Acta Biotechnologica: Volume 9, Number 2 1989 [Reprint 2021 ed.] 9783112581865, 9783112581858

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Acta Blotecfemloflica Volume 9 • 1989 • Number 2

Journal of microbial, biochemical and bioanalogous technology

Akademie-Verlag Berlin ISSN 0 1 3 8 - 4 9 8 8 Acta Biotechnol., Berlin 9 (1989) 2, 9 7 - 200

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Atta Biotecfemlonin Journal of microbial, biochemical and bioanalogous technology

Edited by the Institute of Biotechnology of the Academy of Sciences of the G.D.R., Leipzig and by the Kombinat of Chemical Plant Construction Leipzig—Grimma by M. Ringpfeil, Berlin and G. Vetterlein, Leipzig

Editorial Board: D. Meyer, Potsdam P. Moschinski, Lodz A. Moser, Graz M. D. Nicu, Bucharest Chr. Panayotov, Sofia L. D. Phai, Hanoi H. Sahm, Jülich W. Scheler, Berlin R. Schulze, Halle B. Sikyta, Prague G. K . Skryabin, Moscow M. A. Urrutia, Habana

1989

A. A. Bajev, Moscow M. E. Beker, Riga H. W. Blanch, Berkeley S. Fukui, Kyoto H. G. Gyllenberg, Helsinki G. Hamer, Zurich J. Holló, Budapest M. V. Ivanov, Moscow L. P. Jones, El Paso F. Jung, Berlin H. W. D. Katinger, Vienna K . A. Kalunjanz, Moscow J. M. Lebeault, Compiègne

Number 2

Managing Editor:

L. Dimter, Leipzig

Volume 9

A K A D E M I E -V E R L A G

B E R L I N

"Acta Biotechnologica" publishes original papers, short communications, reports and reviews from the whole field of biotechnology. The journal is to promote the establishment of biotechnology as a new and integrated scientific field. The field of biotechnology covers microbial technology, biochemical technology and the technology of synthesizing and applying bioanalogous reaction systems. The technological character of the journal is guaranteed by the fact that papers on microbiology, biochemistry, chemistry and physics must clearly have technological relevance. Terms of subscription for the journal "Acta Biotechnologica" Orders can be sent — in the GDR: to Postzeitungsvertrieb or to the Akademie-Verlag Berlin, Leipziger Str. 3 - 4 , P F 1233, DDR-1086 Berlin; — in the other socialist countries: to a bookshop for foreign languages literature or to the competent news-distributing agency; — in the FRG and Berlin (West): to a bookshop or to the wholesale distributing agency Kunst und Wissen, Erich Bieber oHG, Postfach 102844, D-7000 Stuttgart 10; — in the other Western European countries: to Kunst und Wissen, Erich Bieber GmbH, General Wille-Str. 4, CH-8002 Zürich; — in other countries: to the international book- and journal-selling trade, to Buchexport, Volkseigener Außenhandelsbetrieb der DDR, P F 160, DDR-7010 Leipzig, or to the Akademie-Verlag Berlin, Leipziger Str. 3—4, P F 1233, DDR-1086 Berlin.

Acta Biotechnologica Herausgeber: Institut für Biotechnologie der AdW der DDR Permoserstr. 15, DDR-7050 Leipzig (Prof. Dr. Manfred Ringpfeil) und VEB Chemieanlagenbaukombinat Leipzig—Grimma Bahnhofstr. 3—5, DDR-7240 Grimma, (Dipl.-Ing. Günter Vetterlein). Verlag: Akademie-Verlag Berlin, Leipziger Straße 3—4, P F 1233, DDR-1086 Berlin; Fernruf: 2236201 und 2236229; Telex-Nr.: 114420; Bank: Staatsbank der DDR, Berlin, Konto-Nr.: 6836-26-20712. Redaktion: Dr. Lothar Dimter (Chefredakteur), Martina Bechstedt, Käthe Geyler, Permoserstr. 15, DDR-7050 Leipzig; Tel.: 2392255. Veröffentlicht unter der Lizenznummer 1671 des Presseamtes beim Vorsitzenden des Ministerrates der Deutschen Demokratischen Republik. Gesamtherstellung: VEB Druckhaus „Maxim Gorki", DDR-7400 Altenburg. Erscheinungsweise: Die Zeitschrift „Acta Biotechnologica" erscheint jährlich in einem Band mit 6 Heften. Bezugspreis eines Bandes 198,— DM zuzüglich Versandspesen; Preis je Heft 33,— DM. Der gültige Jahresbezugspreis für die DDR ist der Postzeitungsliste zu entnehmen. Bestellnummer dieses Heftes: 1094/9/2. Urheberrecht: Alle Rechte vorbehalten, insbesondere der Übersetzung. Kein Teil dieser Zeitschrift darf in irgendeiner Form — durch Photokopie, Mikrofilm oder irgendein anderes Verfahren — ohne schriftliche Genehmigung des Verlages reproduziert werden. — All rights reserved (including those of translation into foreign languages). No part of this issue may be reproduced in any form, by photoprint, microfilm or any other means, without written permission from the publishers. © 1989 by Akademie-Verlag Berlin. Printed in the German Democratic Republic. AN (EDV) 18520 03000

Acta Biotechnol. 9 (1989) 2, 99—110

Akademie-Verlag Berlin

Optimization of Single Cell Protein Production from Cassava Starch (Rhizopus oligosporus) SUKABA, E . 1 , DOELLE, H . W . 2 *

1

2

Pusat Penelitian den Pengembangan Bioteknologi, Lembaga Umu Pengetahuan Indonesia (LIPI), Ji. Raya Juanda No. 18, Bogor, Indonesia Department of Microbiology, MIRCEN-Bioteehnology, University of Queensland, St. Lucia, Brisbane, Qld 4067, Australia

Summary The fermentation pattern of cassava starch utilization was investigated at 37 °C using Rhizopus oligosporus UQM 145F and eight different media. Depending on the medium used, the addition of zinc or zinc plus iron to a combination of calcium plus manganese switches the fermentation from glucose accumulation to biomass (single cell protein) production. Complete starch hydrolyzation was obtained in both cases, with a complete glucose utilization resulting in 24 g biomass containing 30% true protein per 100 g cassava starch ( = 7.45 g SCP/100 g substrate) in 24 hours. In the case of glucose accumulation, biomass was kept low and 15.5 g/1 glucose representing 57.3% of starch supplied were obtained in 36 hours. B. oligosporus UQM 145 F grows well between 30° and 45 °C. At 45 °C and p H 5.0, 7.0 g SCP/100 g substrate were obtained, which rose to 8.6 g if cassava starch is replaced by ground cassava tuber.

Introduction The potential use of single cell protein as an alternative supplement in animal and human diet has promoted research on the microbial fermentation of various substrates including cassava [1, 2]. Apart from the conversion of starchy materials into high quality microbial protein, the major product of starch saccharification, glucose, can also be used for numerous yeast and bacterial fermentations to be used in pharmaceutical industries or for the production of ethanol, acids and other valuable products [2]. Starch saccharification by acid or enzyme hydrolysis is commonly used in t h e industrial ethanol and fructose sweetener production in the USA [3]. I n order to increase the economics of any of these industries, flexibility and simultaneous or alternate multiple product formation will become a future necessity, if natural renewable resources are increasingly used as feedstocks. Although a large variety of fungi have been grown on cassava for single cell protein production [4, 5], the filamentous fungal genus Rhizopus has attracted the attention of researchers owing to its non-toxic nature and high glucoamylase activity [6, 7]. A detailed study into the fermentation pattern of Rhizopus oligosporus [8] indicated the possibility of an economic single cell protein production using cassava starch as a feedstock. * To whom correspondence should be addressed. 1*

100

Acta Biotechnol. 8 (1989) 2

This paper describes the effect of different types of media and the effect of micronutrient supplementation, incubation temperature, and pH on Rhizopus oligosporus, single cell protein. Materials and Methods Microorganism. Rhizopus oligosporus UQM 145 F was obtained from the Culture Collection of the Department of Microbiology, University of Queensland, Australia and was maintained on potato-dextrose agar slants at 5 °C. Preparation of inoculum. The spore inoculum was prepared on polished rice using the solid state fermentation technique of WANG et al. [9]. Cassava Starch Media. The medium composition proposed by BROOK et al. [10], STANTON & WALLBRIDGE [ 1 1 ] , READE & GREGORY [ 1 2 ] , MEIRING e t al. [13], ALAZARD & RAIMBATJLT [ 1 4 ] , SANTOS e t al. [15], RAMOS-VALDIVIA e t al. [ 1 6 ] a n d a m o d i f i c a t i o n [ 1 7 ] w e r e

used. In all cases, cassava starch at the concentration of 30 g/1 was used as a sole source of carbon and energy. In addition to cassava starch, the last medium contains 2.7 g/1 magnesium sulphate (hydrated), 6.8 g/1 sodium chloride, 3.5 g/1 disodium hydrogen phosphate, 2.0 g/1 potassium dihydrogen phosphate and 12 g/1 filter-sterilized urea. The initial pH of the medium was 4.0. Micronutrient supplementation was carried out using 0.001 g/1 of each, hydrated calcium chloride, hydrated manganese sulfate, hydrated zinc sulfate and ferrous sulfate or as further indicated. Cultivation and Harvesting. Flasks (250 ml), each containing 50 ml sterile medium, were inoculated aseptically with a 2.3% (v/v) spore inoculum of R. oligosporus and incubated on a constant temperature water-bath shaker (104 strokes/min) at 37 °C and harvested after 12, 24, 36, 48, 60, and 72 hours fermentation or as indicated. The cultures were filtered, and the filtrate analyzed for starch and glucose, whereas the biomass was thoroughly washed with distilled water, oven-dried at 60 °C to a constant weight. Analytical Determination. The true protein of microbial biomass was estimated with the biuret method [18]. Glucose was estimated using the modified method of NELSON [19]. Starch was estimated using a slight modification of the method of SMITH & ROSE [20]. Approprietly diluted samples containing 10—100 (xg/ml starch were made up to 5.0 ml with distilled water. One ml of a K I - I solution (30 g K I and 3.0 g I in 1.01 distilled water) was added to the above sample and the colour intensity was read at 580 nm against a reagent blank in a BAUSCH-LOMB SPECTRONIC 20 spectrophotometer. The starch concentration in the sample was obtained by extrapolating against a standard curve. Experimental Design for Computation. The experiments to evaluate the effect of micronutrient requirements were designed according to MCDANIEL et al. [21]. The following regression equation was used: y = K + M i + b2x2 + bnxn

+ bnxl2

+ b22x22

where y = predicted yield, x1 and x2 = levels of the two variables tested, bx and b2 = linear coefficients, bn and b22 = quadratic coefficients, bl2 = cross product coefficients, b0 = constant. For the second design involving as variables cassava starch, 3.0—6.0% (w/v), urea 0.5—1.5% (w/v) and magnesium sulfate, 0.05—0.35% (w/v), the following regression equation was used: V — b0 + blx1 + b2x2 + 63X3 + b12XiX2 + b^x^ -f" bll%12

&22-C22 "I" b33X32 -f- 6223^1^2*^3

+ b23x2x3

SUKABA, E., DOELLE, H. W., Single Cell Protein Production

101

Results The cassava starch utilization patterns in relation to single cell protein production using Rhizopus oligosporus under the influence of a number of micro- and macro-nutrients have been investigated. Growth Pattern of Rhizopus oligosporus The growth behaviour of R. oligosporus under different nutritional conditions using cassava starch as the sole carbon and energy source is given in Fig. 1. Biomass production was greatly affected by the nutritional composition of the medium. The medium composition proposed by [12], containing Ca, Fe, Zn and urea, was found to be the best among all of the media tested for biomass production. The amount of the biomass being recovered after 44 hours fermentation from this medium was 18.0 g/100 g of initial substrate. Close observation indicated that the biomass production using the media without Zn supplementation, with the exception of the S U K A B A & D O E L L E medium, was always much lower compared to that obtained using R E A D E & G R E G O R Y medium. R E A D E & G R E G O R Y medium together with other media containing Zn were also found to be suitable media for the synthesis of protein. The highest protein contents of the biomass observed were 39.71,39.68, and 32.15% obtained from the media described by [11, 12, 15], respectively. The newly formulated medium and the medium proposed by M E I R I N G [13] and his associates, using urea as a sole source of nitrogen at the concentration of 1.2 and 0.35% favour the production of glucose. The addition of Zn, Fe, Mn and Ca to the S U K A R A & D O E L L E medium resulted in the production of 24 g biomass/100 g initial substrate, containing 30.14% protein ( = 7.45 g single cell protein/100 g initial substrate). The rate of the hydrolysis of cassava starch by Rhizopus oligosporus was positively correlated with the production of biomass, protein and glucose. Using [11, 12] and the newly formulated medium, more than 80% of the cassava starch was utilized after 44 hours fermentation. This utilization was increased to 100% in 36 hours after the addition of Ca, Fe, Mn and Zn to the S U K A R A & D O E L L E medium. Although the experiments were started with the same initial pH of 4.0, the final pH observed varied considerably. A high final pH value enhanced the production of biomass and protein synthesis and the utilization of cassava starch. A slightly lower final pH seems to be better for accumulation of glucose. If the final pH falls below 3.0, however, the utilization of cassava starch was significantly inhibited. In order to explore this differential behaviour of cassava starch fermentation further and with an aim towards the improvement of the single cell protein production and complete starch utilization, the second medium developed in our laboratory was used. Significance of Micronutrient Addition Regression analysis of the data obtained indicated that cassava starch concentrations between 3 and 6% (w/v) do not affect single cell protein production. Similarly, urea between 0.5 and 1.5% (w/v) and magnesium sulphate between 0.005 and 0.35% (w/v) are not significantly important for the production of single cell protein by Rhizopus oligosporus grown on the newly formulated S U K A R A - D O E L L E cassava starch medium. In the case of zinc and calcium, however, a positive effect was obtained (Tab. 1) with the influence of zinc on the process being more significant compared to the effect of calcium. In terms of g of protein/100 g initial substrate, an increase in the concentration of zinc from 0 to 0.005% (w/v) resulted in an increase in single cell protein production

102

Acta Bioteohnol. 9 (1989) 2 ¿0 36 32 28

2i r 20 16 12

8

U 0

J

24 20 16

f.

12

8 U 0

m

H

ti Fig. 1. The growth behaviour of Rhizopus oligosporus UQM 145 F under different nutritional conditions using cassava starch as the sole carbon and energy source A. Biomass production [g/100 g initial substrate] B. True protein content of biomass [%] C. Protein production (g/100 g initial substrate) D. Glucose production [g/1 filtrate] E. Starch utilization [%] F. Final p H \///\

[10] without trace metal [11] with Fe, Mn and Zn [12] with Ca, Fe and Zn [13] without trace metal

mrm

[14] with trace metal [15] with Zn [16] with Ca and Fe [17] without trace metal

E S3 E m

103

Sukaba, E., D o e l l e , H. W., Single Cell Protein Production

Tab. i . Experimental design and results of the effect of calcium and zinc on the production of biomass and protein synthesis by Rhizopus oligosporus U Q M 145 F grown on cassava starch medium* at an initial p H of 4. The culture was inoculated with 2.28% (v/v) of spore suspension and incubated in a water bath shaker at 37 °C for 24 h with the agitation of 76 strokes per minute. 1 — Biomass (g/100 g initial substrate) 2 — True protein content of biomass ( % ) 3 — Protein yield (g/100 g initial substrate) Trials

Variables

Yield

ZnS0 4 • 7 H 2 0 [ % • w/v]

CaCl2 • 2 H 2 0 [ % • w/v]

1

0.0015

0.0015

2

0.0085

0.0015

3

0.0085

0.0085

4

0.0015

0.0085

5

0.0000

0.0050

6

0.0100

0.0050

7

0.0050

0.0000

8

0.0050

0.0100

9

0.0050

0.0050

1 21.53 22.21 23.38 22.37 21.81 20.52 21.43 22.89 22.11 17.25 21.71 21.47 8.67 7.40 7.37 22.35 21.59 21.21 21.75 21.97 22.50 23.79 23.03 23.37 24.93 23.15 21.97

30.26 33.00 29.00 21.74 20.83 27.68 22.56 19.09 21.35 33.50 41.67 34.50 21.10 16.79 17.37 31.84 24.18 33.13 32.69 30.71 30.51 26.61 . 35.09 29.38 30.28 31.51 37.79

6.51 7.33 6.78 4.86 4.54 5.68 4.83 4.37 4.72 5.78 9.05 7.41 1.83 1.24 1.28 7.12 5.22 7.03 7.11 6.75 6.86 6.33 8.08 6.87 7.55 7.29 8.30

Regression analysis Parameter

Computed't' value for protein production

ZnS0 4 -7H 2 0(X 1 ) CaCl 2 -2H 2 0 (X 2 ) X j square X 2 square X j X 2 (cross product)

3.034** 0.585 2.986 0.4197 0.4600

* Medium composition (w/v): cassava starch 3%, urea 1.2%, MgS0 4 • 7 H 2 0 0.27%, Na 2 HP0 4 0.35%, K H 2 P 0 4 0.20%, NaCl 0.68%, FeS0 4 • 7 H 2 0 0.001%; and MnS0 4 -5H 2 0 0.001%. The medium was supplemented with CaCl2 • 2 H 2 0 and ZnS0 4 • 7 H 2 0 at indicated levels. ** Significant at P = 0.05

10 8

I

6

U 2 0

SL

LIidi H J

• - 30° C

L i

35° C

CL

-

¿0° C

E-

¿5°

Fig. 2. The effect of temperature and different inocula on the growth behaviour and SCP production of Rhizopus oligosporus UQ.M 145 F at 24 h [I] and 30 h [II], with inoculum size of 1 % (1); 2 % (2); 3 % (3) and 4 % (4) A. Biomass production [g/100 g initial substrate] D. Glucose production [g/1 filtrate] B. True protein content biomass [%] E. Starch utilization [%] C. Protoin production [g/100 g initial substrate F . pH profile

SUKABA,

E.,

DOELLB,

H. W., Single Cell Protein Production

105

from 2.7 to 7.2 g. The addition of 0.007% of calcium lead to a further increase to 8.13 g protein/100 g initial substrate. Using these optimal concentrations of zinc (0.005%) and calcium (0.007%), magnesium exhibited a positive effect at 0.2% (w/v), leading to a single cell protein value of 8.6 g/ 100 g initial substrate. Effect of Physical

Factors

After optimization of the single cell protein formation at a growth temperature of 37 °C it was decided to test a higher temperature range for the use of single cell protein production in tropical countries. A temperature range of 30° to 45 °C was chosen for our experiments and the results are outlined in Fig. 2. In raising the temperature from 30° to 40 °C, an increase in biomass production was noted after 24 hours incubation with almost all levels of inocula. The highest biomass yield of 22.22 g/100 g initial substrate containing 31.56% true protein was obtained at 40°C with an inoculum size of 3% (v/v). If the temperature was increased to 45°C, the biomass decreased in general. However, an increase in inoculum size from 1 to 4 % (v/v) resulted in an increase in biomass yield at 45 °C. In contrast to the biomass yield, the true protein of the biomass at 45 °C was higher in all cases of inoculum compared to lower temperatures. The highest true protein content of 37.28% was obtained with an inoculum size of 4% (v/v) with a concomitant yield of 18.86 g biomass/100 g cassava starch. Despite the fact that the medium used was optimized for protein and biomass production, glucose accumulation could still be detected at 30° and 45 °C. At 30 °C the accumulation of glucose decreased by increasing the size of the inoculum. For example, the highest glucose accumulation of 7.55 g/1 occurred with a 1% inoculum, it decreased to 1.7 g/1 using a 4% inoculum. The highest amount of glucose accumulated at 45°C with a 2% inoculum (9.62 g/1) decreased to 5.3 g/1 by increasing the inoculum size to 4%. In all cases of our experiments, more than 95% of the starch was utilized after 24 h •and not less than 98% at 30 h. At 35° and 40 °C, a total utilization of cassava starch was achieved after 24 h, especially by using high levels of inocula. I n almost all cases, especially after 24 h, the final p H of the growth culture increased with increasing inoculum size. The highest final p H measured was 8.06 at 40 °C with a 4 % inoculum size. I n order to explore this potential of biomass and protein correlation further, the combined effect of initial p H and incubation time on the fermentation pattern of B. oligosporus was further investigated, using the optimal conditions of 45 °C and 4 % inoculum size. These studies were carried out using the special experimental design with the computation of the final regression coefficient. The results are illustrated in Fig. 3. An initial p H between 5 and 6 appears to favour biomass formation, whereas an initial p H of 3 and 7 appears to favour glucose accumulation. Although the synthesis of protein was better in the initial stages of incubation at lower initial pH, it decreases sharply with further incubation time. The lowest protein synthesis was observed at an initial p H of 7, whereas an initial p H of 5.0 produced the best value with 7.8 g protein per 100 g initial cassava starch after 36 h incubation. In modifying the regression equation fitted for the protein production with simple calculus, the optimum initial p H and time of incubation could be calculated as p H 5.21 and 35.13 h respectively with an expected protein production of 7.93 g per 100 g initial cassava starch. These calculations were confirmed, as the amount of 7.9 g protein per 100 g cassava starch were obtained after 27 h incubation. I t was shown further that the protein content can be increased from 7.9 to 8.6 g per 100 g initial substrate if ground cassava tuber replaced the cassava starch.

