Acta Biotechnologica: Volume 7, Number 3 1987 [Reprint 2021 ed.] 9783112581483, 9783112581476

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Acta BlMiologica Journal of microbial, biochemical and bioanalogous technology

Akademie-Verlag Berlin ISSN 0138-4988 Acta Biotechnol., Berlin 7 (1987) 3, 207- 292

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Acta Biotutooloiica Journal of microbial, biochemical and bioanalogous technology

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

Editorial Board: P. Moschinski, Lodz A. Moser, Graz M. D. Nicu, Bucharest Chr. Panajotov, Sofia L. D. Phai, Hanoi H. Sahrn, Jülich W. Scheler, Berlin R. Schulze, Halle B. Sikyta, Prague G. K . Skrjabin, Moscow M. A. Urrutia, Habana J . E. Zajic, El Paso

1987

A. A. Bajev, Moscow M. E. Beker, Riga H. W. Blanch, Berkeley S. Fukui, Kyoto H. G. Gyllenberg, Helsinki G. Hamer, Zurich J . Hollo, Budapest M. V. Iwanow, Moscow F. Jung, Berlin H. W. D. Katinger, Vienna K . A. Kalunjanz, Moscow J . M. Lebeault, Compiegne D. Meyer, Leipzig

Number 3

Managing Editor:

L. Diinter, Leipzig

Volume 7

AKADEMIE-VERLAG

B E R L I N

"Acta Biotechnologica" publishes original papers, short communications, reports and reviews from t h e whole field of biotechnology. The journal is to promote t h e establishment of biotechnology as a new and integrated scientific field. The field of biotechnology covers microbial technology, biochemical technology and t h e technology of synthesizing and applying bioanalogous reaction systems. The technological character of t h e journal is guaranteed by t h e f a c t t h a t papers on microbiology, biochemistry, chemistry and physics must clearly have technological relevance. Terms of subscription for t h e journal "Acta Biotechnologica" Orders can be sent — in the GDR: to Postzeitungsvertrieb or to t h e Akademie-Verlag Berlin, Leipziger Str. 3—4, P F 1233, D D R -1086 Berlin; — in the other socialist countries: to a book-shop for foreign languages literature or to t h e competent news-distributing agency; — in the FRG and Berlin (West) : to a book-shop or to t h e wholesale distributing agency Kunst uild Wissen, Erich Bieber, Wilhelmstr. 4 — 6, D-7000 S t u t t g a r t 1; — in the other Western European countries: to K u n s t 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 D D R , P F 160, D D R - 7 0 1 0 Leipzig, or to t h e AkademieVerlag Berlin, Leipziger Str. 3 - 4 , P F 1233, D D R -1086 Berlin.

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Acta Biotechnol. 7 (1987) 3, 209—219

Formation of Higher Alcohols and Phenol by Strains of Zymomonas sp. R A O , S . C., JONES, L . P .

Department of Biological Sciences University of Texas a t El Paso Texas 79968, U.S.A.

Summary Strains of the bacteria Zymomonas sp. were studied for their ability to form higher alcohols. In a complex growth medium, six strains were shown to produce significant amounts of 1-propanol, 1-butanol, 2-methyl-l-butanol, 3-methyl-l-butanol, 2-methyl-2-butanol, pentanols, secondary hexyl-alcohols, and trace amounts of n-hexanol. When resting cells of these organisms were placed into a fermentation medium containing glucose and Tris-buffer, Z. mobilis 8938 produced increased levels of 1-butanol, and secondary hexyl-alcohols a t concentrations of 13.5 mg/liter and 5.8 mg/ liter, respectively. Another strain, Z. mobilis subsp. mobilis B 806, stimulated t h e formation of 1-propanol and 1-butanol a t concentrations of 14.9 mg/liter and 23.52 mg/liter, respectively. Amino acids or amino acid precursors were then added to the fermentation medium. The presence of threonine and a-ketobutyric acid stimulated Z. mobilis 8938 to produce 82.6 mg/liter secondary hexyl-alcohols and 8.0 mg/liter n-hexanol, respectively. Isoleucine and valine increased the production of 2-methyl-l-butanol (394.0 mg/liter) and 3-methyl-l-butanol (113.4 mg/liter), respectively, by Z. mobilis subsp. mobilis B 806. Glutamine enhanced the formation of 2-methyl-2-butanol production to concentrations 38.8 mg/liter in Zymomonas strain B 806. Additional experiments suggested t h a t higher alcohol production could also be accomplished in the absence of glucose when cells were allowed to metabolize t h e precursors only. The effect of aromatic amino acids on phenol production was determined using resting cells of Zymomonas sp. The maximum yield of phenol (111.6 mg/liter) was found b y Zymomonas strain 8938 in the presence of tyrosine. The addition of phenylalanine also stimulated this strain to form 71.4 mg/liter of phenol.

Introduction The genus Zymomonas is a Gram-negative, mostly nonmotile, nonsporing, anaerobic rod occurring singly and in pairs. It can be found in spoiled beer [1] fermented apple juice, and in sugar cane sap [2], Historically, these beers and ciders have developed "fruity" aromas due to the production of aldehydes, esters, and fusel alcohols. Very limited information is available with respect to high molecular weight alcohol production by Zymomonas sp. B E V E R S and VERACHTERT [3] did report on several strains of Zymomonas sp. capable of producing n-propanol, iso-butanol, D-amyl- and iso-amylalcohols by both growing and resting cells. The resting cell studies resulted in an increased yield of higher alcohols in the presence of amino acids and/or their precursors. The mechanisms by which these precursors support the production of higher alcohols have been studied in yeast and bacteria [3, 4], 1*

Acta Bio techno]. 7 (1987) 3

210

Since high molecular weight alcohols are commercially important, it was of interest to study their synthesis in the bacteria Zymomonas sp. This communication describes the conversion of amino acids and/or their precursors in the presence and absence of glucose for the production of higher alcohols. The synthesis of phenol by Zymomonas sp. using aromatic amino acids is also reported.

Materials and Methods Bacteria The six strains of Zymomonas sp. used in this study and their sources were as reported by et al. [6] (Table 1).

ZAJIC

Table 1. The production of higher alcohols by growing and resting cells in strains of Zymomonas sp. Higher alcohols [mg/liter] Glucose 1-Pro- 1-BuStrain Dry biomass utiliza- panol tanol tion [g / 100 ml] [%] 0.178

8938

—

B 806

0.198 —

B 4490

0.138 —

10988

0.150 —

B 1073

0.133 —

8227

0.166 —

a b c d

2-Me3-Me2-MePenta- Sec. n-Hexathyl-1- thyl-1- thyl-2- nols Hexyl nol butanol butanol butanol alcohols®

88.015

9.20

7.60

27.70

32.80

0.60

75.0 d 89.0

12.30 8.00

13.50 18.00

1.40 3.80

34.40 30.00

65.0 86.0

14.90 4.50

23.52 2.52

5.60 1.20

70.0 99.0

31.80 51.60

4.20 3.80

98.0 99.0

8.80 Trace

99.0 90.0 80.0

Phe nol

c

4.30

Trace

Trace

0.20 Trace

4.79 10.40

5.80 Trace

Trace Trace

32.70 1.30

Trace Trace

12.30 Trace

Trace Trace

Trace Trace

Trace Trace

Trace 46.00

Trace Trace

3.70 Trace

6.30

—

—

—

—

—

2.40 37.90

Trace 6.30

48.60

Trace Trace

—

7.40 6.50

1.80 4.80

Trace 4.80

40.50 32.20

•Trace 1.20

2.57 Trace

Trace

60.50

10.80

9.60

21.60

1.30

16.25

9.10

Trace

—

—

1.25

— —

— —

—

—

—

—

—

—

—

—

Trace

—

—

Includes the following alcohols: 2-Methyl-l-pentanol and/or 4-Methyl-2-pentanol Growing cells Not detected Resting cells

Chemicals Amino acids and/or their precursors were obtained from Sigma Chemical Co. Alcohol standards were purchased from Supelco, Inc. All other chemicals were analytical reagent-grade quality. Media All organisms were maintained on agar slants of a basal medium composed of 0.5% yeast extract (w/v), 2.0% glucose and 2.0% agar with transfers to fresh media every three weeks. A liquid medium composed of 5.0% glucose, 1.0% yeast extract, 0.5% KH 2 PO, and 2.0% mineral salts solu-

211

RAO, S. C., JONES, L. P., Formation of Higher Alcohols

tion (v/v), as described by G I B B S and D E M O S S [ 7 ] , was used as t h e growth medium. A resting cell medium, composed of 5.0% glucose in 0.1 M Tris-HCl buffer (pH 6.4), was used as the fermentation medium. Experiments also involved t h e addition or replacement of glucose in the fermentation medium by 300 u Moles of amino acids and/or their precursors. Culturing

Procedures

Seed cultures were obtained by inoculating 5 ml of growth medium in a screw-cap test tube from agar slants and incubating in anaerobic jars for 72 h a t 30 °C. One ml of seed culture was used t o inoculate 50 ml of growth medium in a 125 ml E R L E N M E Y E R flask and incubated as before. After 72 h, cultures were centrifuged (24,000 x g) for 20 min a t 28°C. The cells were then washed and resuspended in 10 ml of saline. Two ml of this cell suspension was used to inoculate 750 ml of growth medium in a 1 liter Erlenmeyer flask. After 72 h of incubation, 50 ml of this cell suspension was centrifuged; t h e supernatant was analyzed for sugar consumption and higher alcohol production. The pellet was used to determine the biomass which was recorded in g dry wt/100 ml. The cells remaining from the growth medium were collected by centrifugation, washed, and used to inoculate 100 ml of fermentation medium in a 125 ml E R L E N M E Y E R flask for experiments using zesting cells. All experiments were stopped after 72 h or after most of the glucose had been utilired. All test systems were monitored for sugar consumption, higher alcohol production, and dry biomass. Analytical

Methods

Utilization of sugar was determined according to a method described by M I L L E R et al. [8]; t h e values expressed as percent glucose consumed. To determine biomass, a known volume of cell suspension (routinely 50 ml) was centrifuged and the pellet dried to constant weight in a hot-air oven a t 105 °C. Higher alcohols were determined on a Varian Model 1400 gas chromatograph equipped with an 80/100 Carbopack C/0.3% Carbowax 20 M glass column. Column, injector, and d e t e c t o r temperatures were maintained a t 175, 150 and 250°C, respectively. Nitrogen was used as t h e carrier gas a t a flow rate of 60 ml/min. The volume of sample injected into the gas chromatograph was 0.5 —1.0 (xl. Higher alcohols in the medium were determined using the retention times of peaks and normalized using the retention times of internal standards. All standard deviations were performed using regression analyses.

Results Growth and Substrate

Utilization

S i x isolates of Zymomonas sp. were a n a l y z e d for their ability t o p r o d u c e higher a l c o h o l s i n b o t h a g r o w t h m e d i u m a n d in a f e r m e n t a t i o n m e d i u m c o n t a i n i n g 5 % g l u c o s e ( w / v ) . R e s u l t s f r o m t h e s e a n a l y s e s are s u m m a r i z e d in T a b l e 1. T h e b i o m a s s f r o m t h e g r o w t h m e d i u m s h o w e d little d i f f e r e n c e b e t w e e n strains. B i o m a s s i n t h e f e r m e n t a t i o n m e d i u m d i d n o t increase a p p r e c i a b l y f r o m t h e i n o c u l u m , i n d i c a t i n g g l u c o s e w a s b e i n g utilized either for m a i n t e n a n c e e n e r g y a n d / o r for t h e p r o d u c t i o n of precursors a n d co-factors in t h e d e n o v o s y n t h e s i s of higher alcohols. S u g a r c o n s u m p t i o n for all strains in t h e g r o w t h m e d i u m w a s similar, ranging f r o m 8 5 % t o 9 9 % g l u c o s e utilization. I n t h e f e r m e n t a t i o n m e d i u m , h o w e v e r , g l u c o s e u t i l i z a t i o n v a r i e d g r e a t l y f r o m strain t o strain. The Production

of Higher

Alcohols

by Growing

and Resting

Cells

T a b l e 1 s h o w s t h a t of t h e strains of Zymomonas s t u d i e d , all p r o d u c e d s i g n i f i c a n t a m o u n t s of 1-propanol a n d 1-butanol. F i v e - c a r b o n alcohols, i n c l u d i n g 2 - m e t h y l - l - b u t a n o l , 3m e t h y l - l - b u t a n o l , 2 - m e t h y l - 2 - b u t a n o l a n d p e n t a n o l s (including t h e p r i m a r y a n d s e c o n d a r y p e n t a n o l s ) , were f o u n d t o b e p r o d u c e d b y several strains; n o t a b l y , 8938, B 8 0 6 ,

212

Acta Biotechnol. 7 (1987) 3

B 4490, 10988, B 1073, and B 8227. Strains 8938, B 806, 10988, and 8227 synthesized greater than 30 mg/liter 3-methyl-l-butanol. Significant amounts of pentanols were produced by strains 8938, B 806, and 8227. Several of these organisms, including strain B 4490, were also capable of forming six-carbon secondary hexyl-alcohols, and n-hexanol. The Screening of Zymomonas Strains for their Ability to Form Higher Alcohols from Amino Acids and/or their Precursors. Three strains demonstrated potential for higher alcohol production. These strains were: Z. mobilis 8938, Z. anaerobic 8227, and Z. mobilis subsp. mobilis B 806. Two strains were chosen for further study to analyze the effect of amino acids and/or their precursors on higher alcohol production in the presence and absence of glucose. Table 2. The production of higher alcohols by Z. mobilis 8938 in the fermentation medium using amino acids and/or their precursors in the presence of glucose Amino acids and/or their precuisors

Aspartate Family Oxaloacetate Aspartic acid Homoserine Threonine a-Ketobutyric acid Isoleucine Lysine

Higher alcohols [mg/liter] l pro

2

-Methyl- 3-Methyl- 2-Methyl- Penta- Sec. n-Hexa- Phenol 1-butanol 1-butanol 2-butanol nols Hexylnol alcohols'5

tanol

5.6 24.3 37.5 24.5 30.9

3.0 14.0 12.0 14.5 20.6

8.2 128.5 35.2 58.2 40.0

14.5 11.5

13.9 56.0

10.3 3.0

54.5

68.9 150.0 128.0

18.0 72.8 12.5

Trace 35.1 76.8

15.0 32.1

2.1 34.0

15.0

Pyruvate Family 178.0 Alanine Valine 18.5 a-Ketoisovaleric 25.5 acid Leucine 19.5 a-Ketoisocaproic 13.0 acid Glutamate Family 32.7 a-Ketoglutaric acid Histidine Family 125.0 Histidine Aromatic Family Tyrosine Phenylalanine Tryptophane

1 B u

panol

17.2 17.0 15.2

c

Trace

6.9 1.2 13.4

5.5 Trace 5.4 11.3 9.1

62.3 18.6 82.6 Trace

1.2 30.2

21.0 6.2

41.4 Trace

4.5 4.5 2.5

Trace 12.0 15.0

Trace 2.5 36.5

Trace 4.6

44.3 64.2

1.7 1.8

8.3 6.2

3.0 8.3

0.9 Trace

—

35.1

4.5

71.8

12.0

12.5

8.0

—

23.5

16.0

34.5

2.2

Trace

Trace

—

—

42.6 Trace 4.6

Trace Trace Trace

—

—

23.4 31.2

-

22.3 13.6 Trace

—

— —

—

Trace Trace Trace

—

-

—

—

—

—

8.0

—

Trace

—

-

—

—

—

—

—

—

—

—

—

—

The amount of amino acid or precursor added was 300 ¡x Moles *> Includes the following alcohols: 2-Methyl-l-pentanol and/or 4-Methyl-2-pentanol c Not detected a

—

— —

—

111. 71.

