are directly secreted into the growing medium, which makes their separation easier. They are mostly produced by SF methods. However, SSF was found to enhance the yield of enzyme, thus reducing the cost (Aliyah et al., 2016). Colla et al. evaluated the effectiveness of the two methods in producing lipase from two strains of Aspergillus (O-8 and O-4) isolated from dieselcontaminated soil. The results indicated that enzymes produced by submerged fermentation were stable at a high temperature of 90 °C, with loss of 28% activity after one hour at 90 °C. Also, the lipases produced were stable in acidic conditions, whereas the lipases by SSF were stable in alkaline environment (Colla et al., 2015). The steps involved in the production process and the extent of purification depends primarily on the intended endues of lipase. The extracellular lipases are gen­ erally isolated from the growth medium by a centrifugation/filtration process (Chandra et al., 2020). Several chromatographic methods and their combinations were used for further purification of lipase (Saxena et al., 2003). Membrane filtration, immunopurifcation, chromatographic separations, etc. are also used for further refining of the enzyme according to end use (Chandra et al., 2020). Immobilization is performed to enhance the long-term operational stability of enzymes; it also prevents the enzyme from structural denaturation upon deviation from optimum conditions of pH and temperature. Immobilization also allows easy separation of enzymes from the reaction medium, which can be easily washed and reused (Hwang et al., 2012). The method commonly employed in immobilization can be classified into physical and chemical methods. In physical methods, adsorption, entrapment/encapsulation, and confinement are employed (Homaei et al., 2013).

Microbial Lipases

83

An adsorbent (usually activated carbon, silica gel, alumina, cellulose etc.) is used to collect enzymes from the reaction medium in physical adsorption, whereas in encapsulation method, shear force is used to entrap the enzymes within a carrier molecule. The chances for enzyme inactivation are more in an encapsulation method due to the application of shear force (Nguyen and Kim, 2017). In the confinement method, the enzymes are isolated by making of liposomes or microcapsules (Datta et al., 2013). Cross-linking and covalent binding comes under the chemical methods of immobilization. An excellent review by Coelho and Orlandelli (2021) evaluated the relevant re­ searches addressing various immobilization procedures employed for food applications of lipases (Coelho and Orlandelli (2021).

5.5.1 SCREENING

FOR

LIPOLYTIC ACTIVITY

The basic principle in determining lipase activity is by measuring the release of free fatty acids and glycerol from triacylglycerol and expressed as unit (U) of lipase activity, as defined by the quantity of enzyme to produce 1 μmol of fatty acids per unit time under standard conditions. The enzyme activity is also expressed as units per mL or units per g (Nema et al., 2019). The assays performed for analyzing lipolytic activity includes fluorescence, chromatography, HPLC, immunochemistry, monomolecular film technique, oil drop method, atomic force microscopy, and IR spectroscopy (Nagarajan et al., 2012). The titrimetry assay and spectrophotometric assay weremost commonly used for screening the lipolytic activity of isolated lipase (Singh et al., 2012).

5.6 APPLICATIONS Microbial lipases have found wide applications in diverse industrial sectors. Table 5.2 shows some of the most salient applications of lipases, some of which have been detailed below for the un­ derstanding of the readers.

5.6.1 APPLICATION

IN

FOOD

AND

NUTRACEUTICAL INDUSTRY

5.6.1.1 Food Products Microbial lipases are generally used as part of a green technology in the industrial production of several food stuffs. Due to mild reaction conditions over traditional methods, immobilized lipases have often been used for the synthesis and enrichment of mono- and polyunsaturated fatty acids (PUFA) (Coelho and Orlandelli, 2021). In the dairy industry, lipases are used for facilitating the ripening of cheese, hydrolysis of milk fat and enhancing cheese flavor. Microbial lipases can also catalyze the formation of food ingredients and additives, which finds wide applications in the synthesis of flavoring agents, short-chain esters or fragrances. Lipases cat­ alyse the biotransformation of various compounds containing a carboxyl group, such as ester­ ification, transesterification, and aminolysis, in which the typical nucleophile (water) is replaced by alcohol or an amine, due to their previously mentioned substrate acceptance and stability in many organic solvents. The synthesis of aromatic food additives, which commonly contain esters or lactone structures and are used not only in the food business but also in the cosmetic industry, is a well-known use of lipases. Biocatalysis is a helpful and promising alternative green method for producing low-molecular weight esters, such as flavour molecules, with high yields and gentle reaction conditions (Zieniuk et al., 2022). In addition, these lipases can also prevent the autooxidation of fats. 5.6.1.2 Pharmaceuticals and Nutraceuticals In the pharmaceutical industry, the use of enzymes via biocatalysis is highly preferred over chemical synthesis. Lipases have the ability to modify the structure of bioactive compounds to make them compatible for use in the nutraceuticals industry (Ferreira-Dias et al., 2013). Lipases

