Zeitschrift für Allgemeine Mikrobiologie: Band 22, Heft 8 1982 [Reprint 2021 ed.] 9783112538920, 9783112538913

Citation preview

ZEITSCHRIFT FÜR ALLGEMEINE MIKROBIOLOGIE AN INTERNATIONAL JOURNAL ON MORPHOLOGY, PHYSIOLOGY, GENETICS, AND ECOLOGY OF MICROORGANISMS HEFT 8 • 1982 BAND 22

AKADEMIE-VERLAG • BERLIN EVP 20,— M

ISSN 0044-2208

34112

CONTENTS OF NUMBER 8

Preparation and characterization of proteases from Thermoactinomyces vulgaris. V. Investigations on autolysis and thermostability of the puriiied protease

U . BEHNKE, KLEINE

Seasonal fluctuations of freshwater fungi in River Nile (Egypt)

F . T . E L - H I S S Y, A . H . MOUBASHER AND M . A . E L - N A G D Y

521

Effect of nalidixic acid on the activation of RNAsyntheses in outgrowing Bacillus subtilis spores

M . HECKER

529

Theoretical estimation of energetic efficiency of microbial carbon source conversion and comparison with experimental results on phased cultures

B . HEINBITZ, E . STICHEL, G . ROGGE, T . B L E Y AND F . GLOMBITZA 535

Degradation of mixed substrates by yeasts. I. Substrates of waste sulfite liquor

UTE KRAUEL, H . WEIDE

Genetic segregation in a high-yielding streptomycinproducing strain of Streptomyces griseus

M . ROTH, B . SCHWALENBERG, R. REICHE, D . NOACK, R . GEUTHER AND I . E R I T T

557

M.SUEKANE

565

M . S U E K A N E AND H . I I Z U K A

577

U . GRÄFE

591

Immobilization of glucose isomerase Production of glucose isomerase by genus myces

Strepto-

Short Notes Effect of short-chain alcohols on production of NADP-glycohydrolase by Streptomyces griseus Effect of L-valine and L-isoleucine on fatty acid composition of Streptomyces hygroscopicus and S. griseus Book

H . RUTTLOFF

AND

R. 511

H . H . KRAUEL

AND 545

U . GRÄFE, M . ROTH a n d D . KBEBS

595

601

Reviews INHALTSVERZEICHNIS

HEFT 8

Gewinnung und Charakterisierung von Proteasen aus Thermoactinomyces vulgaris. V. Untersuchungen zur Autolyse und Thermostabilität der gereinigten Protease

U . BEHNKE, KLEINE

Jahreszeitlich bedingtes Pilze im Nil

F . T . E L - H I S S Y , A . H . MOUBASHER UND M . A . E L - N A G D Y

521

Einfluß von Nalidixinsäure auf die Aktivierung der RNA-Synthesen auswachsender Sporen von Bacillus subtilis

M . HECKER

529

Theoretische Bestimmung energetischer Wirkungsgrade der mikrobiellen Kohlenstoffsubstratwandlung und Vergleich mit experimentellen Werten an Phasenkulturen

B . HEINRITZ, E . STICHEL, G . R O G GE, T . B L E Y UND F . GLOMBITZA

535

Abbau von Mischsubstraten durch Hefen. I. Substrate der Sulfitablauge

U T E KRAUEL, H . WEIDE

545

Genetische Segregation in einem Streptomycin-produzierenden Hochleistungsstamm von Streptomyces griseus

M . ROTH, B . SCHWALENBERG, R. REICHE, D . NOACK, R . GEUTHER UND I . E R I T T

Immobilisierung von Glucose-Isomerase

M.SUEKANE

565

M . S U E K A N E AND H . I I Z U K A

577

U . GRÄFE

591

U . GRÄFE, M . ROTH u n d D . KREBS

595

Vorkommen

Bildung von Glucose-Isomerase durch

aquatischer

Streptomyces

Kurze Originalmitteilungen Einfluß von Cj- und C 2 -Alkoholen auf die Bildung von NADP-Glycohydrolase in Streptomyces griseus Einfluß von L-Valin und L-Isoleucin auf die Fettsäurezusammensetzung von Streptomyces hygroscopicus und S. griseus Buchbesprechungen