213

RAO, S. C., JONES, L. P., Formation of Higher Alcohols

Z. mobilis

8983

Amino acids and/or their precursors serve as key substrates to form higher alcohols through intermediate metabolic products in many microorganisms. Table 2 summarizes the results for the synthesis of higher alcohol production from these precursors in the presence of glucose by Z. mobilis 8938. All flasks contained 250—350 mg of cells. Sugar consumption ranged from 81—89%. The formation of 1-propanol, 1-butanol, branched derivatives of butanol and pentanols, was greatly enhanced by the addition of most amino acids and/or their precursors when compared to Table 1 in the absence of precursors by this strain. The aspartate family of amino acids and/or their precursors, when added to resting cells of Z. mobilis 8938, stimulated higher alcohol production. Most notable was the fact that this strain produced 128.5 mg/liter 2-methyl-1-butanol from aspartate, 54.5 ing/liter 3-methyl- 1-butanol and 21.0 mg/liter pentanols from isoleucine. Of the pyruvate family of amino acids, valine stimulated Z. mobilis 8938 to produce 150.0 mg/liter 1-butanol and alanine stimulated the production of 178.0 mg/liter of 1-propanol. It was also noted that the intermediate ¿x-ketoiso valeric acid and a-ketoisocaproic acid stimulated the formation of 76.8 mg/liter and 64.2 mg/liter 3-methyl-lbutanol, respectively. Table 3. The production of higher alcohols by Z. mobilis 8938 in the fermentation medium using amino acids and/or their precursors in the absence of glucose Amino acids and/or

Higher alcohols [mg/liter]

their precursors*

1 pro

1 B u

panol

tanol

1-butanol

1.8 0.8 5.6 1.2 18.3

Trace

0.5

Aspartate Family Oxaloacetic acid Aspartic acid Homoserine Threonine »-Ketobutyric acid Isoleucine lysine Pyruvate Family Alanine Valine a-Ketoisovaleric acid Leucine

« JS

T3

s>>

3

« -

.2

"3

•3 O

••!

JS

m

0

pa W

o tí cí

tí m

M E

218

Acta Biotechnol. 7 (1987) 3

B E V E R S a n d V E R A C H T E E T [3] reported t h a t when these precursors were given to resting cells of S. cerevisiae, t h e f o r m a t i o n of n-propanol (9.32 mg/liter), isobutanol (30.83 mg/liter), 2 - m e t h y l - l - b u t a n o l (8.52 mg/liter), a n d 3-methyl-1-butanol (13.05 mg/liter) was observed. T h e y also showed t h a t a-ketoisovaleraldehyde increased t h e f o r m a t i o n of 3-methyl- 1-butanol (25.2 mg/liter) in Zymomonas strain 8939. However, t h e formation of 1-butanol was n o t detected in t h e study. Obviously, the strains reported herein are more aggressive in their conversions. Figure 1 shows how t h e serine family of amino acids f o r m higher alcohols b y p a t h w a y s involving p y r u v a t e as an intermediate. Of t h e g l u t a m a t e f a m i l y of compounds, notably a-ketoglutaric acid, was shown t o enhance t h e formation of 1-propanol (271.3 mg/liter) a n d 2-methyl-2-butanol (71.8 mg/liter) by Zymomonas strains B 806 a n d 8938, respectively. This precursor can be converged to oxaloacetic acid a n d t h e n to higher alcohols. This was also t r u e in t h e case of g l u t a m i n e with respect to branched-butanols. Other amino acids, like glutamic acid, can be converted either to p y r u v a t e a n d t h e n to 1-butanol or t o b u t y r a t e , ultimately forming 1-butanol via 1 - b u t y r a l d e h y d e (Figure 1). This was observed, a n d resulted in t h e stimulation of 1-butanol (118.0 mg/liter) in strain B 806. This indicated Zymomonas sp. utilize g l u t a m a t e more efficiently as compared t o strains of Clostridium, which produced only t r a c e a m o u n t s of 1-butanol f r o m g l u t a m a t e [9]. Finally, t h e stimulation of higher alcohols by Zymomonas sp. was observed in t h e presence of histidine. This can be accomplished by converting histidine, either to p y r u v a t e or to glutamic acid, which may act as a s u b s t r a t e to higher alcohol formation (Figure 1). An increased effect on t h e production of secondary hexyl-alcohols was also noticed in t h e presence of most of t h e compounds of a s p a r t a t e , p y r u v a t e , a n d g l u t a m a t e families of amino acids a n d / o r their precursors. The f o r m a t i o n of n-hexanol was shown to be enhanced with t h e addition of isoleucine, a-ketoisovaleric acid, leucine, a n d a-ketoglutaric acid. The mechanism for t h e f o r m a t i o n of these alcohols is not k n o w n a t t h e present time. These are noval reactions. A possible mechanism, however, m a y be t h a t t h e synthesis of n-hexanol a n d / o r secondary hexyl-alcohols is due to t h e f o r m a t i o n of b u t y r a l d e h y d e a n d acetaldehyde which are intermediates in t h e f o r m a t i o n of higher alcohols via t h e m e t a bolism of amino acids (Figure 1). These two compounds can be condensed using aldolase enzymes which t h e n undergo t h e following series of reactions to form n-hexanol or secondary hexyl alcohols:

Butyraldehyde dehydration + -> /j-Hydroxyaldehyde —\—, . , , , • Acetaldehyde alcohol dehydrogenase ->• n-hexanol and/or secondary alcohol This conclusion can also be s u p p o r t e d f r o m t h e results obtained f r o m precursors by Zymomonas strains in t h e absence of glucose. Trace a m o u n t s of higher alcohols were synthesized b y Zymomonas sp. I t a p p e a r s t h a t these organisms are capable of converting amino acids a n d / o r their precursors into their corresponding higher alcohols b y using several enzymatic reactions. A similar observation also was reported previously b y B E V E R S a n d VERACHTERT [3].

F o r m a t i o n of phenol f r o m aromatic amino acids has been reported previously in m a n y microorganisms [10—12] including Clostridium sp. [13, 14]. I t has been previously reported t h a t t h e e n z y m e tyrosine phenollyase catalyzes t h e conversion of tyrosine to phenol. This reaction has not been observed previously in Zymomonas sp. This s t u d y shows, for t h e first time, t h a t Zymomonas sp. can produce phenol using tyrosine as a s u b s t r a t e a n d resulted in t h e f o r m a t i o n of phenol u p to 111.6 mg/liter f r o m tyrosine b y strains of Zymomonas tested.

RAO, S. C., JONES, L. P., Formation of Higher Alcohols

219

A similar result was also obtained with the addition of phenylalanine. A possible explanation is that phenylalanine can be converted to tyrosine by the enzyme phenylalanine hydroxylase. Tyrosine m a y then be converted to phenol. This conclusion can be supported b y several observations. These strains were not able to form phenol from fermentation medium in the absence of these precursors. This clearly indicated that the aromatic amino acids play an important role in Zymomonas sp. to synthesize phenol. These results can be compared well with the results obtained by SPOELSTRA [10], ELSDEN et al. [13], and CHEN and L e v i n e [12], The phenol-producing capability of Zymomonas strains establishes a need for improved control of these cultures in beer and wine making. Tyrosine and phenylalanine should be kept at low levels in the fermentation medium. T h e Zymomonas sp. tested also have in common the ability to form 1-propanol, 1-butanol and branched derivatives of butanol in the presence of aromatic amino acids. A n explanation is that the released pyruvate during aromatic amino acid metabolism m a y serve as a precursor to form these higher alcohols or b y using de novo synthesis of glucose.

Acknowledgement A Portion of this study was supported by a grant from the University of Texas at El Paso. Received July 17, 1986

References [1] AULT, R. G.: J. Inst. Brew. (London) 71 (1965), 376. [ 2 ] SWINGS, J., D B L E Y , J . : B a c t e r i o l . R e v . 41 (1977), 1.

[3] BEVERS, J., VERACIITERT, H.: J. Inst. Brew. (London) 82 (1976), 35. [4] PRESCOTT, C. S., DUNN, C. G. : Industrial Microbiology, 3rd ed. New York: McGraw-Hill Book Company, Inc., 1959, 10. [5] MONICK, A. J. : Alcohols, Their Chemistry, Properties and Manufacture, New York : Rheinhold Book Corporation, 1968, 88. [ 6 ] ZAJIC, J. E., M A D D U X , N . , JONES, L . P . : A c t a B i o t e c h n o l . 2 (1982), 307.

[7] GIBBS, M., DEMOSS, R. D.: J. Biol. Chem. 207 (1954), 689. [ 8 ] MILLER, G. L . , B L U M , R . , GLENNON, W . E . , BURTON, A . L . : A n a l y t i c a l B i o c h e m . 1 ( 1 9 6 0 ) , 127.

[9] GOTTWALD, M., HIPPIE, H., GOTTSCIIALK, G.: Appi. Envt. Microbiol. 48 (1984), 573. [10] SPOELSTRA, F. S.: Appi. Envt. Microbiol. 36 (1978), 631. [11] CHEN, T. C., NAWAR, W. W., LEVIN, R. F.: Appi. Microbiol. 28 (1974), 679. [12] CHEN, T . C., LEVIN, R . E . : A p p i . M i c r o b i o l . 30 (1975), 120. [13] ELSDEN, S. R . , HILSTON, M . G., WALLER, J. M . : A r c h . M i c r o b i o l . 107 (1976), 283. [14] BROT, N . , SMIT, S., WEISSBACK, H . : A r c h . B i o c h e m . B i p h y s . 112 (1965), 1.

Acta Biotechnol. 7 (1987) 3,220

Book Review H . K L E I N K A U F , H . V. D Ö H R E N , H . D O R N A U E E , G .

NESEMANN

Regulation of Secondary Metabolite Formation Proceedings of the Sixteenth Workshop Conference Hoechst, Gracht Castle, May 1 2 - 1 6 , 1985 Weinheim: VCH Verlagsgesellschaft m b H , 1986 402 S., 142 Abb., 53 Tab., 108 DM

Das Buch ist das Ergebnis der 16. Workshop Conference Hoechst a t Gracht Castle. Wissenschaftler aus Berlin-West, der B R D , CSSR, D D R , aus England, Israel, J a p a n , K a n a d a , Spanien und den USA haben mit 23 Vorträgen, die von 64 Autoren verfaßt worden sind, zum Gelingen dieser Konferenz beigetragen. Auf 367 Seiten erfährt der Leser Neues und Interessantes über die Genetik der Mycotoxinbildung (J. D. BU'LOCK), über multiple Formen der RNA-Polymerase bei Streplomyces (Janet W E S T P H E L I N G et al.), über die Organisation und Expression von Genen der Antibiotikasynthese bei Streptomyces (D. A. H O P W O O D et al.), über Vektoren f ü r eukaryotisches Klonieren (K. ESSER), über die Regulation der /?-Lactam- Bi osynthe.se durch Kohlenstoff und Stickstoff (J. F. M A R T I N et al.), über die Regulation der Cephamycin-Bildung durch Stickstoffverbindungen (A. L. D E M A I N & A. F. B R A N A ) , über den Einfluß des Lysin-Stoffwechsels auf die Cephamycin C-Biosynthese (Y. A H A K O N O W I T Z et al.), über die Genetik von Penicillium chrysogenum und die Biosynthese des jS-Lactam Antibiotikums (G. H O L T et al.), über Enzyme der Penicillin- und Cephalosporinbildung (Sir E. P. ABRAHAM), über die Beteiligung der CyclopeninCyclopenolbiosynthese am Entwicklungsprogramm von Penicillium, cyclopium (M. L U C K N E R et al.), über Ergot Peptid Alkaloid-Synthese bei Claviceps purpurea (U. KELLER), über Enzymsysteme, die Peptidantibiotika synthetisieren ( H . K L E I N K A U F & H . V. D Ö H R E N ) , über die Regulation der Chloramphenicolbildung (L. C. V I N I N G et al.), über Faktoren, die die Synthese von Polyketiden und Glycopetiden bei Streptomyceten regulieren (U. G R Ä F E et al.), über die Regulation der TetracylinSynthese und von Enzymen des Sekundärstoffwechsels (V. BEHAL), über die Biosynthese von Polyketid-Antibiotika (H. G. F L O S S et al.), über die Synthese von Tylosin und ihre Regulation durch N H , + und P O , 3 - (S. O M U R A & Y. T A N A K A ) , über Beziehungen zwischen der Produktivität der Aminoglycosid-Antibiotika und der Antibiotika-Resistenz des Produzenten (Y. OKAMI) sowie über Induktion und Regulation der Phytoalexinsynthese bei Sojabohnen (H. GRISEBACH). Die letzten 20 Seiten des insgesamt 388 Seiten umfassenden Buches gehören H a n s von D Ö H R E N mit dem Thema „Current Research on Secondary Metabolism — A General Discussion" und Arnold L. D E M A I N mit „Giesing R e m a r k s : Regulation of Secondary Metabolite F o r m a t i o n : Progress and Challenges". Das Buch vermittelt einen guten Einblick in den Stand der Entwicklung und in die gegenwärtig bestehenden Probleme auf dem Gebiet der Sekundärmetabolit-Produktion. So gesehen ist es jedem Experten wärmstens zu empfehlen. Aber es wird sicher auch von nicht auf diesem Gebiet tätigen Biowissenschaftlern m i t Gewinn zu lesen sein, wobei insbesondere die zuletzt genannten Beiträge helfen, in die Problematik einzuführen. W. Babel

Acta Biotechnol. 7 (1987) 3, 2 2 1 - 2 2 5

D-Amino Acid Oxidase, Aromatic L-Amino Aminotransferase, and Aromatic Lactate Dehydrogenase from Several Yeast Species: Comparison of Enzyme Activities and Enzyme Specificities BODE, R . BIBNBAUM, D .