106

Acta Biotechnol. 9 (1989) 2

_

12

18

2U 30

36

12

18

2 U 30

36

hi A8 Time Ch]

12

18

1U

30

36

U2 U8 Time Chi

12

18

24

30

36

¿2 ¿8 Time Ch]

C ¿2

¿8

Time Ch]

Initial pH

ZU

30

36

42 48 Time Ch3

O

30

•

4-0

•

5-0

•

6-0

A

7-0

Fig. 3. The effect of initial pH and incubation time on SCP production of Rhizopus UQM 145 F at 45 °C using a 4 % inoculum A. Biomass production [g/100g initial substrate] B. Glucose production [g/1 filtrate] C. True protein content of biomass [%] D. Protein production [g/100 g initial substrate] B . Starch utilization [%]

oligosporus

107

SUKAKA, E., DOELLB, H. W., Single Cell Protein Production

Bubble Fermenter The fermentation was now transferred from shaken flask to a bubble fermenter equipped with a hollow draft tube and a working volume of 1.5 1. Using 1 % initial substrate, the highest biomass produced was 28.65 g per 100 g cassava starch containing 25.12% true protein (Tab. 2). This gives a total single cell protein of 7.2 g per 100 g initial substrate. In altering the composition of the medium to contain (w/v): 1 % ground cassava tuber, 0.3% urea, 0.05% magnesium sulphate, 0.001% calcium chloride and 0.2% NaCl, the amount of 50.54 g dry biomass/lOOg initial substrate could be harvested after only 12 h. Although the protein content of the biomass was only 15.4%, the value of single cell protein was 7.87 g/100 g substrate (Tab. 3). I t is of interest to observe, that the significant effects of micronutrients on cassava starch did not apply in the case of cassava tubers. We have therefore demonstrated that it is possible to produce a single cell protein of high quality (8 g/100 g cassava tuber) in 12 h using a bubble fermenter. Work is progressing for further scale-up of this process using ground cassava tuber. Tab. 2. Fermentation pattern of ground cassava tuber by Rhizopus oligosporus UQM 145 F cultivated in bubble column fermenter equipped with a hollow draft tube with an initial substrate concentration of 1% (w/v) 1 — Biomass (g/100 g of initial substrate); 2 — True protein content of biomass (%); 3 — Protein yield (g/100 g of initial substrate); 4 — Glucose (g/1 filtrate); 5 — Starch utilization (%); 6 — pH profile; 7 — Dissolved oxygen profile (ppm) Time [h]

1

2

4

3

0 3

—

—

6

It). SO

9.09

1.53

9

22.55 ±1.20 28.65 21.70 22.60

14.98 ±1.82 25.12 29.82 33.66

3.37 ±0.22 7.20 6.47 7.61

12 15 18

0.32 ±0.11 0.54 ±0.23 0.96 ±0.18 1.47 ±0.03 0.91 0.29 0.36

5 0 21.49 23.09 51.71 41.87 71.70 75.16

6

7

5.25 ±0.01 5.28 ±0.11 5.45 ±0.07 5.60 ±0.23 6.20 5.92 6.63

6.95 ±2.05 6.71 ±3.24 4.90 ±3.54 2.30 ±0.14 2.40 0.50 —

Medium composition (w/v): 1.0% of ground cassava tuber, 1.0% of urea, 0.50% of (NH 4 ) 2 S0 4 , 0.30% of MgS0 4 -7H 2 0, 0.15% of KH 2 P0 4 , 0.10% of Na 2 HP0 4 , and 0.005% of ZnS0 4 • 7 HaO. The medium was inoculated with 8% (v/v) of spore suspension. The culture was incubated at 40 °C and aerated with sterile filtered air at the flow rate of 50 1/h.

Discussion

Although the conversion of cassava starch into biomass and other valuable products by microfungi attracted many scientiests, few literature reports are available on nutritional studies for S C P and glucose production. In [22], G B A Y and ABOTX-EL-SEOUD reported that the addition of vitamins and minerals may enhance or inhibit the protein production depending upon the microorganisms used. Using Cladosporium cladosporioides 1-83, the addition of vitamins and minerals into the medium containing 50 g/1 of unpeeled cassava root, 1.0 g/1 of ammonium chloride, and 2.0 ml/1 corn steep liquor could improve the

Acta Biotechnol. 9 (1989) 2

108

Tab. 3. Fermentation pattern of ground cassava tuber by Rhizopus oligosporus UQM 145F cultivated in bubble column fermenter equipped with a hollow draft tube with an initial substrate concentration of 1% (w/v) in the presence of sodium chloride and calcium (CaCl 2 -2H 2 0) with low levels of urea (0.30%, w/v) and MgS0 4 -7H a 0 (0.05% w/v) without supplementation of (NH 4 ) a S0 4 and Z n S 0 4 - 7 H a 0 i — Biomass (g/100 g of initial substrate); 2 — True protein content of biomass (%); 3 — Protein yield (g/100 g of initial substrate); 4 — Glucose (g/1 filtrate); 5 — Starch utilization (%); 6 — pH profile; 7 — Dissolved oxygen profile (ppm) Time [h]

1

2

3

0 6 9 12

4

5 0.01

20.02 ±2.09 31.58 ±4.24 50.54 ±9.17

6.72 ±0.91 12.30 ±3.84 15.42 ±1.68

1.22 ±0.05 3.96 ±1.73 7.87 ±2.26

0.13 ±0.01 0.22 ±0.00 0.26 ±0.08

0 16.77 30.99 43.40

6

7

5.36 ±0.12 5.32 ±0.20 5.72 ±0.09 6.02 ±0.07

5.15 ±0.21 2.80 ±0.57 1.85 ±0.35 1.30 ±0.28

Medium composition (w/v): 1.0% of ground cassava tuber, 0.30% of urea, 0.005% of MgS0 4 -7H 2 0, 0.15% of KH 2 P0 4 , 0.20% of NaCl, 0.01% of CaCl 2 -2H a O. The medium was inoculated with 8% (v/v) of spore suspension. The culture was incubated at 40 °C and aerated with sterile filtered air at the flow rate of 50 1/h.

protein production from 1.59 to 3.04 g/1 after 4 days incubation. In contrast, when Gladosporium sp. 1—75 was used, the addition of the same vitamins and minerals reduced the production of protein from 2.32 to 1.76 g/1 after the same time of incubation. In [ 1 1 ] , STANTON and W A L L B R I D G E found that various trace metals are important for biomass production from cassava by microfungi. Their results show that Fe, Mn, and Zn may inhibit or stimulate the production of biomass by R. oligosporus depending on the particle size of cassava flour used. Our studies show that trace metals are not only important for the production of biomass, but also play an important role on the synthesis of protein and glucose production. Depending on the medium supplementation used, the addition of Zn or Zn and F e to a combination of Ca plus Mn could switch the fermentation from glucose accumulation to biomass production. R. oligosporus was also used by R A M O S - V A L D I V I A and his coworkers in [ 1 6 ] for the production of S C P from cassava. Their process using 20 g/1 cassava flour was able to produce 18.5 g of crude protein/100 g initial substrate. When the medium proposed by R A M O S - V A L D I V I A et al. [16] was used using cassava starch as carbon source for the production of SCP by R. oligosporus UQM 145 J 1 , the protein produced was only 1.8 g/100 g initial substrate. Our studies using R E A D E and G R E G O R Y medium and S U K A R A and D O E L L E medium showed that the presence of Ca in combination with F e (the two micronutrients present in R A M O S - V A L D I V I A medium) contribute to the detrimental effect on the protein production. The inhibitory effect, however, can be nullified by the addition of Mn and Zn. Studies on the direct saccharification of cassava starch to glucose by microfungi are very scarce. TAN and his co-workers [1] are probably the only one's who studied the conversion of cassava starch into biomass, reducing sugars and organic acids using Aspergillus niger. The highest biomass produced was 27.14 g/100 g cassava starch with a total accumulation of reducing sugars of 3.426 g/1 after 42 h and a 7 0 % starch utilization. With the right combination of micronutrients in our investigations, complete starch and glucose utilization can be obtained with the formation of 24 g biomass/100 g cassava

STTKABA, E., DOELLE, H. W., Single Cell Protein Production

109

starch containing 30% true protein with an incubation time of only 24 h, or alternatively, 15.5 g/1 glucose representing 57% of the initial starch. I n regard to environmental conditions the presented results confirm the finding of BERY [23], that temperature of incubation and p H control the growth of fungi. They are, however, in contrast to BROOK et al. [10], who found that the optimum temperature for the growth of R. oligosporus and R. stolonifer on cassava flour was 30 °C with the initial p H of 5.5. READE and'GREGORY [12], who worked with Aspergillus fumigatus reported an optimum temperature of 45 °C and an optimum pH of 3.5 for protein production. The presented results indicated that the optimum temperature for SCP production was 40 °C, whereas at 45-°C, protein production may be improved by increasing the inoculum size from 1 to 4%. The optimum p H for an incubation temperature of 45 °C was found to be 5.0. The amount of SCP being produced using 18 strains of microfungi including 4 strains of R. oligosporus [10] is reported to vary between 3.13 and 6.13 g/100 g of initial substrate after 3 days incubation. This is considerably lower to the ones reported here. I t has also been reported [15] that using urea as a nitrogen source, the p H of the growth culture would remain stable during the course of fermentation by Aspergillus fumigatus I-21A. I n our experiments, the pH increased rather sharply after 24 h fermentation from 4.0 to more than 6.0, especially in the cases of high inocula. The final p H could in fact reach a value of 8.06 at 40 °C and an inoculum size of 4%. READE and GREGORY [12] reported that in the case of A. fumigatus, only a slight fluctuation in p H around the optimal 3.5 would be detrimental to the protein yield. Our results, on the other hand showed, that R. oligosporus UQM 145 F seem to be more resistant to the change of pH. However, with an initial p H of less than 4 and higher than 7, the fermentation pattern of R. oligosporus switches from protein to glucose production [24]. The possibility to obtain higher protein yields with cassava tuber than with cassava starch confirms earlier reports [10, 22] that the cruder the flour, the better the fermentation and lesser needs for nutrient supplementation. The presented investigation revealed that there are several advantages of using R. oligosporus UQM 145 F for the production of SCP from cassava. Firstly, the fungus is able to grow directly on cassava and no additional enzymes are required to hydrolyze the starch. Secondly, this fungus is also able to grow at elevated temperatures and reasonably low pH, which are extremely valuable characteristics for a fermentation application in tropical countries. Finally, the fungus is known to be free of toxin production and has actually been incorporated into the daily diet of many people, especially in Indonesia. Received June 1, 1988

Acknowledgement We are grateful for the financial assistance received from the University of Queensland and the Australian Development Assistant Bureau (ADAB) under the Commonwealth Scholarship Plan.

References [1] TAN, K. H., FERGUSON, L. B., CARLTON, C.: J. Appl. Biochem. 6 (1984), 80. [2] TUSE, D.: CRC Crit. Rev. Food Sci. Nutr. 19 (1984), 273. [3] TORREY, S. — In: Microbiological Synthesis. Ed. TORREY, S. NOYES, Data Corp Park Ridge, N.S., 1983. 1 7 4 - 2 0 1 .

Acta Biotechnol. 9 (1989) 2

110 [4] OPOKU, A . R . , ADOGA, P . A . :

E n z y m e Microb. Technol.

2 (1980), 241.

[5] SMITH, R . E . , OSOTHISILP, C., BICHO, P . , GREGORY, K . F . : [6] UEDA, S., KANO, S . :

Starch

Biotechnol. L e t t .

8 (1986), 31.

27 (1975), 123.

[7] UEDA, S., SAHA, B . C., KOBA, Y . : M i c r o b i o l . Sci. 1 ( 1 9 8 4 ) , 2 1 .

E., D O E L L E , H . W . : M I R C E N J . Appi. Microbiol. Biotechnol. 1 9 8 8 . [9] W A N G , H . L., S W A I N , E. W . , H E S S E L T I N E , C. W . : J . Food Sci. 40 (1975), 168. [10] B R O O K , E . J . , S T A N T O N , W. R., W A L L B R I D G E , A.: Biotechnol. Bioeng. 9 (1969), 1271. [11] S T A N T O N , W . R., W A L L B R I D G E , A.: Process Biochem. April (1969), 45. [12] READE, A. E., GREGORY, K . F . : Appi. Microbiol. 30 (1975), 897. [13] M E I R I N G , A . G., A Z I , F. A . — I n : Proc. Workshop "Cassava as animal feed". Eds. N E S T E L , B., G R A H A M , M . O t t a w a : Univ. Gueph, 1 DRC-095c, 1977. 7 9 - 8 4 . [14] ALAZARD, D., RAIMBAULT, M.: E u r . J . Appi. Microbiol. Biotechnol. 12 (1981), 113. [ 1 5 ] S A N T O S , J . , G U I L L E R M O , G . , A L E X A N D E R , G . C . : Anim. Feed Sci. Technol. 8 ( 1 9 8 3 ) , 3 1 3 . [ 1 6 ] R A M O S - V A L D I V I A , A., D E LA T O R R E , M . , C A S A S - C A M P I L L O , C . — I n : Production and feeding of single cell protein. Eds. FERRANTI, P., FIÉCHTER, A. Essex U K : Applied Sci., 1983. 104 bis 111. [17] SUKARA, E., DOELLE, H . W. : Proc. V l l t h Austral. Biotech. Conf. Melbourne Australia, 1986. 356-360. [ 1 8 ] H E R B E R T , D . , P H I P P S , P . J . , S T R A N G E , R . E. — I n : Methods in Microbiology. Eds. N O R R I S , J . R . , R I B B O N S , D . W . , Vol. 5 B . London: Academic Press, 1 9 7 1 . 2 0 9 — 3 4 4 . [19] N E L S O N , N . : J . Biol. Chem. 158 (1944), 375. [20] SMITH, B. W., RHOSE, J . H . : J . Biol. Chem. 179 (1949), 53. [21] M C D A N I E L , L. E . , B A I L E Y , E . G., E T H I R A J , S., A N D R E W S , H. P . — I n : Developments in Industrial Microbiology. Ed. UNDERKOFLER, L. A., 17, Virginia, USA: Soc. Industrial Microbiology, 1976. 9 1 - 9 8 . [ 2 2 ] G R A Y , W . D., A B O U - E L - S E A U D , M. O. : Econ. Bot. 2 0 ( 1 9 6 6 ) , 2 5 1 . [ 2 3 ] B E R R Y , D . R . — I n : The filamentous fungi Vol. I : Industrial mycology. Eds. S M I T H , J . E., [ 8 ] SUKARA,

BERRY, D . R . L o n d o n : E d w a r d A r n o l d , 1975. 1 7 - 3 2 .

[24]

GARG, S.

K.,

DOELLE,

H . W . : MIRCEN J . Appi. Microbiol. Biotechnol. 1989 (in press).

Book Review 0 . KANDLER, W .

ZILLIG

Archaebakterien '85 Stuttgart, New York: Gustav Fischer Verlag, 1986. 430 S., 284 Abb., DM 158,— ISBN 3-437-11057-8 Das Buch „Archaebakterien '85" f a ß t die Vorträge u n d Poster des EMBO Workshop „Molekulare Genetik der Archaebakterien" und des Internationalen Workshop „Biologie und Biochemie der Archaebakterien" (München, 1985) zusammen. Die vorgestellten Ergebnisse reichen von den molekularbiologischen Grundlagen des Lebens u n t e r extremen physiko-chemischen Bedingungen, bis hin zur Mikrobiologie u n d Biochemie der Archaebakterien. Beeindruckend sind die in den letzten J a h r e n gemachten Fortschritte auf dem Gebiet der Gentechnik. Eine Vielzahl von Genen wurde aus Archaebakterien isoliert, charakterisiert u n d in Eubakterien kloniert. Diese Untersuchungen sind auf die Konstruktion von Hochleistungsstämmen zur effektiven Synthese thermostabiler/thermophiler Enzyme gerichtet. Das Buch ist Naturwissenschaftlern, die sich über den internationalen S t a n d der Erforschung und praktischen N u t z u n g von Archaebakterien informieren wollen, zu empfehlen. B . HEINIÜTZ

Acta Biotechnol. 9 (1989) 2, 1 1 1 - 1 2 1

Akademie-Verlag Berlin

Macromixing Characteristics of Gas-Liquid Jet Loop Reactors WABNECKE, H . - J .

Technische Chemie und Chemische Verfahrenstechnik Universität-Gesamthochschule Paderborn Warburger Str. 100, D-4790 Paderborn, PEG

Summary Measurements of liquid macromixing characteristics are reported for a half industrial scaled jet loop reactor operating with air-water mixtures. Based on a model of loop reactors with sections of different mixing behavior the single circulation dispersion coefficient can be split into its components caused by the riser and the downcomer. The dispersion coefficient of the riser is about 100 times greater than that of the downcomer. The addition of gas involves greater dispersions coefficients. The comparison of the mixing times of the JLR with those of stirred vessels leads to the conclusion that the JLR is equivalent or even superior to stirred vessels.

Introduction Because of their simple construction and operation and their defined mixing and intense dispersing effects with relatively low power requirements loop reactors are frequently used in chemical process technology and biotechnology. They are particularly suitèd for processes which demand rapid and uniform distribution of the reaction components, e.g. neutralization of waste water, and for multiphase systems for which high mass and heat transfer are necessary, e.g. fermentation processes [1—3]. If zones of high turbulence including microshear fields are required, e.g. for generation of high interfacial areas or for disruption of cell agglomerates, the jet loop reactor (JLR) with hydrodynamic jet flow drive is of special interest. The liquid jet causes the circulation drive and adequately fixed convective streams as well as turbulent backmixing and in heterogenous gas-liquid systems additionally an efficient primary dispersion and redispersion of the gasphase. The circulation can be altered over a wide range by adapting the geometry of the nozzle for the jet drive. A new method [5] based on a general model of loop reactors with sections of different mixing characteristics [4] is being applied to determine the mixing characteristics of the liquid, the single circulation BoDENSTEiNnumber Bo c and the recycle factor r from pulse response curves in the liquid phase of a gas liquid J L R . The BoDENSTEiNnumber Bo c can be split up into its components caused by the riser and the downcomer. Furthermore it allows the calculation of the mixing time required to achieve a certain degree of mixing. The mixing time is a suitable mixing parameter to compare the J L R with other reactors, e.g. stirred vessels.

112

Acta Biotechnol. 9 (1989) 2

Model and Parameter Estimation The general flow pattern of the model employed is shown in Fig. 1. The reactor consists of two parts, the riser and the downcomer with its corresponding residence times and variances. The variance is a measure of the BoDENSTEiNnumber. The molar feed flow Vf • Cf is mixed with the molar circulation flow V2 •c/ the flow of the downcomer.

Fig. 1. Plow pattern of loop reactor

The molar flow V f • c t leaves the reactor after the volumetric flow of the riser is split up into volumetric outlet flow F/and volumetric circulation flow F 2 . The characteristic parameters are summarized in Fig. 2. recycle factor

, =

F, r =

circulation number

nc =

residence times

tt = r

nc ~ 0.5 nc -f 0.5

Vi

- l i

circulation time

tu = tt +

mean residence time

r =

BoDENSTHiNnumber

Boi = ^ ¿ p

t x + rr 2 1 - r

Ffi Vj i = c, 1, 2

*

c: circulation Fig. 2. Model parameters JLR

1: riser

2: downcomer

WARNECKE, H.-J., Gas-Liquid Jet Loop Reactors

113

The characteristic shape of a response at the exit of a loop reactor subsequent to the addition of an approximately ideal Dirac impulse is shown in Fig. 3 by the solid line. The residence time distribution ( R T D ) can be understood phenomenally as follows. Because of the circulation the input peak is reproduced, diminished by the output at each circulation. The distance between two consecutive peaks is determined approximately by the time of each circulation, i.e. the mean circulation time tc. The recycle factor r characterizes the exponential decay of the concentration of the tracer. The greater r, the smaller is this decay. The relative height of the peaks and their number is determined by the axial dispersion of the reactor. The better the reaotor is mixed during one circulation, the stronger is the decay of consecutive peaks.

-_L =

tc

tn A E " n l

" AE(tn.|)

E(tm+At) Fig. 3. Residence time distribution J L R parameter estimation

T o understand the R T D quantitatively the tubular reactor with axial dispersion model including DANKWERTS boundary conditions is applied to the riser and the downcomer. This model is used as experimental data on axial mixing of multiphase flow systems evaluated on its basis agree well with those resulted from theoretical approaches. Then, a useful asymptotic formula for the R T D of the J L R is the sum of the first three eigenfunctions [5] E(t) ~ a 0 e»«' + 2Sl e~x>' cos (yj

+ f j ,

(1)

where s0, s t = —xl + iyu s2 = are the first three eigenvalues, which in turn are related to the main system parameters according to the approximations s0 = t f 1 In r

-

y1 ~ 2jrr c -1 x1 ^

-S

0

+

The circulation variance xi and y1 from the experimental RTD-Kurve. To achieve this, in principle, any curve fitting method can be used. However, there is a very simple procedure [5], which is demonstrated in Fig. 3. The solid line represents the experimental RTD, the dashed line the extrapolation of the exponential decay versus t = 0. The circulation time tc approximately equals the period of oscillation in RTD, hence it can be determined directly from the distance of two consecutive maxima in the middle range of t. The real part of the second eigenvalue emerges from the logarithmioal difference between consecutive peaks, diminished by the decay line. The first eigenvalue can easily be obtained by linear regression of the non oscillating part of the RTD. To obtain the system parameters of the individual sections the RTD at the end of the riser as well as at the end of the downcomer has to be measured. In Fig. 4 the former is shown by a solid line and the latter by a dashed line. The curves are dislocated according to the residence time T2 of the downcomer.