H . RUTTLOFF

UND

R. 511

H . H . KRAUEL

UND

557

601

ZEITSCHRIFT FÜR ALLGEMEINE MIKRO- BIOLOGIE AN INTERNATIONAL

JOURNAL

ON

MORPHOLOGY, PHYSIOLOGY, GENETICS, AND ECOLOGY OF MICROORGANISMS

H E R A U S G E G E B E N VON

F. Egami, Tokio G. F. Gause, Moskau 0 . Hoffmann-Ostenhof, Wien A. A. Imseneckii, Moskau R. W. Kaplan, Frankfurt/M. F. Mach, Greifswald 1. Mälek, Prag C. Weibull, Lund

unter der Chefredaktion von W. Schwartz, Braunschweig und U. Taubeneck, Jena

UNTER MITARBEIT VON

J . H. Becking, Wageningen H. Böhme, Gatersleben M. Girbardt, Jena S. I. Kusnecov, Moskau 0 . Necas, Brno C. H. Oppenheimer, Port Aransas N. Pfennig, Göttingen I. L. Rabotnova, Moskau A. Schwartz, Wolfenbüttel REDAKTION

HEFT 8

1982

BAND 22

AKADEMIE-VERLAG • BERLIN

U. May, Jena

Die Zeitschrift für Allgemeine Mikrobiologie soll dazu beitragen, Forschung und internationale Zusammenarbeit auf dem Gebiet der Mikrobiologie zu fördern. Es werden Manuskripte aus allen Gebieten der allgemeinen Mikrobiologie veröffentlicht. Arbeiten über Themen aus der medizinischen, landwirtschaftlichen, technischen Mikrobiologie und aus der Taxonomie der Mikroorganismen werden ebenfalls aufgenommen, wenn sie Fragen von allgemeinem Interesse bebehandeln. Zur Veröffentlichung werden angenommen: Originalmanuskripte, die in anderen Zeitschriften noch nicht veröffentlicht worden sind und in gleicher Form auch nicht in anderen Zeitschriften erscheinen werden. Der Umfang soll höchstens 1 1 / 2 Druckbogen (24 Druckseiten) betragen. Bei umfangreicheren Manuskripten müssen besondere Vereinbarungen mit der Schriftleitung und dem Verlag getroffen werden. Kurze Originalmitteilungen über wesentliche, neue Forschungsergebnisse. Umfang im allgemeinen höchstens 3 Druckseiten. Kurze Originalmitteilungen werden beschleunigt veröffentlicht. Kritische Sammelberichte und Buchbesprechungen nach Vereinbarung mit der Schriftleitung. Bezugsmöglichkeiten der Zeitschrift für Allgemeine Mikrobiologie: Bestellungen sind zu richten — in der DDR an eine Buchhandlung oder an den Akademie-Verlag, DDR-1086 Berlin, Leipziger Straße 3 - 4 — im sozialistischen Ausland an eine Buchhandlung für fremdsprachige Literaur oder an den zuständigen Postzeitungsvertrieb — in der BRD und Berlin (West) an eine Buchhandlung oder an die Auslieferungsstelle KUNST UND WISSEN, Erich Bieber OHG, D-7000 Stuttgart 1, Wilhelmstraße 4 - 6 — in den übrigen westeuroäpischen Ländern an eine Buchhandlung oder an die Auslieferungsstelle KUNST UND WISSEN, Erich Bieber GmbH, CH-8008 Zürich, Dufourstraße 51 — im übrigen Ausland an den Internationalen Buch- und Zeitschriftenhandel; den Buchexport, Volkseigener Außenhandelsbetrieb der Deutschen Demokratischen Republik, DDR-7010 Leipzig, Postfach 160, oder an den Akademie-Verlag, DDR-1086 Berlin, Leipziger Straße 3—4. Zeitschrift für Allgemeine Mikrobiologie Herausgeber: Im Auftrag des Verlages von einem internationalen Wissenschaftlerkollektiv herausgegeben. Verlag: Akademie-Verlag,DDR-1086Berlin, Leipziger S t r a ß e 3 - 4 ; Fernruf: 2236229 oder2236221 Telex-Nr.: 114420; Bank: Staatsbank der DDR, Berlin, Kto-Nr.: 6836-26-20712. Chefredaktion: Prof. Dr. U D O T A U B E N E C K Prof. Dr. W I L H E L M S C H W A B T Z Anschrift der Redaktion: Zentralinstitut für Mikrobiologie und experimentelle Therapie der Akademie der Wissenschaften, DDR-6900 Jena, Beutenbergstr. 11; Fernruf: Jena 885614; TelexN r . : 058621. Veröffentlicht unter der Lizenznummer 1306 des Presseamtes beim Vorsitzenden des Ministerrates der Deutschen Demokratischen Republik. Gesamtherstellung: VEB Druckerei „Thomas Müntzer", DDR-5820 Bad Langensalza. Erscheinungsweise: Die Zeitschrift für Allgemeine Mikrobiologie erscheint jährlich in einem Band mit 10 Heften. Bezugspreise je Band 250,—M zuzüglich Versandspesen (Preis für die DDR 200,-M). Preis je Heft 2 5 , - M (Preis für die DDR 2 0 , - M ) . Urheberrecht: Alle Rechte vorbehalten, insbesondere die der Übersetzung. Kein Teil dieser Zeitschrift darf in irgendeiner Form — durch Photokopie, Mikrofilm oder irgendein anderes Verfahren — ohne schriftliche Genehmigung des Verlages reproduziert werden. — All rights reserved (including those of translations into foreign languages). No part of this issue may be reproduced in any form, by photoprint, microfilms or any other means, without written permission from the publishers. Erscheinungstermin: November 1982 Bestellnummer dieses Heftes 1070/22/8 © 1982 by Akademie-Verlag Berlin. Printed in the German Democratic Republic. AN (EDV) 75218