Emst-Moritz-Arndt-Universität Greifswald Sektion Biologie, WB Molekularbiologie Jahnstraße 15 a, Greifswald, 2200 G.D.R.

Summary Production of D-amino acid oxidase, L-aromatic aminotransferase and aromatic lactate dehydrogenase by several yeast species was examined. Of 16 strains tested, Trigonopsis variabilis and Rhodosporidium toruloides were found to be most suitable for D-amino acid oxidase production, T. variabilis and Brettanomyces anomalus for L-aromatic aminotransferase production, and Hansenula polymorpha, Cryptococcus terreus, and Candida maliosa for aromatic lactate dehydrogenase production. This selection is based on a high amount of enzyme activity as well as a broad enzyme specificity. The data will be reported here.

Introduction During t h e last years t h e knowledge accumulated by intensive enzyme studies in vitro became more a n d more relevance t o biotechnological aspects. Besides traditionally industrial applications of yeasts as in brewery, bakery, winery, in distillation, food and feed production various yeast species play an increasing p a r t for production of enzymes. R e c e n t studies with t h e fodder yeast Candida maltosa [1, 2] have provided circumstantial evidence for t h e involvement of three enzyme activities (D-amino acid oxidase, arom a t i c L-ainino aminotransferase, aromatic lactate dehydrogenase) in t h e catabolism of a r o m a t i c amino acids which m a y be of practical importance. D-Amino acid oxidase (EC 1.4.3.3) catalyzes t h e oxidative deamination of a variety of D - a m i n o acids with t h e production of t h e respective 2-keto acids and ammonia. This enzyme from b o t h microbial and m a m m a l i a n sources is useful for d e t e r m i n a t i o n and degradation of D - a m i n o acids, testing optical p u r i t y of L - a n u n o acids and production of 2-keto acids [3]. I n addition, S W A J C E R a n d M O S B A C H [ 4 ] showed t h a t D-amino acid oxidase f r o m t h e yeast Trigonopsis variabilis possesses a great industrial importance for t h e preparation of semisynthetic cephalosporins. L-Aromatic aminotransferase (EC 2.6.1.57), which catalyzes t h e oxidative t r a n s a m i n a t i o n of aromatic amino acids, can serve as a useful tool in t h e determination of L-aromatic amino acids, production of L-aromatic amino acids f r o m t h e respective 2-keto acids and vice versa. Aromatic lactate dehydrogenase (EC 1.1.1.110) catalyzes a N A D H d e p e n d e n t reaction of aromatic p y r u v a t e derivatives t o aromatic l a c t a t e derivatives. This e n z y m e m a y be used for t h e production of aromatic lactates as well as for d e t e r m i n a t i o n and degradation of aromatic 2-keto acids. In t h e present p a p e r we report on our investigations of b o t h a c t i v i t y and specificity of these three enzymes f r o m 16 yeast species.

222

Acta Biotechnol. 7 (1987) 3

Materials a n d M e t h o d s Yeasts T h e following yeast strains, obtained f r o m t h e N o r t h e r n Regional L a b o r a t o r y ( N R R L Y ) , Czechoslovak Collection of Yeasts (CCY), I n s t i t u t e of F e r m e n t a t i o n of Osaka (IFO), Centralbureau voor Schimmelcultures of Delft (CBS) or Section of Biology of University a t Greifswald (SBUG), were used: Brettanomyces anomalus SBUG 289, Candida maltosa S B U G 700, Candida utilis I F O 0576, Cryptococcus terreus CBS 1895, Hansenula anomala CCY 38-1-13, Hansenula henricii SBUG 155, Ilansenula polymorpha S B U G 500, Kluyveromyces marxianus S B U G 6702, Pichia guilliermondii SBUG 50, Pichia pinus S B U G 304, Rhodosporidium toruloides I F O 0559, Saccharomyces cerevisiae S B U G 312, Saecharomycopsis capsularis SBUG 241, Sporobolomyces salmonicolor CCY 19-4-4, Trigonopsis variabilis SBUG 703, Yarrowia lipolytica N R R L Y 423-12. Growth

Conditions

Strains were grown in minimal salt medium [5] supplemented with 1 mg biotin a n d 1 mg thiamine per litre. Glucose (10 g/1) was used as carbon source. Yeasts were grown a t 30°C in a r o t a r y shaker. Enzyme

Preparation

W h e n t h e cultures h a d reached t h e s t a t i o n a r y phase of growth, t h e cells were harvested a n d washed with 100 m i l Tris/HCl buffer (pH 8). The washed cells, resuspended in t h e same buffer, were disr u p t e d b y passing t h e m twice t h r o u g h a X-pressure cell. The homogenate was centrifuged a t 20,000 X g for 20 min. T h e cell-free e x t r a c t was passed t h r o u g h a Sephadex G-25 column equilib r a t e d with either 50 mM K - p h o s p h a t e buffer (pH 6.5) (aromatic lactate dehydrogenase) or 100 mM Tris/HCl buffer ( p H 8.5) (D-amino acid oxidase, L-aromatic aminotransferase). To estim a t e t h e substrate specificity of these enzymes t h e cell-free extracts were chromatographed on a DEAE-cellulose column (2 x 2 0 cm) equilibrated with 50 mM Tris/HCl buffer ( p H 8). The columns were washed with equilibration buffer, a n d a 300-ml linear gradient (0—0.5 M KC1 in t h e same buffer) was applied. Fractions of 3.5 ml were collected with a flow r a t e of 60 ml/h. Enzyme

Assays

D-Amino acid oxidase was assayed according to t h e m e t h o d s described b y W E L L N E R a n d L I C H T E N B E R G [ 6 ] a n d b y F R I E D E M A N N [ 7 ] . L-Aromatic aminotransferase was assayed as described b y B O D E a n d B I R N B A U M [8] a n d aromatic lactate dehydrogenase was assayed b y t h e m e t h o d of J E A N a n d D E M O S S [9]. Protein concentrations of cell-free extracts were determined according to L O W R Y et al. [10] using bovine serum albumin as s t a n d a r d .

Results T h e i n i t i a l p u r p o s e of t h i s s t u d y w a s t o e s t i m a t e a n d t o c o m p a r e t h e a c t i v i t y of D - a m i n o acid oxidase, L-aromatic aminotransferase a n d aromatic lactate dehydrogenase within v a r i o u s y e a s t s t r a i n s w i t h t h e a i m t o f i n d a g o o d m i c r o b i a l s o u r c e of t h e s e e n z y m e s . T a b l e 1 s h o w s t h e e n z y m e a c t i v i t i e s e s t i m a t e d i n s e v e r a l y e a s t s . A h i g h p r o d u c t i v i t y of D - a m i n o a c i d o x i d a s e w a s e x h i b i t e d b y C. maltosa, Gr. terreus, Rh. toruloides, a n d T. variabilis. S o m e y e a s t s t r a i n s (Br. anomalus, P. guilliermondii, Sacch. cerevisiae a n d s o m e Hansenula s p e c i e s ) w e r e s h o w n t o p r o d u c e o n l y a s m a l l a c t i v i t y of t h i s e n z y m e w h e r e a s all s t r a i n s d i s p o s e d of a h i g h a c t i v i t y of L - a r o m a t i c a m i n o t r a n s f e r a s e . T. variabilis, in t h i s c a s e , p r o d u c e d t h e h i g h e s t e n z y m e a c t i v i t y . G o o d s o u r c e s f o r p r o d u c t i o n of a r o m a t i c l a c t a t e d e h y d r o g e n a s e m a y b e Br. anomalus, C. maltosa, Or. terreus, H. anomala, H. polymorpha, P. guilliermondii, P. pinus, or F . lipolytica. Based on these d a t a the best p r o d u c e r of t h e s e e n z y m e s w e r e u s e d a s t e s t o r g a n i s m s i n t h e s u b s e q u e n t e x p e r i m e n t s .

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Bode, R., Birnbaum, D., Enzyme Production by Several Yeast Species

Table 1. Enzyme activities of D-amino acid oxidase, L-aromatic aminotransferase, and aromatic lactate dehydrogenase from several yeast strains Strain

Specific enzyme activity [pkat/mg] D-amino acid oxidase a

2 264 11 284 3 1 3 33 9 38 591 1 12 48 575 15

Brettanomyces anomalus Candida maltosa Candida utilis Cryptococcus terreus Hansenula anomala Hansenula henrieii Hansenula polymorpha Kluyveromyoes marxianus Pichia guilliermondii Pichia pinus Rhodosporidium toruloides Saccharomyces cerevisiae Saccharomycopsis capsularis Sporobolomyces salmonicolor Trigonopsis variabilis Yarrowia lipolytica a

b

c

d

L-aromatic aminptransferase b 1020 650 550 340 900 540 370 ND d 470 530 510 550 ND 370 2400 530

aromatic lactate dehydrogenasec 1570 1250 860 1340 1040 70 1790 120 1670 1490 160 70 180 120 575 1470

Assayed in the presence of 5 mM D-tryptophan, 0.01 mM FAD, 100 mM Tris/HCl buffer (pH 8.5) at 37°C Assayed in the presence of 5 mM L-tryptophan, 5 mM 2-oxoglutarate, 0.1 mM PLP, 100 mM Tris/HCl buffer (pH 8.5) at 37°C Assayed in the presence of 1.0 mM p-hydroxyphenylpyruvate, 0.5 mM NADH, 0.2 mM MnCl2, 50 mM K-phosphate buffer (pH 6.5) at 25 °C Not determined

Table 2. Substrate specificity a of D-amino acid oxidase from several yeasts Substrate

D-tryptophan D-tyrosine D-phenylalanine D-isoleucine D-valine D-leucine D-threonine D-serine D-methionine D-cysteine D-alanine a

2

Strain C. maliosa

Cr. terreus

Rh. tornloides

Sp. salmonicolor

1.0 0.68 0.59 0.12 0.07 0.91 0.03 0.13 1.90 0.27 0.19

1.0 0.95 0.82 0.65 1.08 2.31 0.68 0.72 2.13 0.38 1.33

1.0 0.61 0.38 0.40 0.41 1.69 0.28 0.59 2.02 0.26 1.94

1.0 1.04 0.98 1.71 1.41 4.70 1.59 5.46 6.26 0.64 3.18

T. variabilis 1.0 2.24 1.85 3.95 4.39 7.14 0.82 0.96 13.18 0.55 4.80

D. lipolytica 1.0 2.67 1.36 4.46 3.17 5.26 3.33 2.85 5.90 1.08 4.62

Relative to D-tryptophan; assayed in the presence of 5 mM D-amino acid, 0.01 mM FAD, 100 mM Tris/HCl buffer (pH 8.5)

Acta Biotechnol. 7 (1897) 3

224

Acta Biotechnol. 7 (1987) 3

After chromatographic separation of the enzyme activities on DEAE-cellulose it became possible to study their substrate specificities. The broad substrate specificity is one of the major advantages of D-amino acid oxidase and, as can be seen in Table 2, several amino acids can be converted to the corresponding 2-keto acids with relatively high activity by enzymes of the investigated yeasts. Substrate specificities of enzymes differed within the yeast sources but D-methionine and D-leucine were mostly the best substrates. Whereas the enzyme from C. maltosa oxidized the D-isomers of isoleucine, valine, threonine, serine and alanine at a slow rate, these amino acids were good substrates for the enzyme from other yeast species. The substrate specificity of the isolated aromatic aminotransferase from various sources is presented in Table 3. In each case the highest reaction rate was observed for L-tryptophan. The activity of aromatic lactate dehydrogenase in the reduction reaction of p-hydroxyphenylpyruvate, phenylpyruvate, and indolepyruvate to the corresponding lactates is shown in Table 4. The reaction rate for the 2-keto acids indicates that in the most cases Table 3. Substrate specificity of L-aromatic aminotransferase from several yeasts Strain

Br. anomalus C. maltosa H. anomala H. henricii T. variabilis a

Relative activity 8 L-tryptophan

L-phenylalanine

L-tyrosine

1.0 1.0 1.0 1.0 1.0

0.85 0.67 0.82 0.62 0.81

0.55 0.70 0.43 0.55 0.70

Relative to L-tryptophan; assayed in the presence of 5 mM aromatic L-amino acid, 5 mM 2-oxoglutarate, 0.1 mM PLP, 100 ml Tris/HCl buffer (pH 8.5)

Table 4. Substrate specificity of aromatic lactate dehydrogenase from several yeasts Strain

Br. anomalus C. maltosa C. utilis Cr. terreus H. anomala H. henricii H. polymorphs P. guilliermondii P. pinus Rh. toruloides Sacch. cerevisiae Sp. salmonicolor T. variabilis Y. lipolytica a

Relative activity 11 p-Hydroxyphenylpyruvate

Phenylpyruvate

Indolepyruvate

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

0.07 0.14 0.08 0.29 0.05 1.00 0.06 0.02 0.08 0.58 1.88 1.08 0.50 0.05

0.07 0.12 0.11 0.18 0.07 0.71 0.13 0.00 0.09 1.05 0.78 2.17 0.33 0.05

Relative to p-hydroxyphenylpyruvate; assayed in the presence of 0.5 mM p-hydroxyphenylpyruvate, phenylpyruvate or indolepyruvate, 0.5 mM N A D H , 0.2 mM MnCl2, K-phosphate buffer (pH 6.5)

BODE, R., BIRNBAUM, D., Enzyme Production by Several Yeast Species

225

p-hydroxyphenylpyruvate was the best enzyme substrate. Aromatic lactate dehydrogenase from Rh. toruloides, Sacch. cerevisiae, and Sp. salmonicolor, which is synthesized by these yeasts only in a weak degree, was especially active for indolepyruvate and phenylpyruvate, respectively. Discussion We have investigated several yeast strains as potential sources of D-amino acid oxidase, L-aromatic aminotransferase and aromatic lactate dehydrogenase. On the basis of their enzyme activity and relatively broad specificity we could show t h a t as best producers for D-amino acid oxidase appear both T. variabilis and Rh. toruloides, for L-aromatic aminotransferase T. variabilis and Br. anomalus, and for aromatic lactate dehydrogenase H. polymorpha, Cr. terreus and C. maltosa. I n contrast to studies about L-aromatic aminotransferase as well as aromatic lactate dehydrogenase a lot of results have been published about D-amino acid oxidase from yeasts with respect to their biotechnological relevance [3, 11—16]. On the basis of these investigations including enzyme formation in several strains, regulation of enzyme synthesis, enzyme purification and cell immobilization, T. variabilis appears to be the fitest producer of D-amino acid oxidase. We suggest here that the enzyme from Rh. toruloides may be a good source for this enzyme, too. Studies about purification and properties of both L-aromatic aminotransferase and aromatic lactate dehydrogenase from the best producers presented here have been described for T. variabilis [17] and for C. maltosa [1, 2, 18]. The purification procedures have been reported for both enzymes are rapid, simple and highly reproducible. For this reason an effective isolation and economical application seems to be possible. Further investigations about these problems are now in progress and will be reported elsewhere. Received March 3, 1986

References Basic Microbiol. 2 6 ( 1 9 8 6 ) , 1 4 5 . BODE, R., LIPPOLDT, A., BIRNBAUM, D.: Biochem. Physiol. Pflanzen 181 (1986), 189. B R O D E L I U S , P . , NTLSSON, K., MOSBACH, K.: Appl. Biochem. Biotechnol. 6 (1981), 293. SZWAJCER, E., MOSBACH, K.: Biotechnol. Lett. 7 (1985), 1. T A N A K A , A . , OHISHI, N . , F U K U I , S . : J . Ferment. Technol. 4 5 ( 1 9 6 7 ) , 6 1 7 . W E L L N E R , D . , LICHTENBERG, L . A.: Meth. Enzymol. 1 7 B ( 1 9 7 1 ) , 5 9 3 . FRIEDEMANN, T. E.: Meth. Enzymol. 3 (1957), 414. BODE, R., BIRNBAUM, D.: Biochem. Physiol. Pflanzen 173 (1978), 44. J E A N , M., D E M O S S , R . D . : Can. J . Microbiol. 1 4 ( 1 9 6 8 ) , 4 2 9 . LOWRY, 0 . H . , ROSEBROUGII, N . J . , F A R R , A . L . , R A N D A L L , R . J . : J . Biol. Chem. 1 9 3 ( 1 9 5 1 ) ,

[ 1 ] LIPPOLDT, A . , B O D E , R . , B I R N B A U M , D . : J .