Fig. 4. Residence time distribution at the end of the riser ( —), and at the end of downcomer ( — ) parameter estimation

The logarithmical difference between the height of the j-th peaks of both curves placed on the corresponding decay function is the decisive measure of the variance in the downcomer, reps, of its BoDENSTEiNnumber Bo 2 . The variance of the riser follows from

« = »\ •*. "v* \\ ^ ^ \\ ^ ^ • •.s*. 1 \ oi

1 1 I I 1 0.001 0.1 1.0 100.0 ¿00.0 0 0.001 0.1 1.0 100.0 ¿00.0 0.0001 0.01 10.0 200.0 0.0001 0.01 10.0 200.0

0

Organic growth factor concentration CppmD

0.6

r

0^ ni

£

Q.

0.2

o

'

I

'

'

i

'

.'. N

I

Organic growth factor concentration CppmU

Fig. 1. Growth of Candida species on different concentrations of essential organic growth factors A — C. curvata Thiamine — Biotin Pyridoxine — Inositol - . - . B — C. steatolytica Thiamine — Biotin Pyridoxine • • • • Inositol C — C. curiosa Thiamine — Biotin Pyridoxine — Inositol D — C. catenulata Thiamine — Biotin Pyridoxine — Ascorbic acid A — A Xanthine • — • E — C. tropicalis Biotin

M A D AN,

M.,

K A M R A ,

N., Organic Growth Factor Requirements

147

brevis [7], E. ashbyii [8], C. parakrusei [9] and in filamentous fungi like G. cruenta [10], Rm. sacchari and Rm. sorghii [11]. However, the adverse effects of thiamine on the growth of certain yeasts have also been reported [5, 12—15]. The complete deficiency for biotin was observed in 5 auxotrophic yeasts while 2 yeasts species were partially deficient for it. Biotin auxotrophy is more common among the yeasts than in the filamentous fungi. In yeasts, it is reported in T. sphaerica, Sacch. logas, C. pseudotropicalis, K. brevis, Z. prioriamts and Z. japonicus [7], E. qshbyii [8], H. anómala [3], G. parakrusei [9] species of Rhodoturula [16], C. albicans [17], C. parapsilosis [5]. Four yeasts species viz. C. catenulata, C. curiosa, C. curvata and C. steatolytica were found to be wholly auxotrophic whereas partial deficiency has been found in G. tropicalis only. In requiring an external supply of pyridoxine for their optimum of these yeasts resemble Kloeckera, Torulopsis, Pichia and Brettanomyces [7], Sacch. carlsbergensis [18], C. parakrusei [9]. Among the filamentous fungi pyridoxine heterotrophy has been reported in Ophistoma spp. [6, 19]. C. pilifera [20], C. capsii [21] and R. sacchari [11], Inositol auxothrophy is almost always associated with a requirement for thiamine or biotin as it has been reported here in G. curiosa, G. curvata, C. steatolytica. Partial deficiency of inositol is observed for C. tropicalis only. Our findings are in a agreement with those of Z. priorianus, Z. japonicus and K. brevis [7], E. ashbyii [8], Schizosaccharomyes octosporus [22], Sacch. cerevisiae [22—24] and Sacch. carlsbergensis [25, 26], Among the filamentous fungi total or partial deficiency of inositol has been recorded in C. lindemuthianum [4], D. phaseolorum var. bataticola [27] and C. cruenta [10]. Partial deficiency for pantothenic acid has been reported in G. humicola only. Pantothenic acid auxotrophy is more frequent among the yeasts than in filamentous fungi. In yeasts it is reported in Saccharomyces species [7, 15, 28, 29] and Hausenia spora valbyensis [5]. Among the filamentous fungi, partial or absolute deficiency, for this vitamin has been reported in P. digitatum [30], P. texanus [31] and F. coeruleum [32], More complex and diversified requirements such as complete deficiency for ascorbic acid and xanthine have been repórted here in CT catenulata for the first time. All the species of yeasts studied here have been found to require different concentrations of growth factors for their optimum growth. There is an increase in the biomass production of all the yeasts after the addition of optimum amount of essential vitamins. So by the addition of optimum amounts of all the essential growth factors, the growth of yeasts was tremendously increased which is evident from Fig. 1. Concentrations higher than the optimum were found to be inhibitory for the growth of the yeasts. Acknowledgement Acknowledgements are due to the Council of Scientific and Industrial Research, New Delhi for the award of fellowship to Neelam K A M R A . Received February 18, 1988 Revised April 24, 1988

References L O B D B R , J . : The yeasts. A texonomic study. North Holland Publishing Co., Amsterdam: The Netherlands, 1970. [2] B A B N E T T , T. A., P A N K H U B S T , R. J.: A new key to the yeasts, North-Holland and Publishing Co., Amsterdam: The Netherlands, 1974.

[1]

4*

Acta Biotechnol. 9 (1989) 2

148 [3]

W I C K E R H A M , L. J . : Taxonomy of yeasts. U.S. Department of Agric. Tech. Bull. 1029, Washington, D.C., 1951. [ 4 ] MATHTJB, R . S . , B A R N E T T , H . L . , L I L L Y , V . G . : Phytopathology 40 ( 1 9 5 0 ) , 1 0 4 . [ 5 ] A H R E A M , D. G . , R O T H , P. J . J r . : Dev. Ind. Microbiol. 8 (1962), 163. [ 6 ] R O B B I N S , W . J . , M A , R . : Science 100 ( 1 9 4 4 ) , 8 5 . [ 7 ] B U R K H O L D E R , P . R . , M C V E I G H , I . , M O Y E R , D . : J . Bacteriol. 4 8 ( 1 9 4 4 ) , 3 8 5 . [8] S C H Ö P F E R , W. H., G U I L L O U D , M.: Z. Vitaminforsch. 16 (1945), 181. [9] M I Y A S H I T R A , S . , M I W A T A N I , T., F U N J I N O , T.: Biken's J . 1 (1958) 45. [10] JANDIAK, C. L., KAPOOR, J . N.: Indian Phytopathol. 25 (1972), 563. [ 1 1 ] R A W L A , G . S., C H A H A L , S. S.: Trans. Br. Mycol. Soc. 6 4 ( 1 9 7 5 ) , 5 3 2 . [12] B U R K H O L D E R , P. R., M O Y E R , D.: Bull. Torr. Bot. Club 70 (1943), 372. [13] M I L L E R , G., A S C H N E R , M.: J . Gen. Microbiol. 6 (1952), 361. [ 1 4 ] R A B I N O W I T Z , J . C . , S M E L L , P . : Arch. Biochem. Biophys. 3 8 ( 1 9 5 1 ) , 4 7 1 . [ 1 5 ] W I L L I A M S , R . J . , E A K I N , R . E . , S M E L L , E . S.: J . Am. Chem. Soc. 6 2 ( 1 9 4 0 ) , 1 2 0 4 . [16] H A N S E G A W A , T., B A N N O , I., Y A M U C H I , S.: J . Gen. App. Microbiol. 5 (I960), 200. [ 1 7 ] F U N J I N O , T . , MIWATANI, T . , AKAGI, M . , MIYASHITA, S., TAKAGI, S., KIMUBA, K . , KAMIKA, T . , YEMURA, T . , ISHIGAMI, T . :

I n : Studies on candidiasis in Japan. Publik Education Ministry,

Japan. 1961, 27. B. L . : Biol. Chem. 1 6 0 ( 1 9 4 5 ) , 1 . N.: The chemical environment for fungal growth 3. Vitamin and other organic growth factor. In A I N S W O R T H , G . C . and A. S . STTSSMAN (Eds.) The fungi. An Advance Treatise, Vol. 1. Academic Press, New York, 1965, 491. [20] LEAPHART, C. D.: Mycologia 48 (1956), 25. [21] M I S R A , A . P., M A H M O O D , M . : Indian Phytopathol. 14 (1961), 20. [22] N O R T H A M , B. E., N O R R I S , F. W.: J . Gen. Microbiol. 5 (1951), 502. [ 2 3 ] C H A L L I N O R , S . W . , P O W E R , D . M . , T O N G E , R . J . : Nature ( 1 9 5 5 ) , 2 0 3 .

[ 1 8 ] MELNIOK, D . , MOCHBERG, M . , H I M E S , H . W . , OSER,

[19]

FRIES,

[ 2 4 ] D O M I N Q U E Z , A . , E L O R J A , M . V . , SANTOZ, E . , V I L L A N U E V A , J . R . , S E N T A N D R E W ,

[25] [26] [27] [28] [29]

113 (1959),

134.

Arch. Biochem. 8 ( 1 9 4 5 ) , 3 1 1 . H.: Bull. Torr. Bot. Club 80 (1953), 43. S A T Y A V I R and G R E W A L , G . S . : Indian Phytopathol. 26 (1973), 274.

[30] WOOSTER, R . C., CHELDELIN, V . H . :

[31] [32]

R.:

Ant.

Leeuw. J . 44 (1978), 341. G H O S H , A., B H A T T A C H A R Y A , S. N.: Biochim. Biogphys. Acta 186 (1967), 19. L E W I N , L . M.: J . Gen. Microbil. 441 (1956), 215. T I M M I C K , M. B . , L I L L Y , V. G., B A R N E T T , H. L.: Phytopathology 41 (1951), 327. L E O N I A N , L . H., L I L L Y , V. G.: Am. J . Bot. 29 (1942), 459. W E I G N F T J R T H E R , F . , E S C H E N F E C H E R , F . , B O R Y E S , W . D . : Zbl. Bakt. Parasit. Kde. (Abt.

YUSEF,

II)

Acta Biotechnol. 9 (1989) 2, 149—156

Akademie-Verlag Berlin

Growth of Pellets of a Basidial Fungus Pleurotus ostreatus under Various Cultivation Conditions STYOGANTSEVA, J . M . 1 , A E B , R . J . 1 , V I E S T U K S , U . E . 2

1

2

August Kirchenstein Institute of Microbiology, Latvian SSR Academy of Sciences, A. Kirchenstein Street, 226067 Riga, Latvian SSR, U S S R Institute of Wood Chemistry, Latvian SSR Academy of Sciences, 27, Akademijas Street, 226006 Riga, Latvian SSR, U S S R

Summary Studies on the pelletization of fungus Pleurotus ostreatus have established the pellets to differ as to the size, density and chemical composition. Changes in the pellets, taking place during culture growth, are helpful in studying the mode of their development. We have studied various conditions of cultivation, and established the factors enhancing culture growth. Prehomogenization of seeds, velocity of stirrer revolution, partial pressure of dissolved oxygen and that of other factors was established to affect the structure of pellets and parameters of culture growth.

Pelletization is a form of mycelial culture growth under submerged conditions. Pellets consist of densely intertwined mycelial hyphae and can reach upto 10 mm in size. Most often they are spheric. Since new pellets are formed from mycelium separated from the old ones, pellets of all sizes are present in the culture liquid at a time. An important and well-studied process, using pelletal growth, is production of citric acid based on Aspergillus niger cultivation. The concentration and pellet size optima have been established for the process, ensuring a maximum yield of the product [1, 2]. Pelletal growth has been studied in various species of mycelial organisms — Morchella sp. [3], Penicillum chrysogenum [4], Agaricus bisporus [5] and others. Cultivation of higher fungi by submerged fermentation for the obtaining of foodstuffs or biologically active substances is also connected with biomass growth, mainly in the form of pellets [5—8]. The object of the present studies was a basidial fungus Pleurotus ostreatus, cultivated for the obtaining of biomass [9, 10]. The fungus forms big spheric pellets on all the media under study. The task of the work was a quantitative and qualitative study of the changes taking place in the structure and composition of pellets during growth and development, depending on the effect produced by the velocity of stirrer shaft revolution, partial pressure of oxygen and quality of the seeds. The results were interpreted in keeping with the diagram, reflecting the effect of external medium factors on the formation and state of the pellets (Fig. 1). Dependences, used to determine the optimum parameters of cultivation yielding a maximum of biomass, were studied as well.

150

Acta Biotechnol. 9 (1989) 2

Materials and Methods Mycelium was grown on a medium of the following composition [g/1]: glucose — 20; yeast extract - 8; NH4C1 - 2.4; K 2 H P 0 4 - 3 H 2 0 - 0.5; K H 2 P 0 4 - 0.5; MgS0 4 -7H 2 0 — 0.25. Initial p H 5.2—5.4, temperature 28°C. The fungus was cultivated in 750 ml flasks (100 ml filling) on a shaker (200 rpm) and on a laboratory scale fermentation equipment FU-8 [11] — 3 1 working volume, doubletier turbine stirrers. During the cultivation the following parameters were monitored: pH, temperature, velocity of stirrer revolution (rpm), aerating air inlet, partial pressure of dissolved oxygen (p02, %).

Pig. 1. Factors affecting the structure of pellets

The seeds was prepared as follows: 7 days of mycelium cultivation on slant agar, 6 to 7 days of surface cultivation of mycelium in flasks with baffles on liquid wort, disruption of surface mycelium by baffles and seeding of submerged inoculation medium, seeding of 3—4 day old submerged inoculation mycelium (10% of seeding medium) into test flasks. In some experiments, discussed in the present paper, the seeds were prehomogenized on a homogenizer MPW-302 (Poland) at 1/4 of the maximum rate for 30 s. Each day the biomass was separated from the culture liquid and the pellets were fractioned on sieves with the 1 —4 mm grating diametre at 0.5 mm intervals. We determined the number of pellets and their dry weight on the sieve. Thus the medium weight of a pellet and, consequently, its density in each fraction could be determined. Results and Discission I t was established that during the cultivation of Pleurotus ostreatus in shaker flasks the mass fraction of pellets 1 — 1 . 5 mm in diameter increases. At the beginning of the stationary growth phase (towards the 4th day) the fraction constituted 28.6% (Fig. 2).

STYOGANTSEVA, J . M., ABE, J . R . et al., Growth of Pellets

30

151

i

20 F—I

o

I I

10

,

0

1 2 3 Diameter CmrrG

4

Fig. 2. Dependence of mass fraction (0.5 mm interval) on culture age 1, 2, 3,4 — days of cultivation

With regard to a dependence of density on the diameter and growth stage, it was established that pellet density increases with culture age and diminishing of pellet diameter (Fig. 3). A relatively constant density of pellets 1.5 to 3.0 mm in diameter testifies to a homogeneity of the structure. A zone of lysis is observed in the centre of big pellets (3—4 mm in diameter), hence their density decreases. 80

20

-

0 " 1

2 3 Diameter [mm]

U

Fig. 3. Changes in pellet density depending on their diameter 1, 3, 4, 5 — days of cultivation

At the stationary growth phase towards the 5th day the density falls in practically all the pellets exceeding 1.5 mm in diameter. Studies on the pellet age- and diameter-dependent changes in nucleic acid content demonstrated that the peak of nucleic acid content during culture growth shifts towards pellets with smaller diameter. The maximum content of nucleic acids (6.7%) is observed on the second day of growth in 1.5—2 mm pellets. Since substrate and oxygen concentration per unit of biomass decreases during cultivation, and the pellets themselves become denser, the smaller ones, who are not subjected to limitation, grow more actively.

152

Acta Biotechnol. 9 (1989) 2

The shifts of the average content of nucleic acids in the biomass are negligible: from 4.86% on the first day of cultivation to 4.38% on the fourth. The total nitrogen content also depends on the size of pellets; in all the experiments the 2.5—3.5 mm pellets contained more total nitrogen than the others (Fig. 4).

Diameter

[mm]

Fig. 4. Changes in nucleic acid and total nitrogen content depending on pellet diameter nucleic acids, - - - total nitrogen 1, 2, 3, 4 — d a y s of cultivation

Studies on the effect of seeds homogenization revealed that towards the second day of culture growth the nucleic acid content in the biomass notably increases, reaching 6.15%. This is not observed with non-prehomogenized seeds. Since a large number of growth zones are formed after homogenization (all the pellets under 1 mm), the growing

Fig. 5. E f f e c t of seeds homogenization on the changes in mass fraction (0.5 m m interval) 1, 2, 3, 4 — d a y s of cultivation

STYOGANTSEVA, J . M., AKE, J . R. et al., Growth of Pellets

J 53

pellets simultaneously enter the active growth phase, characterized by a high nucleic acid content. I f the seeds were not prehomogenized, the nucleic acid content normalizes, due to the presence of pellets of various age and size. Homogenization of the seeds causes the biomass to consist practically mostly of pellets more of the same size (Fig. 5), which increases with culture age. The rate of pellet diameter increase in the maximum fraction is 0.36 mm/day. During the growth there can be observed a certain desynchronization and the portion of the maximum fraction decreases. Towards the fourth day the distribution of pellet mass as to their sizes nears that of non-homogenized pellets. Homogenization of the seeds causes the formation of looser pellets, during the process they become denser. Experiments in fermenters have demonstrated, that acceleration of stirrer revolution causes the formation of more compact pellets, their medium size decreases (Fig. 6).

Fig. 6. Dependence of mass fraction (0.5 mm. interval) on the velocity of stirrer revolution (on the third day)

Thus, upon 100 rpm of stirrer revolution the main mass portion consists of 3.0—3.5 mm pellets, upon 200 rpm the biomass consists of smaller pellets — 2.0—3.0 mm on the third day of growth. Consumption of air maintains p02 at the level of 2 0 % of saturation in both variants. The density of pellets in both, fermenter and shaker flasks grows during the growth process. Upon a high velocity of revolution there can be observed a notable compression of pellets of all sizes (Fig. 7). Partial pressure of medium oxygen is another factor determining the density and distribution of pellets as to their size. Experiments were carried out with three p02 levels — 15, 40 and 6 5 % of saturation. The given p02 level was maintained by air consumption, the stirrer revolution velocity was constant — 150 rpm. Higher p02 levels (40 and 65%) are characterized by a more even distribution of pellets as to their size (Fig. 8). The mass fraction of big pellets increases (over 2.5) as compared with the fraction of these pellets at p02 = 15%. Consequently, a high p02 level ensures the growth of big pellets, suffering from oxygen limitation at lower p 0 2 values. The effect of p02 (Fig. 9) level on density can be exemplified on 2.5—3.0 mm pellets. The higher the p02 level, the denser the pellets and the more intensive their compression during the process. Changes in medium composition and concentration of the main carbon source—glucose, did not affect pellet density and their distribution. The results confirm the literature data [12] on oxygen as the main limiting factor upon pelletal growth.

154

Acta Biotechnol. 9 (1989) 2 901-

70

E o> 50

30

10

1

2 3 Diameter Cmm]

U

Fig. 7. E f f e c t of stirrer revolution velocity on changes in pellet density depending on their diameter 2, 3 — days of cultivation

¿Or

1

2 3 Diameter Emm]

Fig. 8. Dependence of mass fraction (0.5 m m interval) on p02 level (on the third day)

Compression of pellets, caused by acceleration of stirrer revolution, negatively affects oxygen diffusion within the pellets, the biomass yield decreases, hence mass transfer improvement does not take place on account of stirring intensity. If the level of p 0 2 is raised, there is an increase in pellet density and critical radius, beyond the latter the lysis of the centre sets in and the concentration of biomass increases, yet the bulk of it consists of big pellets (2.5—4 mm) with a marked lytic zone. Upon a high level of p02 it is important to decrease pellet size, it is ensured by prehomogenization of the seeds. 36% of the obtained biomass consists of 1.5—2.0 mm pellets, no pellets exceeding 3.0 mm are observed (Tab. 1).

155

STYOGANTSEVA, J . M . , A R E , J . R . e t a l . , G r o w t h of P e l l e t s

60 p 0 2 = 65 7. 50 E en ¿0

p02=

40

%

p02=

15

%

30

Fig. 9. Changes in pellet density (2.5- -3.0 mm fraction) depending on pO , level

20 Time

Cd3

Tab. i. Characteristics of the maximum fraction of pellets on the third day of growth depending on cultivation regime Cultivation regime

Biomass

Characteristics of maximum fraction

n [rpm]

p 0 2 [%]

[g/1]

[%]

Size [mm]

Density [kg/m 3 ]

100 200 150 150 150 100 100

20 20 15 40 65 50* 50

6.8 6.3 4.4 6.2 8.5 11.5 7.6

25 24 33 28 23 36 24

2.5-3.5 2.0-3.0 1.5-2.0 1.5-2.5 2.0-3.0 1.5-2.0 2.5-3.0

35 56 29 44 56 30 43

* Homogenization of the seeds

Conclusion Studies on the changes of pellet density and mass fractions established the critical sizes of pelletts to exceed which causes a lysis of the centre, as well as determined the conditions favourable for the growth of various fractions. Accumulation of pellet-forming culture biomass is ensured not only on account of an increase in their size and number, but also due to their compression, which is most marked in the second half of the process. The sensitivity of pellet sizes and properties to cultivation paramétrés enables to use the pellet-forming culture as a model for equipment and cultivation regime studies. The obtained data were further used to select the equipment and elaborate the technology of a mass cultivation of Pleurotus ostreatws for practical purposes. Received February 29, 1988

References [ 1 ] K I S S E R , M . , KTTBICEK, C . P . , R O H R , M . : A r c h . M i c r o b i o l . 1 2 8 ( 1 9 8 0 ) , 2 6 . [2] MITARD,

A.,

RIBA, I. P . :

Appi. Microbiol. Biotechnol.