84

Microbial Enzymes and Metabolites for Health and Well-Being

are the enzymes that have been widely used in medicines for several ailments, including digestion, hypolipidemic, local anti-inflammatory, clinical treatment of lipemia, and pancreatic diseases (Kato et al., 1989; Majtán et al., 2002; Xiao et al., 2017). Lipases can also be used for the synthesis of drugs, such as antihypertensive drug (captopril), anti-cholesterol drugs (squalene synthase inhibitor), anticancer drugs (Taxol®, spergualin), anti-Alzheimer disease drug ([S]-2-pentanol), antiviral drug (lobucavir), anti-inflammatory analgesic drug (ibuprofen, naproxen), and vitamin A (Bonrath et al., 2002; Houde et al., 2004). In th e pharmaceutical industry, the lipases play an important role in enantio selective inter-esterification and transesterification reaction and also in modification of monoglycerides as an emulsifier (Andualema and Gessesse, 2012). Lipases have the ability to resolve racemic mixture by the synthesis of an enantiomer that has been implemented for the production of drugs (Houde et al., 2004). This enzyme behaves as a tumor necrosis factor (TNF) activators that can be used to cure malignant tumors (Hasan et al., 2006). The application of lipases has also been studied for the treatment of hair loss and skin scalp disease and also for the diagnosis of tuberculosis. Several studies had suggested various applications of lipases from microbial sources in pharmaceutics. It has been reported that lipases from Candida rugosa and S. marcescens has been widely used for the synthesis of lovastatin drug that reduces the serum cholesterol level and diltiazem hydrochloride that helps the vasodialation of coronary, respectively (Andualema and Gessesse, 2012). The lipases from Staphylococcus were also studied for the synthesis of antioxidants such as tyrosol acetate, eugenol benzoate, and propyl gallate (Chandra et al., 2020). Several researches have been done using immobilized lipase from different species such as Pseudomonas aeruginosa and Pseudomonascepacia for the synthesis of fluoxetine inter­ mediates and fungicide intermediates, respectively (Xiao et al., 2017). It has also been reported that Galleria mellonella (wax moth) lipases have bactericidal activity on Mycobacterium tuberculosis (MBT) H37Rv. A patent have been filed against the medicine for skin inflammation containing enzymes such as hyaluronidase and/or thiomucase enzymes with lipases (Hasan et al., 2006).

5.6.2 APPLICATION

OF

LIPASE

IN

TEXTILE INDUSTRY

Use of wet processes for the removal of the lipid barrier from the surface of wool is inefficient due to the hydrophobic nature of wool. To overcome this limitation, conventionally alkaline solutions were used, but these have a major drawback in that these methods are deteriorative to the fiber (Ismail et al., 2021; Kabir and Koh, 2021; Shanmugasundaram and Pandit, 2022). Therefore, the application of enzymes in the textile industry is more preferred owing to its non-toxic and ecofriendly characteristics (El-Fiky et al., 2021). In the textile industry, lipases have been widely used for degreasing textile raw materials and enhancing the performance. Lipases have also been used to remove the sizing lubricants to add more to the fabric absorbancy while improving the levelness during dyeing and also used to reduce the frequency of streaks and crevises in the denim wear system. The physical and chemical modifications in treated wool fibers and its commercialization have been studied. It was found that fatty acids on the surface wool fiber treated with anhydrous alkali can be removed by lipases thus improving the quality of wool. Lipases have applications in the leather factory in the removal of collagen fiber from leather and fat from the fur, thus improving the product quality (Xiao et al., 2017). Polyesterases (closely related to lipases) in textile industry also enhances the ability of polyester fiber to uptake chemical components, such as anti-staining, fabric-finishing compositions, anti-static, and cationic compounds, dyes, anti­ microbial, deodorant, and antiperspirant compounds (Andualema and Gessesse, 2012). It has been also reported that the lipases can be widely used for improving the wettability, dyeing, gloss, and absorbance of polyester fabric. The application of microbial lipases from Pseudomonas spp. in degrading the polymers of aliphatic polyethylene were also documented (Hasan et al., 2006). Furthermore, the thermophilic lipase derived from Bacillus aerius strain 24 K showed good activity (150 U/mL) and was found applicative in scouring and dyeing of wool (El-Fiky et al., 2021). It was found that this lipase exhibited effective scouring activity

Microbial Lipases

85

even at acidic pH (6.0). Interestingly, it was reported that the solubility and chemical compo­ sition of bio-scoured wool was similar to untreated wool.

5.6.3 COSMETICS Lipases have wide applications in the cosmetics industry as it shows surfactant activities by emulsifying glycerol into mono- and di-acylglycerols. Instead of conventional catalysis, the use of enzymes has been favored as it produces high quality products and requires minimum down­ stream refining. However, according to the manufacturers, the production cost is higher than that of conventional method but can be indemnified due the production of upgraded and high-quality end products (Andualema and Gessesse, 2012). Lipases from microbial sources such as C. cylindracea have been used for the production of personal-care products. Immobilized lipase from Rhizomucormiehei has been widely used as biocatalyst. Furthermore, it can be used for curling of hairs and as an element of topical anti-obese emulsion (Andualema and Gessesse, 2012). Lipases are characterized to be well suited with fatty, non-aqueous media, and emulsions that are relatively essential for the production of cosmetics products with high process stability. Lipases can act both as an active ingredient in the formulation of cosmetics and as a biocatalyst for the synthesis of certain cosmetics chemicals. Various applications of active lipases in cosmetics have been studied such as a facial cleanser by Juju cosmetics, Silhouette Sculptant Exfoliating Mousse by Maria Galland for anti-cellulite treatment, ‘Bath Additive with Fat Dissolving Enzymes’ by Ishizawa Laboratories for slimming body fat and also can be used for beauty masks, hair care, and makeup (Ansorge-Schumacher and Thum, 2013; Huber et al., 2004; Kellis and Lad, 2006). Therefore, the use of lipases in manufacturing of cosmetics products plays an important role in increasing a demand for the green chemical process and natural products. Water-soluble retinol derivatives prepared via catalytic reaction of immobilized lipase can be commercially used in cosmetics products. Unichem International (Spain) initiated the production of isopropyl myristate, isopropyl palmitate and 2- ethylhexylpalmitate in application of soothing cosmetics products like skin and sun tan ointments, bath oils, etc. (Chandra et al., 2020).