Zeitschrift für Allgemeine Mikrobiologie

22

1982

511-519

(Zentralinstitut für Ernährung in Potsdam-Rehbrücke, Forschungszentrum für Molekularbiologie und Medizin, Akademie der Wissenschaften der DDR, Direktor: Prof. Dr. H. HABBEL1 und Physiologisch-Chemisches Institut der Martin-Luther-Universität Halle-Wittenberg, Direktor: Prof. Dr. H. AURICH2)

Preparation and characterization of proteases from Thermoactinomyces vulgaris V. Investigations on autolysis and thermostability of the purified protease U . BEHNKE 1 , H . RUTTLOFF 1 a n d R . KLEINE 2

(Eingegangen am

11.12.1981)

Thermitase, the main component of the proteases of the culture medium from Thermoactinomyces vulgaris, is degraded by autolyses (increase of liberated amino groups) and thereby inactivated especially at elevated temperature, at alkaline pH-values and in the absence of added substrates. As shown by Polyacrylamide gel electrophoresis autolysis is an essential part during heat inactivation (complete disappearance of the thermitase band after heating the enzyme at 85 °C for 5 min). The quantitative comparison of autolysis and heat inactivation as well as the kinetics of reversible inhibition of the enzyme by HgCl2 at different temperatures showed that above 60 °C thermal denaturation of the enzyme protein contributes to thermitase inactivation. Ca 2+ -ions (20 mM) have a stabilizing effect against both autolysis and thermal denaturation (inactivation) of thermitase.

Thermitase is an extracellular endopeptidase from Thermoactinomyces vulgaris, which is well suited for application in different fields of food industry because of its special properties. It is applied in the cereal processing industry, e. g. for saving energy by the use of low water doughs, due to partial hydrolysis of gluten during production of bread crumbs, instant flour, instant grits and waffle (DRECHSEL and RUTTLOFF 1976, TÄUFEL et al. 1978, DBECHSEL et al. 1974), and for the reduction of residual protein in the production of pure wheat starch (TÄUFEL et al. 1979b). Moreover, because of its relatively low cleaving specificity towards peptide bonds (SCHALINATUS et al. 1979) thermitase has many applications. Thus it is suitable for producing partially hydrolysed proteins for health and other special diets. In highly purified form it can be used for special investigations as fine biochemical. Besides its well-known ability to hydrolyse soluble proteins (BEHNKE et al. 1978 a) thermitase is capable of liquifying the unsoluble proteins elastin and collagen (KLEINE et al. 1981, KLEINE 1982) efficiently. At the same time thermitase is free of endonuclease activity (KLEINE and ROTHE, unpublished) and therefore suitable, for D N A isolation. Previous publications described the production (RUTTLOFF et al. 1978, KLINGENBERG et al. 1979, LEUCHTENBERGER et al. 1979, TÄUFEL et al. 1979a), purification (KLEINE et al. 1981, KLEINE and ROTHE 1977, BEHNKE et al. 1978b, FRÖMMEL et al. 1978) as well as some properties (BEHNKE et al. 1978a, 1978c KLEINE et al. 1981, KLEINE 1982) of this enzyme. The temperature optima of thermitase towards peptide esters and peptide p-nitroanilides under conditions of initial hydrolysis are 60 °C and 65 to 75 °C, respectively. However, with casein or azocasein as substrates the temperature optimum for initial hydrolysis is near 90 °C, despite rapid inactivation of the enzyme at this temperature. 35*