2 5 (1986), 245.

[3] LITCHFIELD, I . H . : D e v . I n d . Microbiol. 1 1 (1970), 341.

156

Acta Biotechnol. 9 (1989) 2

[ 4 ] WITTLER, R . , BAUMOARTL, H . , LUBBERS, D . W . , (1986), 1024.

SCHUGERL, K . : B i o t e c h n o l . B i o e n g .

[5] LITCHFIELD, I. H., OVERBECK, R . C., DAVIDSON, R. S.: J . Agric. Food. Chem. Ill

28

(1963), 158.

[6] KAPICH, A . I . , STAKHEEV, I . V . , VAZHYNSKAYA, I . S . : M i k o l o g i a i p h y t o p a t o l o g i y a 2 0 ( 1 9 8 6 ) ,

199 (in Russian). [7] WHITAKER, A . , LONG, P . A . : P r o c . B i o c h e m . 1 1 ( 1 9 7 3 ) , 2 7 . [ 8 ] METZ, B . , KOSSEN, N . W . F . : B i o t e c h n o l . B i o e n g . 1 9 ( 1 9 7 7 ) , 7 8 1 .

[9] STYOGANTSEVA, J . M., ARE, R. J . : Production of Higher Edible Fungi in the USSR. Kiev, Naukova Dumka, 1985, 126 (in Russian). [10] SOLOMKO, E. F., ARE, R. J., STYOGANTSEVA, E. M. : Biotekhnologiya (1988) (in press). [11] VIESTURS, U. E., KUZNETSOV, A. M., SAVENKOV, V. V. : Fermentation Systems. Riga, Zinatne, 1986, 368 p. (in Russian). [12] PIRT, S. J . : Proc. Roy. Soc. 166 (1966), 369.

Book Review BAJAJ, Y . P . S. (Editor)

Biotechnology in Agriculture and Forestry 3 Berlin, Heidelberg, New York: Springer-Verlag, 1987 509 S., 155 Abb., DM 3 2 8 , - ISBN 3-540-17966-6

Innerhalb der Reihe „Biotechnology in Agriculture and Forestry" liegt nunmehr der Band 3 vor, der sich der Züchtung, Vermehrung und Nutzung der Kartoffel unter dem Aspekt biotechnologischer Betrachtungsweise widmet. Ausgehend von einer Analyse traditioneller Methoden zur Kartoffelvermehrung wird in der vom Herausgeber verfaßten Einleitung die Vorstellung der Kartoffel des 21. Jahrhunderts beschrieben. Dabei wird festgestellt, daß die Kartoffel eigentlich die erste wichtige Frucht war, die „biotechnologisch eingehend untersucht wurde (und wird)". Neben der Erzeugung resistenter Sorten gegenüber Viren, Nematoden, Herbiziden werden dabei auch Aspekte der Klimaresistenz angesprochen. I n der ersten Sektion des Buches werden die unterschiedlichsten Forschungsergebnisse aus verschiedenen Ländern der Erde zur in-vitro-Vermehrung dargestellt. Die Sektion I I befaßt sich mit physiologischen, biochemischen und ernährungsphysiologischen Studien, während in den Sektionen I I I und IV Selektionsprobleme im Vordergrund stehen. In der abschließenden V. Sektion geht es um Konservierungsprobleme des vermehrungsfähigen Materials. Allgemein wird eingeschätzt, daß der vorliegende dritte Band sehr umfassend in 35 Artikeln von Experten aus 20 Ländern ein f ü r unsere Ernährung wichtiges Problem behandelt. Die biotechnologischen Erfolge am Versuchsobjekt Kartoffel, weltweit erzielt, führen zu verbesserten und schädlingsresistenteren Nutzkartoffeln und tragen damit wesentlich zur Lösung des Welternährungsproblems bei. Deshalb wird dieses Buch nicht nur all denen empfohlen, die direkt auf dem Gebiet der biotechnologischen Pflanzenforschung arbeiten; vielmehr ist es auch ein geeignetes Nachschlagewerk f ü r Forscher auf dem Gebiet der Schädlingsbekämpfung und der Ernährung. R . PÄTZ

Acta Biotechnol. 9 (1989) 2, 1 5 7 - 1 7 2

Akademie-Verlag Berlin

Streptomyceten als Bildner industriell und diagnostisch bedeutsamer Enzyme HÄUTUNG, B .

Akademie der Wissenschaften der DDR, Zentralinstitut für Mikrobiologie und experimentelle Therapie, Beutenbergstraße 11, Jena, 6900-DDR

Summary This review deals with known natural enzyme activities of streptomycetes and their applications. This knowledge may help potential users to exploit these organisms for individual purposes. The data given in tables are based on scientific publications and the IUPAC recommendations regarding nomenclature are followed. Included are only enzymes already utilized, with those of the primary metabolism being neglected.

Einleitung Wesentlicher Bestandteil moderner biotechnologischer Verfahren der Stoffwandlung ist die Verwendung von Enzymen, entweder in gelöster oder immobilisierter Form. Etwa 20 vorwiegend hydrolytische Enzyme (Proteasen, Glucosidasen, Isomerasen) werden weltweit im großen Maßstab produziert und für vielfältige Zwecke, von der Verwendung in Waschmitteln bis hin zur Stärke- und Zuckerverarbeitung eingesetzt. Weitere Anwendungsgebiete von Enzymen sind Diagnostik und Therapie, wobei hohe Ansprüche an die Reinheit gestellt werden. Thermoatabilität, langzeitiger Erhalt der Enzymwirkung, hohe Reaktionsgeschwindigkeit bei entsprechendem pH-Wert sind Kriterien, die über die Anwendbarkeit enzymatischer Verfahren entscheiden. Da enzymatische Umsetzungen preislich mit konventionellen chemischen Verfahren konkurrieren müssen, spielen die Kosten für die Enzymgewinnung bzw. die Immobilisation an Trägermaterialien eine wesentliche Rolle. Man ist daher weiterhin bemüht, die Palette von Bildnern bereits bekannter Enzyme zu erweitern, um die Ökonomie enzymatischer Stoffwandlungsverfahren weiter verbessern zu können. Eine andere Zielrichtung ist es, neue enzymatische Reaktionen aufzufinden, die für die Ausführung spezieller Biotransformationen bzw. stereo- und regiospezifischer Synthesen und Stoffwandlungen geeignet sind. Bei der vergleichenden Betrachtung potentieller technischer Enzymbildner verdienen die Streptomyceten und andere Actinomyceten aus folgenden Überlegungen heraus besonderes Interesse: — Streptomyceten sind grampositive filamentös wachsende Bodenbakterien, die seit langem ' für die Produktion von Antibiotika eingesetzt werden. Vielfältige stoffwechselphysiologische 1 Untersuchungen haben gezeigt, daß diese biotechnologisch genutzten Mikroorganismen eine (Reihe von Enzymen bilden und zahlreiche Synthese- und Abbauleistungen ausführen. GlucoseIsomerase ist ein Streptomycesenzym, das in die technische Anwendung gelangt ist.

158

Acta Biotechnol. 9 (1989) 2

— Die flockige filamentöse Mycelstruktur erleichtert die Aufarbeitung der Biomasse für die Enzymextraktion (Endoenzyme) bzw. die Gewinnung immobilisierter Biokatalysatoren. — Enzyme aus Biomasse oder Kulturlösung können als Nebenprodukte von Produktionsverfahren für Antibiotika gewonnen werden, die im großen Maßstab in der Pharmaindustrie durchgeführt werden. — Die Anwendung moderner gentechnischer Methoden auf Streptomyceten hat inzwischen einen hohen Reifegrad erreicht und zu Prinziplösungen geführt, die sowohl zur Erhöhung der natürlichen Enzymproduktion durch Genamplifikation als auch zum Einsatz von Streptomyceswirten für die Expression heterologer Enzymgene und den Export der entsprechenden Genprodukte genutzt werden können.

Nach Angaben von Alan W I S E M A N [75] sind 60% der Enzyme, die verwendet werden, proteolytische, 30% sind Carbohydrasen (a-Amylase, Glucose-Isomerase), nur ca. 20 Enzyme werden in merklichen Mengen verwendet. W I S E M A N unterscheidet 3 Klassen der kommerziell anwendbaren Enzyme in bezug auf Nutzbarkeit, Preis, Reinheit: 1. Enzyme im großen Maßstab (large scale) wie Glucose-Isomerase sind relativ nutzbar in großen Mengen, aber relativ unrein in bezug auf biochemische Standards.

billig,

2. Enzyme, die in kleinen Mengen verwendet werden, z. B. bei klinischer Analyse, wie Glucoseund Cholesterol-Oxidasen, sind rein, in geringen Mengen verfügbar, sehr teuer wegen der hohen Kosten der Labor-Aufarbeitungs-Reinigungsverfahren. 3. Spezielle Enzyme, die für Forschung^wecke produziert werden, oft auf ad hoc Basis, sind von variabler Reinheit, von sehr begrenzter Anwendbarkeit, gewöhnlich extrem teuer, so daß large scale-Versuche oft unzulässig sind. Für Prüfung auf Anwendbarkeit und Toxikologie aber ist eine bestimmte Menge nötig.

Die Anforderungen richten sich besonders auf die Bereitstellung billiger(er), stabiler(er) und überhaupt neuartiger Enzyme mit technologisch relevanten Eigenschaften. Enzyme für die Nahrungsmittelindustrie unterliegen besonders strengen Auswahlkriterien. Gegenwärtig sind nur 10 Mikroorganismenarten als Produzenten zugelassen [98]. Die Anwendungsmöglichkeiten von Streptomyceten-Enzymen liegen im medizinischen, zahnmedizinischen (Therapie und Diagnostik), pharmazeutischen, kosmetischen und Waschmittelsektor (seit der Entwicklung von Pellets sind allergische Reaktionen zurückgegangen), außerdem in der Textilindustrie, im Leder- und Rauchwarensektor, in der Nahrungsmittelindustrie (herkömmliche lebensmitteltechnologische Verfahren zu rationalisieren, die Qualität zahlreicher Produkte zu verbessern, das Sortiment zu erweitern, neue Nahrungsquellen zu erschließen), in der Landwirtschaft (Tierernährung, Futtermittelindustrie), im biochemischen, analytischen und genetischen Labor.

Streptomycesenzyme und ihre Anwendung Oxidoreduktasen Oxidoreduktasen sind Wasserstoff- und elektrönenübertragende Enzyme. Sie sind für die Oxidations- und Reduktionsprozesse verantwortlich. Die hier genannten Oxidoreduktasen werden in der medizinischen Diagnostik und für Forschupgszwecke angewendet. Die Uratoxidase wird in immobilisierter Form verwendet.

Häutung, B., Streptomyceten als Enzymbildner

159

Tab. 1. Enzym

Bildner

Literatur/Bemerkungen/ Anwendung

Cholesteroloxidase (EC 1.1.3.6), intrazellulär MG 44000; 22000 Daltons, Flavoproteid UV-Adsorbtionsmax. bei 279, 380, 470 nm isoelektr. Punkt bei pH 5,9. Michaeliskonstante 5,6 x 10"6 mol/1

Streptomyces sp.

med. Diagnostik, quantitative Cholesterolbestimmung im Blut, large scale-Produktion [1], [2], [106].

Str. griseus

Xanthin-Dehydrogenase (EC 1.1.1.204 Xanthin-NAD+ oxidoreduktase) Bildung kontrolliert durch An- u. Abwesenheit von C- u. N-Quellen im Medium.

Str. cyanogenes Str. spec.

[3], [4] (Xanthin + NAD+ + H 2 0 = Urat + NADH) wirkt auch auf D-glucuronat; Diagnostik.

Uratoxidase = Uricase (EC 1.7.3.3) Urat-oxygen-oxidoreduktase ein Kupfer-Protein

Str. spec.

isoliert von Boden [5]

Str. Str. Str. Str. Str.

Spuren von Enzym genetische Optimierung UV

nigrificans aureofaciens nigrifaciens cyanogenes gannmycicus

[6]

[3] [95] Anwendung: medizinische Diagnose, biochemische Diagnose von Gicht und einigen Formen von Rheumatismus durch Nachweis von Harnsäure (urid acid) im Urin-Serum [75]; immobilisiert.

L-Glutamat-Oxidase (EC 1.4.3.11) MG 140000 neues Flavoproteinenzym = L-Glutamat-oxygenoxidoreduktase

Str. sp. X-119-6

[7] kann auch Phenazin-Methosulfat und Eisencyanid als Elektronenakzeptoren benutzen.

l-Alanin-Dehydrogenase (EC 1.4.1.1)

Str. phaeochromogenes Str. clavuligerus

[8] enzymat. Analyse von 1-Alanin; Pyruvat-Bildung (aus 1-Alanin: 1-Alanin + H 2 0 + NAD+ = pyruvat + NH„ + NADH), wichtige Rolle im C- u. N-Stoffwechsel = 1-Alanin-NAD+ oxidoreduktase.

160

Acta Biotechnol. 9 (1989) 2

Tab. i . (Portsetzung) Enzym

Bildner

Literatur/Bemerkungen/ Anwendung

Tyrosinase (EC 1.10.3.1/1.14.18.1) intrazelluläre und extrazelluläre Formen der Tyrosinase sind identisch im MG 2 9 0 0 0 Kupferprotein (Cu 0,21%) durch Aminosäuren induzierbar. = Catecholoxidase Diphenyloxidase Phenolase Monophenoloxidase

Str. glaucescens (Mel-, mel A, B , C) Str. antibioticus Str. lividans

[9], [9 b] [9 a] [9a] Genetische Untersuchungen mit Mutanten mit Tyrosinase Mangel (Mel - ). Tyrosinase an Melaninbildung beteiligt (taxonomische Bedeutung).

Transferasen Transferasen sind bei der Übertragung einer chemischen Gruppe (Ein-Kohlenstoffverbindungen, Aldehyd- oder Ketogruppe, Acylreste, Zucker sowie stickstoff-, phosphoroder schwefelhaltige Verbindungen) von einem Donor auf ein Akzeptorsubstrat beteiligt. Tab. 2. Enzym

Bildner

Literatur/Bemerkungen/ Anwendung

Ornithin-Carbamoyltransferase (EC 2.1.3.3)

Str. fradiae Str. clavuligerus NRRL-3585 Str. wadayaminsis Str. lactamdurans Str. albogriseolus Str. jumonjinensis Str. lactamgenes

[10]

intracellular

bevorzugter Stamm

[U]

Enzym spielt Rolle bei Differenzierung von Prokaryoten. Synthese von semisynthet. Cephalosporinantibiotika, -CH 2 OCONH 2 = Carbamoyloxymethylgruppe

Hydrolasen Die Hydrolasen katalysieren hydrolytische, also Spaltungs- und Kondensationsreaktionen unter Beteiligung von Wasser. Die hier erwähnten Hydrolasen finden Verwendung in der Medizin und Zahnmedizin, in Kosmetik und Pharmazie, in Leder- und Nahrungsmittelindustrie und Textilindustrie, Landwirtschaft und in der Forschung. Pronase, neutrale Protease, Pullulanase, a-D-Galaktosidase, Cellulase, a-Amylase, D-Aminoacylase können in immobilisierter Form verwendet werden.

HÄRTUNG, B., Streptomyceten als Enzymbildner

161

Tab. 3. Enzym

Bildner

Literatur/Bemerkungen/ Anwendung

neutrale Protease = Hygrolytin (EC 3.4.24.4) MG 37000 isoelektrischer Pkt. p H 8,8, optimale Aktivität bei 40—45 °C, schnell inaktiviert bei 50 °C, hydrolytische Aktivität, Ca 2+ stabilisiert die Aktivität, EDTA (bindet Ca ++ ) inaktiviert; stabil in wäßrigen Lösungen bei p H 6,0 bis 8,0.

Str. hygroscopicua

[12]

neutrale Protease (EC 3.4.24.4)

Str. tanakasis sp. 4680

(JEKSEN 1931)

Nr. 1913 Sammlung Moskau deposit Nr. 997

Str. fradiae Str. caespüosua Str. griseus

medizin.: Entfernung von Heilung verzögerndem Schorf auf Verbrennungswunden; bei Erkrankung der Atmungsorgane, Schleimhäute von entzündlichem Exsudat befreit; thrombolytische Wirkung in Kombination mit Antikoagulans; niedrige Kosten, da produziert mit Antibiotikum Hygromycin B (Trennung über Kationenaustauscher Carboxyresin). Lederindustrie: Gerben, Weichmachen von Häuten u. Fellen; als Protohydrolytin in der Landwirtschaft: Hydrolyse von Futterkomponenten zur besseren Verwertbarkeit. [13] angemeldet durch NISSIN SHOKUHIN KAISHA, Ltd. Japan [97]

[106] [106]

thermostabil effizient. Abbau von ProteinEinheiten, bes. bei Bereitung von Nahrungsmitteln (z. B. Spaghetti), immobilisiert. neutrales proteolytisches Enzym (EC 3.4.24.4)

Str. hygroscopicus DS 14.649 (NRRL 3,999) Str. naracusis

[14] Brotherstellung

alkal. Proteinase (EC 3.4.21.14)

Str. rectus var. proteolytieus, thermophil

[15]

thrombolytisch u. fibrinolytisch wirksames Enzym (EC 3.4.21.7) ' MG 40000 ± 5000 22,750 R P

Str. venetus DS 24,288 (NRLL 3,987)

Anwendung medizinisch

chymotrypsin- u. trypsinähnl. Proteinase-Mischung: jetzt (EC 3.4.21.4), früher (3.4.4.4)

Str. rimosics

[17], [19]

5

Acta Biotechnol. 9 (1989) 2

[16]

Acta Biotechnol. 9 (1989) 2

162 Tab. 3. (Fortsetzung) Enzym

Bildner

Literatur/Bemerkungen/ Anwendung

= Trypsin alkal. Zn-abhängige MetallProteinase (EC 3,4.24.4) neutrale Metallproteinase (EC 3.4.24.4) Ca-aktivierte Leucin-Aminopeptidase (EC 3.4.11.1)

Str. rimoms

[17], [19]

Pronase (EC 3.4.21.4) greift ähnlich wie Trypsin u. Subtilisin das PolysaccharidDerivat Chitosan an, wird aktiviert durch Behandlung mit Glutaraldehyd.

Str. griseus

[18], [19], [105], [106] immobilisiert

Serratia-lytisches Enzym (177) = neues lytisches Enzym! eines der wenigen Enzyme, die gram-negative Bakterien lysieren.

Str. sp. Stamm Nr. 177

[20] klinische Bedeutung bei Infektionskrankheiten.

D-CarboxypeptidaseTranspeptidase (EC 3.4.17) extrazellüläres Penicillinbindungsprotein hat penicillinabbauende Aktivität, bildet andere Spaltprodukte des Penicillins (Phenylacetylglycin + N-formylD-Penicillamine) als die /9-Lacta-

Str. griseus K-l Str. R 39 Mol.-Gew. ca. 53300, eine Polypeptidkette; Str. R 61 Mol.-Gew. ca. 38000

Desoxyribonuklease (EC 3.1.21.1) extrazellulär

Str. rimoms

[22] antiviral

N-acetylmuramidase (EC 3.2.1.17)

Str. globisporus 1829 Str. rutgersensis H-46

[23] Ml-Enzym: für Studien der bakteriellen Zellwandstruktur, Präparation typ-spezif. Antigene, Plasmid DNA, Protoplasten. SR-l-Enzym: Nahrungsmittelindustrie: Konservierung von Käse, Wurst, Salaten gegen Streptococcus /aecaJis-Infektionen [24], [25]. Streptococcus faecalis ist resistent gegen Hühnereiweiß-Lysozym, Ersatz: SR-l-Enzym.

[21], [100], [101]

Autoren folgern aus Experimenten, daß die extrazelluläre DDCarboxypeptidase-Transpeptidase eine lösliche Form der „physiologischen" membrangebundenen Transpeptidase ist.

HÄRTUNG, B., Streptomyceten als Enzymbildner

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Tab. 3. (Fortsetzung) Enzym

Bildner

Literatur/Bemerkungen/ Anwendung

„Mutanolysin" enthält Endopeptidase, extrazell.

Str. globisporus 1829 Str. albus G Str. e.g. S-l strain {Str. diastatochromogenes ATCC 21481, FER5f-P-Nr. 326) H-191 strain (Str. farinosus, ATCC 21482 FERM-P-Nr. 327) H-402 strain (Str. griseus var. H-402 ATCC 21483, F E R M P-Nr. 328) B-1829 strain (Str. globisporus ATCC 21553 FERM-P-Nr. 596)

[27], [26] lyt. Enzyme gegen ZahnkariesErreger Streptococcus mutans — salivarius — sangius Lactobacillus acidophilus, Actinomyces viscosus.

«-1,3-GIucanase (EC 3.2.1.59)

Str. sp. KI-8 Str. griseus H-402

[28], [29] gegen Zahnkaries-Erreger: versch. Typen von Streptococcus mutans (bildet exträzellulär wasserunlösl. Glucan).

/3-1,3-Glucanase (EC 3.2.1.59) produziert Gentiobiose aus Curdlan (/3-1,3-glucan)

Str. spec.