5.6.4 BIODIESEL PRODUCTION The increasing environmental pollution including greenhouse gases, climate changes, and eleva­ tion in the prices of fossil fuels desired to look for alternatives or improvements in the biodiesel technology (Klek, 2016). In this context, the use of lipolytic enzymes for the utilization of lipid rich waste as a fossil fuel substitute offers a sustainable strategy that challenges energy safety (Dauvergne and Neville, 2009). The thermostability and short-chain thermotolerant ability of lipase makes it a suitable candidate for lipase production. Lipases and triacylglycerol acylhy­ drolases exhibit superlative physiochemical features and act as good catalysts. Lipases derived from several microbial species, including Candida antarctica, Pseudomonas cepacia, Aspergillus niger, Mucormiehei, Rhizopusoryzae, Chromobac-teriumviscosum, Pseudomonas fluorescens, Bacillus subtilis, and Burkholderiacepacian, has been employed for biodiesel production (Chandra et al., 2020; Stefanovic et al., 2018). Candida guilliermondii, an endophytic yeast-derived extracellular lipases, was reported to be employed for biodiesel production by oleic acid esterification. Interestingly, the lipase was produced by employing agro-industrial residues as a low-cost substrate. The C. guilliermondii derived lipase was partially purified, and it exhibited higher activity of 26.8 U mL−1 at pH 6.5 and 30 °C. Under different solvent and acid alcohol molar ratio, the enzyme was tested for oleic acid esterification potential. It was found that at 1:9 molar ration, and with hexane, the rate of ester conversion was 81%. This suggested the effective biocatalyst application of this lipase (Caroline Defranceschi Oliveira et al., 2014). The immobilization of lipases has also proven to be efficient for the production of biodiesel (Arumugam and Ponnusami, 2017;

86

Microbial Enzymes and Metabolites for Health and Well-Being

Chandra et al., 2020). The activated carbon-immobilized lipase enzyme was found to be a good catalyst for biodiesel production by using fish-industry waste sardine oil. Immobilization of lipase was also found to increase the usability, and it was found that reusing the activated carbon immobilized lipase for five cycles, decreasing the FAME yield by 13%. The biodiesel ester content was recorded to be Hexadecanoic acid (19.41%), 9-Hexadecenoic acid (13.2%), 10-methyl octadecenoic acid (19.13%), Eicosapentaenoic acid (16.25%), Octadecanoic acid (10.49%), Tridecanoic acid14.95%). The biodiesel derived by this process exhibited 891 kg/m3 density at 15 °C, 56 cetane number and 161 °C flashpoint. The cetane number was high of biodiesel as compared to the diesel fuel and showed better ignition performance (Arumugam and Ponnusami, 2017). These findings from different research groups suggested that lipase can be efficiency used for sustainable biodiesel production by using different waste types.

5.7 CONCLUSION Recent advances in bioprocessing strategies have enabled the production of well-characterized lipase on a large scale. From the different classes of lipases, extracellular lipases have found wider applicability but needs further purification by suitable techniques depending on its source and composition. The use of microbial lipases has opened up a plethora of applications in the food, beverage, detergents, paper and pulp, textiles, pharmaceutical and nutraceuticals, cosmetics, bio­ diesel production, biosensor and other sectors. The contribution of microbial lipases in these sectors have been mainly attributed to its potential to catalyse the biotransformation of various compounds through esterification, transesterification, and aminolysis. Overall, microbial lipases provide a viable green alternative to existing chemical based bioprocesses and is therefore rec­ ommended for industrial use.

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Health Benefits of Bioactive Compounds Produced Using Microbial Enzymes Swati Sharma School of Skill Buildings, Shri Ramasamy Memorial (SRM) University, Sikkim, Gangtok, India

Loreni Chiring Phukon and Rounak Chourasia Institute of Bioresources and Sustainable Development, Regional Centre, Tadong, Sikkim, India

Ranjana Sirohi School of Health Sciences and Technology, University of Petroleum and Energy Studies, Dehradun, Uttarakhand, India

Binod Parameswaran Microbial Processes and Technology Division, CSIR-National Institute for Interdisciplinary Science and Technology (NIIST), Thiruvananthapuram, Kerala, India

Ashok Pandey CSIR-Indian Institute of Toxicology Research and Centre for Energy and Environmental Sustainability, Lucknow, Uttar Pradesh, India

Dinabandhu Sahoo Department of Botany, University of Delhi, New Delhi, India

Amit Kumar Rai Food and Nutritional Biotechnology Division, National Agri-Food Biotechnology Institute, Mohali, Punjab, India

CONTENTS 6.1 6.2 6.3

Introduction.............................................................................................................................92 Microbial Enzymes for Production of Bioactive Compounds..............................................92 Health Benefits of Bioactive Compounds .............................................................................93 6.3.1 Antioxidant Compounds............................................................................................. 93 6.3.2 Antihypertensive Compounds ....................................................................................94 6.3.3 Antidiabetic Compounds............................................................................................97 6.3.4 Antimicrobial Properties ............................................................................................97 6.3.5 Anticancer Compounds ..............................................................................................98 6.3.6 Production of Prebiotic Molecules ............................................................................99

DOI: 10.1201/9781003369295-7

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6.3.7 Immunomodulatory Properties...................................................................................99 6.3.8 Reduction of Antinutritional Factors .......................................................................101 6.4 Conclusions........................................................................................................................... 101 References...................................................................................................................................... 101