[2] [3] [4] [5] [6] [7] [8] [9] [10]

265.

[11] BERG, C. P., RODDEN, F. A.: Anal. Biochem. 71 (1976), 214. [ 1 2 ] KAWAMOTO, S . , K O B A Y A S H I , M . , T A N A K A , A . , F U K U I , S . : J . Ferment. Technol. 5 5 ( 1 9 7 7 ) , [ 1 3 ] K U B I C E K - P R A N Z , E . M . , R Ö H R , M . : Biotechnol. Lett. 7 ( 1 9 8 5 ) , 9 . [ 1 4 ] K U B I C E K - P R A N Z , E . M., R Ö H R , M.: Can. J . Microbiol. 3 1 ( 1 9 8 5 ) , 6 2 5 . [ 1 5 ] SIMONETTA, M . P . , V A N O N I , M. A . , CURTI, B . : F E M S Microbiol. Lett. 1 5 ( 1 9 8 2 ) , 2 7 . [16] ZWART, K. B., OVERMARS, E. H., HARDER, W.: FEMS Microbiol. Lett. 19 (1983), 225. [17] SHENTESHANMUGANATHAN, S . , NICKERSON, W. J.: J . Gen. Microbiol. 27 (1962), 465. [ 1 8 ] B O D E , R . , B I R N B A U M , D . : Z . Allg. Mikrobiol. 2 4 ( 1 9 8 4 ) , 6 7 .

2*

13.

Acta Biotechnol. 7 (1987) 3, 226

Book Reviews S.

OLSEN

Biotechnology An Industry Comes oi Age Washington: Academic Press, 1986 10.50 L Dieser Titel stellt die Zusammenfassung der Resultate einer im Februar 1985 durchgeführten Konferenz über Gentechnologie dar, die vom Academy I n d u s t r y Program der National Academy of Sciences, der National Academy of Engineering und des I n s t i t u t e of Medicine organisiert war. Die daraus resultierende E r w a r t u n g des Biotechnologen, eine Bestandsaufnahme moderner Biotechnologie vorzufinden, ist erfüllt worden. Nicht durch erschöpfendes Angebot detaillierter F a k t e n — das ist bei 116 Seiten Textumfang beim heutigen Stand der Biotechnologie nicht möglich — sondern durch klare Herausarbeitung der einzelnen Entwicklungslinien und deren Zusammenhang. I m Mittelpunkt der Betrachtungen stehen die Möglichkeiten der Gentechnologie, die in Kapiteln gegliedert auf den Gebieten mikrobieller Produkte, Biotechnologie in der Landwirtschaft und GenTherapie vorgestellt werden. Der Biotechnologe vom Fach mag sich nur bestätigt finden, vorzüglich lesbar ist der Titel aber auch f ü r alle mittelbar an Biotechnologie Interessierten: Der Autor h a t mit großem Geschick zum Verständnis notwendiges Grundlagenwissen eingebaut. Einen breiten Umfang nehmen die Fragen sowohl der ethischen Verantwortung gegenüber der Entwicklung der Gentechnologie, als auch der staatlichen Förderung, der aktuellen Situation auf dem Firmensektor, der Verbindung zwischen Industrie und Universitäten u n d des Rechtsschutzes ein. Auch hier bleibt der Autor überwiegend im Faktischen, das, dem Wesen der Probleme entsprechend, wesentlich mehr gesellschaftliche Bezüge aufweist. Das zehnte und letzte Kapitel des Titels „Biotechnology in J a p a n " ist in diesem Sinne k a u m als Fremdkörper anzusehen. Die Frage ,,A Challenge to the U.S. Leadership?" demonstriert das bereits in seiner Überschrift. Der analytische Leser, dem auch diese Abschnitte nützlich sein werden, wird das berücksichtigen. Auf diese Weise ist der Titel einem breiten Leserkreis ausdrücklich zu empfehlen. Sogar demjenigen, f ü r den er keine neuen Informationen brächte. Denn — das sollte man, wenn es schon zut r i f f t , ruhig aussprechen — er liest sich mit Genuß. M. Prause E . HEITZ, J . C. ROWLANDS, F . MANSFELD

Electrochemical Corrosion Testing with Special Consideration of Practical Applications DECHEMA Monographie Vol. 1 Weinheim: VCH Verlagsgesellschaft, 1980. 352 S. Die vorliegende Dechema Monographie Nr. 101 enthält die Vorträge und Diskussionsbeiträge eines internationalen Workshops in Ferrara, Italien, vom 11. —13. 9. 1985. Dargestellt werden neueste Erkenntnisse der Ermittlung von Ursachen der elektrochemischen Werkstoffkorrosion durch Einflüsse unterschiedlichster Medien. Neueste Meßverfahren zur Ermittlung von korrosionsinduzierten bzw. -inhibierten Einflußfaktoren und Methoden der mathematischen Auswertung u n d Modellierung werden beschrieben. Beispiele aus der Biotechnologie sind nicht enthalten, aber Ableitungen f ü r bestimmte Prozeßzustände aber gegeben. Die Besprechung in der „Acta Biotechnologica" ist nach Ansicht des Rezensenten dadurch gerechtfertigt, daß sowohl auf dem Gebiet der Reaktorentwicklung als auch der Aufarbeitung eine Fülle noch ungeklärter Probleme der optimalen Werkstoffauswahl als auch des Korrosionsschutzes bestehen. Besonders trifft dies auf den in Vorbereitung befindlichen Einsatz extremophiler Mikroorganismen in Pilot- und großtechnischen Anlagen zu. F ü r die auf diesem Fachgebiet — bedauerlicherweise in viel zu geringer Anzahl — tätigen Spezialisten gibt diese Monographie eine Fülle wertvoller Hinweise. D. Beck

Acta Biotechnol. 7 (1987) 3, 2 2 7 - 2 3 5

Continuous Production of Protein Hydrolysates in Immobilized Enzyme Reactors LASCH, J . , KOELSCH, R . , KRETSCHMER,

K.

Martin-Luther-Universitat Halle Institut fur Biochemie, Bereich Medizin HollystralBe i , PSF 184, Halle/Saale, 4020 G.D.R.

Summary The digestion of several proteins, casein, cc-lactalbumin, human serum albumin and a mixture of whey proteins by immobilized pronase, thermitase and leucine aminopeptidase was studied on various conditions in five types of enzyme reactors. Reactors and operating conditions were designed to maximize the extent of hydrolysis and to minimize the adverse effects of the macromolecular nature of the substrates. A simple analytical method was developed to follow routinely the extent of hydrolysis. Substrate proteins were subjected to various pretreatments intended to disturb their native structure. The maximum feasible extent of hydrolysis in the reactor effluent, which is an average quantity, clustered around the magic figure of 33% in all systems studied. Protein digestion in bubbled column reactors charged with the polyaminomethylstyrene-fixed thermostable proteinase "thermitase" and operated at 50 to 60°C turned out to be the most efficient setup to produce continuously amino acid/peptide mixtures.

Introduction There is ample evidence now that di- and tripeptides are absorbed by the small intestine at least as efficiently as free amino acids [1 — 5]. This peptide uptake is not dependent on a sodium ion concentration difference across the luminal enterocyte membrane but is probably driven by an inward proton gradient [3, 4], The transmembrane peptide transport does not compete with the absorption of free amino acids [6]. These findings entailed worldwide an increasing interest in short-chain peptide mixtures as ingredients of fully balanced ballast-free diets. Mixtures of synthetic amino acids are practically no longer in use for this purpose. Compared to amino acids, peptide mixtures are cheaper to produce, have a higher nutritional value and are better tolerated [7, 8], mainly because of their lower osmolarity at a given nitrogen content (an osmolarity above 800 mosmol/1 causes diarrhoe). Peptide diets have a wide range of applications extending from clinical nutrition during different kinds of malabsorption in children, treatment of protein malnutrition and postoperative diets to fortifiers for sportsmen. Furthermore, oligopeptides are ideally suited as additives to beverages to improve their nutritional value. The use of immobilized proteases of broad specificity ,for'the continuous production of protein hydrolysates containing mainly small peptides is a very promising way to respond to the increasing demand for oligopeptide diets [9].

228

Acta Biotechnol. 7 (1987) 3

Matrix-bound proteases have been introduced as an analytical tool into protein chemistry a decade ago by B E N N E T T et al. [ 1 0 ] , C H I N and W O L D [ 1 1 ] and D E T A R et al. [ 1 2 ] , I t was shown in analytical batchtype experiments t h a t peptides and some pretreated proteins (S-aminoethylated or performic acid oxidized) can be completely hydrolyzed [10] or nearly so [11]. But already this early work revealed severe limitations of the concept of total protein hydrolysis by immobilized enzymes as demonstrated by us [13] and others [11, 12], who found a very low recovery of certain amino acids, mainly proline and cysteine/cystine or even remaining peptides [12], I n fact, we were unable to obtain total hydrolysis of unmodified proteins even if mixtures of matrix-bound endo- and exopeptidases were employed [13]. In the light of what has been said above about peptide diets the goal of a technical scale protein hydrolysis need not be complete hydrolysis though this is mandatory for analytical purposes. The design of a proteolytic reactor, whatever its precise configuration, has to overcome five serious problems: (i) very low mass transfer coefficients due to the macromolecular nature of the substrate, (ii) protection of peptide bonds buried in the native structure from proteolytic attack even though intrinsically susceptible to enzymatic cleavage, (iii) biocatalyst stability under operating conditions, (iv) viscosity and solubility problems and (v) enzyme leakage. This communication describes results obtained with different reactor configurations, various substrate/enzyme combinations and substrate pretreatments intended to disturb the native substrate structure. Materials and Methods Materials Enzymes used were pronase (Serva, Heidelberg, FRG), thermitase — a proteinase from the culture filtrate of the thermophilic microorganism Thermoactinomyces vulgaris [14] purified by adsorption chromatography on porous glass [15] (kindly provided by Dr. U. KETTMANN, this institute) and leucine aminopeptidase (a-aminoacylpeptide hydrolase, EC 3.4.11.1) prepared from bovine eye l e n s e s a c c o r d i n g t o HANSON a n d FROHNE [16],

Enzymes were immobilized on DEAE Sephadex A-50 (Pharmacia, Uppsala, Sweden) and/or on the macroporous polyaminomethylstyrene Wofatit Y-58 (pilot product of the CK Bitterfeld, GDR) [17]. Substrate proteins tested were human serum albumin (Institute of Vaccination Research, Dessau, GDR), A-lactalbumin (kindly provided by Prof. H. RTTTTLOFF, Central Institute for Nutrition, Potsdam-Rehbriicke, GDR), Hammarsten casein (Merck, Darmstadt, FRG) and spraydried whey proteins (VdGB Kombinat Milchwirtschaft Riigen, Bergen, GDR). All other reagents were analytical grade and used without further purification. Reactor configurations run were the continuous stirred tank reactor (CSTR), the packed bed column reactor (PBCR), the fluidized bed column reactor (FBCR), i.e. fluidized by an increased feeding rate, and a bubbled column reactor (BCR). The laboratory scale reactors were home-made and will not be described here in detail. All column reactors were fed or bubbled from the bottom. Columns with moving beds (FBCR, BCR) were open at the top and equipped with a lateral siphon at the upper end the orifice of which was covered with a nylon net. The feeding solution was either degassed by pumping it through a 2 m long gas-permeable fluid-tight tubing positioned in a vacuum chamber (PBCR, FBRC) or mixed with N2 at the column entrance (BCR) — cf. Fig. 1. Methods Enzymes were either crosslinked on the surface of DEAE Sephadex (procedure A) or bound to activated polyaminomethylstyrene (procedure B). Procedure A: DEAE Sephadex A-50, swollen in 12 mM citrate/veronal buffer, pH 8.5, ionic strength 0.04, was mixed with enzyme dissolved in the same buffer (10 to 50 mg/ml) and shaken at room temperature for 30 minutes. The buffer was sucked off and the preparation thoroughly wash-

LASCS, J., KOELSCH, R. et al., Proteolytic Enzyme Reactors

Mixer

^

N

Buffer Substrate

11 -

229

Flow Meter

Fig. 1. Schematic diagram of experimental setup for the hydrolysis of proteins in immobilized protease column reactors showing in particular the bubbled column reactor (magnified encircled part of the drawing); IMP — immobilized protease.

ed. Then, glutardialdehyde was added (final concentration 5%) and the preparation shaken for 4 h. Thereafter, the immobilized enzyme was transferred to the reactor and washed slowly with 40 reactor volumes of buffer before feeding with substrate solution. Mean enzyme load: 10 mg/g dry carrier. Procedure B: 200 g (wet weight) polyaminomethylstyrene (Wofatit Y-58) were shaken with 1 liter glutardialdehyde (final concentration 2.5%) a t p H 8.3 (phosphate buffer, 10 mM) at room temperature for 3 h. The preparation was then thoroughly washed, 1 g enzyme in phosphate buffer added and shaken for another 3 h period. Final washings were with 0.9% NaCl and phosphate buffer. Mean enzyme load: 70 mg/g dry carrier. Retention of specific catalytic activity toward low molecular substrates was around 80% and toward azocasein 20 to 40%. The product mixture at the reactor drain was analyzed for free amino groups with 2,4,6-trinitrobenzene sulfonic acid essentially as described by OKTJYAMA and SATAKE [18] and total nitrogen after ashing with 10 N H 2 S0 4 at 300 °C with the phenol/hypochlorite reaction (indophenolblue formation) according to a modified micromethod [19]. In addition, samples of hydrolysates were applied to a Sephadex G-15 column in order to analyze their peptide spectra. There were no TCA-precipitation steps (cf. results). Substrate pretreatments, intended to improve their susceptibility toward proteolytic attack, were done with organic solvent (10 vol% ethanol), by reduction with thiols (cysteine, dithiothreitol, 2 mM) at 60°C and by sonication (Branson sonfier B-12, power setting 6). The mixture of whey proteins was chromatographed on Sephadex G-25 to remove lactose.