[30], [31]

Chitinase I und II (EC 3.2.1.14/3.2.1.30) = /S-N-Aoetyl-D-glusaminidase

Str. Str. Str. Str. IFO Str.

[32], [33], [34], [106] Enzym löst Rhizopus-Zellwand; produziert außerdem noch /S-N-Acetylgalactosaminidase.

ß-Mannanase (EC 3.2.1.78) extrazellulär

Str. sp. St. Nr. 17

[35], [36], [37], [38] gereinigt

Phospholipase B (EC 3.1.1.5)

Str.

hiroshimensis

[39]

Phospholipase A (EC 3.1.1.32)

Str.

cinnamonens

[40]

Phospholipase D (EC 3.1.4.4) MG 16000 max. Akt. pH 7,5 bei 37 °C

Str. Str.

hachijoensis chromofuscus

[41], [42], [105] Kosmetik: Entfettungscreme, Salbengrundlage; geeignet zum Studium der Membranstruktur.

5»

orientalis spec. ATCC 11238 L-19 albus 3710 griseus

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Tab. 3. (Fortsetzung) Enzym

Bildner

Literatur/Bemerkungen/ Anwendung

Pullulanase (EC 3.2.1.41) a-Dextrin endo-l,6-a-GIucosidase = a-Dextrin 6-Glucanohydrolase pH-Optimum 5—6. Temperatur Optimum 50 °C.

Str. sp. Nr. 280 Hydrolyse von 1,6-a-Dglukosidischen Bindungen in Pullulan, Amylopektin u. Glykogen

[43] Pullulanase zerlegt das lineare Polysaccharid Pullulan; kommerziell interessant, Nahrungsmittelindustrie (Brauerei) ; zerlegt Stärke und produziert Glucose-Sirup; immobilisiert.

Xylanase (EC 3.2.1.32/3.2.1.8) = ß-1,4-xylanxylano-hydrolase, extrazellulär, MG 45000, isoelektr. Punkt 4.0, Induktur nötig (Methyl-/3-xylosid).

Str. Str. Str. Str.

[44], [45], [46], [52] Nahrungsproduktion (Tee, Getreide, Kaffee, Kakao, Schokolade).

«-D-Galaktosidase (EC 3.2.1.22)

Str. 9917 S,

[47], [48] Blutgruppe B-Substanz abbauendes Enzym, wandelt B u. AB in O u. A um; Oligosaccharid-Hydrolyse, Zucker-Raffinade; immobilisiert.

Laktase (EC 3.2.1.108) = /5-Galaktosidase Laktose -> Glucose + Galaktose, thermostabil (70 °C)

Str. coelicolor ATCC 21666 Str. griseus

[49] (ROHM u. HAAS-Comp.) in der Milchwirtschaft, Hydrolyse von Laktose in Milch, bes. MolkeHydrolyse, auch pharmazeutisch.

Keratinase (EC 3.4.99.11.) alte Nr. 3.4.4.25 extrazellulär

Actinomyces, nicht identifiziert Str. fradiae

[50], [51] Dermatologie u. Kosmetik, Enthaarungsmittel, Beseitigung von Überschuß-Keratin als Folge von Psoriasis; Präparation von Leder und Wolle.

Cellulase (EC 3.2.1.4) Endoglucanase, extrazellulär ; MG 45000 isoelektr. Pkt. 4,0 Substrat : Carboximethylcellulose, Baumwolle

Str. Str. Str. Str.

[52], [53], [18], [92], [96] Enzyminduktion mit Wei^enstroh und Baum wollstielen; Fruchtund Gemüse-Bearbeitung; immobilisiert (schneidet ^-1,4-Ketten von Zellulose); „Induktion" von Cellulase dauert sehr lange Zeit, rel. ineffiziente Enzyme, Ertrag v. extrazell. Enzymen beträgt 20% des Gewichtes d. konsumierten Zellulose. Preis der Cellulase z. Z. noch so hoch im Vergleich mit dem der reinen Zellulose, daß Großproduktion noch nicht lohnt.

ostregriseus sp. Nr. 3137 xylophagus lividans

lividans sp. B 814 afghaniensis viridosporus

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165

Tab. 3. (Fortsetzung) Enzym

Bildner

Literatur/Bemerkungen/ Anwendung

Exoglucanase Bestandteil des ZellulaseSystems, (EC 3.2.1.91) 1,4-/?-glucancellobiohydrolase MG 46000/45000 isoelektr. Pkt. 4,15

Str. flavogriseus NRCC Nr. 23591

[53 a] Exoglucanase setzt Cellobioseoder Glucose-Einheiten vom nicht reduzierenden Ende der Zellulose frei. Die Produktion dieses Enzyms wurde bisher bei prokaryotischen Zellulose-Zersetzern nicht demonstriert,

cc-Amalyse (EC 3.2.1.1) MG 40000 extrazell. Stärke-Substrat opt. pH 4,5—5,5 Temp. opt. 40°C akt. durch Ca++ u. Cl~

Str. aureofaciens F R I 606 Str. flavus F R I 605 Str. hygroscopicus F R I 602 ATCC 21722 Str. angustemyceticus* (Str. hygroscopicus var. angustemyceticus F R I 607) Str. viridochromogenes F R I 603 Str. albus F R I 604 Str. tosaensis nov. spec. F R I 601 Str. rimosus Str. olivaceus

[54], [55], [56], [18], [17] Brauen, Backen, Textilindustrie, Pharmazeutika, Tierfutter, Kleber, Detergentien, kann Stärke in Sirup umwandeln, Klärung von Fruchtsäften, Alkoholproduktion. immobilisiert

Str. olivaceus

[57] (D-Aminosäuren sind Bausteine für physiologisch aktive Peptide, £f-Laetam-Antibiotika, halbsynthetische Penicilline u. Cephalosporine), immobilisiert.

Hydantoinase (EC 3.5.2.2) = Dihydropyrimidinase MG 190000 Enzym stabil bei pH 6—7 bis 65 °C, breite Substratspezifität, spaltet DL-5-substituierte Hydantoine in N-Carbamyl-Daminosäuren, die chemisch in D- Aminosäuren umgewandelt werden.

Str. albus — almquistii — aureus — flaveolus — griseus — griseoruber — griseolus — mikataensis Pseudomonas striata bester Produzent

[58], [59], [60], [61] Gewinnung von D-Aminosäuren als Intermediate für Synthese von /J-Lactam- und Peptid-Antibiotika sowie semisynthetischen Penicillinen und Cephalosporinen.

^-Lactamase (EC 3.5.2.6.) ß-Lactamhydrolase Penicillinase Cephalosporinase MG 25000 bis 30000

Str. coelicolor — fradiae — lavendulae subsp. — lavendulae — phaeochromogenes — diastatochromogenes subsp.

D-Aminoacylase (EC 3.5.1.14) N-acetyl-DL-Aminosäuren 4

D-Aminosäuren

* hat auch schaften.

Pullulanase-Eigen-

=

[64], [65], [66] Str. lavendulae 130 U/ml = Aktivität von Bac. cereus u. licheniformis, Str. diastatochr. 150 U/ml, Screening v. 100 Stämmen 72 positiv [18] auße medizin. Gesichtspkt. für Rou

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Acta Biotechnol. 9 (1989) 2

Tab. 3. (Fortsetzung) Enzym

Bildner

Literatur/Bemerkungen/ Anwendung

Wirkung: /S-Lactam + H 2 0 4 substituierte /3-Amidosäure. konstitutiv, extracellulär.

— — — — — —

tineuntersuchung zur Identifikation v. Penicillin in Nahrung (Milch), Blutserum. Verhütung von allergischen Reaktionen

diaatatochromogenes cellulosae diastaticus gougeroti hygroscopicus cacaov

— albus — nmosus Agarase (EC 3.2.1.81) extrazellulär (Dag + phenotyp) Plasmid SCPI: insert. I n a k t . : Dag A Dag AI ungewöhnliches genetisches Element beteiligt.

Str. coelicolor A3(2)

[9] einzige C-Quelle = Agar (ungewöhnliches Verhalten dieses Streptomyceten). Gen-FusionsTechniken können dazu beitragen, daß in Zukunft mehr Informationen über extrazelluläre Enzyme gesammelt werden können. Geeignet als Vektorplasmid f ü r die gentechnische Stammkonstruktion.

Restriktion»- und Modifikationsenzyme [62] (EC 3.1.21.4) Tab. 4. Enzym

Sequenz

Stamm

SstIV SphI Spa I

TGATCA GCATGC/C GCATGC

Sac I

GAGCT/C

Sst I Stu I Sac I I

GAGCT/C AGG/CCT CCGC/GG

Sbo I Sfr I Shy I Sst I I Sal P I Ska I I Ska I

CCGCGG CCGCGG CCGCGG CCGC/GG CTGCA/G CTGCAG GCCGGC

Sail

G/TCGAG

Str. stanfordii Str. phaeochromogenes Str. phaeochromogenes I F O 3108 Str. achromogenes ATCC 12767 Str. stanfordii Str. tubercidicus Str. achromogenes (ATCC 12767) Str. bobili (ATCC 3310) Str. fradiae (ATCC 3355) Str. hygroscopicus Str. stanfordii Str. albus (CMI 52766) Str. karnatakensis Str. karnatakensis (ATCC 25463) Str. albus G

kommerziell vertrieben durch

B, C, E, G, H —

A, B, E, G, H, I prod. kohesive Enden C A, B, E, I E, G — — —

C, prod. kohesive Enden — — —

A, B, C, D, E, F , G, H, I [89] prod, kohesive Enden

HÄUTUNG, B . ,

Streptomyceten als Enzymbildner

167

Tab. 4. (Fortsetzung) Enzym

Sequenz

Stamm

Scul

CTCGAG

Sex I

CTCGAG

Sgal

CTCGAG

Sgol

CTCGAG

Slal

C/TCGAG

Slu I

CTCGAG

Spa PI

CTCGAG

Seal Sgrll

AGT/ACT CC(A)GG (T) CC/TNAGG GGCC(N)4 NGGCC

Str. eupidosporus (KCCS 0316) Str. exfoliatus (KCC SO 030) Str. ganmycicus (KCC SO 759) Str. goshikiensis (KCC SO 294) Str. lavendulae (ATCC 8664) Str. luteoreticulus (KCC SC 788) Str. albus subspee. pathocidicus (KCC SO 166) Str. caespitosus Str. griseus Kr. 20

Sau I Sfil Sae I I I

?

?

Sal I I Sex I I Sgrl Shy I I SodI Sod I I Sst I I I A B C D E F G H I

— -

Str. aureofaciens I K A 18/4 Str. fimbriatus (ATCC 15051) Str. achromogenes (ATCC 12767) Str. albus G Str. exfoliatus (KCC SO 030) Str. griseus (ATCC 23345) Str. hygroscopicus Str. odorifer (ATCC 6246) Str. odorifer (ATCC 6246) Str. stanfordii

kommerziell vertrieben durch —

—

—

—

—

—

—

B, E, G, [84], [85] - [87] B E [86] —

— —

—

— —

—

—

AMERSHAM BUCHLER BOEHRINGER Mannheim BETHESDA RESEARCH Labs. MILES LABORATORIES NEW ENGLAND BIOLABS NEW ENGLAND NUCLEAR PROMEGA BIOTEC PHARMACIA TAKARA-SHUZO Co.

2 spezifische Endonucleasen mit einer möglichen Rolle bei der Restriktion Anwendung:

Str. antibioticus ATCC 11891 und ETH 7451

[88]

Restriktions-Endonukleasen sind cloning-Technologie-wirksame moderne Werkzeuge, um genetische Probleme in Medizin, Landwirtschaft und industrieller Mikrobiologie anzugehen.

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Acta Biotechnol. 9 (1989) 2

Viele Typ-II-Restriktions-Endonukleasen wurden aus Bakterien isoliert und ihre Spezifitäten charakterisiert. Sie erkennen eine spezifische Nukleotidsequenz von 4 bis 8 Basenpaaren in doppelsträngiger DNA und schneiden innerhalb oder nahe der Erkennungssequenz. Diese Enzyme sind unentbehrlich für das genetic engineering, aber der Mechanismus der Erkennung und des Schneidens ist noch nicht vollständig aufgeklärt [63]. Immobilisierung ist eine günstige, aber teure Möglichkeit, um diese kostbaren Enzyme zu konservieren [75]. (Tab. 4) Lyasen

Lyasen trennen auf nichthydrolytischem Wege bestimmte Gruppen von ihren Substraten ab bzw. fügen diese an, und zwar unter Zurücklassen einer Doppelbindung bzw. unter Addition von Gruppen an Doppelbindungen.

Tab. 5. Literatur/Bemerkungen/ Anwendung

Enzym

Bildner

Histidase

Str. coelicolor Str. griseus

[67]

Str.

[68]

(EC 4.3.1.3) ( = Histidin-Ammoniak-Lyase)

Pektat-Lyase (EC 4.2.2.9) Exopolygalakturonat Lyase setzt ungesättigte DigalakturonatEinheiten v. Oligo- u. Polygalakturonsäuren frei; neuer Typ setzt ungesättigte Trigalakturonat-Einheiten frei; Keine mazerierende Aktivit. auf Pflanzen

Endopektat-Lyase (EC 4.2.2.2) pH-Optimum 9,1 mazerierende Aktivität auf Pflanzengewebe

massasporeus

Str. nitrosporeus KA 37

[68], [69], [70]

Str. fradiae IFO 3439

[71]

'

Isomerasen

Isomerasen katalysieren die reversible Umwandlung isomerer Verbindungen. Die Glucoseisomerase wird in immobilisierten Formen verwendet. Tab. 6. Enzym Xylose-Glucose-Isomerase (EC 5.3.1.5) intrazellulär optimaler pH 8—8,5, optimale Temperatur 80 °C, stabil über weite Strecken (pH 4,5—11), inhibiert durch Ag+, Hg++, Cu++; aktiviert durch Co++, Mg++, MG 165000 bis 191000 MG 187000; 165000

HÄUTUNG, B., Streptomyceten als Enzymbildner

169

Tab. 6. (Fortsetzung) Bildner/Literatur Str. nigrificans, phaeochromogenes1 [72, 74a, 80, 94], frädiae1 [94], albus ATCC1-2 21132, 21175 [94], achromogenes ATCC1-2 1*2767 [73, 94] immobilisiert, echinatus ATCC1-2 21933 [94], wedmorensis ATCC1 21230 [94], flavovirens ATCC1-2 3320, olivochromogenes ATCC1 21114 [94], olivaceus N R R L B 3583 [74, 74 a, 81, 94], glaucescens NRRL 3 B 2900 [82, 94], thermovvlgaris, violacoeniger [79], violaceus-ruber [18], platensis, kananyceticus, rubiginosus [91], olivocinereus, nigrifaciens, niveus, rnatensis, griseoflavus, gracilis, galbus, flaveus, acidourans, albogriseolus, atroviolaceiLS, venezuelae [74a], purpeofuscus [74a], hygroscopicus [74a], scabies [74a], calijornicus [94], bobilai [94], roseochromogenes [94], venuceus [94], virginial [94], bikiniensis [94], rubiginosus [94], olivochromogenes ATCC 21114 [74], flavovirescens, flavogriseus, violaceus-ruber, a/ghaniensis [92], griseofuscus S-41, olivochromogenes ATCC 21114 - Mutanten: CPC 3 (ATCC 21713) CPC 4 (ATCC 21714) CPC 15 (ATCC 21715) [93] Bemerkungen/Anwendung Fructose-Sirup-Produktion, Isomerase®, Isosyrup®, Cornsweet, Isosweet® USA. Keine Beziehung zwischen taxonomischer Position und Enzymaktivität [74 a] 1 2 3

ganze Zellen immobilisiert [80] gefrorene flokkulierte ganze Zellaggregate (Zellimmobilisierung), Separieren des Enzyms vom Mikroorganismus ist unnötig, Anwendung zur Glucoseisomerisierung [83] hat keine Tyrosinase-Aktivität [82]

Folgerungen Die bereits aufgeführten Literaturangaben demonstrieren die Eignung der Streptomyceten als Enzymbildner. Die Nutzung des natürlichen Potentials der Actinomyceten erscheint damit weiterhin als eine vielversprechende Aufgabe. So darf angenommen werden, daß weitere enzymatische Leistungen bei der Abwandlung spezieller chemischer Strukturen entdeckt und einer Anwendung zugeführt werden. Es ist erwiesen, daß sich gentechnische Entwicklungen nur in wenigen Organismen, die genetisch, physiologisch und biochemisch ausreichend erforscht sind, verwirklichen lassen. Dazu gehören neben Escherichia coli, Hefe und Bacillus, auch Streptomyces. Der Trend geht in Richtung einer Beschränkung und zweckentsprechenden Spezialisierung von wenigen industriell geeigneten Wirtssystemen [98]. Neuere Untersuchungen von YOKOZEKI et al. 1987 führten zu dem erstaunlichen Ergebnis, daß das neu isolierte Bakterium Pseudomonas sp. AJ-11220 eine direkte Umwandlung von DL-HPH 1 in D-HPG 2 mit einer molekularen Ausbeute von 9 4 % bewirken kann [102]. Verantwortlich dafür sind zwei Hydrolasen [104]. Hier eröffnen sich möglicherweise neue Wege, über den Gentransfer auch einen Streptomyces-Stamm mit diesen Eigenschaften zu konstruieren, um den Prozeß zur Gewinnung von D-Aminosäuren zu beschleunigen. Die Fähigkeit zur Bildung von 1-Aminosäuren, wie es YOKOZEKI et al. 1987 [103] bei Flavobacterium feststellten, ist bisher allerdings bei Streptomyceten noch nicht entdeckt worden. Andererseits zeigen sich bei der Betrachtung der Originalliteratur aber auch einige Grenzen für die Anwendung natürlicher Streptomycesenzyme. Während einige Enzyme 1 2

DL-HPH = DL-5-(p-Hydroxyphenyl)hydantoin D-HPG = D-p-Hydroxyphenylglycin

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Acta Biotechnol. 9 (1989) 2

t r o t z der mesophilen N a t u r der P r o d u z e n t e n eine h o h e T h e r m o s t a b i l i t ä t aufweisen (z. B. Glucose-Isomerase), e n t s p r e c h e n a n d e r e in ihren E i g e n s c h a f t e n d e n t a t s ä c h l i c h e n p h y siologischen B e d i n g u n g e n u n d sind, verglichen m i t P r ä p a r a t e n a u s a n d e r e n O r g a n i s m e n , hinsichtlich der T h e r m o s t a b i l i t ä t als weniger g u t geeignet einzuschätzen. A b e r a u c h hier ist sicherlich d u r c h die bereits e r w ä h n t e n Möglichkeiten d e s P r o t e i n - E n g i n e e r i n g n o c h n i c h t d a s l e t z t e W o r t gesprochen, so d a ß die Z u k u n f t eine b r e i t e r e N u t z u n g v o n Actinom y c e t e n als E n z y m b i l d n e r e r w a r t e n l ä ß t . Eingegangen: 16. 2. 1988 Überarbeitet: 12. 4. 1988

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J.

Gen. Microbiol. 128 (1982), 371. [ 1 0 ] S Z I L Á G Y I , I., V A R G H A , G., SZABÓ, G. In: Proc. 6th Int. Symp. on Actinomycetes (Debrecen, Ed. SZABÓ, G. et al.) Budapest 1 9 8 6 , 4 1 5 . [ 1 1 ] F L E M I N G , I . D . , T U R N E R , M . K . , B R E W E R , S. J . : U. S. Pat. 4 . 1 6 4 . 4 4 7 , Glaxo Laboratories Limited, England ( 1 9 7 9 ) . [12] B A S H K O V I C H , A. P.: U. S. Pat. 4.075.322, U. S. Pat. 4.066.503 (1978). [13] T A N A K A , T . et al.: U. S. Pat. 3.827.939 (1974). [14] B E L L O C , A. et al.: U. S. Pat. 3.875.006 (1975). [15] M A T S U E , M . : Agrie. Biol. Chem. 46 (1982), 2485. [16] B E L L O C , A. et al.: U. S. Pat. 3.875.005 (1975). [17] V I T A L E , L J . , T U R K , V . , P O K O R N Y , M . I n : Proc. 6th Int. Symp. on Actinomycetes (Debrecen, Ed. SZABÓ, G . et al.) Budapest 1986, 418. [18] ROSE, A. H. (Ed.): Economic Microbiology, Vol. 5, Microbial Enzymes and Bioconversions, Academic Press, London, New York 1980. [ 1 9 ] T A S H I R O , M . , S U G I H A R A , N . , M A K I , Z . , K A N A M O R I , M . : Agrio. Biol. Chem. 4 5 ( 1 9 8 1 ) , 5 1 9 . [ 2 0 ] S U Z U K I , K . , U Y E D A , M . , S H I B A T A , M . : Agrie. Biol. Chem. 4 9 ( 1 9 8 5 ) , 3 0 4 9 . [ 2 1 ] N A R A H A S H I , Y., H A T T O R I , K . , M U R A K O S H I , H . : Agrie. Biol. Chem. 4 4 ( 1 9 8 0 ) , 1 6 6 1 . [ 2 2 ] Y A N A G I D A , T . , O G A W A R A , H . : J . Antib. 3 3 ( 1 9 8 0 ) , 1 2 0 6 . [ 2 3 ] K A W A T A , S., T A K E M U R A , T . , Y O K O G A W A , K . : Agrie. Biol. Chem. 4 7 ( 1 9 8 3 ) , 1 5 0 1 . [ 2 4 ] H A Y A S H I , K . , K A S U M I , T . , K U B O , N . , T S U M U R A , N . : Agrie. Biol. Chem. 4 5 ( 1 9 8 1 ) , 2 2 8 9 . [25] H A Y A S H I , K . , K A S U M I , T . , K U B O , N., T S U M U R A , N.: Agrie. Biol. Chem. 48 (1984), 465. [26] Y O S H I M U R A , Y . et al.: U. S. Pat. 3.929.579, DAINIPPON Pharmaceutical Co (1975). [27] K A W A T A , S., T A K A H A S H I , E., T A K A S E , Y . , Y O K O G A W A , K . : Agrie. Biol. Chem. 47 (1983), 2801. [ 2 8 ] I M A I , K . , K A B A Y A S H I , M . , M A T S U D A , K . : Agrie. Biol. Chem. 4 1 ( 1 9 7 7 ) , 1 8 8 9 . [ 2 9 ] Y O K O G A W A , K . , K A W A T A , S., Y O S H I M U R A , Y . : Agrie. Biol. Chem. 4 0 ( 1 9 7 6 ) , 6 6 1 . [ 3 0 ] K U S A M A , S., K U S A K A B E , I . , M U R A K A M I , K . : Agrie. Biol. Chem. 4 8 ( 1 9 8 4 ) , 2 6 5 5 .