6.1 INTRODUCTION Microorganisms have been used for many generations for the production of functional foods and nutraceuticals (Kumari et al., 2022). Lactic acid bacteria, yeasts, Bacillus spp., and fungi have been used to produce enzymes for application in the functional food industry (Kumari et al., 2021; Sanjukta et al., 2021). Microbial processes depend on specific and efficient biocatalysts for the production of nutraceuticals and functional foods (Phukon et al., 2022; Chourasia et al., 2020). These enzymes are preferred over enzymes from plant and animal enzymes due to the microbes’ short generation time, high yields and consistency, and economic feasibility; they also can be supplied throughout the year, no matter the seasonal fluctuations (Gurung et al., 2013). Further, their higher production can be optimized to improve their yield and activity, making the product economical. The types of microbial enzyme applied for the development of nutraceuticals are lipases, proteases, pectinases, β-glucosidase, β-galactosidase, xylanase, glutamate decarboxylase, etc. (Thakur et al., 2021; Hathwar et al., 2011; Kumari et al., 2022). In recent years, the combination of advanced techniques for the identification of metabolites produced during enzymatic hydrolysis and computational approach has led in the identification of novel bioactive molecules (Padhi et al., 2021; Sanjukta et al., 2021). Further, predicted bioactive peptides are synthesized, and their activities are validated using specific invitro and invivo models (Karami et al., 2019). The health benefits reported by enzymatically hydrolysed/transformed food metabolites are antioxidants, antihypertensive, antimicrobial, immunomodulatory, anticancer, and antidiabetic properties (Chourasia et al., 2020; Li et al., 2020; Phukon et al., 2022; VillanuevaLazo et al., 2021). Apart from improving health benefits, microbial enzymes also play a significant role in the reduction of antinutritional factors (Jatuwong et al., 2020; Vohra et al., 2003). Several studies have reported application of recombinant enzymes for improved biocatalytic systems for nutraceutical production (Kaushal et al., 2021; Thakur et al., 2021; 2022). Several commercial enzymes are available in markets having application in the functional food industry (Kumari et al., 2021). Microbial enzymes have been widely applied for the recovery of nutraceuticals, including lipids, peptides, carotenoids, and chitin (Hathwar et al., 2011; Lasrado and Rai, 2018). Due to increasing awareness of functional food additives and nutraceuticals, microbial enzymes appli­ cations are gaining popularity globally. This book chapter describes the role of microbial enzymes in improving the health benefits of food components.

6.2 MICROBIAL ENZYMES FOR PRODUCTION OF BIOACTIVE COMPOUNDS Several hydrolytic enzymes compounds are known for the production of functional food additives, nutraceuticals, and the reduction of antinutritional factors. Microbial enzymes have also been applied for the production and transformation of bioactive compounds, such as polyphenols (Sarkar et al., 2022; Tang et al., 2016), bioactive peptides (Abedin et al., 2022; Li et al., 2020), oligosaccharides (Khangkhachit et al., 2021; M. Yu et al., 2021), and functional lipid moieties (Ferreira-Dias et al., 2013; Zieniuk et al., 2022). Microbial proteases have been extensively applied for the production of protein hydrolysates and bioactive peptides having different health benefits (Hathwar et al., 2011; Zhang et al., 2019; Zhao et al., 2021). These enzymes are specific and are applied against a wide range of food proteins, resulting in the production of bioactive peptides with unique sequences exhibiting functional properties. The functional properties exhibited by bioactive peptides produced using microbial proteases include antihypertensive, antioxidant, anticancer, immunomodulatory, antidiabetic, and antimicrobial properties (Abedin et al., 2022; Li et al., 2020;

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Sanjukta and Rai, 2016; Sharma et al., 2021; Zhao et al., 2021). Carbohydrate active enzymes are an important group of enzymes responsible for the production of different types of oligo­ saccharides (Khatun et al., 2021; Kumari et al., 2021). The carbohydrate active enzymes are xy­ lanase, pectinase, β-galactosidase, pullunase, β-glucosidase, α-amylase, and cellulase, etc.; they have been applied for the production of different types of nutraceuticals (Rakariyatham et al., 2020; Thakur et al., 2022). β-galactosidase have been reported for the hydrolysis of lactose resulting in products (milk and whey) for lactose-intolerant subjects (Thakur et al., 2022). Some enzymes are responsible for the production of antinutritional factors, such as phytases known for reducing phytate in food and feed (Handa et al., 2020).

6.3 HEALTH BENEFITS OF BIOACTIVE COMPOUNDS Bioactive compounds produced on enzymatic hydrolysis and transformations are responsible for different types of health benefits (Figure 6.1). The health benefits are antioxidants, antimicrobial, antihypertention, anticancer, immunomodulatory and prebiotic properties. The different types of health benefits produced using microbial enzymes are described in this section.

6.3.1 ANTIOXIDANT COMPOUNDS In recent years, increasing health complications associated with oxidative stress have resulted in demand for natural antioxidants (Sharma et al., 2022). Biotechnological approaches are becoming popular for the production of antioxidant molecules because they enhance the value of foods as well as underutilized raw material by improving their antioxidant potential (Chourasia et al., 2022; Hathwar et al., 2011). Microbial enzymes have been applied for the production of antioxidant compounds such as peptides, polyphenols, and oligosaccharides (Hathwar et al., 2011; Rai et al., 2017; Valls et al., 2018). Microbial proteases have been applied for the production of bioactive peptides using food protein substrates and food-processing by-products (Abedin et al., 2022;

FIGURE 6.1 Diagrammatic representation of production of health beneficial molecules using microbial hydrolytic enzymes. Xylo-oligosaccharides (XOS), galacto-oligosaccharides (GOS), isomalto-oligosaccharides (IMOS), fructo-oligosaccharides (FOS), mannose-oligosaccharides, and malto-oligosaccharides (MOS), poly­ unsaturated fatty acid (PUFA), monounsaturated fatty acid (MUFA).