512

U. BEHNKE, H . RTJTTLOFF a n d R . R L E I N E

Substrate-dependent autolysis or heat denaturation are considered to be responsible for this inactivation. The sensitivity of thermitase to autolysis was published elsewhere ( B E H N K E etal. 1978b). Knowledge on the inactivation of thermitase at increased temperatures is of importance for scientific and practical applications as well as for the f u r t h e r characterization of the enzyme. Therefore, experiments on t h e thermal stability of the enzyme in the absence of substrate depending on pH, time and temperature of preincubation, and enzyme concentrations were carried out. In addition the influence of Ca 2+ -ions on thermitase activity in the presence or absence of subs t r a t e are reported. The influence of various substrates on thermitase stability will be discussed in a following paper. Materials

and

methods

Enzyme preparation: Thermitase was purified subsequently by column chromatography onSephadex G-75,DEAE-cellulose, and on Sephadex G-75 (BEHNKE et al. 1978b). The preparation is applied as an aqueous solution in concentrations of 0.025 to 5 mg/ml (for details see BEHNKE et al. 1978a). Enzyme assays: Protease activity or the residual activity after preincubation under different conditions is determined either with casein as substrate by measuring the trichloroacetic acid (TCA)-soluble peptides at 274 nm liberated during incubation (0.75% casein, Britton-Robinson buffer, pH 8.0, 55 °C, 20 min incubation time) (BEHNKE et al. 1978c) or by the pH-stat technique with N-acetyl-L-tyrosine ethyl ester (ATEE) as substrate (0.02 M ATEE, pH 8.0, 55 °C) (BEHNKE et al. 1978a). Determination of NH 2 -groups: NH 2 -groups liberated during the autolysis of thermitase are determined according to the trinitrobenzene sulfonic acid (TNBS) method (LANGNER et al. 1971). Polyacrylamide gel electrophoresis (PAGE): PAGE is carried out in 7.5% gels according to JVIAURER (1971), in Tris/HCl, pH 8.9. After fixation of the protein bands with TCA the gels are stained with Coomassie blue G-250 (0.1% TCA) according to DIEZEL et al. (1972). For detecting proteolytic activity after electrophoresis untreated gels are divided into equal longitudinal sections, the cut layed on photo paper and incubated at 30 °C for 20 min in a moist chamber. Proteolytically active bands can be recognized by brightening to transparent zones (TAUFEL et al. 1974). Determination of protein concentrations: The protein content of thermitase solutions is assayed either spectrophotometrically ( E ^ N M , 1% = 15.5) or according to a KJELDAHL micro procedure (LANGE et al. 1 9 7 9 ) .

Results and

discussion

Inactivation, autolysis and denaturation at increased temperatures I n order to determine the participation of autolysis during thermal inactivation of thermitase, the enzyme is preincubated at different temperatures (20—95 °C), pH-values (pH 5.0—9.0), and concentrations (0.1—2 mg/ml) for 0 to 8 h in the absence of substrate. Within this time liberated NH 2 -groups (autolysis) and the residual activity are assayed. The results for hydrolysis of casein are summarized in Fig. 1 — 3 and those for hydrolysis of A T E E in Fig. 4. Autolysis and inactivation increase with rising temperature, p H and enzyme concentration while the residual activity decreases towards high as well as low molecular weight substrates. Based upon these results both autolysis and thermal denaturation could be responsible for the inactivation of thermitase, especially at elevated temperatures, b u t it cannot be concluded which of the two processes prevails. Electrophoretic analysis of preincubated samples of thermitase indicates autolysis t o be important during thermal inactivation. When heating these samples for 5 min a t different temperatures (20—100 °C) the protease migrating to the cathode is increasingly degraded at temperatures above 55 °C. At temperatures near or above 85 °C

Proteases from T. vulgaris.