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171

B., Streptomyceten als Enzymbildner

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6 3 ] K I T A , K . , HIRAOKA, N . , KIMIZUKA, F . , OBAYASHI, A . , K O J I M A , H . , TAKAHASHI, H . , SAITO, H , : N A R 1 3 (1985), 7015. 6 4 ] OGAWARA, H . : 6 5 ] OGAWARA,

Chemother.

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Antimicr. Ag. Chemother. HORIKAWA,

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[75] WISEMAN, A. (Ed.): Handbook of Enzyme Biotechnology, Ellis Horwood Ltd., Chichester, N.Y., Ontario, 1985. [76] 3 r d Symposium of Socialist Countries on Biotechnology, Bratislava (1983). [77] HALPERN,. M. G. (Ed.): Industrial Enzymes from Microbial Sources, Recent Advances, ndc Park Ridge, New Jersey, U.S. A., 1981. [79] MARCEL, T . , DROCURT, D . , TIRABY, G . : M o l . G e n . G e n e t . 2 0 8 ( 1 9 8 7 ) , 1 2 1 . [80] YOSHIKAZU, N . , KAZUMASA, S., TSUYOSHI, M . : U . S. P a t e n t 4 . 1 9 1 . 8 1 0 , M i t s u i S u g a r CÔ., L t d .

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[81] SNELL, R. L.: U. S. Pat. 3.974.036 MILES Laboratories Inc. (1976). [82] WEBER, P . : U . S. P a t . 4 . 1 3 7 . 1 2 6 G i v a u d a n C o r p o r a t i o n (1979). [83] L E E , C. K . , LONG, M. E . : U . S. P a t . 3 . 9 8 9 . 5 9 7 R . J . REYNOLDS T o b a c c o C o m p a n y ( 1 9 7 6 ) . [84] KITA, K . , HIRAOKA, N . , KIMIZUKA, F . , OBAYASHI, A . , KOJIMA, H . , TAEAHASHI, H . , SAITO, H . : N A R 1 3 (1985), 7 0 1 5 . [85] TAKAHASHI, H . , KOJIMA, H . , SAITO, H . : B i o c h e m . J . 2 8 1 (1985), 2 2 9 .

[86] QIANO, B. Q., SCHILDKRAUT, I.: NAR 12 (1984), 4507. [87] ORECHOW, A . W . , REBENTIS, B . A . , DEBABOV, V . G . : D o k l . A k a d . N a u k . S S S R 2 6 3 ( 1 9 8 2 ) , 217. [88] SANCHEZ, J . C., BARBES, K . , HERNADEZ, C . - R . , G., GAVILAN, R . , HARDISSON, C. : C a n . J .

Microbiol. 81 (1985), 942. [89] ARRAND, J . R . , MAERS, P . A . , ROBERTS, R . J . : J . M o l . B i o l . 1 1 8 ( 1 9 7 8 ) , 127.

[90] Enzyme Nomenclature IUB Recommendations of the Nomenclature committee of the international Union of Biochemistry on the nomenclature and classification of enzyme catalyzed reactions. Academic Press, Inc., New York, London, San Diego, Toronto, Montreal, Sydney, Tokyo, Orlando (1984). [91] SUOMEN, SOKARI Oy (VIZURI, K . J . ) : E u r . P a t . App. E P 166.427; CA 104 (1986), 87145.

[92] YUSUPOVA, I. KH.: Uzb. Biol. Zh. 5 (1985), 9; CA 104 (1986), 87083. [93] ARMBRUSTER, P . C., HEADY, R . E . , CORY, R . P . : U . S. P a t . 3 . 9 5 7 . 5 8 7 , a s s . t o C P C I n t e r n a t i o n a l I n c . (1976). [94] ANTRIM, R . L . , COLILLA, W . , SCHNYDER, B . J . I n : WINGARD, C. B . , GOLDSTEIN, L . : A p p l .

Biochem. & Bioengin. 2 (1979), 97. [95] NAKANISHI, T., SHIGEMASA, Y . I n : WISEMAN, A. (Ed.) : H a n d b o o k of E n z y m e Biotechnology,

Ellis Horwood Ltd. Chichester, New York, Ontario, 1985. [96] DEOBALD, LEE A., CRAWFORD, DON L. : Appl. Microbiol. Biotechnol. 26 (1987), 158. [97] KOKUBU, T., KARUBE, I., SUZUKI, S . : Biotechnol. Bioengin. 23 (1981), 29. [98] HOPEMEISTER, J . : S p e c t r u m 1 8 ( 1 9 8 7 ) , 2 2 .

[99] GHUYSEN, J.-M. et al.: Annals of the N. Y. Acad. Sciences 235 (1974), 236. [100] FRÈRE, J . - M . , MORENO, R . , GHUYSEN, J . - M . , PERKINS, H . R . , DIERICKX, L . , DELCAMBE, L . : B i o c h e m . J . 1 4 3 (1974), 2 3 3 .

[101] FRÈRE, J.-M., GHUYSEN, J.-M., PERKINS, H. R., NIETO, M.: Biochem. J . 186 (1973), 463. [102] YOKOZEKI, K . , NAKAMORI, SH., EGUCHI, CH., YAMADA, K . , MITSUGI, K . : A g r i c . B i o l . C h e m . 5 1 (1987), 355. [103] YOKOZEKI, K . , SANO, K . , EGUCHI, CH., YAMADA, K . , MITSUGI, K . : A g r i c . B i o l . C h e m . 5 1 (1987), 3 6 3 .

[104] YOKOZEKI, K . , KUBOTA, K . : Agric Biol. Chem. 5 1 (1987), 721.

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Acta Biotechnol. 8 (1989) 2, 1 7 3 - 1 7 7

Akademie-Verlag Berlin

Production of. Lysine by Mutants of Escherichia coli K 12 in a Medium with Lactose SOBOTKOVA, L . 1 , SlKYTA, B . 1 , SMEKAL, F . 2

1

2

Czechoslovak Academy of Sciences Institute of Microbiology, Prague CS-14220 Prague 4, Videnska 270 Research Institute of Antibiotics and Biotransformations CS-25263 Roztoky near Prague

Summary In an effort to use whey for lysine production, we isolated from a jS-galactosidase-hyperproducing strain of E. coli K 12 multiple mutants — auxotrophic, regulatory and penicillin-resistant. These mutants exhibited for the most part a high reversion rate but some of them produced about 2 mg/ml lysine in an enriohed fermentation medium.

The production of amino acids in traditional producers, Corynebacterium and Brevibacterium, has been increased by selection of mutants with suitably altered regulatory mechanisms of the appropriate biosynthetic pathways and amino acid excretion [1], Increased lysine production was particularly prominent in mutants resistant to analogues of S-(2-aminoethyl) L-cysteine (AEC) and NPG exhibits a split kinetics characterized by high and low substrateconcentration kinetics which are differentiated by different values of V and of Km. In addition, jS-glucosidase-II is shown to be an exo-glucohydrolase as deduced from experiments with MUcellobiopyranoside. Experimental features should be emphasized; usual soft-gel ion-exchange materials did not work in the chromatofocusing separation of the two /3-glucosidases, in contrast to the 10 rj.-Si 500 = DEAE exchange material (Serva) typically used in HPLC-experiments. Furthermore, protein content determinations based on different procedures yielded widely differing values.

Introduction Crystalline cellulose is hydrolyzed by the synergistic action of three different types of cellulases denoted as endoglucanases (1.4-(1,3; l,4)-/9-D-glucan 4-glucanohydrolase, E.C.3.2.1.4), exoglucanases (exo-cellobiohydrolase, 1,4-/?-D-glucan cellobiohydrolase, E.C.3.2.1.91), and /3-glucosidases (cellobiase, /5-D-glucoside glucohydrolase, E.C.2.3.1.21) [1—6]. Attack on crystalline cellulose begins with the action of endoglucanases and is continued by the action of exoglucanases [4,5,7—11], However, crystalline cellulose may also be decomposed by exoglucanases only [1,4, 7]. Exoglucanases are competitively inhibited by the reaction product (cellobiose) [3, 6, 12, 13]. Therefore, a strong continuous reaction of the exoglucanase requires a simultaneous decomposition of cellobiose by /3-glucosidase [6, 7, 14, 15]. Just like the exoglucanases, the endoglucanases (acting randomly on the glucoside bonds of the cellulose chains and, thereby, forming cellodextrins of different sizes) are inhibited by cellobiose [3, 6]. The endoglucanase reaction may require the removal of cellobiose as well. * To whom correspondence should be addressed. 6*

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Strains of Trichoderma reesii (formerly Tr. viride) were shown to be highly active with regard to cellulose degradation [15, 16]. Although they produce high amounts of exoglucanases and endoglucanases, strains of this species fail to secrete sufficient amounts of /3-glucosidases [15,16] or they secrete /S-glucosidases which are unstable at low pH-values [14]. Therefore, when cellulose is degraded by Tr. reesii, cellobiose is accumulated thereby inhibiting further saccharification of cellulose. In contrast with Tr. reesii, remarkable amounts of /S-clucosidases are produced by Aspergillus niger and other species of Aspergillus [15—17]. It has been recommended [14, 15] that one can obtain a more complete saccharification of cellulose through applying mixtures of the cellulases from Tr. reesii and A. niger. As a consequence, the cellulase complex of A. niger deserves increased interest, although up to now it has been little investigated compared with the cellulose complexes of Tr. reesii and other cellulase-active species. It is interesting to note in this connection that commercial cellulases are often based on enzymes from A. niger. The strain of A. niger (CBS 554.65) has been shown to grow on insoluble cellulose (like paper) and to degrade it by 56% of dr.w. within 28 days. In our publication we report on the purification of two /S-glucosidases produced by this strain and on some of their properties.

Materials and Methods Culture Conditions and Extraction of the Enzymes Aspergillus niger (strain CBS 554.65 = ATCC 16888) was cultivated in 11 culture flasks with 300 ml of a modified Norkrans medium [18]. Instead of the cellulose powder of this medium, we added 12 g of "PROFIX-Cellulose-Vlies" separated into single sublayers. The sterilized flasks were inoculated and incubated for 28 days at 33 °C. The mycelium and the non-decomposed cellulose were removed from the solution with the help of vacuum filtration funnels. The dialyzation (against a 1% (w/v) glycin solution) and concentration (hundredfold) were performed with a MINITAN-Tangential Filtration System (ultrafiltration packet iS 10 kDa), Millipore. The solution containing the non-diffusible material was subsequently filtered with a membrane filter (Millipore, pore size 0.45 ¡xm, type HAWP). Chromatographic Fractionation The initial gel filtration of the crude filtrate was carried out with a 20 X 1000 mm SEPHACRYL S 200 HR column (Pharmacia Fine Chemicals) using a buffer system of 0.15 M NaCl and 0.02 M KH 2 P0 4 , pH 5.6. Flow rate 20 ml/h ^ 6.35 cm/h; 6.65 ml/ fraction. Chromatofocusing was carried out with a 150 X 20 mm column (KRANNICH) with DEAE = Si 500 0.01 mm (SERVA) in order to fractionate the reconcentrated and deionized fraction of the /S-glucosidase peak of the primary gel filtration. The start buffer was 0.025 M GABA, pH 4.6, and the limit buffer was 0.2% (v/v) SERVALYT 3 - 5 (80% (v/v)) + SERVALYT 3 - 1 0 (20% (v/v)), pH 3.0. Flow rate 12 ml/h ^ 3.8 cm/h; 2 ml/fraction. Anion exchange chromatography was performed using a 150 X 10 mm column (KRANNICH) with DEAE = Si 300 material 0.01mm (SERVA). Gradient: 0 . 0 2 - 0 . 6 M KH 2 P0 4 (pH 5.6). Flow rate 40 ml/h ^ 51 cm/h. The protein detection was carried out by a photometer UV-VIS (KNAUER, type 9700)

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181

(280 nm). A KNICK pH-meter (type 540) with a flow-through cell equiped with an INGOLD pH-electrode and a CORNING digital conductivity meter (type PTI-8) were

employed for detection in the chromatofocusing. Analytical Isoelectric Focusing A MULTIPHOR I I chamber was equipped with a MACRODRIVE 5 power supply (LKB). The electrophoresis was carried out with SERVALYT» PRECOTES» 3—10 or 3—6 (SERVA) applying 1800 V and 3.5 W. Samples of 10 (xl protein solution each (equivalent to 1—10 ¡xg protein) were applied. A solution of 200 g trichloroacetic acid/1 of H 2 0 was employed for the protein fixation. Staining: Coomassie-Brilliant Blue R-250 (== Serva Blue R) 400 mg/1 of destaining solution, 10 min; destaining: 2(to 3) X 5 min in methanol/acetic acid/water (40:10:50, v/v). The pi-values were estimated from isoelectric focusing calibrated with Protein Test Mix 9 (SERVA No. 39206) and cellulase "Onozuka", 5 ¡J, 0.6% (w/v) solutions. In addition, pi-values were measured with an INGOLD flat-surface combination electrode ( ± 0 . 1 pH-units). The PAS-test was executed using the method of ZACHARIAS et al. [19]. Thin Layer Chromatography HPTLC Si60 F254 (10 X 20 cm), provided with a concentration zone, were used in the experiment. Development: butan-l-ol/ethanol/water (5:3:3, v/v) recycled twice. MUglycopyranosides and MU were detected by absorption of UV (A = 254 nm) and fluorescence induced by UV (A = 365 mm), respectively. Detection: anisaldehyde-H 2 S0 4 , heated to 110°C. Protein Content and Carbohydrate Content Protein contents for specific activity calculations were determined by measuring the absorbance at 280 nm of the sample solutions using a Zeiss DMR 10 photometer. The measured E 280 -values were converted into protein content values by using values of extinction coefficient e280. These values were determined with standard solutions of the fractionated enzymes which had been lyophilized and subsequently weighed for standardization. Constancy of £280-values was detected over the range of protein concentration from 0.01 up to 0.2% (w/v). Protein contents determined by other methods resulted in values about/three times lower. The methods of LOWRY et al. [20] and BRADFORD [21] gave only 3 3 and 3 8 % (w/v) of the weight determined from the E 280 -values, respectively. Carbohydrate contents of the freeze-dried samples of enzymes were determined using the method published by DUBOIS et al. [22]. Enzyme Assays — Activity towards pNPG. The pH-optima of /9-glucosidases-I and - I I were determined at the pH-range of 3.6 to 5.6 at appropriate concentrations (50 and 3.3 mM, respectively) of pNPG (SERVA). The kinetics of /9-glucosidases-I and -II with pNPG were determined using mixtures containing 1.66 to 80.0 mMpNPG in 0.05 M sodium acetate buffer' (pH 4.1, 4.6, 5.1) and 50 ¡xl of /?-glucosidase-I solution (1 mg/ml) or 10 fxl of /?-glucosidase-II solution (1 mg/ml). During incubation at 40°C, samples of 1.1 ml were withdrawn after 2, 4, 6, 8, 10, 12, 15, and 20 min and each mixed with 2 ml 1 M sodium carbonate solution. Each sample was diluted with 5 ml H a O dest. and measured with a ZEISS Elko I I photometer at 420 nm. — Activity towards Cellobiose. The activity was measured as the amount of released glucose (FIM/min). Glucose was determined using the UV-test of BOEHRINGER. The incubation mixture contained 10 to 576 mg and 2 to 432 mg cellobiose in 20 ml of 0.05 M

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sodium acetate buffer p H 5.1 (/?-glucosidase-I) and 4.1 (/9-glucosidase-II), respectively. 10 fxl (/8-glucosidase-I) or 50 [j.1 (/?-glucosidase-II) of a enzyme solution (0.1% (w/v)) were added to 20 ml of the incubation mixture. Samples of 0.2 ml were withdrawn and added to 2 ml of solution 1 (pH 7.6) of the test set. The release of glucose was stopped completely by the addition of solution 1. — Activity towards MU-Olucopyranoside and MU-Cellobiopyranoside. The reaction mixture contained 0.03 to 0.3 mM of MU-glucopyranoside or MU-cellobiopyranoside in 0.05 M sodium acetate buffer pH 4.1. Specifications of incubation and the times for taking the samples were the same as those used in the pNPG assay. Each sample of 0.5 ml was diluted with 3.5 ml glycine buffer (0.5 M, adjusted to pH 10.4 with NaOH) and measured with the ELKO I I photometer at 365 nm. Results and Discussion The crude enzyme concentrate (a hundredfold concentration increase) obtained from the filtrate by dialyzation and ultrafiltration was fractionated by gel filtration (Fig. 1). Only proteins from the fraction corresponding to the first peak (96 kDa), proved to be

20

25

30 Fraction

35

Fig. i . Primary gel filtration of the crude enzyme concentrate yielded from Aspergillus niger The peaks range from 96 kDa (peak 1) to 15 k D a (peak 5). /?-Glucosidase activity was determined only for the proteins of the hatched 96 kDa peak. Experimental conditions of this and the following figures were as decribed in the Materials and Methods section.

active as /?-glucosidases using pNPG as a substrate. This fraction was further fractionated by chromatofocusing (Fig. 2). The two resulting main peaks both showed /S-glucosidase activity towards pNPG. No other protein from any other fraction of the gel filtration showed activity towards pNPG. Each of these /S-glucosidases, tentatively named /?-glucosidase-I and -II, behaved homogeneously with respect to isoelectric focusing (Fig. 3). The pi-values of /?-glucosidases estimated from isoelectric focusing are given in Tab. 1. Additionally, anion exchange experiments revealed different elution profiles for the two /J-glucosidases and thereby different charge distributions along their molecules chains. Compared with the anion exchange experiment the chromatofocusing gave much better homogeneity of the proteins. Fractions 26 to 27 (from chromatofocusing) corresponding to jff-glucosidase-I and 32 to 33 corresponding to /?-glucosidase-II have been used for kinetic investigations. -

WITTE,

K.,

WARTENBERG,

A., /?-Glucosidase

183

Fractiorr Fig. 2. Further fractionating by chromatofocusing of the 96 kDa-peak obtained from the primary gel filtration The hatched fractions 26 to 27 corresponding to /9-glucosidase-I and 32 to 33 corresponding to /8-glucosidase-II have been used for kinetic investigations.

f r a c t i o n 2 5 to 3 4 Fig. 3. Analytical isoelectric focusing of the 0-glucosidase peak of the chromatofocusing of Fig. 2 The bands of fraction 26 to 28 and 31 to 34 correspond to /3-glucosidase-I and /?glucosidase-II, respectively. Tab. 1. Extinction coefficients, carbohydrate contents and pi-values of the /S-glucosidases-I and -II from Aspergillus niger 280,0.1%

e

Carbohydrate content [ % ]

Pi

/5-Glucosidase-I

10.89

17.5

4.6

jS-Glucosidase-II

10.67

17.0

3.8

/?-Glucosidase fraction from the gel filtration

—

27.0

Acta Biotechnol. 9 (1989) 2

184

I t should be emphasized that usual soft-gel ion-exchange materials in the chromatofocusing separation process gave no separation with regard to activities in contrast to the 10 [i.m-Si500-DEAE exchange material (Serva). This exchange material is typically used in HPLC experiments. Its main advantage with respect to this separation problem is that it may be used in short columns (15 cm) without loosing its separation power. The extinction coefficients (e280) of the two /S-glUcosidases deviated from each other only to a small extent (Tab. 1). The carbohydrate content of the /J-glucosidases was high (Tab. 1) when the determination was performed with the peak 1 fraction of the primary gel filtration (cf. Fig. 1). However, both /?-glucosidases obviously lost equally sized portions of their carbohydrates when the fraction of peak 1 of the gel filtration was further fractionated by the chromatofocusing experiment of Fig. 2. We tentatively explain these observations by postulating that each one of the two /?-glucosidases has two different portions of carbohydrates at the start which are bound to the enzyme molecules with different degrees of attachment. The bigger portion of the carbohydrate content in each case (16.5 to 17.0% (w/v)) is more greatly bound to the protein than the smaller portion (9.5 to 10.0% (w/v)) is.