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Hathwar et al., 2011). Antioxidant properties of the peptides depend on the presence of specific amino acids, such as tyrosine, trptophan, cystiene, methionine, histidine, and phenylalanine (Liu et al., 2016; Sanjukta et al., 2021). The antioxidant potential of the peptide will depend on the composition and sequence of these amino acids (Sanjukta and Rai, 2016). High antioxidant activity was observed in protein hydrolysates prepared from fish-processing by-products (fish visceral waste) using different enzymes (Hathwar et al., 2011). Four antioxidant peptides identified from soybean alcalase hydrolysate exhibiting antioxidant activity were VVFVDRL, VIYVVDLR, IYVVDLR, and IYVFVR (Zhang et al., 2019). Three bioactive peptides, namely SLPY, QYPPMQY, and EYEA, were identified in protein hydrolysate prepared on alcalase hydrolysis of antarctic krill proteins (Zhang et al., 2021). Four peptides produced using hydrolysis of casein protein and microbial proteases exhibiting antioxidant activity were identified as FSDIPNPIGSE, YFYP, YQLD, and FSDIPNPIGSEN (Zhao et al., 2021). Wheat-germ protein hydrolysates produced using alcalase and proteinase K exhibited DPPH and ABTS radical scavenging activity in a dose-dependent manner (Karami et al., 2019). Enzymatic hydrolysis of grape pomace (by-product of wine production) using different en­ zymes, including tannase, pectinase, and cellulase-enhanced antioxidant potential due to an increase in free polyphenols (Martins et al., 2016). β-glucosidase from Alicyclobacillus herbarius was found to transform the bioconversion of bound isoflavones to free form (Delgado et al., 2021). Treatment of quinoa seeds using different carbohydrate active enzymes resulted in the conversion of bound phenolics to their free form along with enhancement of antioxidant properties (Tang et al., 2016). Pretreating longan peel using enzymes such as α-amylase, cellulase, β-glucosidase, and protease resulted in enhancement of total phenolics, DPPH scavenging activity, ferrous-ion chelating activity, and oxygen-radical absorbance capacity (Rakariyatham et al., 2020). The ex­ traction yield and associated antioxidant properties depend on the type of enzymes applied for the hydrolysis process. The different types of bioactive compounds and their health benefits are given in Table 6.1. Xylooligosaccharides (XOS) released from different xylan substrates using GH10 (Xyn10A) and GH30 (Xyn30D) xylanases exhibited high antioxidant activity (Valls et al., 2018). XOS having xylobiose content produced from corncob using xylanase_1948 exhibited excellent DPPH and ABTS radical scavenging activity (Boonchuay et al., 2018). Similarly, XOS mixture rich in arabinosyl-XOS produced from empty fruit bunch xylan using purified xylanase ME742 resulted in high antioxidant activity (Khangkhachit et al., 2021). In another study, pectic oligo­ saccharides (POS) produced from finger citron using pectinase exhibited high antioxidant potential proven by multiple antioxidants mechanisms (M. Yu et al., 2021). Ulvan oligosaccharides pro­ duced on hydrolysis of algal polysacchardies using enzymes from Aeromonas salmonicida MAEF108 and Pseudomonas vesicularis MA103 exhibited high antioxidant properties. The an­ tioxidant compounds produced using microbial enzymes treatment can be applied in as nutra­ ceuticals or production of functional foods.

6.3.2 ANTIHYPERTENSIVE COMPOUNDS Microbial enzymes have been applied to produce bioactive compounds that are known for anti­ hypertensive properties (Hung et al., 2021; Villanueva-Lazo et al., 2021; Yogeswara et al., 2021). Bioactive peptides produced using microbial enzymes have been shown to inhibit angiotensinconverting enzymes (ACE) inhibition (Li et al., 2007; Martínez-Montaño et al., 2022). ACE is the key enzyme responsible for the conversion of angiotensin I to angiotensin II, playing a major role in controlling blood pressure (Chourasia et al., 2022; Sanjukta and Rai, 2016). Bioactive peptides exhibit an antihypertensive effect, depending on their amino acid sequence, and composition in­ hibits the activity of ACE (Chourasia et al., 2022). Bioactive peptides were produced in horse gram hydrolysate exhibited ACE inhibitory property using alcalase (Bhaskar et al., 2019). Rice protein hydrolysate generated using alcalase exhibited high ACE inhibitory activity (IC50–0.14 mg/mL)

Aspergillus fumigatus RSP-8

Trichoderma reesei

Penicillium funiculosum

Lecanicilliumfungicola

Xylanase

Endo-xylanase

GH 10 xylanase

Endochitinase and Nacetylhexosaminidase Glucansucrase E81, and 4,6‑α‑glucanotransferase

Aspergillus fumigatus R1

Bacillus subtilis GA2

Acremonium sp. L1–4B

Aspergillus niger, Rhizopus javanicus YarrowialipolyticaIMUFRJ 50682

Streptomyces sp. strain W007

Xylanase

Mannanase

Protease

Lipase

Lipase

Lipase

Pseudomonas vesicularis MA103 and Aeromonas salmonicida MAEF108

Ulvanolytic enzymes, amylase, cellulase, and xylanase

Lactobacillus reuteri E81

Pichia stipitis

Microbial Source

Xylanase

Enzymes

Arabino-xylooligosaccharide hydrolysate

Xylooligosaccharides (X2-X7)

Xylooligosaccharides (X2-X4)

Bioactive Compounds

Conjugated linoleic acid (CLA) and glycerol

Soyabean oil and sardine oil Olive oil

Bovine and caprine caseinate

Spent coffee

Wheat husk

Ulvan from green seaweed

CLA-rich triacylglycerols

( Wongsiridetchai et al., 2021)

( Jagtap et al., 2017)

( Hung et al., 2021)

( Ramírez-Coutiño et al., 2006) ( İspirli et al., 2019a; İspirli et al., 2019b; İspirli and Dertli, 2019)

( Lafond et al., 2011)

( Kale et al., 2018)

( Ravichandra et al., 2022)

( Yang et al., 2011)