Data Treatment The percentage of hydrolysis in the reactor effluent was calculated from the ratio of free amino groups to total nitrogen. Analyzing sequence data of 20 globular proteins including the substrate proteins used in our studies it was found that their peptide nitrogen amounted rather exactly to 8.7 mmol/g of protein and their total nitrogen to 11.4 mmol/g of protein (KJELDAHL factor). From these figures it follows that 76% of the protein nitrogen are peptide nitrogen and, correspondingly, 24% side chain plus N-terminal nitrogen. TT Hence

, i • = a; mmol NH,/e/v ... % ,hydrolysis ^ ^ mmol N/e — • 100

(1)

230

A c t a B i o t e c h n o l . 7 (1987) 3

T h i s e q u a t i o n was r e c a s t into a form which is directly applicable to t h e d a t a as afforded b y t h e analytical methods degree of hydrolysis ( % ) = ^ ( h y d r o l y s a t e ) - A 3 3 4 ( b l a n k ) ] • 9 2 1 A 3 3 l (leucine standard) -(¡ig N/50 ¡xl)

(2)

A 3 3 1 ( b l a n k ) is t h e a b s o r b a n c e of t h e unhydrolyzed s u b s t r a t e a f t e r t r e a t m e n t with trinitrobenzene sulfonic acid. T h e f a c t o r 9 2 1 derives from 7 • (100/0.76) where t h e F i g u r e 7 converts [xg N/50 ¡xl into [xmol N/100 ¡xl. Using this e q u a t i o n , control hydrolyses of s u b s t r a t e proteins with 6 N hydrochloric acid a t 1 1 0 ° C for 24, 4 8 and 72 h yielded percentages of hydrolysis in t h e range of 9 8 to 105%.

Results Results obtained with different kinds of reactors under a variety of conditions are assembled in Tables 1 and 2. In most cases reactors were run at elevated temperatures (50 to 60 °C). Substrate residence times varied from 8 to 20 h. Collection of reactor effluents was started after a time lapse of one residence time to ensure steady state conditions. The hydrolysates contained in no case TCA-precipitable material. The results shown in Tables 1 and 2 demonstrate that for all substrates in spite of various pretreatments arid addition of an exopeptidase the mean fractional degradation never exceeded a value of about 0.33. As the degree of hydrolysis of the reactor effluent is an average quantity which, for a given value, can correspond to quite different amounts of various peptides and amino acids, gel filtration with Sephadex G-15 was performed in order to estimate the size of

T a b l e 1. M a x i m a l e x t e n t of hydrolysis of t h r e e s u b s t r a t e proteins in t h e p a c k e d bed column reactor under a v a r i e t y of conditions. T h e first half of t h e column was filled with D E A E Sephadex-pronase and t h e second half with D E A E S e p h a d e x - l e u c i n e aminopeptidase. Substrate" (cone.)

Temperature [°C]

Substrate pretreatment

Hydrolysis" [%]

Casein (0.5%)

45

-

33

a-Lactalbumin (0.2%)

37 37 37 50 50

35 33 39 34

50

— sonication sonication plus 10 v o l % ethanol -

37 45 50 50

reduction with D T E b reduction with D T E b reduction with D T E b

36 35 31 33

H u m a n serum albumin (1%)

a

b

39 34

all s u b s t r a t e s were dissolved in nitrogen-free buffers (phosphate a n d c a r b o n a t e buffer, p H 8.0) D T E , d i t h i o t h r e i t o l ; c calculated from eq. (2)

LASCH, J . , KOELSCH, R . et al., Proteolytic Enzyme Reactors

231

Table 2. Extent of hydrolysis of casein (5 g/1) and whey proteins (5 g/1) by immobilized thermitase in various types of reactors Support

Reactor type a

Temperature r° C 1

D E A E Sephadex Polyaminomethylstyrene Polyaminomethylstyrene Polyaminomethylstyrene Polyaminomethylstyrene Polyaminomethylstyrene a

PBCR CSTR PBCR FBCR FBCR (recycled) BCR

Hydrolysis [ % ] Casein

Whey proteins

55 35 60 60 60

33 19 25 10 35

28 12 27 9 30

55

35

33

L

J

for abbreviations of reactor types see: Materials

and

Methods

peptides. Some typical elution pattern are shown in Figs. 2 to 5. Usually, one milliliter fractions were collected and analyzed as described under Methods. Then, the fractional degradation was calculated from Eq. (2) and its reciprocal taken as a measure of peptide size. E.g.., a degree of hydrolysis of 55% corresponds to dipeptides (peptide size: 1/0.55 as 2). In this way, the absorption peaks were assigned to peptides of different size.

"core" n >10

n = t>-9

tr ¡peptides

dipeptides

20%

10%

25%

20%

amino

acids

25%

Fig. 2. Elution pattern of a casein hydrolysate on Sephadex G-lo. Casein (0.5%) was passed at 45 °C through a packed bed column reactor (residence time 12 h) containing pronase immobilized on polyaminomethylstyrene. Three milliliters of the effluent were applied to a 1.5 X 15 cm Sephadex column and developed with phosphate buffer, pH 8.4. Assignement of peaks and estimation of relative amounts of peptides were done as described in the text.

I t is clear from the elution pattern shown in Figs. 2 to 5 that this approach, though quite useful for the analysis of reactor performance, because of overlapping peaks, has its limitations. Notice that the percentages of material in differently sized peptide fractions (cf. Figs. 2 and 3) do not reflect fractional areas under the absorption curve but represent percentages of total nitrogen.

232

Acta Biotechnol. 7 (1987) 3

The decay of enzyme activity under operating conditions was always monoexponential. Half lives, depending on the particular system studied, fell in the range of 8 to 14 days. Reactors were usually run for 2 to 3 half lives. Freeze-dried product mixtures were also evaluated with respect to their taste properties. With the exception of casein hydrolysates, which had a slightly bitter taste, all digestion mixtures were bland or palatable.

"core" n > 10 20%

n=t,-9 tnpep tides 26%

10%

dipeptides '

20%

amino

acids

24 %

Fig. 3. Elution pattern of a human serum albumin hydrolysate on Sephadex G-15. Details of the experiment are the same as described in Pig. 2.

1

2

3

i

5

Fig. 4. Peptide spectrum of a casein hydrolysate obtained by digestion with polyaminomethylstyrene-fixed thermitase in a CSTR at 35 °C. Residence times: 2 h (A) and 14 h (B). The second line on the abscissa designates peak numbering: 1 and 2 — "core' ' (n > 10), 3 — tetra- to decapeptides, 4 — tripeptides, 5 — dipeptides, 6 — amino acids.

LASCH, J., KOELSCH, R. et al., Proteolytic Enzyme Reactors

14.4 15.6 21.0 3

12 O « «s»

24.0 27.0 4

5

31.5 6

36.9 7

A

N I L 15.1 17.6 21.0

233

imi]

B

25.6

I 37.6

I 42.9

[ml]

Fig. 5. A. Peptide spectrum of a casein hydrolysate obtained with polyaminomethylstyrene-fixed thermitase in a packed bed column reactor at 60 °C. Residence time: 9.6 h. Peaks: 1 and 2 — "core" (n > 10), 3 — tetra- to decapeptides, 4 and 5 — tripeptides, 6 and 7 — dipeptides. B : Peptide spectrum of a casein hydrolysate obtained with polyaminomethylstyrenefixed thermitase in a fluidized recycled column reactor at 60 °C. Total residence time: 12 h. Peaks :1 and 2 — "core", 3 — tetra- to decapeptides, 4 — tripeptides, 5 and 6 — dipeptides.

Discussion

I t is well established that the activity of immobilized proteases toward protein substrates is appreciably less than that of their free counterparts. This is also born out by our data, though a remarkably high specific activity toward azocasein was retained (s. Methods). As expected, the five problems with proteolytic reactors mentioned in the introduction could be solved only partly. Operating the reactors at 55 or 60 °C reduced the adverse effects of high viscosity. Cleavage of macromolecular substrates by immobilized enzymes is considerably impaired by (i) slow pore diffusion, (ii) low external mass transfer coefficients and (iii) size exclusion effects of the support which are proportional to M}' 3 or log M r [21, 22] (Mr — molecular mass of substrate and intermediate split products). The influence of pore size and geometry was kept small by either fixing the enzyme in pellicular form ( D E A E Sephadex) or by selecting a carrier with large pores (Wofatit Y-58, porosity 0.8 cm 3 /g, nominal mean pore diameter 70 nm). The external mass transfer coefficient was increased by fluidizing column reactor beds. This prevented also channel formation which is a serious problem with long operation times. We conclude from our experience with 5 reactor types that the bubbled column reactor (Fig. 1) is the most efficient setup because with it the substrate residence time, in contrast to reactors fluidized by increased pumping speed, can be varied over a wide range. Using glutardialdehyde as coupling reagent which can form a variety of stable bonds including cross-links [23, 24] no leakage was detected up to a month by monitoring proteolytic activity in the reactor effluent with azocasein.

234

Acta Biotechnol. 7 (1987) 3

Perhaps the most surprising result of our studies was the finding t h a t the maximum feasible degree of hydrolysis clustered around the magic figure 0.33 (cf. Tables 1 and 2). The explanation is straightforward for thermitase reactors because the split products of an endopeptidase are a t most dipeptides but mainly tripeptides. The very small amount of free amino acids found in some experiments (cf. Fig. 4 A) is due to an exopeptidase activity of thermitase which is several orders of magnitude lower than its endopeptidase activity. The same figure of 33% final hydrolysis was found by others with a mixture of soluble thermitase and trypsin [20]. Arrest of reactor proteolysis at 33% is less obvious when pretreated proteins were digested with pronase and leucine aminopeptidase. We surmise that there is in all proteins a substantial number of unfavorable and proteinase-resistent peptide bonds. X-pro-, X-gly-Y-, cys-cys-, -pro-cys-, pro-pro-bonds and bonds in the neighborhood of disulfide bridges can be expected to belong to this category. For instance, in human serum albumin there are 25 proline residues, 17 disulfide bridges, one -cys-pro-sequence, eight -cys-cys- and two -pro-cys-sequences. Depending on the protease mixture employed there might be yet more unfavorable sequence clusters (protease-resistent core). In addition, as the digestion proceeds, proteases will be progressively inhibited by split products which, eventually, will switch them off. There is, for instance, only a slight increase in the amount of dipeptides if the residence time of casein in the thermitase CSTR is increased from 2 to 14 h (Fig. 4). In the light of what has been said above, the widespread practice of taking as 100% digestion that of a reference sample extensively hydrolyzed by free enzyme should be discontinued and replaced by standard acid hydrolysis with 6 N HC1. Depending on wheter 100% digestion is achieved by extensive enzymatic or total acid hydrolysis it will have a different meaning. Only the latter procedure yielding complete hydrolysis and thus a true 100% point is suited for the assessment of protein hydrolysates if nutritional applications are envisaged. The same problem arises if only a few selected amino acids of the product mixture are determined. Hydrophobic amino acids which are preferred points of attack of proteases might be split off quantitatively (100% hydrolysis) whereas amino acids of unfavorable sequences, as defined above, would reflect an extent of hydrolysis of, say, 1 0 — 2 0 % . This is nicely illustrated by the finding of C H I N and W O L D [11] t h a t the disulfide loop Cys-Cys-Ala-Ser-Val-Cys of insulin was not cleaved at all by a mixture of proteases with broad specificity. In view of the complexity of reactions in proteolytic reactors and the uncertainty of several parameters, no attempt was made to model and simulate mathematically reactor performance. Most of the reactors were run for 3—4 weeks without troubles which is considerably longer than reported for other types of laboratory-scale proteolvtic reactors [25, 26]. Received July 2, 1986

References [1] MATTHEWS, D. M.: Physiol. Rev. 55 (1975), 537. [2] GANAPATHY, V., LEIBACH, P. H.: J. Biol. Chem. 258 (1983), 14189. [3] GANAPATHY, V . , LEIBACH, F . H . : L i f e Sci. 8 0 ( 1 9 8 2 ) , 2 1 3 7 . [4] TAKTJWA, N . , SHIMADA, T., MATSUMOTO, H . , HOSHI, T . : B i o c h i m . B i o p h y s . A c t a 8 1 4 (1985),

186. [5] FRIEDRICH, M., NOACK, J., NOACK, R . : Biomed. Biochim. Acta 4 3 (1984), 117.

[6] WARD, M. R., BOYD, C. A. R.: Nature 287 (1980), 157. [7] IMONDI, A. P . , STRADLEY, R . P . : J . N u t r . 1 0 4 ( 1 9 7 4 ) , 7 9 3 . [8] LALASIDIS, G., BOSTROM, S., SJOBERG, L . - B . : J . Agric. F o o d C h e m . 2 6 ( 1 9 7 8 ) , 7 5 1 .

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R. et al., Proteolytic Enzyme Reactors

[ 9 ] LASCH, J . , KOELSCH, R . , HABERLAND,

Patent WP C 12 P/264 292/5, 1985. [10] B E N N E T T , H, P. J . , E L L I O T T , D. E . , 129 (1972), 695.