0.6 "pH5.1 L o w concentration plot

Z.0J 3. ff/pH 5,1

S

I

H i g h concentration plot

0.1

0.3

J

L

0.5 1/S [mM-'] .

Fig. 4. L i n e w e a v e r - B u r k plots of /?-glucosidase-I reacting with pNPG as a substrate Measurements of activity were performed (1) at the optimum pH (5.1) of /?-glucosidase-I and (2) at that pH which is accurately situated between the optimum pHs of /i-glucosidase-I and -II (pH 5.1 and 4.1). Protein concentration 50 ¡xg/ml

From the enzymatic reaction rates of the two /S-glucosidases with pNPG or cellobiose as substrates pH-optima were determined, yielding about 5.1 and 4.1 for /?-glucosidase-I and -II, respectively. When incubated at the optimum pH (5.1) /3-glucosidase-I showed two different linear sections in Lineweavek-Btjrk plots (fading into one another, Fig. 4), one in the high concentration range (10 to 80 MM) and the other in the low concentration range (1.66 to 3.3 mM) of ^NPG. The rate in the high concentration range of /?-glucosidase-I is distinguished by a gréât iTm-value, equivalent to a low affinity (Tab. 2). In contrast to this, the rate in the low concentration range of this enzyme is marked by a small if m -value, equivalent to a high affinity (Tab. 2). Hence, these split kinetics of /3-glucosidase-I may be described hypothetically by a change of the conformation of the enzyme induced by change of substrate concentration or by reactions on two different reaction centers acting at different concentrations of pNPG. Because of the two different linear Leneweaveh-Burk plots we do not conclude a simple inhibition by excess of substrate in the case of the split kinetics of /?-glucosidase-I. The corresponding experi-

WITTE, K., WARTENBEEG, A., jS-Glucosidase

185

Tab. 2. Values for V and Km of the /3-glucosidases-I and -II from Aspergillus niger pH 4.1 V

pH 4.6 Km

f?-Glucosidase-I ^>NPG, high conc. pNPG, low con. cellobiose

V

5800 -

,3-Glucosidase-II MU-glucopyranoside cellobiose

311 15.7

pH 5.1 Km

69.8 -

V

7710 814 0.247

63.0 0.616 0.446

3.09 0.201

F[[xM x min- 1 x mg- 1 ], i J m M ]

merit performed at the non-optical p H (4.6) shows only the high-concentration range of the split kinetics. Consequently, the LINEWEAVEE-BTJRK plot of this experiment exhibits only one linear section (Fig. 4). The low-concentration behaviour at p H 4.6 could not be observed because it fell short of the detection limit. The kinetics of the /?-glucosidase-I when reacting with cellobiose as a substrate is different when compared with the kinetics of the PNPG experiment (Fig. 5). A linear LINEWEAVER-BUBK plot results in the range of given reciprocal values of substrate concentration, 0.2 to 0.75 mM - 1 , fading into an inhibition behaviour in the range 0.1 mM - 1 . Additionally, the 1/v-values of the cellobiose experiment are shown to be 300 times higher than those of the experiment executed with pNPG, indicating a much smaller reactivity with cellobiose when compared with jpNPG. The smaller reactivity with cellobiose is presented more explicitely in Tab. 2: The V related to cellobiose as a substrate is 29,000 and 3,800 times smaller than the values for V related to the high and

150 ?

50

0.1

0.3

0.5 l/SCmM"']

Fig. 5. LINEWEAVER-BDRK plot of /J-glucosidase-I reacting with cellobiose as a substrate Measurements of enzyme activity were performed at pH 5.1. Protein concentration 50 ¡xg/ml

186

Acta Biotechnol. 9 (1989) 2

low concentration ranges of pNPG, respectively. Corresponding differences were reported between enzymatic activities of /S-glucosidases with regard to cellobiose and pNPG as substrates [23, 24]. With respect to the enzymatic activity caused by our /3-glucosidase-I, cellobiose is obviously a less suitable substrate than pNPG, in spite of the corresponding iLm-values of /9-glucosidase-I which are shown to be approximately equal (Tab. 2.). A strong substrate-induced inhibition of reaction rate resulted from the experiments with /S-glucosidase-II (Fig. 6 A). Within the range of used concentrations of pNPG (0.5 to 12.0 mg/ml), the greatest reaction rate occurred at the lowest concentration of the substrate, and vice versa. Consequently, it is not possible to determine V and Km for

£ 1.0

a 2 3.

30 1/S [mM ]

0

0.1

0.3

0.5 1/S [mM*^

Fig. 6. LINEWEAVEB-BURK plots of ^-glucosidase-II A. Measurements with pNPG as a substrate, performed at pH 4.1 (optimum pH of the enzyme) and pH 4.6 (cf. the legend of Pig. 4). B. Measurements with MU-/5-Dglucopyranoside as a substrate, performed at pH 4.1. Protein concentration 10 (xg/ml

pNPG as a substrate and /3-glucosidase-II as an enzyme. The shape of the curve of Fig. 6A suggests that the range of used concentrations covers only the region where asymptotic saturation and inhibition by excess of substrate coexist. However, one should consider the scale of the reaction rate of /?-glucosidase-II acting to wards ^NPG (Fig. 6 A). I t was equal to that of /3-glucosidase-I when reacting on the same substrate (Fig. 4) but was shown to be very high compared with the rate of reaction of /9-glucosidase-II with cellobiose (Fig. 7). A similar experiment was performed with MU-glucopyranoside as a substrate (Fig. 6B). MU-glucopyranoside as a substrate for /3-glucosidases can be employed at concentrations a hundred times lower than £>NPG due to the stronger optical absorption of MU. On the other hand, MU-glucopyranoside must be used in a lower concentration range due to its smaller solubility compared with that of pNPG. A LINEWEAVER-BURK plot applied in the concentration range 0.03 to 0.3 mM of MU-glucopyranoside (Fig. 8B) indicates the usual MiCHAELis-MENTEN-type kinetics. A relatively small iTm-value resulted from the plot (cf. Tab. 2) equivalent to a high affinity of /3-glucosidase-II with MU-glucopyranoside. When /S-glucosidase-II was incubated with cellobiose as a substrate the resulting values yielded a linear LINEWEAVER-BURK plot in the low concentration range of the substrate (Fig. 7). Corresponding to the similar plot of the experiment with /S-glucosidase-I (Fig. 5)

187

W i t t e , K . , W a r t e n b e r g , A . , /S-Gluoosidase

3.0 VS CmM+l Fig. 7. Lineweavbe-Burk plot of ^-glucosidase-II reacting with cellobiose as a substrata Measurements were performed at pH 4.1. Protein concentration 10 [ig/ml 4-Methylumbelliferone [Mil]

* #

M U - Glucopyranoside M U - Cellobiopyranoside

Glucose

•

Start

0

10

20

40

• 60 min

Pig. 8. Thin layer chromatography reflecting the lapse of time of the reaction of /?glucosidase-II towards MU-cellobiopyranoside Incubation at pH 4.1, protein concentration 15 jig/ml, substrate concentration 5 mg/ml. Because of the different methods of detection (absorption, fluorescence) the chromatography is outlined as a computer graphic.

plotted values show a positive deviation from the linear behavior at low 1/S-values indicating an inhibition of the enzymatic reaction. The kinetics of the cellobiose experiment yields a lower X m -value and a lower V-value (cf. Tab. 2) compared with the kinetics of the MU-glucopyranoside experiment of /?-glucosidase-II (cf. Fig. 6 A). These differences mean that /S-glucosidase-II has a greater affinity towards cellobiose but at the same time a smaller reaction rate with this substrate compared with MU-glucopyranoside. Hence, our experiments with /J-glucosidase-II just as with ^-glucosidase-I confirm the previous report [24] that /3-glucosidases from A. niger are ty picall aryl-/?-glucosidases and show low activity with cellobiose as a substrate. This contrasts with the recommendation [14, 15] to decompose cellulosic materials with mixtures of the cellulases from T. reesii and A. niger in order to take advantage of the /3-glucosidases from the latter fungus. The fractions of the two /9-glucosidases yielded by the chromatofocusing (Fig. 2) were tested additionally with MU-cellobiopyranoside as a substrate which is known as a sub-

188

Acta Biotechnol. 9 (1989) 2

strate for exo-cellobiohydrolases [25]. The /?-glucosidase-I proved to be inactive with regard to MU-cellobiopyranoside. By contrast, /S-glucosidase-II is active in decomposing MU-cellobiopyranoside. The time dependence in the release of MU from MU-cellobiopyranoside yields a sigmoid curve, indicating a two-step reaction in which the second reaction step consumes the product of the first reaction step. The assumed two-step reaction could be proved through a thin-layer chromatographic determination of the reaction products of the decomposition of MU-cellobiopyranoside catalyzed by /3-glucosidase-II (Fig. 8). During an initial period of this decomposition experiment, glucose was released from the MU-cellobiopyranoside molecules, yielding MU-glucopyranoside molecules. In the following period of the experiment MU-glucopyranoside was decomposed into glucose and MU. Therefore, with respect to decomposition of MU-cellobiopyranoside, /J-glucosidase-II behaves like an exo-glucohydrolase. The same is supposed for ^pNPG or cellobiose where the enzyme is able to split off a glucose molecule from 2?NPG or from the non-reducing end of the disaccharide molecule. Current investigations are directed to the question whether /?-glucosidase-II acts as an exo-cellodextrinase, similar to Tr. koningii cellulase [3], or as an exo-glucanase which might catalyze the destruction of highly ordered celluloses. The present result confirms the report of Heft i n s t a i l et al. [25], namely that the decomposition of MU-cellobiopyranoside is specific for exo-glucanases. Tab. 3. F-values and Km -values of /S-glucosidases published in the literature Cellobiose

i>NPG F Aspergillus niger Aspergillus wentii Pyricularia oryzae Thermoascus aurantiacus Trichoderma viride Trichoderma viride Trichoderma viride Trichoderma viride Trichoderma viride Trichoderma viride F(|xM x min- 1 x mg-1),

Km

3.76 200.0 2410.0 45880.0 32000.0 —

1.02 3.00 1.43 0.52 0.28 —

—

—

—

—

125.0 270.0

0.049 0.12

F

Km

118.0 1150.0

0.15 0.91

—

33.0 66.2 116.0 44.6 90.0 67.0

! .50

2.65 2.50 2.74 0.23 1.80

Lit. [26] [27] [28] * [29] [23] [30] [30] [30] [31] [31]

Km(mM)

For comparison with our /?-glucosidases, Tab. 3 shows kinetic constants of /9-glucosidases published in the literature. The F-values resulting from the jS-glucosidases-I and -II which used pNPG as a substrate (Tab. 2) correspond with the respective F-values from the literature (Tab. 3). It should be mentioned that in the cited experiments the determinations of enzyme proteins were performed with the method of L o w k y et al. [20], which when applied to our /J-glucosidases gave protein contents that were about three times lower (cp. Materials and Methods). Consequently, an even better agreement could be attained between the high-concentration F-values of our experiments (Tab. 2) and the highest of the 7-values cited in Tab. 3 (Thermoascus aurantiacus [29], Trichoderma viride [23]) when divided by the factor 3. In contrast to the F-values, the resulting respective Zm-values (Tab. 2, 3) differed greatly. Indeed, thepNPG low-concentration iTm-value of the /5-glucosidase-I is placed within the respective literature values, but the ifm-value of thc^NPG high concentration range of enzymatic behaviour of the ^-glucosidase-I is shown to be unusually high. Therefore,

WITTE, K . , WARTENBERG, A.,

189

/S-Glucosidase

the affinity of the /3-glucosidase-I is extraordinarily low for highly concentrated p N P G . We thereby have to deduce t h a t the /3-glucosidase-I, with its two ranges of ^ N P G applied kinetic constants, covers a wide range of affinity and enzymatic reactivity even though the substrate is artificial. — Kinetik constants for the /S-glucosidase-II could not be determined due to the strong substrate induced inhibition of the enzyme. With regard to cellobiose as a substrate, the /?-glucosidase-I and /?-glucosidase-II both resulted with high affinities corresponding to low X m -values (Tab. 2). These affinities to cellobiose equal (or exceed) the respective affinities of the /?-glucosidases of A. wentii [27] and Tr. viride [31] which are marked by the lowest Km-values and therefore by the highest affinities of Tab. 3. However, the V-values of the /9-glucosidases-I and - I I determined for cellobiose as a substrate (Tab. 2) fell far below the V-values of the /S-glucosidases reported in the literature (Tab. 3). I n spite of their high affinity to cellobiose as a substrate the /S-glucosidase-I and - I I are unable to decompose high amounts of cellobiose. I t has thereby been confirmed t h a t the /3-glucosidase-I and - I I of the used strain of A. niger acted well as aryl-/?-glucosidases but failed to be potent cellobiases. I t is not possible for us at this time to determine whether the two /S-glucosidases are independent enzymes or two types of one enzyme. On the one hand, the different kinetics and the different behaviours on a column running in the anion exchange mode indicate different enzymes. On the other hand, the results from the gel filtration experiment, the results from the carbohydrate content determination, and the very similar values for £2SO show some evidence for one enzyme acting differently in two different contexts.

Abbreviations MU MUj>NPG oNPG

— — — —

4-methylumbelliferon 4-methylumbelliferyl-residue 4-nitrophenyl-/S-D-glucopyranoside 2-nitrophenyl-/S-D-glucopyranoside

Received April 4, 1988

Acknowledgements The authors are grateful to Mr. P . M E I E R S for his assistance in carrying out the experiments and to Mr. J . POCKLINGTON for proofreading the manuscript.

References [ 1 ] B E R G H E M , L . E. R . , P E T T E R S S O N , L . G . : Eur. J . Biochem. 3 7 ( 1 9 7 3 ) , 2 1 . [2] E R I K S S O N , K . - E . : Biotechnol. Bioeng. 20 (1978), 317. [3] HALLIWELL, G.: Proc. Symp. Enz. Hydrol. Cellulose, Aulanko, Finland, 1975, p. 319. [4] PETTERSSON, L. G.: Proc. Symp. Enz. Hydrol. Cellulose, Aulanko, Finland, 1975, p. 255. [ 5 ] W O O D , T . M . , M C C R A E , S . I . : Biochem. J . 1 2 8 ( 1 9 7 2 ) , 1 1 8 3 . [6] WOOD, T. M., MCCRAE, S. I.: Proc. Symp. Enz. Hydrol. Cellulose, Aulanko, Finland, 1975, p. 231. [ 7 ] B E R G H E M , L . E. R . , P E T T E R S S O N , L . G . , A X I Ö - F R E D R I K S S O N , U . - B . : Eur. J . Biochem. 5 3 (1975), 55.

[ 8 ] BERGHEM, L . E . R . , PETTERSSON, L. G., AXIÖ-FREDRIKSSON, U . - B . : E u r . J . B i o c h e m .

(1976), 621. [9] ERIKSSON, K.-E., PETTERSSON, B.: Eur. J. Biochem. 51 (1975), 312. i I

61

190 [10] [11] [12] [13]

Acta Biotechnol. 9 (1989) 2 F U J I I , M . , SHIMIZU, M . : B i o t e c h n o l . B i o e n g . 2 8 ( 1 9 8 6 ) , 8 7 8 . WOOD, T . M . , MCCRAE, S . I . : B i o c h e m . J . 1 7 1 ( 1 9 7 8 ) , 6 1 . HALLTWELL, G., RIAZ, M . : A r c h . Microbiol. 7 8 ( 1 9 7 1 ) , 2 9 5 . HALLIWELL, G . , GRIFFIN, M . : B i o c h e m . J . 1 3 5 ( 1 9 7 3 ) , 5 8 7 .

[14] STERNBERG, D.: Appl. Environ. Microbiol. 31 (1976), 648.

[15] STERNBERG, D . , VIJAYAKTTMAR, P . , R E E S E , E . T . : Can. J . Microbiol. 2 3 ( 1 9 7 7 ) , 1 3 9 .

[16] RYU, D. D. Y., MANDELS, M.: Enzyme Microb. Technol. 2 (1980), 91. [17] WOODWARD, J . , WISEMAN, A.: Enzyme Microb. Technol. 4 (1982), 1289. [18] ERIKSSON, K.-E., PETTERSSON, B . : Eur. J . Biochem. 51 (1975), 193. [19] ZACHARIAS, R . M . , ZELL, T . E . , MORRISON, J . H . , WODDLOCK, J . J . :

(1969), 148.

Anal. B i o c h e m .

30

[20] LOWRY, O. H . , ROSEBROUGH, N . J . , FARR, A. L . , RANDALL, R . J . : J . B i o l . Chem. 1 9 3 ( 1 9 5 1 ) , 265.

[21] BRADFORD, M.: Anal. Biochem. 72 (1976), 248.

[22] DUBOIS, M . , GILLES, K . , HAMILTON, J . K . , R E B E R S , P . A . , SMITH, F . : A n a l . Chem. 2 8 ( 1 9 5 6 ) , 350. [23] BERGHEM, L . E . R . , PETTERSSON, L . G . : E u r . J . B i o c h e m . 4 6 ( 1 9 7 4 ) , 2 9 5 .

[24] KING, K. W., SMIBERT, R. M.: Appl. Microbiol. 11 (1963), 315.

[25] HEPTINSTALL, J . , STEWART, J . C., SERAS, M . : E n z y m e Microb. T e c h n o l . 8 ( 1 9 8 6 ) , 7 0 .

[26] DEKKER, R. F. H.: Biotechnol. Bioeng. 28 (1986), 1438. [27] LEGLER, G.: Hoppe-Seyler's Z. Physiol. Chem. 348 (1967), 1359.

[28] HIRAYAMA, T . , HORIE, S . , NAGAYAMA, H . , MATSUDA, K . : J . B i o c h e m . 8 4 ( 1 9 7 8 ) , 2 7 . [ 2 9 ] TONG, C. C., COLE, A . L . , SHEPHERD, M. G . : B i o c h e m . J . 1 9 1 ( 1 9 8 0 ) , 8 3 . [30] GONG, C . - S . , LADISCH, M. R . , TSAO, G. T . : B i o t e c h n o l . B i o e n g . 1 9 ( 1 9 7 7 ) , 9 5 9 .

[31] WILHELM, M., SAHM, H.: Acta Biotechnol. 6 (1986), 115.

Acta Biotechnol. 9 (1989) 2, 191-195

Akademie-Verlag Berlin

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Summary Three cellulase components and one xylanase of Trichoderma sp. M-17 have been immobilized on a soluble high molecular weight polymer ( P V A ) , using carbodiimide. The immobilized enzymes retained about 80% of the cellulase, cellulose l,4-/9-cellobiosidase, ¡S-glucosidase and 60% endol,4-/S-xylanase activities. The bound enzymes catalyzed the hydrolysis of alkali-treated cornstalks with a higher efficiency than the free cellulase. The potential for reutilization of the immobilized enzymes was studied using membrane filters and the system was found to be active for three cycles.

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j i H a n p o B a T b o ö p a ö o T a H H M e MEJIOHBIO K y K y p y a H t i e C T e ß j i H ö o j i e e 3eKTHBHO, HGM H e c B H s a H H H i i i I^EJIJIROJIAAHHIT KOMNJIENC H HCN0JIB30BATBCH B T E ^ E M I E T p e x U,HKJIOB .

IIocTynnjia B peaarajHio 9 . 3 . 1988

JlHTepaTypa [1] KOSARIC, N., NG, D . C. M., RUSSELL, I., STEWART, G. S. : Adv. Appi. Microbiol. 26 (1980), 147.

[2] MANDELS, M.: Annu. Rep. Ferment. Processes 34 (1981), 423. [3] [4] [5] [6]

VIETH, W . R . , WANG, K A R U B E , I . , TANAKA, LINKO, Y . - Y . , L I N E O , FADDA, M . B . , D E S S I ,

SH. S . , GILBERT, S . G . : P a t e n t U . S . A . 3 9 7 7 9 4 1 ( 1 9 7 6 ) . S H . , SHIRAI, T . , SUZUKI, S H . : B i o t e c h n o l . B i o e n g . 1 9 ( 1 9 7 7 ) , 1 1 8 3 . P . : B i o t e c h n o l . L e t t . 1 (1979), 489. M . R . , MANRICI, R . , R I N A L D I , A . , SATTA, G . : A p p i . M i c r o b i o l . B i o -

technol. 19 (1984), 306. [7] WOODWARD, J . , ZACHRY, G. S . : E n z y m e Microb. Technol. 4 (1982), 245. [8] MITZ, M. A., SUMMARIA, L . : N a t u r e 189 (1961), 576. [9] MISHRA, CH., DESHPANDE, V., RAO, M.: E n z y m e Microb. Technol. 6 (1983), 342. [ 1 0 ] RUZIEVA, D . M . , CHERNYAVSKAYA, G . N . : B i o t e c h n o l o g i a 2 ( 1 9 8 6 ) , 7 1 .

{11] RLYOSOV, A. A., DEVDARIANI, T. G., MUDJIRI, L. A.: Appi. Biochem. Microbiol. 17 (1981), 216. {12] KUMAKURA, M., KAETSU, I . : B i o t e c h n o l . L e t t . 7 (1985), 733. { 1 3 ] O ' N E I L L , S . P . , W Y K E S , J . R . , DUNNILL, P . , LILLY, M . D . : B i o t e c h n o l . B i o e n g . 1 3 ( 1 9 7 1 ) , 3 1 9 .