References

Nutritional and therapeutic properties

(Continued )

( Lian et al., 2018)

( Akil et al., 2020)

( Araújo et al., 2016)

Antioxidative and ( Nascimento et al., 2021) antihypertensive properties

Prebiotic and antioxidative properties Prebiotic properties

Antioxidant and Angiotensin-Converting Enzyme-Inhibitory Activities

Prebiotic and immunemodulatory properties

Prebiotic properties

Prebiotic properties

Prebiotic properties and fiber fortification in food

Prebiotic properties

Prebiotic properties

Activity

Triacylglycerols enriched in n-3 Clinical nutrition fatty acids Bioactive structured lipids Immunomodulatory effects

Bioactive peptide rich hydrolysates

Mannooligosaccharides

Xylooligosaccharides

Ulvan Oligosaccharides

Commercial xylan and Xylooligosaccharides arabinoxylan α-chitin from shrimp Oligosaccharide waste Commercial sucrose Malto-oligosaccharides and and mannose mannose containing oligosaccharides

Bio-Fiber Gum from corn barn

Xylan from Populas tomentosachips Sorghum xylan

Substrate

TABLE 6.1 Health Beneficial Compound Produced Using Microbial Hydrolytic Enzymes

Health Benefits of Bioactive Compounds 95

Pseudomonas aeruginosa

Aspergillus japonicus Kluyveromyces lactis

Aspergillus oryzae, Lactobacillus acidophilus ATCC 4356

Protease

β-fructofuranosidase β-galactosidase

β-galactosidase

Microbial Source

Rhizomucormiehei

Lipase

Enzymes

Lactose

Sucrose Soft cheese

Wheat gluten

Microbial oil from Mortierellaalpina

Substrate

GOS

Fructooligosaccharides (FOS) Galactooligosaccharides (GOS)

Bioactive peptides

Structured lipids enriched with medium-chain fatty acids

Bioactive Compounds

TABLE 6.1 (Continued) Health Beneficial Compound Produced Using Microbial Hydrolytic Enzymes Activity

Prebiotic properties

Prebiotic properties Prebiotic properties

Angiotensin-Converting Enzyme-Inhibitory Activities

Nutritional and therapeutic properties

References

( Carević et al., 2018; Wang et al., 2021),

( Sheu et al., 2001) ( Vénica et al., 2020)

( Zhang et al., 2020)

( Abed et al., 2018)

96 Microbial Enzymes and Metabolites for Health and Well-Being

Health Benefits of Bioactive Compounds

97

(Li et al., 2007). An ACE inhibitory peptide (Thr-Gln-Val-Tyr) purified from the hydrolysate lowered blood pressure in spontaneously hypertensive rats at dose of 30 mg/kg body weight. Chia protein hydrolysate produced using alcalase and flavarozyme exhibited promising ACE inhibitory effect (Villanueva-Lazo et al., 2021). Apart from food proteins, the enzymatic hydrolysis approach has been applied for the production of ACE inhibitory peptides from protein-rich food-processing by-products (Martínez-Montaño et al., 2022). Apart from bioactive peptides, γ-aminobutyric acid (GABA) enriched foods are known for antihypertensive effect, which is proven on clinical trials (Shimada et al., 2009). Conversion of glutamate to GABA is catalyzed by the action of glutamate decarboxylase (GAD) (Chang et al., 2017; Yogeswara et al., 2021). Several lactic acid bacteria from fermented food products are known for the production of GAD, which during food fermentation catalyses the production of GABA. GABA has been purified and characterized from different lactic acid bacteria. Enzymatic production of GABA has been carried out on the bioconversion of monosodium glutamate by the application of recombinant GAD having higher efficiency in comparison to fermentation approach (Yogeswara et al., 2021). Apart from bioactive peptides and GABA, ulvan oligosaccharides produced using enzymatic hydrolysis of polysaccharides from Ulva lactuca exhibited ACE inhibitory effect (Hung et al., 2021). There is a possibility of finding novel ACE inhibitory mo­ lecules on enzymatic hydrolysis.

6.3.3 ANTIDIABETIC COMPOUNDS Microbial enzymes have been applied for the production of hydrolysates rich in molecules having the ability to inhibit key enzymes responsible for diabetes, including α-amylases, α-glucosidases, and dipeptidyl peptidase-IV (DPP-IV) (Jin et al., 2020; Rivero-Pino et al., 2021). Many proteolytic enzymes lead to the production of peptides having different ability to inhibit these metabolic enzymes. Higher DPP-IV inhibitory activity was observed in salmon skin collagen hydrolysate produced using trypsin in comparison to other proteolytic enzymes such as pepsin, papain, or Alcalase 2.4 L (Jin et al., 2020). Among different protease employed, walnut protein hydrolyzed using alcalase exhibited higher DPP-IV inhibitory activity (Kong et al., 2021). On further detail analysis of bioactive peptides responsible for inhibition of DPP-IV, it was found that peptides with presence of basic amino acid residues had higher inhibitory potential. Bioactive peptides identified in soybean protein hydrolysate prepared using alkaline proteinase exhibited high α-glucosidase inhibitory activity in comparison to hydrolysate prepared using other enzymes (Wang et al., 2019). On purification and characterization of α-glucosidase inhibitory pep­ tides, three novel peptides (SWLRL, LLPLPVLK, and WLRL) were found to be present in soy protein hydrolysate. Enzymatic treatments (subtilisin and trypsin) were applied for the production of bioactive peptides rich chickpea protein hydrolysates exhibiting α-glucosidase inhibitory activity (Rivero-Pino et al., 2021). Luffa cylindrica seed hydrloysates prepared using alcalase demonstrated a strong α-amylase and α-glucosidase inhibitory activity (Arise et al., 2019). Apart from peptides, free phenolic formed on the enzymatic treatment of quinoa seeds was found to exhibit α-glucosidase inhibitory properties (Tang et al., 2016). There are possibilities for the production of novel bioactive peptides with higher antidiabetic properties using novel protein sources.