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MCMARTIN, C . :

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A., K L I N G E N B E R G , P . , R U T T L O F F , H . : Z . allgem. Mikrobiol. 1 9 ( 1 9 7 9 ) , 2 7 . U., K E T T M A N N , U., S C H E L L E , H. — I n : Proteinases and their inhibitors. Eds.: T U R K , V . , V I T A L E , S . Oxford: Pergamon Press. 1 9 8 1 , 2 0 1 . H A N S O N , H . , F R O H N E , M.: Methods Enzymol. 4 5 B ( 1 9 7 6 ) , 5 0 4 . K A U F M A N N , R . , H Ä U P K E , K . , F I S C H E R , J . , S C H E L L E N B E R G E R , A.: Acta Polymerica 3 1 ( 1 9 8 0 ) ,

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Biochem. (Tokyo) 4 7 ( 1 9 6 0 ) , 4 5 4 . physiol. Chem., 3 4 8 ( 1 9 6 7 ) , 1 0 0 . [ 2 0 ] F R I E D R I C H , M . , N O A C K , J . , N O A C K , R . : Nahrung 2 6 ( 1 9 8 2 ) , 8 1 1 . [21] C R E S S W E L L , P., S A N D E R S O N , A. R . : Biochem. J . 1 1 9 (1970), 447. [ 2 2 ] G E M E I N E R , P . , P O L Ä K , C . , B R E I E R , A . , P E T R U S , L . , B E N E S , M. J . : Enzyme Microb. Technol. 8 (1986), 109. [ 2 3 ] F O R D , D . J . , P E S C E , A . J . — In: Enzyme Immunoassay. Eds.: I S H I K A W A , E., K A W A I , T., M I T Y A I , K . Tokyo, New York: Igaku-Shoin Ltd. 1 9 8 1 , 5 4 - 6 6 . [ 2 4 ] K O E L S C H , R., F U S E K , M., H O S T O M S K A , Z . , L A S C H , J . , T U R K O V A , J . : Biotechnol. Lett. 8 ( 1 9 8 6 ) , [ 1 8 ] OKUYAMA, T . , SATAKE, K . : J .

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Acta Biotechnol. 7 (1987) 3, 236

Book Review W . J . BEEK

Chemran III (Chemical Research Applied to World Needs) World Conference on Resource Material Conversion (Bio-) Chemical Process Bridges to meet Future Needs Perspectives and Recommendationes The Hague, The Netherlands, June 2 5 - 2 9 , 1984 Amsterdam: I U PAC Stichting Chemische Congressen 14, KNCV QLT Convention Services, 1985. 164 S.

I m Gegensatz zur Zusammenfassung der Chemrawn I I Konferenz bietet dieses Material einen nahezu vollständigen Überblick über die Konferenzbeiträge einschließlich Schlußfolgerungen und Empfehlungen. Chemrawn I I I orientierte sich weniger auf die Erschließung alternativer Energiequellen gegenüber Erdöl, sondern auf die Möglichkeiten der Entwicklung von Verfahren der effektiven Konversion von organischen Rohstoffen in Futtermitteln, Zwischenprodukte und Hochtechnologie-Chemikalien. Dramatische Konsequenzen in bezug auf die Rohstoffsituation werden nach der Einführung von effizienten Verfahren zur Nutzung von zellulosehaltigen Abprodukten als Rohstoffquelle f ü r Basischemikalien gesehen. Der Entwicklung und E i n f ü h r u n g biotechnologischer Arbeitstechniken wie DNA-Insertion und -deletion; Protoplastfusion; Kultivierung tierischer und pflanzlicher Zellen; Enzymtechnologie wird eine besondere Bedeutung zugemessen. Die gegenwärtige Nutzung biotechnologischer Prozesse zur Produktion von Hefen, Primärmetaboliten wie Alkohol, Ketosäuren und Aminosäuren, Sekundärmetaboliten wie Antibiotika und Enzyme mit einer weltweiten jährlichen Zuwachsrate zwischen 6 — 10% werden in Z u k u n f t durch die Einführung neuer Erkenntnisse der Genmanipulation und Protoplastenfusion erweitert durch Produkte wie Chymosin und Humanpeptide, Hormone, Blutfaktoren und Blutproteine; pflanzlicher und tierischer E n z y m e ; Sekundärmetabolite wie Fette, öle und Alkaloide; Biofeinchemikalien. Von Interesse ist der Hinweis, daß in Z u k u n f t der Einsatz von Enzymen f ü r die Stoffwandlung aus Kostengründen nahezu ausschließlich in immobilisierter Form mit entsprechenden Konsequenzen f ü r die Reaktorentwicklung erforderlich sein wird. Die Auswertung des Konferenzmaterials bestätigt ferner die Erkenntniss, daß der Entwicklung des down-stream-processing durch Einf ü h r u n g bekannter bzw. Suche nach neuen Wirkprinzipien außerordentliche Beachtung geschenkt werden m u ß . Der Rezensent schätzt ein, daß das vorliegende Büchlein eine Fülle interessanter Informationen f ü r auf dem Gebiet der Biotechnologie in Forschung und Lehre tätigen Wissenschaftler enthält, aber auch f ü r prognostische Zwecke und Marktbearbeitung von Interesse ist. D. Beck

Acta Biotechnol. 7 (1987) 3, 237—245

Continuous Fermentation in High Flow Rate Fermenter Systems RICHTEK, K . , BECKES, U . , MEYER, D .

Academy of Sciences of the G.D.R. Institute of Biotechnology, Leipzig PermoserstraBe 15, Leipzig, 7050 G.D.R.

Summary In commercial batch processes the productivity of product formation is low. But a significant increase of productivity can be achieved in continuous fermentations. By using high flow rate fermenter systems characterized by a relatively long retention time of biomass in comparison with the retention time of the liquid we can realize a high-performance fermentation. The problem of holding back the biomass within the reactor could be solved by means of membranes being impenetrable to the cells, but permeable to the hydraulic phase. Such a process technology was successfully tested for its applicability in alcoholic and lactic acid fermentations. The maximum productivities obtained on this way were P = 120 g/1 • h for ethanol production and P = 51 g/1 • h for lactic acid fermentation, respectively.

Introduction During the last years an extensive field of application for membranes has evolved in microbial product formation processes. The possibilities for its use include microfiltration, ultrafiltration, reverse osmosis, dialysis, electrodialysis, pervaporation, and cell or enzyme immobilization [1], I n regard to the problem of maintaining high cell concentrations within the fermenter system the procedures of microfiltration and ultrafiltration are suitable in particular [2], For instance the use of U F membranes in ethanolic fermentation is known from bibliography [3—14] in which different variants (rotorfermenter [3], rotating membrane in the bypass [4], membrane bottom fermenter [5], hollow fiber fermenter [6—10], crossflow membrane module [11 —14] are described. Using membranes in continuous product formation we can realize a high-performance fermentation characterized by very great hydraulic dilution rates and high productivities. Such a high flow rate fermenter system was a research object of our investigations, the results of which are obliged to present in this paper. Materials and Methods Apparatus A diagram of the used high flow rate fermenter system is shown in Pig. 1. The fermenter had a working volume of 1.0 1 and was connected with a membrane module in the bypass. As membrane

238

Acta Biotechnol. 7 (1987) 3

Fig. 1. D i a g r a m of t h e used high flow r a t e f e r m e n t e r system. 1 — m e d i u m reservoir, 2 — alkali reservoir, 3, 4, 6 — p u m p s , 5 — f e r m e n t e r , 7 — m e m b r a n e module, 8, 12 — valves, 9 — biomass recycle, 10 — reservoir for cell-free culture f i l t r a t e , 11 — reservoir for f e r m e n t e r effluent containing biomass modules a horizontal filter plate u n i t (filter a r e a : 0.03 m 2 , CA U F m e m b r a n e ) a n d a commercial hollow fiber module (MLW 1.3 Artificial K i d n e y , V E B M L W K e r a d e n t a - W e r k R a d e b e r g , G.D.R.) were used for alcoholic f e r m e n t a t i o n a n d lactic acid production, respectively. Microorganisms T h e ethanolic f e r m e n t a t i o n s were carried o u t w i t h t h e y e a s t s t r a i n Saccharomyces F o r t h e production of lactic acid a strain of Streptococcus thermophilus was used.

cerevisiae Sc 5.

Medium, As feed a mineral salt m e d i u m containing sucrose (80—150 g/1) a n d y e a s t e x t r a c t (5 g/1) was used in ethanolic f e r m e n t a t i o n [15]. T h e cultivation of lactic acid bacteria took place w i t h t h e MRSmedium [16]. Process

Parameters

Temperature: pH: Biomass concentration: H y d r a u l i c dilution r a t e : P r o d u c t concentration :

Ethanolic fermentation

Lactic acid f e r m e n t a t i o n

33 °C 4.5 1---110 g/1 0.1—3.9 b - 1 7 — 70 g/1

40 °C 6.5 5—35 g/1 0.35-3.0 h-1 10 • • • 60 g/1

Theoretical Considerations A simplified scheme of the mass flow relationships in the used fermenter system is given in Fig. 2. It follows that the question is of a combination of a chemostate and a turbidostate. In regard of the product concentration the system reacts as a chemostate. On ideal steady-state conditions the product concentration in the medium is determined by the

239

RICHTER, K . , BECKER, U. et al., High Flow R a t e Fermenter

Input

Fermenter

Output 1

Membrane module P

F,

C

Fig. 2. A simplified scheme of mass flow in a continuous high flow rate fermenter system.

biomass concentration and the hydraulic dilution rate above all. To obtain a steady product concentration the both parameters must be kept constantly during the fermentation. The major part of the product-containing liquid leaves the fermenter system as a cellfree filtrate (output 1). If cell growth takes place the biomass excess formed permanently within the fermenter system must be eliminated by flowing out (output 2). Relating to the biomass concentration the fermenter system acts as a turbidostate. For the used high flow rate fermenter system the following assumtions were made: — Cell growth takes place, the specific growth rate is greater than the specific death rate. Therefore the efflux of output 2 is permanently existing, but it is much less than the efflux of output 1:

f,' " t

; gg | | - •

l ^ a r i r C.

'

- Ev •*(*•" 'V

^

" ¿ f .

. D • . ' ^ ) ^ J _ MI * .1 ,}'* WKMiPSSiSsW^^&wS>? • fflimi{" t'

fr '

WM

h

& '

*

Abb. 2. Konidien des Aspergillus rugrer-Stammes Z nach 12stündiger Inkubationszeit im antischaumhaltigen Nährmedium (20 ¡¿1: 100 ml; Vergrößerung 600 X)

Eine weitere Reihe biologischer Prüfungen von Gemischen, welche Mineralöl, Emulgator bzw. beide Substanzen enthielten, bestätigt die Richtigkeit dieser Schlußfolgerung (Tab. 2). Die Zugabe des Emulgators hemmte nur im kleinen Maß das normale Wachstum der Mycelschicht auf der Oberfläche des Mediums (daher soll er nicht zu den für Aspergillus niger ganz neutralen Substanzen gezählt werden). Für das Nährmedium, das außer Emulgator noch Öl enthielt, wurde eine Verzögerung des Mycelwachstums beobachtet. J e größer deren Konzentrationen waren, um so insel- und punktartiger sah die Mycelschicht aus, was eine geminderte Säurebildung zur Folge hatte. Der Hemmungseffekt der im Nährmedium dispergierten n-Alkane macht sich am deutlichsten während der Quellung und Keimung der Konidien bemerkbar. Eine Bestätigung dafür brachten Versuche, bei denen ausgekeimte Konidien der Stämme R 16 und Z nach mehrstündiger Inkubationszeit auf das öl- bzw. antischaummittelhaltige Nährmedium verlegt wurden (Tab. 3). Die besten Ergebnisse wurden mit der Mycelschicht erhalten, die erst nach 24 h Inkubationszeit in die toxische Umgebung gebracht wurde. Unsere Beobachtungen lassen schließen, daß die inhibierende Wirkung von n-Alkanen aufs Mycelwachstum von Aspergillus niger bei der Citronensäurefermentation durch eine mechanische Blockierung wichtiger Lebensfunktionen während der ersten Phase des Konidienwachstums verursacht wird (Zufuhr von Sauerstoff und Wasser, Enzymaktivierung). Die sich in Antischaummittel befindenden kohlenwasserstoffhaltigen Emulgatoren erleichtern die Dispersion des Öles im Nährmedium, wodurch die Berührungsfläche mit Konidien vergrößert und der Hemmungseffekt verstärkt werden. Eine unkontrollierte Anwendung von Antischaummitteln in verschiedenen Stadien der

¿AKOWSKA, Z . , DBITRI, M . , C i t r i c - A c i d

283

Fermentation

Tabelle 3. Einfluß der frühen Inkubation des Mycels von Aspergillus »igrer-Stämmen R 1 6 und Z im öl- und antischaummittelhaltigen Nährmedium (100 ml) auf die Säurebildung (ml 0,1 N NaOH/2 ml Fermentationslösung) Stamm

Rl6

Z

Antischaummittel

Öl

Säurebildung [ml] F r ü h e Inkubation

[¡xl]

0 [h]

_

38,0

10

—

—

—

10

—

20 -

20

30,0 20,0 23,0

12 [h]

24 [h]

10,0 12,0

36,0 36,0

26,0 27,0

35,0 36,0

Zuckertechnologie kann eine zu große Konzentration von diesen Stoffen in der Melasse bewirken und sie teilweise bzw. gar vollständig für die Biosynthese unbrauchbar machen. Bemerkenswert sind dabei die beträchtlichen Unterschiede im Verhalten einzelner A. m'j/er-Stämme in Anwesenheit von Antischaummitteln im Nährmedium. Daraus ergibt sich die Notwendigkeit, in Zukunft die für die Citronensäurefermentation verwendeten Stämme auch auf ihre Empfindlichkeit gegen Hilfsmittel der Zuckerindustrie zu charakterisieren. Dieser Vorschlag könnte einen wesentlichen Beitrag zur besseren Nutzung von Melasserohstoffen in vielen Biotechnologiebereichen leisten. Diese Arbeit wurde im Rahmen der Problematik MR I I . 17.2.3.2. durchgeführt. Eingegangen: 7. 1. 1986

Literatur [1] 2AKOWSKA, Z., DRURI, M.: XV Symposium d. Pol. AdW, Fortschritte in Analytik d. Lebensmittel Warszawa 1984. [2] DRURI, M., ZAKOWSKA, Z.: Acta Biotechnol. 7 (1987) 3, 275. [ 3 ] ZAKOWSKA, Z . , DRURI, M . , NOWAKOWSKA-WASZCZUK, A . : T a g u n g B i o t e c h n o l o g i e u . L e b e n s -

mittelchemie, Technische Universität Lodz, 1985. [4] JAKUBOWSKY, J . : Postepy Mikrobiol. 16 (1975), 37. [5] Indisches P a t e n t : 1187334 (1970).