Preincubation time (hi

513

V.

Preincubation time iti)

Fig. 1. Autolysis and residual activity of thermitase (th.) after preincubation a t different temperatures (Preincubation: 1 mg of th./ml, Britton-Robinson buffer p H 7.0) Residual activity was a s s a y e d with 0 . 7 5 % casein a t p H 8.0 and 55 °C. 20 °C«, 37 °Co, 55 °C 9, 70 °C A, 9 5 ° C n

Preincubation time (h)

Preincubation time (h)

Fig. 2. Autolysis and residual activity of thermitase after preincubation a t different p H values (Incubation temperature: 55 °C. For further details see Fig 1). p H 5,0 p H 7,0 9 , p H 9.0 O

neither proteolytic activity (Fig. 5) nor inactivated enzyme protein or their degradation products (staining of the electropherogram with Coomassie blue) can be detected. Furthermore, a thermal protein denaturation is considered to be responsible for the loss of activity at increased temperatures. In Fig. 6 the residual activities after preincubation of thermitase (without subs rate) at different temperatures are plotted versus the degree of autolysis of the enzyme. Since the curves above 55 °C decline more steeply than at lower temperatures at a

514

U. Behnke, H. R u t t l o f f and R. Kleine

Preincubation time (h)

Preincubation time (h)

Pig. 3. Autolysis and residual activity of thermitase after preincubation at various enzyme concentrations (Incubation temperatures: 55 °C. For further details see Fig. 1). • 0,1 mg/ml, 9 1,0 mg/ml, O 2.0 mg/ml

Preincubation

time

(min)

Fig. 4. Residual activity of thermitase after preincubation at different temperatures and pH values (Preincubation: 0.10 mg of th./ml; 0.25 mg of th./ml; - - - 0.35 mg of th./ml; 0,025 M phosphate buffer pH 5.5, ph 7.1 and pH 7.4, 0.025 m Tris/HCl buffer pH 8.0. Residual activity was assayed with A T E E at pH 8.0 and 55 °C) 55 °C p H 5.5 pH 7.1 O, pH 8.0 O, 60 °C pH 7.4 x, 70 °C p H 5.5 A, pH 7.4 A, pH 8.0 •

P r o t e a s e s f r o m T. vulgaris.

V.

515

F i g . 5. P o l y a c r y l a m i d e gel electrophoresis of t h e r m i t a s e a f t e r p r e i n c u b a t i o n a t d i f f e r e n t t e m p e r a tures ( P r e i n c u b a t i o n : 3 m g of th./ml, B r i t t o n - R o b i n s o n - b u f f e r p H 8.0, 5 min. A c t i v i t y was d e t e c t e d on p h o t o p a p e r ; T ä u f e l et al. 1974)

P i g . 6. A u t o l y s i s a n d residual a c t i v i t y of t h e r m i t a s e a f t e r p r e i n c u b a t i o n a t d i f f e r e n t t e m p e r a t u r e s a n d t i m e i n t e r v a l s ( F o r f u r t h e r details see F i g . 1.) • 20 °C (up t o 192 h), O 37 °C (up to 72 h), 3 5 5 °C (up t o 8 h), A 70 °C ( u p to 3 h), A 85 °C (up t o 0 . 1 h)

given value of autolysis the residual activity is the more reduced, the higher the temperature was during preincubation. Protein denaturation is the only plausible reason for this additional inactivation. Accordingly, the incubation times had to be varied greatly with different temperatures to reach a range of autolysis between 0.2 and 2.4 [¿moles NH 2 /mg. The participation of both processes, autolysis and heat denaturation, during thermitase inactivation can also be detected by heating the enzyme inhibited reversibly with HgCl2. For this purpose native and Hg-treated thermitase are premcubated for 5 mm at different temperatures in the absence of substrate. Sub-