6.3.4 ANTIMICROBIAL PROPERTIES Enzymatic treatments have resulted in the production of molecules that have been effective an­ timicrobial agents (Silva et al., 2021). These molecules include bioactive peptides, oligosacchar­ ides, and lipid metabolites (Kallel et al., 2015; Song et al., 2020). Antibacterial peptides were identified from cottonseed protein hydrolysate that was produced using alcalase (Song et al., 2020). Among nine peptides, KDFPGRR exhibited the highest antibacterial effect by destroying the cells. Degradation of chitosan using chitosanases enzyme extract resulted in the production of chitosan

98

Microbial Enzymes and Metabolites for Health and Well-Being

oligosaccharides exhibiting antimicrobial properties against Candida albicans and selected grampositive bacteria (Silva et al., 2021). Xylooligosaccharides produced using xylanase from Bacillus mojavensis exhibited antibacterial effect against Pseudomonas aeruginosa, Enterococcus faecalis, and Klebsiella pneumonia (Kallel et al., 2015). Protein hydrolysate produced from camel milk whey using alcalase and protease exhibited promising antifungal properties against the Candida species (Mudgil et al., 2022). Enzymatic treatment of lipids results in production of molecules that have been reported to exhibit antimicrobial properties (Ferreira-Dias et al., 2013; Zieniuk et al., 2022). Enzymatically hydrolysed metabolites need to be explored for novel antimicrobial com­ pounds using different protein rich substrates.

6.3.5 ANTICANCER COMPOUNDS Bioactive compounds produced on hydrolysis using microbial enzymes of food substrate, and their by-products have been shown to possess anticancer properties (Ghosh et al., 2021; Hasani et al., 2022). A higher anticancer property was observed in Indian mackerel protein hydrolysate produced using alcalase in comparison to flavourzyme, which was due to a higher degree of protein hydrolysis (Hasani et al., 2022). A peptide SSDEEVREEKELDLSSNE produced on enzymatic hydrolysis of wheat germ protein decreased viability of A549 cells in a dose-dependent manner (Karami et al., 2019). Similarly, rice husk protein hydrolysate demonstrated cytotoxicity of A549 and MCF7 cells with IC50 values of 1.98 and 3.58 µg/mL, respectively (Ilhan-Ayisigi et al., 2021). Protein hydrolysis obtained upon proteolysis of chia expeller by the Alcalase-Flavourzyme sequential system resulted in antithrombotic activity against both intrinsic and extrinsic coagula­ tion pathways (Ozón et al., 2022). Hydrolysis of the Japanese carpet shell (Ruditapes philippi­ narum) using α-chymotrypsin, followed by purification, resulted in the identification of the novel peptide AVLVDKQCPD that successfully induced apoptosis on lung, breast, and prostate cancer cells and not on normal liver cells (Kim et al., 2013a). Enzyme hydrolysate of sorghum kafirin exhibited antioxidant potential in meat and emulsion system, and inhibited HepG2 cell growth via nontoxic mechanisms, indicating its anticancer activity potential (Xu et al., 2019). In another study, eel (Monopterus sp.) protein hydrolysate (EPH) demonstrated potential primary antioxidant activity with 3–4, 5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide (MTT) assay of EPH using MCF-7 cell lines demonstrating its potential anticancer effect (Halim et al., 2018). Acetone extract of egg white hydrolysates, prepared using alcalase exhibited the maximum antiproliferative activity in a dose dependent manner in mouse lymphoma cell line (P388D1) (Yi et al., 2003). Polysaccharides and their derivates have been studied extensively for their potential bioactiv­ ities, and researchers have reported anticancer potential of several polysaccharides and their en­ zyme modified derivatives (Lin et al., 2022; Ouyang et al., 2021). Moreover, algal polysaccharides (APs) have demonstrated several bioactivities against cancerous cells and their associated toxi­ cities. APs have demonstrated reduced toxicity of chemotherapy against cancer (Ouyang et al., 2021). Besides, APs induce apoptosis, anti-angiogenesis, and cell-cycle arrest, and regulate intestinal microflora and functions of the immune system (Ouyang et al., 2021). The endo-acting fucoidan sulfatase SWF5 enzyme of the marine bacterium Wenyingzhuangia fucanilytica CZ1127has been used for the preparation of 4O-desulfated derivative of fucoidans (fucosecontaining bioactive polysaccharides), produced by brown algae (Silchenko et al., 2021). The 4Odesulfated fucoidans exerted inhibition of colony formation by DLD-1 and MCF-7 cells (Silchenko et al., 2021). Xylooligosaccharides (XOS), derived from the hydrolysis of plant carbohydrates by microbial xylanase enzyme, have demonstrated anticancer effects (Ghosh et al., 2021; Sharma et al., 2020). The mixed XOS produced using endo-xylanase from Bacillus velezensis AG20 in­ hibited proliferation of the human colorectal cancer (HT-29) cells and Caco-2 cells (Ghosh et al., 2021). Similarly, hydrolysis of neem (Azadirachta indica) xylan by a recombinant endo-β-1, 4-xylanase resulted in the generation of a mixture of XOS that ranged from the degree of polymerization (Sharma et al., 2020). The XOS mixture inhibited the growth of HT-29 cells but