Acta Biotechnol. 7 (1987) 3, 284

Book Reviews H . F . LINSKENS, J. F.

JACKSON

Modern Methods of Plant Analysis New Series, Vol. 1. Cell Components Berlin, Heidelberg, New York, Tokyo: Springer-Verlag, 1985. 399 S., 98 Abb., 23 Tab., 238 DM Die Herausgeber haben sich das Ziel gestellt, die erfolgreiche Serie „Modern Methods of P l a n t Analysis" (Eds. M. V. T R A C E Y und K . P A E C H ) durch eine neue Reihe von Monographien dem Erkenntniszuwachs seit 1964, dem Erscheinungsjahr des letzten Bandes der alten Reihe, anzupassen. Der vorliegende Band 1 der „ M o d e m Methods of P l a n t Analysis, New Series" beschäftigt sich ausführlich mit der Isolation und den Eigenschaften von Bestandteilen pflanzlicher Zellen sowie der Anwendung von Präparationen von Zellbestandteilen und Organellen in der Forschung. Die 18 Kapitel des Buches behandeln folgende Schwerpunkte: Zellwand (Isolation, Wachstum, Chemie, Struktur); Protoplasten als Modelle f ü r Studien zur K o m p a r t i m e n t i e r u n g ; Das Markerkonzept bei der Zellfraktionierung; Plasma-Membranen; Vakuolen; Protein-, Lipid-Bodies; Chloroplasten (Gesamtisolation, Membrangewinnung; Markerproteine) sowie nicht grüne Piastiden; Mitochondrien, Polyribosomen, endoplasmatisches Retikul u m ; Kern (cytologische Methoden, Isolation); Mikrotubuli. Alle Kapitel sind klar aufgebaut und berücksichtigen sowohl die Fragen der Isolierung, der P r ü f u n g der einzelnen Präparationen auf Reinheit als auch deren Anwendungsmöglichkeiten in der Forschung. Spezielle Aufarbeitungsmethoden werden ebenso beschrieben wie Besonderheiten, die sich aus der Arbeit m i t einzelnen Spezies ergeben können. Die Beiträge sind durch ausführliche Literaturverzeichnisse ergänzt, die die Literatur bis 1984/1985 erfassen. Auf Grund der sehr guten Qualit ä t kann der vorliegende Band den interessierten Wissenschaftlern empfohlen werden. E r sollte in keiner Fachbibliothek fehlen. H.-P. LEVIN, S. Vol.

ScHMAUDER

A . : Biomathematics

16. CASTI, J . S . , KARLQUIST,

A.

Complexity, Language and Life: Mathematical Approaches Berlin, Heidelberg, New York, Tokyo: Springer-Verlag, 1986. 281 S., 128 DM Das IIASA und die Schwedische Akademie der Wissenschaften h a t t e n im Mai 1984 einen kleinen Kreis hervorragender Wissenschaftler (die Bezeichnung Biomathematiker wäre sicherlich zu eng) versammelt. Es wurde ohne thematische Einengung über theoretische Fragen der Evolution komplexer Systeme gesprochen. Die Namen der Geladenen können f ü r die Breite und Tiefe des dort Gedachten stehen: D. BERLINSKI, J . L . CASTI, P . GOULD, U . G R E N A N D E R , J . J O H N S O N , H . H . P A T T E E , R . R O S E N , K .

SIG-

MUND, N . C. STENSETH u n d R . THOM.

Wenn J . L. CASTI seinem Beitrag als Motto voranstellt: " I have yet to see any problem, however complicated, which when you looked a t it in the right way, did not become still more complicated." (P. A N D E R S O N ) , SO k o m m t darin einiges über den Gegenstand der Einzelbeiträge des Buches zum Ausdruck. P. G O U L D bringt in dem einleitenden Artikel: "Allowing, Forbidding, b u t not Requiring: A Mathematics for a H u m a n W o r l d " sehr viel zum Stand und zur Einordnung der Mathematik heute; einer Mathematik, die nicht mehr gefordert ist, einfache Systeme (oder einfach zu machende Systeme) zu beschreiben, sondern die sich einer Komplexität, wie sie lebenden Systemen zu eigen ist, zuwenden muß und zuwendet, und die damit neue Methoden, neues Denken erfordert. Wenn wir lebende Systeme technisch nutzen wollen, und das nicht nur mit traditionellen Verfahren, so ist es wichtig, an dem neuen Denken über komplexe Systeme teilzuhaben. Dieses Teilhaben wird durch das vorliegende Buch leichter gemacht. Th. Bley

Acta Biotechnol. 7 (1987) 3, 2 8 5 - 2 8 8

Short Communications A Procedure for Gluconic Acid Synthesis with Bacteria Made Continuous by Means of an Auxiliary Substrate* B A B E L , W . , MTJLLER, I I . H .

Academy of Sciences of the G.D.R. Institute of Biotechnology, Leipzig PermoserstraBe 15, Leipzig, 7050 G.D.R.

Summary A method is introduced which makes a continuous oxidation of glucose to gluconic acid possible. This method is based on the auxiliary-substrate concept and co-metabolism, respectively. Microorganisms (e.g. Acinetobaeter caleoaceticus), which cannot assimilate glucose, but merely oxidize it, are grown continuously on a heterotrophic substrate (e.g. acetate). While growing they simultaneously synthesize gluconic acid. The productivity of the gluconic acid synthesis with a given strain depends on the dilution rate and the mixing proportion. Since growth and product synthesis are closely connected and growth yield is very much higher due to an auxiliary substrate effect in the presence of glucose than on the heterotrophic substrate alone, this method is suitable for SCP production as well. The productivity of gluconic acid production is controlled at a certain dilution rate by the mixing proportion of the growth substrate and glucose.

Gluconic acid (or gluconate) is mainly used in t h e food, pharmaceutical and chemical industries. I t can be produced by electrochemical oxidation of glucose, biochemically by using free or immobilized cells of fungi a n d bacteria or by m e a n s of enzymes, respectively. The large-scale production of gluconic acid takes place almost exclusively by microbes, with strains of Aspergillus niger, being used, b u t recently bacteria of t h e genera Pseudomonas, Gluconobacter a n d Acetobacter have been b r o u g h t into action as well [1, 2].

Microbial production of metabolites of t h e p r i m a r y a n d i n t e r m e d i a r y metabolism occurs as a result of stress in t e r m s of life [3—5]. W i t h a balanced s u b s t r a t e supply microorganisms grow a n d m u l t i p l y w i t h o u t forming a m a j o r a m o u n t of carbon-containing intermediates except for carbon dioxide. F a c u l t a t i v e anaerobes produce for instance ethanol f r o m glucose in t h e absence of oxygen, some yeasts a n d fungi form citric acid a t a certain oxygen tension a n d in t h e absence of a m m o n i a , a n d some fungi a n d bacteria oxidize glucose t o gluconic acid a t a high glucose concentration. If this capability of microorganisms is t o be used in a n industrial procedure, t h e highest possible p r o d u c t yield is desired. K n o w i n g t h e metabolic sequence f r o m t h e s u b s t r a t e t o * Shortened version of the paper given on the 4th Symposium of the Socialist Countries on Biotechnology, Varne (Bulgaria) May 2 6 - 3 0 , 1986.

286

Acta Biotechnol. 7 (1987) 3

the product wanted the maximum yield can be predicted. In the case of ethanol fermentation it amounts to 0.51 g, in citric acid synthesis to 1.07 g, and in gluconic acid production to 1.09 g per gram of glucose, provided glucose is only converted into the product according to the metabolic scheme, glucose is not wasted in byproducts and cannot be further metabolized. Hence, the cells operate only as a catalytic system. All known processes operate discontinuously in two stages. In the first phase the producer, i.e. the microbial biomass, is generated by growth and in the second phase the biomass is employed as a catalyst for the syntheses of metabolites. Under the conditions of overflow production of metabolites the catalyst is labile, and denaturation and degradation processes take place, thus product syntheses diminish and eventually stop unless the catalyst can be stabilized. The inactivation processes may be counteracted by immobilization if isolated enzymes are applied; if whole cells are used as catalysts this method fails in its effect. In this case the catalyst can be stabilized best by regeneration, which is aimed at, for instance, by alternating frequently between the growth and production phases [6]. Nevertheless such a procedure remains periodic and the overall yield is markedly smaller than the metabolically determined possible yield because part of the substrate consumed is used for the synthesis of the catalyst. On the basis of the auxiliary substrate concept, which has been developed to improve the growth yield of SCP synthesis [5, 7 to 9], the gluconic acid production can be made continuous in a one-stage process according to the chemostat principle. This can be done by means of microorganisms which grow continuously on a heterotrophic substrate and simultaneously oxidize the glucose present but cannot assimilate it due to phenotypic measures or genotypically determined defects. Some species of the genera Acinetobacter, Acetobacter ... fulfil these conditions. For instance Acinetobacter calcoaceticus 69-V is able to grow on acetate, however, it is not capable of growing on glucose [10]. With acetate as the sole carbon source for growth the overall process is to be described as follows: Glc 4-1 Acetate

Ae netob cter '' ^ calcoaceticus

y GlcA +1 A. calcoaceticus +1 CO,^

A mutant of Acinetobacter calcoaceticus 69-V, named RAG 111-21 [11], which is characterized by lower emulsifying activity was used. The strain was cultivated with acetate limitation at 30 °C and a pH of 6.8 f 0.1 in a LF-2 laboratory fermenter (CSAV, Prague, Czechoslovakia) having a working volume of 1.51. Dissolved oxygen was kept at least at 20% saturation. Growth was performed on 2.6 g-l _1 acetate in a minimal medium, which contained (in mg • 1 _1 ): NH4C1, 3400; KH 2 P0 4 , 310; MgS0 4 • 7 H 2 0, 15; CaCl2 • 2 H a O, 20; FeS0 4 • 7 H 2 0, 5; ZnS0 4 • 7 H 2 0 , 0.44; MnS0 4 X 4 H 2 0, 0.82; CuS0 4 • 5 H 2 0, 0.78; Na 2 Mo0 4 • 2 H 2 0, 0.25. Foaming was suppressed by adding 0.01% (v/v) of antifoaming emulsion (Serva, Heidelberg, FRG) to the growth medium. The experiments aimed at gluconic-acid production were started and performed with "the transient state cultivation technique [12], After having reached steady state conditions during cultivation on acetate, glucose was added to this culture in a linearly increasing concentration gradient generated in the medium reservoir flasks as described elsewhere [13]. Glucose and gluconic acid were determined by enzymatic analysis using hexokinase/ glucose 6-phosphate dehydrogenase and gluconate kinase/6-phosphogluconate dehydrogenase (Boehringer, Mannheim, FRG), respectively, and acetate was measured gas-chromatographically. Bacterial growth was followed up photometrically or by measuring the biomass weight of 3 ml aliquotes of suspension (4-fold), taken from the fermenter, after centrifugation, washing once with distilled water and drying to constancy at 105 °C.

BABEL, W., MULLEK, R. H., Gluconic Acid Synthesis

287

In Fig. 1 the results obtained for two different dilution rates are shown. At a dilution rate of 0.14 h _ 1 1.05 g biomass are formed from 2.6 g of acetate. This amount of Acinetobacter calcoaceticus cells is able to oxidize simultaneously about 21 g of glucose per liter, hence a productivity of about 3 g • l^ 1 • h 1 results. In another case, at a dilution rate of 0.43 h _ 1 (i.e. 75% of the maximum possible dilution rate), only approximately 13 g of glucose can be oxidized completely to gluconic acid by 1.08 g biomass, but the resulting productivity amounts to about 5.6 g • l" 1 • h 1 . 2l 20 -

15 -

10

1.7 A?

2.6

2.6

10[ B

D 0.1Uh'1 ir Y

2.9bgl~1h~1 0.6 OA g g'1

1.7U

A2 At

1.08

0A3 h~' 5.6 g l'1 h'1 06 O.Ugg'

Tig. 1. Quantitative relations between the amounts of acetate as the growth substrate and gluconic acid produced with simultaneous utilization of acetate and glucose by A. calcoaceticus at two dilution rates. A x : Concentration of acetate as sole carbon and energy source for the formation of A. calcoaceticus cells (B); A 2 : Reduced concentration of acetate as sole carbon source in presence of glucose as an energy donor for the formation of the same amount of A. calcoaceticus cells (B).

Since the productivity is determined by the activity of the glucose dehydrogenase productivity can be increased by enhancing the glucose oxidizing capacity. This can be done, on the one hand, by genetic engineering (amplification) or by increasing the cell concentration in the fermenter, on the other. As during the oxidation of glucose to gluconic acid with Acinetobacter calcoaceticus biologically useful energy is released, which might be consumed for growth, more than 1.05 g or 1.08 g biomass are synthesized or less than 2.6 g of acetate are needed (Fig. 1). In the presence of glucose 1.5 to 1.8 g biomass can be formed from 2.6 g of acetate, that means the growth yield increases from 0.4 g • g" 1 up to 0.6---0.65g-g _1 [11]. This is another positive effect which ought to be exploited in SCP production. Therefore, it is reasonable to combine this kind of gluconic acid synthesis with SCP production. For the production of gluconic acid in an industrial procedure a microorganism strain as a catalyst and a substrate should be chosen, that are of interest for SCP production as well. By varying 6 Acta Biotechnol. 7 (1987)

288

Acta ß i o t e c h n o l . 7 (1987) 3

the mixing proportion of both substrates the combined process can be controlled very easily. A high acetate portion in the mixture favoures growth, and at a high portion of glucose product synthesis predominates. Received J u l y 31, 1986

Keferences [1] KOHR, M., KUIHCKK, C. P., KOMINEK, J . : Gluconic Acid. — I n : Biotechnology. A Comprehensive Treatise in 8 Volumes, E d . : H . D E L L W E G . Vol. 3. E d s . : H . J . R E H M , 6 . R E E D . W e i n h e i m : Verlag Chemie, 1982, 4 5 5 - 4 6 5 . [2] MILSOM, P. E., MEERS, J . L . : Gluconic a n d Itaconic Acids. — I n : Comprehensive Biotechnology.

[3] [4] [5]

[6] [7] [8] [9]

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

E d . M. Moo-YOUNG.

Vol. 3. E d s . :

H. W.

BLANCH, S. D R E W ,

D . I . C.

WANG.