U. Behnke, H. R t j t t l o f f and R. Kleine

516

sequently the NH 2 -groups liberated by autolysis as well as the residual activity are determined. The latter is also determined after reactivation by an excess of cysteine. Because of the increased stability of the enzyme in a weakly acidic medium, 0,05 M acetate buffer, pH 5.5 was used in these experiments. As expected the autolysis of the Hg-treated enzyme is significantly reduced (Fig. 7A, curve 2). On the other hand (Pig. 7B), the residual activity of Hg-thermitase without preheating (25 °C, beginning of curve 4), amounts to less than 2 0 % of the control (curve 3). After reactivation by cysteine about 7 0 — 8 0 % (curve 5 a) of the untreated enzyme (curve 3) can be detected at 25—55 °C preincubation temperature. Due to inhibition of the protease activity of thermitase during preincubation, autolysis and the concomitantly reduced activity are largely excluded. Therefore, the inactivation measured at increased temperatures can be ascribed to thermal denaturation of the enzyme protein. 5b 100

2.0

|>Z5 Ì

3 5a

-6 60

"

t " 0À

20

20 40 SO SO Preincubation temperature (°C)

20 W Preincubation temperature (°C),

Fig. 7. Autolysis (A) and residual activity (B) of thermitase after preincubation at different temperatures with and without inhibition by HgCl2 (Preincubation: 2 mg of th./ml, 0.05 m acetate buffer pH 5.5, 5 min. Inhibition before preincubation: 0.04 mg HgClJmg th., 30 °C, 30 min. Reactivation: 0.576 mg cystein-HCl/mg th., 20 °C, 4 h. Substrate for activity: casein. The treatment of the control sample with and without HgCL and cysteine-HCl, respectively, was analogous. Curves 1 and 3: without HgCl2; curves 2 and 4: with HgCl2; curve 5a: with HgCl2, reactivated after preincubation; curve 5b: identical values as 5a, but in per cent with respect to the value at 25 °C) The slightly increased values of curve 5 a and 5b, respectively, at 40 and 55 °C as compared to those at 25 °C are probably caused by an incomplete reactivation carried out at a lower temperature (20 °C) in order to prevent autolysis. After preincubation at 70 and especially at 85 °C of the inhibited and reactivated samples increased residual activities compared to the untreated sample (curve 5 b) are evident. Starting at about 70 °C, however, in both cases (Fig. 7 B , curves 3 and 5 a and b, respectively) a strong inactivation can be observed mainly caused by thermal denaturation. This inactivation depends on the time of preincubation. Residual activity decreases significantly already at temperatures of 60 °C if the time of preincubation is 20 min instead of 5 min (results of analogous test series are not given in detail in this paper). Preliminary tests on the kinetics of inactivation showed that the course of reaction neither corresponds to a first order (thermal denaturation) nor to a second order reaction (autolysis). Further experiments on this matter are under investigation. All these experiments confirm that both autolysis and thermal denaturation contribute to inactivation of thermitase at increased temperatures (especially above 60 °C). These results

Proteases from T. vulgaris. V.

517

agree with findings of ARCHER et al. ( 1 9 7 3 ) who concluded from their results that in the case of "Monzyme" (a mixture of neutral and alkaline protease and «-amylase from Bacillus subtilis) loss of protease activity caused by autolysis in the absence of substrate may be as significant as loss of activity by thermal denaturation. I n f l u e n c e of C a 2 + - i o n s upon a u t o l y s i s a n d s t a b i l i t y One of the characteristic properties of thermostable proteases, e. g. thermolysin,

is their dependence on Ca 2 + -ions for optimal stability and activity (WEAVER et al. 1976, TONTANA et al.

1976).