{14] NELSON, N. : J . Biol. Chem. 153 (1944), 375. {15] SOMOGYI, M. J . : J . Biol. Chem. 195 (1952), 19. {16] MANDELS, M., WEBER, J . : A d v a n c e s in C h e m . Ser. 9 5 (1969), 391. {17] BERGHEM, L. E . R . , PETTERSSON, L. G,: E u r . J . Biochem. 37 (1973), 21. { 1 8 ] KANDA, T . , WAKABAYASHI, K . , NISIZAWA, K . : J . B i o c h e m . 7 9 ( 1 9 7 6 ) , 9 8 9 .

{19] MILLER, G. L . : Anal. Chem. 3 1 (1959), 426.

7*

Acta Biotechnol. 9 (1989) 2, 196

Akademie-Verlag Berlin

Book Review T. LINDL, J.

BAUER

Zell- und Gewebekultur Einführung in die Grundlagen sowie ausgewählte Methoden und Anwendungen Stuttgart, New York: Gustav Fischer Verlag, 1987 191 S., 41 Abb., 29 Tab., DM 3 6 , - ISBN 3-437-30492-5

Mit der Herausgabe dieses Kompendiums ist ein f ü r jedes Zellkulturlabor unentbehrliches praktisches H a n d b u c h entstanden. Schon auf den Innenseiten der Buchdeckel findet m a n ein alphabetisches Stichwortverzeichnis f ü r alle wichtigen Methoden, die bei der Zellkultivierung eine Rolle spielen. Diese Übersichtlichkeit u n d eine entsprechend logische Gliederung, eine o f t stichwortartige Beschränkung auf das Wesentliche aber mit weiterführenden Quellenangaben, u n d die jeweils durch U m r a h m u n g hervorgehobenen praktischen Methodenangaben sind programmatisch f ü r das Buch. Die Autoren vom I n s t i t u t f ü r angewandte Zellkultur München bzw. von der renommierten Biochemika-Firma Boehringer Mannheim legen d a m i t erstmals in deutscher Sprache ein ausführliches und der Bedeutung der in den letzten J a h r e n in stürmischer Entwicklung begriffenen modernen Zellkultivierung Rechnung tragendes Nachschlagewerk vor. Ausgehend von den räumlichen u n d apparativen Voraussetzungen und den (in der B R D ) geltenden Sicherheitsvorschriften, Angaben über die Kulturgefäße und die notwendige Steriltechnik werden Kapitel über übliche Zellkulturmedien (einschließlich serumfreier Medien und wichtiger Zusätze) und Routinemethoden wie Medienwechsel, Subkultivierung und Aufbewahrung von Zellmaterial angeschlossen. Dabei sind Tips aus vielen Labors und aus der langjährigen praktischen E r f a h r u n g entstandene Verfahrensweisen und Vorschriften einbezogen worden. Entsprechend der Bedeutung stehen K u l t u r e n mit Säugetierzellen im Mittelpunkt; in speziellen Kapiteln werden jedoch auch die entscheidenden Angaben zur Kultivierung von Pflanzenzellen, Insektenzellen und Kaltblüterzellen sowie von speziellen Zelltypen insbesondere Hybridomas dargestellt. An repräsentativen Beispielen werden Organkulturen abgehandelt. Die large-scale-Kultur ist ausschließlich auf den Labormaßstab beschränkt; hier werden Roller-, Spinner- und Hohlfasersysteme beschrieben. Eine Einbeziehung von Bioreaktorsystemen, die gerade in den letzten J a h r e n einen enormen Bedeutungsaufschwung erleben, wäre wohl über die Grenzen des Handbuches hinausgegangen; diese Thematik sollte einem eigenen Buch gewidmet sein. Ein spezielles Kapitel enthält wertvolle Angaben über Tests zur Cytotoxizität, eine Transfektionsmethode und u. a. auch die EBV-Transformation. Ein kleines fachspezifisches Lexikon, eine Übersicht über Hersteller- u n d Lieferfirmen sowie ein Anhang mit Angaben zur „Fehlersuche" sowie Berechnungsbeispielen ergänzen das hervorragende Nachschlagewerk. Es wird erfahrenen Wissenschaftlern auf dem Gebiet der Zellkultur als schnelle Möglichkeit des Auffindens von Daten, aber besonders Studenten, Diplomanden u n d Doktoranden sowie auch technischen Mitarbeitern als Anleitung zum Arbeiten dienen. W . BECHSTEDT

Acta Biotechnol. 9 (1989) 2, 1 9 7 - 2 0 0

Akademie-Verlag Berlin

Konservierung von Mikroorganismen in Polyvinylalkohol RIEDEL, K . 1 , HUBEB, H . 2 , KÜHN, M.1

1

2

Akademie der Wissenschaften der D D R Zentralinstitut für Molekularbiologie Robert-Rössle-Str. 10, Berlin, 1 1 1 5 - D D R Forschungszentrum Biotechnologie Alt-Stralau 62, Berlin, 1 0 1 7 - D D R

Summary A method is described for storage of microbial strains. The microorganisms are mixed with a 10% solution of polyvinylalcohol and stored at 4°C. It is demonstrated that most microbial strains studied remained uncharged were stable for 1 year.

Einführung Für die Sicherung der Konstanz mikrobiologischer Kulturen sind die Methoden der Stammhaltung, insbesondere die der Konservierung, von entscheidender Bedeutung. Neben der Lebenderhaltung der Mikroorganismen spielt die Erhaltung ihrer spezifischen Stammeigenschaften eine dominierende Rolle. Viele Konservierungsverfahren basieren auf einem extrem reduzierten Stoffwechsel der Zellen und einem stark reduzierten Wachstum. Solche Überlebensbedingungen können z. B. durch niedrige Temperaturen oder Entzug des Wassers und/oder des notwendigen Sauerstoffs (zutreffend für Aerobier) erreicht werden. Beispiele dafür sind das Trocknen der Zellen an inerten Trägern, die Dehydratisierung der Zellen, das Lyophilisieren, das Einfrieren in flüssigem Stickstoff [1—3], das Immobilisieren der Zellen in Polymergemischen [4] und die Lipidkonservierung [5]. In der vorliegenden Arbeit wird ein einfaches Verfahren zur Konservierung von Mikroorganismen in einer flüssigen Zubereitung mit Polyvinylalkohol (PVA) beschrieben. Damit wird die Erhaltung der Lebensfähigkeit langzeitig gesichert und der wiederholte Zugang zu den Konserven ermöglicht. Material und Methoden Mikroorganismen und

Kultivierung

Gram-negative aerobe Bakterien der Gattungen Pseudomonas und Enterobacter sowie Arthrobacter (Stammsammlung des Forschungszentrums Biotechnologie: Enterobacter cloacae B l , Escherichia coli 77/24, Klebsiella pneumoniae „C", Serratia marcescens B2, Bhizobium spec. 7/1, Pseudomonasputida, 111, Gluconobacter oxydans subsp. oxydans 3b, Arthrobacter globiformis 71) werden als Submerskulturen in Schüttelkolben angezogen und danach in einem wäßrigen Polymergemisch mit PVA konserviert.

198

Acta Biotechnol. 9 (1989) 2

Die zu konservierenden Stämme werden von Schrägagarkulturen (Nähragar I YEB Immunpräparate, Berlin Weißensee) mittels Impföse in 20 ml Nährlösung der 100 ml fassenden Steilbrustflaschen eingeimpft und 24 h bei 37 °C geschüttelt. Nach dieser Schütteldauer befinden sich die Zellen in der stationären Wachstumsphase, die Glucose ist aufgebraucht und der pH-Wert in den alkalischen Bereich verschoben. Zusammensetzung der Kulturlösimg: 0,5% Bacto-Peptone (Difco), 1,5% Bacto-Tryptone (Difco), 0,2% Hefeextrakt, 1,5% Glucose, 0.5% NaCl und 0.25% K 2 H P 0 4 ; p H vor dem Sterilisieren 6,9. Für die Konservierung aerober Sporenbildner des Genus Bacillus (Forschungszentrum Biotechnologie: Bacillus subtilis C7, B. suhtilis 44, B. lichiniformis 41p, B. cereus J39) werden versporte Zellen verwendet, die von Oberflächenkulturen auf gebräuchlichen Versporungsnährböden entnommen werden. Der Versporungsgrad der Oberflächenkulturen sollte nicht weniger als 60% betragen. Die Sporen werden mit sterilem Leitungswasser abgeschwemmt, zwecks Vereinzelung in EßLENMEYEB-Kölbchen mit Glasperlen geschüttelt und erforderlichenfalls mit sterilem Leitungswasser gewaschen. Diese Sporensuspensionen enthalten 108—1010 Sporen/ml. Die Keimzahlen (KZ) werden nach dem KocHschen Plattengußverfahren bestimmt. In gleicher Weise werden auch Sporensuspensionen mit Streptomyceten (Forschungszentrum Biotechnologie: Streptomyces spec. G43, Str. graminofaciens 15, Str. murnus 18/1) hergestellt, die auf WAKSMAN-Agar gewachsen und stark versport sind. Zusammensetzung des WAKSMAN-Ägars: 0,5% Bacto-Peptone (Difco), 0.3% Fleischextrakt, 1.0% Glucose, 0.5% NaCl, 2,0% Agar; pH,6,8. Neben den Sporen können auch Kulturlösungen mit vegetativen ¿Zellen aus der stationären Wachstumsphase für eine dauerhafte Konservierung verwendet werden. Methode der Konservierung Fester Polyvinylalkohol (Molekulargewicht: 28000—40000; VEB Laborchemikalien Apolda, DDR) wird als 10%ige wäßrige Lösung bei 121 °C 20 min, sterilisiert. 400 ¡xl dieser Lösung werden in 2 ml fassende keimfreie Rundkolbenröhrchen einpipettiert (75 X 7,5 mm; VEB GlaswarenMeuselbach, DDR), mit einem Wattestopfen verschlossen und erneut 20 min autoklaviert. In die PVA enthaltenden Röhrchen werden jeweils 400 [xl Kulturlösung oder das gleiche Volumen von Suspensionen mit Bacillus- oder Streptomyceten-S'poTeii eingetragen und mit einem dünnen Glasstab vermischt. Die Wattestopfen der Röhrchen werden mit Parafilm (Am. Can. Comp., Dixie, Marathon, Greenwich, CT) umwickelt, wodurch eine Verdunstung des Röhrcheninhaltes vermieden wird. Die Aufbewahrung dieser Konserven erfolgt bei 4°C. Enzymbestimmungen Die Bestimmung der Enzymaktivitäten für Lysindecarboxylasen (EC. 4.1.1.18) erfolgte nach [6], für Penicillin-Acylase (EC. 3.5.1.11) nach [7] und für die hydrolytischen Enzyme alpha-Amylase (EC. 3.2.1.1), Glucanase (EC.3.2.1.4) und Protease (EC. 3.4.24.4) nach [8]. Ergebnisse und Diskussion Die erzielten Ergebnisse zur Konservierung von Bakterien unterschiedlicher taxonomischer Zugehörigkeit in PVA gehen aus Tab. 1 hervor. Aus der Bestimmung der Lebendkeimzahlen nach 6 und 12monatiger Aufbewahrung in PVA wird ersichtlich, daß die untersuchten Gram-positiven Bakterien eine höhere Überlebensrate besitzen

R I E D E L , K . , H U B E R , H . U.

a., Konservierung von Mikroorganismen

199

Tab. 1. Lebendkeimzahlen verschiedener Bakterien in PVA-Konserven nach 6 und 12monatiger Aufbewahrung Species

Lebendkeimzahlen [KZ/ml]

Gram-negative aerobe Bakterien Enterobacter cloacae B 1 Escherichia coli 77/24 Klebsielle pneumoniae „ C " Serratia marcescens B 2 Rhizobium spec. 7/1 Pseudomonas putida 111 Gluconobacter oxydans subsp. oxydans 3 b Gram-positive aerobe Bakterien Bacillus subtilis C 7 B. subtilis 44 B. lichiniformis 41p B. cereus J 39 Streptomyces spec. G43 Str. graminofaciens 15 Str. murinus 18/1 Arthrobacter globiformis 71

Ausgangskeimzahlen

nach 6 Monaten

nach 12 Monaten

10" 3 X 10® 10 10 1010 10 9 3 X 108 108

5 X 105 10» 10 4 10 10 0 6 X 107 4 x 105

2 x 103 3 X 107 0 108 0 2 X 105 105

101» 101» 10 10 1010 108 108 109 5 x 108

5 x 109 109 109 5 x 109 8 X 107 5 X 107 108 3 X 106

109 109 5 X 108 109 5 X 107 107 108 8 X 104

als die Gram-negativen. Als besonders stabil unter den gewählten Konservierungsbedingungen haben sich sporenbildende Bacillusund Streptomycetenstämme erwiesen, während für Gram-negative Bakterien wie Klebsiella pneumoniae, Rhizobium spec. und Enterobacter cloacae diese Konservierung offensichtlich weniger geeignet ist. Neben der Bestimmung der Überlebensrate wurde die spezifische Leistungsfähigkeit einiger enzymbildender S t ä m m e untersucht (Tab. 2). Die Ergebnisse zeigen deutlich, daß das Enzymbildungsvermögen der untersuchten S t ä m m e durch eine einjährige Tab. 2. Enzymbildung einiger Produktionsstämme vor der Konservierung und nach einer Aufbewahrungsdauer der PVA-Konserven von 6 bis 12 Monaten Stämme

Klebsielle pneumoniae E. coli 77/24 B. cereus J 3 9 B. licheniformis 41p B. subtilis 07 B. subtilis 44

Enzyme

Lysindecarboxylase Penicillinacylase Metalloprotease Serinprotease alpha-Amylase alpha-Amylase beta-Glucanase Metalloprotease

Enzymaktivitäten nach Konservierung in [ % ] bezogen auf die Kontrolle vor der Konservierung nach 6 Monaten

nach 12 Monaten

98 98 n. b. n. b. n. b. 99 99 n. b.

n. b. 100 100 100 99

/ ) \ )

98-100

200

Acta Biotechnol. 9 (1989) 2

Lagerung in PVA nicht beeinflußt wird. Auch im Fall von Klebsiella pneumoniae bleibt die Lysindecarboxylasebildung trotz Verminderung der Lebendkeimzahlen innerhalb eines halben Jahres erhalten. Die Konservierung von mikrobiologischen Kulturen in PVA zeichnet sich im Vergleich zu herkömmlichen Methoden, wie beispielsweise der Lyophilisierung, durch ihre einfache Methodik aus, die ohne apparativen Aufwand in jedem mikrobiologischen Labor durchführbar ist. Von Vorteil ist weiterhin die Möglichkeit einer wiederholten Nutzung dieser Konserven zur Beimpfung von Kulturen. Da die Konserven unter den gewählten Bedingungen im flüssigen Zustand vorliegen, ist jederzeit eine direkte Abimpfung möglich. Voraussetzung für eine Anwendung von PVA für Konservierungszwecke ist allerdings das Unvermögen zum Abbau und der Nutzung dieses Polymers. Von Pseudomonaden sind Stämme bekannt, die PVA abbauen [9]. Weitere Untersuchungen zur Optimierung dieses Konservierungsverfahrens sind nötig und werden durchgeführt. Eingegangen: 28. i . 1988 Überarbeitet: 10. 5. 1988

Literatur Appl. Microbiol. 3 ( 1 9 6 1 ) , 1. [2] EMAIS, C. C. In: Dechema Monographien, Techn. Biochemie, Tutzing Symposium 3 (1973), 1351. [1] HECKLY, R . J . :

[ 3 ] L A P A G E , S . P . , S H E L T O N , J . E . , MITCHELL, T . G . , MACKENZIE, A . R . — I n : M e t h o d s i n M i c r o -

biology. Eds. NORRIS, J. R., RIBBONS, D. W. London, New York: Academic Press. Vol. 2 (1970) 135-225. [4] Patent: Mittel zur Abgabe von lebensfähigen Mikroorganismen. DE-OS 2608601 (1976). [5] NÖLDECHEN, A . : B i o e n g . 8 ( 1 9 8 6 ) , 4 4 .

[6] HITBER, J., WEISBACH, F.: Acta Biotechnol. 6 (1986), 273. [ 7 ] KUTZENBACH, C., RAUENBUSCH, E . : Z. P h . C h . , 3 5 4 ( 1 9 7 4 ) , 4 5 .

[8] RTJTTLOFF, H. U. a.: Industrielle Enzyme, Fachbuchverlag Leipzig (1979). [9] SAKAI, K., HAMADA, N., WATANABE, Y.: Agric. Biol. Chem. 50 (1986), 989.

Acta Biotechnologica Volume 9

Number 2

1989

Contents E.; D O E L L E , H. W. : Optimization of Single Cell Protein Production from Cassava Starch (Rhizopus oligosporus) SUKARA,

WARNECKE,

H.-J. : Macromixing Characteristics of Gas-Liquid J e t Loop Reactors

99 Ill

K. ; R Ü H L E M A N N , I. ; B E C K E R , U. ; B E R G E R , R . : A Comparison between the Fermentative Activities of Free and Ca-Alginate-Entrapped Cells of Saccharomyces cerevisiae . . 123 RICHTER,

S C H N E I D E R , J . D. ; H A D E B A L L , W. ; F E I L E R , E. : Continuous Fermentation of Methanol and in Mixtures with Grain of Stillage and Molasses with Hansenula polymorpha MH 26 (in German) 131 O N A G H I S E , E. O . ; I Z I J A G B E , Y. S.: Improved Brewing and Preservation of Pito, a Nigerian Alcoholic Beverage from Maize 137 MADAN,

M. ; K A M R A , N. : Comparative Organic Growth Factor Requirements of Nine Candida

species

143

J . M. ; A R E , R. J . ; VIESTTJRS, U. E. : Growth of Pellets of a Basidal Fungus Pleurotus ostreatus under Various Cultivation Condition 149 STYOGANTSEVA,

HÄRTUNG, B .

: Streptomycetes as Producers of Industrial and Diagnostic Important Enzymes

(in German)

157

L. ; S I K Y T A , B. ; S M É K A L , F. : Production of Lysine by Mutants of Escherichia coli K 12 in a Medium with Lactose 173 SOBOTKOVA,

WITTE, K . ; WARTENBERG, A . :

Purification and Properties of two /3-Glucosidases Isolated

from Aspergillus niger

179

; K O L E V , D. ; on a Soluble Polymer (in Russian) SPASOV, S . ; BAKALOVA, N .

NIKOLOV,

T. : Immobilization of Cellulase Complex 191

Short Communications RIEDEL,

K.;

HTJBER, H . ;

KÎTHN,

M.: Storage of Microorganisms in Polyvinylalcohol (in

German) Book Reviews

197 . 110, 122, 129, 130, 142, 148, 156, 177, 178, 196

Acta Biotechnologica is indexed or abstracted in Current Contents/ET & AT; Chemical Abstracts; Biological Abstracts; Biotechnology Abstracts; Excerpta Medica

Acta Biotechnologica Volume 9

Number 2

1989

Contents E.; D O E L L E , H. W. : Optimization of Single Cell Protein Production from Cassava Starch (Rhizopus oligosporus) SUKARA,

WARNECKE,

H.-J. : Macromixing Characteristics of Gas-Liquid J e t Loop Reactors

99 Ill

K. ; R Ü H L E M A N N , I. ; B E C K E R , U. ; B E R G E R , R . : A Comparison between the Fermentative Activities of Free and Ca-Alginate-Entrapped Cells of Saccharomyces cerevisiae . . 123 RICHTER,

S C H N E I D E R , J . D. ; H A D E B A L L , W. ; F E I L E R , E. : Continuous Fermentation of Methanol and in Mixtures with Grain of Stillage and Molasses with Hansenula polymorpha MH 26 (in German) 131 O N A G H I S E , E. O . ; I Z I J A G B E , Y. S.: Improved Brewing and Preservation of Pito, a Nigerian Alcoholic Beverage from Maize 137 MADAN,

M. ; K A M R A , N. : Comparative Organic Growth Factor Requirements of Nine Candida

species

143

J . M. ; A R E , R. J . ; VIESTTJRS, U. E. : Growth of Pellets of a Basidal Fungus Pleurotus ostreatus under Various Cultivation Condition 149 STYOGANTSEVA,

HÄRTUNG, B .

: Streptomycetes as Producers of Industrial and Diagnostic Important Enzymes

(in German)

157

L. ; S I K Y T A , B. ; S M É K A L , F. : Production of Lysine by Mutants of Escherichia coli K 12 in a Medium with Lactose 173 SOBOTKOVA,

WITTE, K . ; WARTENBERG, A . :

Purification and Properties of two /3-Glucosidases Isolated

from Aspergillus niger

179

; K O L E V , D. ; on a Soluble Polymer (in Russian) SPASOV, S . ; BAKALOVA, N .

NIKOLOV,

T. : Immobilization of Cellulase Complex 191

Short Communications RIEDEL,

K.;

HTJBER, H . ;

KÎTHN,

M.: Storage of Microorganisms in Polyvinylalcohol (in

German) Book Reviews

197 . 110, 122, 129, 130, 142, 148, 156, 177, 178, 196

Acta Biotechnologica is indexed or abstracted in Current Contents/ET & AT; Chemical Abstracts; Biological Abstracts; Biotechnology Abstracts; Excerpta Medica