Health Benefits of Bioactive Compounds

99

had no effect on the mouse fibroblast cells that confirmed the XOS mixture’s biocompatibility (Sharma et al., 2020). These studies suggest the potential of using XOS as a potent antiproliferative agent. Fatty acids and their heterocyclic derivatives have been studied over the years for their potential anticancer activities (Jóźwiak et al., 2020). CLA are generated upon treatment of fats by microbial enzymes such as lipase and isomerase (Chourasia et al., 2021). CLA coupled with polymers have been reported to inhibit the proliferation of cancer cells (Guo et al., 2007; Tao et al., 2012). Conjugation of CLA with Pluronic F127 was associated with enhanced apoptosis of MCF-7 breast cancer cells, with dose-dependent upregulation of Bax, p21, and p53, and downregulation of procaspase 9 and Bcl-2 (Guo et al., 2007). In another study, coupling of the nucleoside analog, Gemcitabine (GEM), with CLA resulted in antitumor activity against MCF-7 breast cancer cells, suggesting the use of CLA-GEM conjugate as a prodrug for GEM for clinical use (Tao et al., 2012). Besides carbohydrate, protein, and fat-derived bioactive compounds, polyphenolic com­ pounds released upon hydrolysis of polyphenols by microbial enzymes have also demonstrated anticancer, antiproliferative, and antitumor activities (Attar et al., 2019; Guo et al., 2019; Salama et al., 2021; Sattarinezhad et al., 2021; Yerrabelly et al., 2020). These compounds generated upon treatment by microbial enzymes have high potential to be used for the development of nutra­ ceuticals and functional foods.

6.3.6 PRODUCTION

OF

PREBIOTIC MOLECULES

Prebiotics are nutraceuticals that can stimulate the growth of useful microorganisms in the gut on consumption (Lasrado and Rai, 2022). Many carbohydrate active enzymes have been applied for the production of prebiotic compounds that have shown to stimulate growth of probiotic bacteria (Ghosh et al., 2021). Endoxylanases from microbial sources are excellent tools for the production of prebiotic oligosaccharides that help in stimulation of intestinal bacteria (Karlsson et al., 2018). The XOS mixture released using endo-xylanase from Bacillus velezensis AG20 facilitated probiotic Bifidobacterium, apart from having stability in gastric juice and intestinal fluid (Ghosh et al., 2021). Fructooligosaccharides are known for several health benefits, including the ability to lower tri­ glyceride and cholesterol levels and prebiotic and immunomodulatory properties (Rosa et al., 2019). FOS has also been produced using different types of substrate, such as molasses, coffee by-products, apple pomace, sugar cane bagasse, date by-products, cassava wastes, banana peel-leaf, etc (Khatun et al., 2021; Rosa et al., 2019). The major enzymes responsible for FOS production are β-fructosyltransferases and fructofuranosidases, that are produced by Saccharomyces cerevisiae, Aspergillus niger, Aspergillus flavus, Aspergillus japonicas, and Rhizopus stolonifer (Rosa et al., 2019). FOS, an important prebiotic was produced using sugar cane molasses by the action of transfructosylating enzymes produced by Aureobasidium pullulans FRR 5284 (Khatun et al., 2021). Microbial β-galactosidases have been applied for the breakdown of lactose as well as their transformation of prebiotic molecules (Botvynko et al., 2019; Thakur et al., 2022). Combinations of β-galactosidases were applied for the production of galactooligosaccharides (GOS) from lactose as substrate (Botvynko et al., 2019). Recently, β-galactosidase from Kluyveromyces sp. PCH397 isolated from yak milk resulted in the hydrolysis of lactose, as well as synthesis of lactulose and galacto-oligosaccharides (Nag et al., 2021). Future studies on novel microbial sources for βgalactosidases need to be explored; they can have efficient bioconversion of lactose into prebiotic molecules in milk products.

6.3.7 IMMUNOMODULATORY PROPERTIES Microbial enzymes have been applied for the production of molecules having immunomodulatory properties (Kang et al., 2019; Kim et al., 2013b). Since it encompasses any alteration in innate or adaptive immunity, immunomodulation is considered as a broad term and can be classified as

100

Microbial Enzymes and Metabolites for Health and Well-Being

either anti-inflammatory, inflammatory, antiproliferative, proliferative, cytotoxic, or cytoprotective (Pavlicevic et al., 2022). Bioactive peptides (GVSLLEEFFL) purified from Mytilus coruscus protein hydrolysate was found to have immunomodulatory properties (Kim et al., 2013b). An immunomodulatory peptide (Asp-His-Ala-Val) was purified from alcalase-mediated silkworm pupa protein hydrolysates (molecular weight – 441.06 Da) (Li et al., 2020). Hydrolysis of soybean proteins by microbial proteolytic enzymes is associated with the release of immunomodulatory peptides (Wen et al., 2021). A total of 46 immunomodulatory soybean peptides including NQP­ QGGGNV, EANNQR, and ISTLPA were released by the hydrolysis of soybean proteins using alkaline protease (Wen et al., 2021). The resultant soybean peptides facilitated the proliferation of macrophages, with increase in pinocytotic activity and the levels of nitric oxide (NO) (Wen et al., 2021). The increased production of NO is associated with the induction of inducible nitric oxide synthase (iNOS) mRNA expression by soybean peptides (Wen et al., 2021). In another study, immunomodulatory peptides were obtained by the hydrolysis of soy protein isolate (SPI) (Hsieh et al., 2022). The soy peptide EKPQQQSSRRGS demonstrated phagocytosis and macrophage M1 polarization, suggesting the use of the peptide as a potent immunomodulatory compound (Hsieh et al., 2022). Immunomodulatory soybean peptides can be used for the development of nutraceuticals. Protein hydrolysates and peptides obtained from egg proteins, including ovalbumin, ovo­ transferrin, lysozyme, cystatin, ovomucin, phosvitin, and livetin, have demonstrated significant immunomodulatory activity (Lee and Paik, 2019). A low molecular weight (MW) fraction (