Oxford, New Y o r k , T o r o n t o , Sydney, F r a n k f u r t / M . : P e r g a m o n Press, 1985, 681 — 700. NEIJSSEL, 0 . M „ TEMPEST, D. W . : S y m p . Soc. Gen. Microbiol. 29 (1979), 53. DEMAIN, A. L . : N a t u r w i s s e n s c h a f t e n 67 (1980), 582. B A B E L , W . : A c t a Biotechnol. 6 ( 1 9 8 6 ) , 2 1 5 . ISKE, U . — I n : Proc. 7 t h S y m p . Contin. Cultiv. Microorganisms, P r a g u e , J u l y 10—14, 1978. E d . : 1980, p p . 6 9 9 - 7 0 4 . B A B E L , W . : A b h a n d l . A k a d . Wiss. D D R (Nr. 2), Biotechnologie, 1982. Hrsg. M. R I N G P F E I L . Berlin: Akademie-Verlag, 1982, 1 8 3 - 1 8 8 . BABEL, W . — I n : Proc. 3rd S y m p . Soc. County. Biotechnol., Bratislava, April 25 — 29, 1983. E d . 1984, 1 6 9 - 1 7 6 . B A B E L , W . , M Ü L L E R , R . H . : J . Gen. Microbiol. 1 3 1 ( 1 9 8 5 ) , 3 9 . KLEBER, H . J . , AURICH, H . : Z. Allg. Mikrobiol. 13 (1973), 473. MÜLLER, R . H . , BABEL, W . : Arch. Microbiol. 144 (1986), 62. B A B E L , W . , M Ü L L E R , R . H . , M A R K U S K E , K . D . : Arch. Microbiol. 1 3 6 ( 1 9 8 3 ) , 2 0 3 . M Ü L L E R , R . H . , M A R K U S K E , K . D . , B A B E L , W . : Biotechnol. Bioeng. 2 7 ( 1 9 8 5 ) , 1 5 9 9 .

Acta Biotechnol. 7 (1987) :î, 2 8 9 - 2 9 2

Biochemical Studies on the Fermentation of Cassava (Manihot ntilissima Pohl.) OTENG-GYANG, K . * , ANUONYE, C. C.

Department of Biochemistry University of Nigeria Nsukka, Nigeria

Summary Some original observations have been made on the process of cassava fermentation to produce "foofoo", a local nigérian diet. During the period of fermentation the pH of the fermenting liquor decreases from 6.1 to 3.4 at the end of the 6th day. The change in pH is uniform throughout the fermentation period. Decreases in dry weight of the fermenting cassava have been recorded ; there is a very rapid decline during the third and fourth days of fermentation. Free reducing sugars decrease drastically within the first and second days. Total sugar concentration which is an indication of the starch content of the cassava also declines with fermentation time, and more so during the third and fourth days. Protein concentration in the liquor increases very rapidly during the first and second days of fermentation. It is believed that cassava protein is converted to microbial protein.

v

Cassava, also called manioc, constitutes an important staple food in the tropics [1, 2]. I t is widely grown in many parts of Asia, South America and Africa [3], The cassava plant is of enormous economic importance due to its ability to resist drought and to produce significant yields in low fertility soils [4]. Cassava belongs to the family Euphorbiaceae ; the two species widely cultivated in Africa are Manihot esculenta Crantz or Manihot utilissima Pohl a poisonous species and Manihot dulcis, a non-poisonous species. This classification is based on the hydrogen cyanide content of the roots [5]. A fresh cassava tuber is made up of about 70% moisture, 26% carbohydrates [6]. Over 90% of these carbohydrates are of fermentable sugars [7]. The protein content of cassava is low, about 1.2% [6]; thus the importance of eating cassava with a high protein diet notably meat, fish and vegetables. In Nigeria cassava may also be peeled, grated and fermented directly without grating, and subsequently cooked and pounded to produce " a k p u " or "foofoo", a traditional food of paramount importance. Very little is known of this food, although its economic importance cannot be over-emphasized ; it is locally known as a "famine insurance food". Furthermore, it is suspected that microorganisms and enzymes involved in these fermentation processes could be closely involved in post-harvest spoilage and deterioration * To whom all correspondence should be addressed: CESTAM International, 160 Ave. General Leclerc, 2 res. chateau de courcelle, 91190 Gif-sur-Yvette, France. 6*

Acta Biotechnol. 7 (1987) 3

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of fresh cassava tubers. It should be remembered that post-harvest storage of fresh cassava tubers is very difficult and tubers can generally keep for only a few days after harvest. The aim of this work is not only to initiate research into "foofoo", one of the most important semi-processed nigérian traditional foods, but to contribute towards finding a solution to the problem of post-harvest storage losses of cassava roots arising from microbial spoilage and deterioration. Materials and Methods Freshly harvested cassava Manihot utilissima Pohl roots were obtained from the local University of Nigeria farm at Nsukka. Freshly harvested cassava tubers were peeled, washed and cut into small pieces. A known weight (about 80 g) were put into a large beaker and 100 ml of t a p water of known p H added. The beakers were covered with a fine gauze in a way as to permit the free exchange of air with the surroundings. The cassava was then left to ferment a t ambient temperature in the laboratory. A known weight of cassava (1.0 — 1.3 g) was heated a t 105°C in an oven until a constant weight was observed. The p H was determined using a p H meter Unicam P Y E model 292. The method of 3,5-dinitrosalicylic acid was used with glucose as standard. Complex carbohydrates notably starch were first hydrolysed by hydrochloric acid; the solution obtained was then neutralised by NaOH and the reducing sugar concentration of the hydrolysate determined by the dinitrosalicylic acid method. Quantitative determination of protein was by the F o l i n - C i o c a l t e a u reagent method as decribed in [8] using culture filtrates containing no cassava pieces.

Results and Discussion The results presented here represent the average of 3 trials. The results in Table 1 show a uniform decrease in dry weight from the beginning of the fermentation period. Between the third and fourth days the rate of dry weight decline is very high comparatively, about 10 times what is observed the preceding days. After the fourth day the decrease in dry weight again slows down. Final dry weight at the end of the six day fermentation period is about 50% of the original dry weight of fresh cassava. Two different but related pH values were monitored: the pH of the fermenting liquor and the pH of the fermenting cassava pieces. For this latter operation a piece of fermenting cassava was taken out of the fermentation liquor, crushed in distilled water and the Table 1. Dry w eight and p H changes during the fermentation of cassava Time [d]

Dry weight |g/100 g of fresh cassava]

p H of fermenting cassava

p H of fermenting liquor

(I

26.3 35.2 34.4 33.4 22.7 19.8 18.3

6.7 5.9 5.2 4.5 4.2 4.1 4.1

6.1 5.0 4.2 4.0 3.5 3.4 3.4

2 3 4 5 6

OTENG-GYANG, K., AN U ON YE. C. C., Fermentation of Cassava

291

pH of the mixture measured. The results in Table 1 show that at the start of the experiment the pH of the fermenting cassava is higher than that of the fermenting liquor and that this pH difference of about 0.6 average is maintained throughout the fermentation period. More importantly, there is observed a relatively high acidification of the culture medium during fermentation. It is interesting to note that the rapid decrease in dry weight observed during the third and fourth days is not accompanied by a correspondig drastic pH modification. The experiments on free reducing sugars were conducted on the filtrate obtained after crushing (approximately 1.0—1.3 g) a known weight of fermenting cassava. The concentration of free reducing sugars in the culture medium itself was always negligible. Free reducing sugar concentrations are very low; microbial utilisation could account for these very low values. A slow rate of enzymatic hydrolysis could be the limiting factor to the production of high reducing sugar concentrations. Table 2. Variations in sugar and microbial protein concentrations during the fermentation of cassava. Time [d]

Free reducing sugar [nig/100 g dry wt.]

Total sugars

Total sugars

[g/100 g fresh wt. cassava]

[g/100 g dry weight of cassava]

0 1 2 3 4 5 6

25 20 4.6 4.3 3.7 2.9 1.8

26 23 23 22 12 11 10

72 66 66 (53 52 51 50

Protein in fermentation liquor [g/100 g fresh wt. cassava]

0.02 0.12 0.54 0.55 0.73 0.89 1.08

Total sugar concentrations calculated in grams per 100 g of fresh cassava and also per 100 g dry weight are presented in Table 2. Whereas a uniform decline in sugar concentration is observed, throughout the fermentation period this decline is more pronounced during the third and fourth days. The protein observed in the fermenting liquor is either of microbial origin or to a far less extent of endogenous cassava origin as a result of leaching. The results of protein concentration, calculated per liter of fermentation liquor and then reported to 100 g per fresh cassava weight are presented in Table 2. Protein concentration in the fermentation liquor starts to increase from the first day of fermentation: a rather large increase is observed between the first and second day of fermentation. This rapid increase could correspond to a period of very rapid microbial multiplication. It should be remembered that fermentation is carried out under non-sterile conditions and that the culture is heterogenous. It appears therefore that during the fermentation period there is conversion of cassava protein and other nitrogenous compounds into microbial protein. The conversion of starch to protein involves a first stage of hydrolysis; in a fermentation medium like ours without any external source of enzymes or acid the enzymes involved are endogenous to the cassava product or the microorganisms present. Generally, these enzymes are amylases and amyloglucosidases [9]. The pH of the medium and its composition play a highly significant role in the process [10]. With the endogenous cassava protein concentration of 1.2% (as was the case in the type we used), the turn-over rate of protein conversion is about 90% at the end of the sixth day. It should be noted that 7

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Acta Biotechnol. 7 (1987) 3

microbial numbers continue to increase at the end of the sixth day of fermentation. We have isolated the different microorganisms actively involved in the fermentation process; studies are well under way to determine their properties and characteristics as well as variations in their numbers during the fermentation process.

Conclusion

During the process of fermentation of cassava to produce "foofoo", there is a drastic decline in free reducing sugar concentration associated with a rapid increase in protein concentration in the culture medium from the first day to the second day of fermentation. This could suggest a need to satisfy energy requirements of the rapidly increasing microbial population during the period. This rapid increase in microbial population probably leads to an increase in amylolytic enzyme production; thus the rapid starch hydrolysis between the third and fourth days. The decrease in p H observed could be due to the production of organic acids. Traditionally, the process of cassava fermentation is generally terminated at the end of the fourth day. This period corresponds to the time when dry weight, pH, free reducing sugars as well as total starch concentrations have almost become stable. Our study confirms the low protein content of cassava foods [12]. Although the hydrocyanic acid content was not determined, some quantity of it must be present even though the process of fermentation and rhodanese enzyme detoxification should reduce the amount [1,13]. Cassava is one of the most important staple.foods in sub-saharan Africa; fermented cassava foods are eaten in large parts of the continent. Unfortunately, scientific reports on the nutritive aspects of such foods, apart from "gari", as well as on post-harvest storage of cassava roots are few. Fresh roots when harvested usually deteriorate in a very short time; spoilage and deterioration are due to endogeneous enzymes and more importantly to microorganisms. An understanding of the processes involved in the fermentation of cassava should aid in finding a solution to the problem of post-harvest storage losses. Received December 16. 1985

References [ 1 ] ANOSIKIO, E . 0 . , UGOCHUKWU, E . N . : J . E x p . B o t . 3 2 ( 1 9 8 1 ) ,

1021.

[2] OKE, O. L.: J. Root Crops (India) 1 (1975) 1, 1. [3] LINNEMANN, A. R.: Abst. Trop. Agrio. 7 (1981) 1, 9. [4] PHILLIPS, T. A.: An agricultural notebook. Thetford, London: Lowe and Brydone Printers Ltd., 1977. [5] OYENUGA, V. A.: Nigeria's foods and feeding stuffs. Ibadan University Press Nigeria, 1968, 20. [6] KERR, R. W.: Chemistry and industry of starch. New York: Academic Press, 1950, 659. [7] GRACE, M.: Agric. Services Bull. FAO Rome 8 (1971). [ 8 ] L A N E , E. -- In: Methods in Enzymology, Vol. I l l , KOLOWICK and K A P L A N eds. New York: Academic Press 1957, 447. [ 9 ] O'TENG-GVANG, K . , MOULIN, G . , GALZY, P.: Acta microbiol. Aca. Sei. Hung. 2 7 ( 1 9 8 0 ) , 1 5 5 . [10] O T E N G - G Y A N G , K., M O U L I N , G . , GALZY, P.: Eur. J. Appl. Microbiol. Biotechnol. 9 (1980), 129. [11] M O U L I N , G . , GALZY, P.: Z. Allg. Mikrobiol. 18 (1978), 329. [12] OKE, 0 . L.: Nature 212 (1966) 5066, 1055. [13] UGOCHUKWU, E . N „ OSISTOGU. 1. U . W . : P l a n t a m e d . 3 2 (1977), 105.

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Acta Biotechnologica Volume 7

1987

Number 3

Contents S. C. ; nas sp RAO,

JONES, L .

P. : Formation of Higher Alcohols and Phenol by Strains of Zymomo209

BODE, R.; BIBNBATJM, D.: D-Amino Acid Oxidase, Aromatic L-Aminotransferase, and Aromatic Lactate Dehydrogenase from Several Yeast Species: Comparison of Enzyme Activities and Enzyme Specificities 221 LASOH, J . ; KOELSOH, R . ; RBETSOHMER, K . : Continuous P r o d u c t i o n of P r o t e i n H y d r o l y s a t e s

in Immobilized Enzyme Reactors

227

RICHTEB, K. ; BECKER, U. ; MEYER, D. : Continuous Fermentation in High Flow Rate Fermenter Systems 237 B. ; B Ö H M E , B . ; B L E Y , Th. : Optimal Situation Control for Biosynthetic Process on the Basis of a Prediction Filter 247

MÖCKEL,

REICH, J . G. : Dynamics, Regulation, Block Description and Optimal Performance of Cellular Metabolism in Open Conditions: Outline of a Unifying Concept 257 REIMANN, B. : Application of a Hybrid-Computer System for Processing of Fermentation of Antibiotica in a Scale-up Line (in German) 265 DRURI, M. ; 2AKOWSKA, Z. : Investigation of Components of Beet Molasses having a Toxic Effect on Aspergillus niger at the Citric-Acid Production (in German) 276 M. : Role of n-Alkanes in Molasse-Culture Medium in Surface Process of Citric-Acid Fermentation (in German) 279 ¿AKOWSKA, Z . ; D R U R I ,

Short Communications BABEL, W. ; MULLER, R. H. : A Procedure for Gluconic Acid Synthesis with Bacteria Made Continuous by Means of Auxiliary Substrate 285 OTENG-GYANG, K . ; ANUONYE,

(Manihot utilissima Pohl.) Book Reviews

C. C.: Biochemical Studies on the Fermentation of Cassava

289

220,226,236,246,264,284

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