Table 1 demonstrates the effect of different Ca 2+ concentrations upon both activities of thermitase, the peptide esterase and protease activities. In these experiments Table 1 Effect of CaCI2 on the peptide esterase and proteinase activity of thermitase (substrates: 0.02 M A T E E in 0.02 M Tris/HCl buffer and 0 . 7 5 % casein in Britton-Robinson buffer, respectively; pH 8.0, 55 °C) Relative activity (%)

Ca 2 +-concentration (mole/1) no Ca-+ added 0.01 • IO- 3 0 . 1 0 - IO- 3 0.25 • 10-3 0.50 • 10-3 1 . 0 0 - IO- 3 5.00 • 1 0 " 3 8 . 0 0 - 10"3 10.00 • IO- 3 20.00 • 10"3

10

W

B0

Esterase

Protease

100 114 134 148 190 236 184 116 100 45

100 103 99 94 90 86 73 69 68 63

80

100

110

Preincubation time (mm )

Fig. 8. Residual activity of thermitase after preincubation at 55 °C and 70 °C with and without addition of 6.67 • IO"3 M CaCl„ (Preincubation: 0.35 mg of th./ml; 0.05 M Tris/HCl buffer pH 8.0. Substrate for activity: A T E E in presence of 5 • 10~ 4 M CaCl2) • • 55 °C + 6.67 mM Ca 2 + , 55 °C without Ca 2 + , o o 70 °C + 6.67 mM Ca 2 + o- - - o 70 °C without Ca2+

518

U . B E H N K E , H . RUTTLOFF a n d R . K L E I N E

. Preincubation time (h)

Preincubation

time (h)

Fig. 9. Autolysis and residual activity after preincubation at 55 °C with and without addition of CaCl2 (Preincubation: 1 mg of th./ml; 0,05 M Tris/HCl buffer pH 8.0. Substrate for activity: casein) • without Caa+, 3 5 mM Ca2+, O 20 mM Ca2+

Tris/HCl buffer was used which does not interact with Ca2+. By increasing the Ca2+ concentration up to 1 mmole/1 maximal activation of the peptide esterase (more than 230% compared with the control) occurs. At higher concentrations of Ca2+ a decrease in activity can be observed which turns to inactivation at 10 mmole/1. When using casein as substrate no activation but diminution is to be measured with increasing Ca2+ concentrations from 0.25 mmole/1. In order to decide whether the stimulation by Caa+ is due to inhibition of autolysis the influence of Ca2+ upon thermitase stability was examined. The results summarized in Fig. 8 and 9 show that Ca2+" indeed stabilizes the proteolytic activity of thermitase towards ATEE (Fig. 8) and casein (Fig. 9). Fig. 9 further shows that Ca2+ exerts a distinct, autolysis-diminishing effect. At pH 5.0 and 55 °C or pH 8.0 and 75 °C a corresponding repression of autolysis and thus a stabilisation of activity is found. Increasing temperatures reduce the effect of Ca 2+ ; at 95 °C no effect is detectable. References M. C., RAGNARSSON, J. O., T A N N E N B A U M , S. R . and W A N G , D . I . C., 1973. Enzymatic solubilization of an insoluble substrate, fish protein concentrate: Process and kinetic considerations Biotechnol. Bioengng., 15, 181. B E H N K E , U . , K L E I N E , R., L U D E W I G , M . und RTJTTLOFF, H . , 1978a. Charakterisierung einer Protease aus Thermoactinomyces vulgaris (Thermitase). 3. Substratspezifität und einige Eigenschaften der teilgereinigten Thermitase. Acta biol. med. germ., 37, 1205.

ABCHEB,

B E H N K E , U . , SCHALINATUS, E . , RUTTLOFF, H . , H Ö H N E , W . E . u n d FRÖMMEL, C . , 1 9 7 8 b .

Charak-

terisierung einer Protease aus Thermoactinomyces vulgaris (Thermitase). 1. Untersuchungen zur Reinigung der Thermitase. Acta biol. med. germ., 37, 1185. B E H N K E , TL, T Ä U F E L , A., RUTTLOFF, H . , H Ä F N E R , B. und L E H M A N N , G., 1978 c. Standardisierung der Aktivitätsbestimmung von Proteasen. Lebensmittelind., 25, 485, 545. D I E Z E L , W . , KOPPERSCHLÄGER, G. and H O F M A N N , E . , 1 9 7 2 . An improved procedure for protein staining in Polyacrylamide gels with a new type of coomassie brillant blue. Analyt. Biochem. (New York), 48, 617. D R E C H S E L , W., KRETSCHMER, P., RUTTLOFF, H . und SCHLOBACH, C. R . , 1 9 7 4 . Verfahren zur Verringerung der Viskosität von Cerealienmehlsuspensionen. DDR-Wirtschaftspatent 1 0 6 1 3 0 . D R E C H S E L , W. und RUTTLOFF, H . , 1 9 7 5 . Zum Einsatz von Proteasen bei der Waffelherstellung. Bäcker und Konditor, 23 (29), 212.

Proteases from T. vulgaris. V.

519

C., H A U S D O R F , G., H Ö H N E , W . E., B E H N K E , U . und R U T T L O F F , H . , 1 9 7 8 . Charakterisierung einer Protease aus Thermoactinomyces vulgaris (Thermitase). 2. Einschritt-Feinreinigung und proteinchemische Charakterisierung. Acta biol. med. germ., 87, 1193. K L E I N E , R., 1982. Properties of thermitase, a thermostable serine protease from Thermoactinomyces vulgaris. Acta biol. med. germ., 41, 89-102 K L E I N E , R . und R O T H E , U . , 1 9 7 7 . Isolierung, Kristallisation und teilweise Charakterisierung einer kationischen Protease aus Thermoactinomyces vulgaris.'Acta biol. med. germ., 36, K 27. K L E I N E , R . , R O T H E , U . , K E T T M A N N , U . and S C H E L L E , H . , 1 9 8 1 . Purification, properties and application of thermitase, a microbial serine protease. I n : T U R K , V . , and V I T A L E , L. (Editors), Proteinases and their Inhibitors, p. 201. Pergamon Press, Ljubljana and Oxford. K L I N G E N B E R G , P., Z I C K L E R , F . , L E U C H T E N B E R G E R , A. und R U T T L O F F , H . , 1 9 7 9 . Gewinnung und Charakterisierung von Proteasen aus Thermoactinomyces vulgaris. I I . Selektion von leistungsfähigen Stämmen. Z. Allg. Mikrobiol., 19, 17. L A N G E , R., F R I E S E , R . und L I N O W , F . , 1979. Zur Anwendung der Methodenkombination KjeldahlNaßaufschluß/Berthelot-Reaktion bei der Stickstoffbestimmung in biologischen Materialien. 2. Aufbau und Erprobung einer teilautomatisierten Apparatur zur routinemäßigen Bestimmung des Stickstoffs. Nahrung, 23, 549. L A N G N E K , J . , A N S O R G E , S., B O H L E Y , P . , K I R S C H K E , H . and H A N S O N , H . , 1 9 7 1 . Intracellular protein breakdown. I. Activity determinations of endopeptidases using protein substrates. Acta biol. med. germ., 26, 935. L E U C H T E N B E R G E R , A., K L I N G E N B E R G , P. und R U T T L O F F , H . , 1 9 7 9 . Gewinnung und Charakterisierung von Proteasen aus Thermoactinomyces vulgaris. I I I . Untersuchungen zur Proteasebildung in einer kleintechnischen Versuchsanlage. Z. Allg. Mikrobiol., 19, 27. M A U R E R , H. R., 1971. Disc Electrophoresis and Related Techniques of Polyacrylamid Gel Electrophoresis, 2 n d Edition, p. 44. Walter de Gruyter Berlin-New York.

FRÖMMEL,

RUTTLOFF, H . , KLINGENBERG, P . , BEHNKE, U . , LEUCHTENBERGER, A . u n d TÄUFEL, A . , 1978.

Ge-

winnung und Charakteristik von Proteasen aus Thermoactinomyces vulgaris. I. Proteasen aus thermophilen Mikroorganismen (Literaturüberblick). Z. Allg. Mikrobiol., 18, 437. S C H A L I N A T U S , E., R U T T L O F F , H. und B E H N K E , U., 1979. Untersuchungen zur Substratspezifität einer Protease aus Thermoactinomyces vulgaris. Nahrung, 23, 275. T Ä U F E L , A., B E H N K E , U. und R U T T L O F F , H . , 1979a. Gewinnung und Charakterisierung von Proteasen aus Thermoactinomyces vulgaris. IV. Extrazelluläres Proteasespektrum im Verlauf der Kultivierung. Z. Allg. Mikrobiol., 19, 129. T Ä U F E L , A., F R I E S E , R . and R U T T L O F F , H . , 1 9 7 4 . Rapid assay of proteolytic enzymes on film material. J . Chromatogr. (Amsterdam), 93, 487. T Ä U F E L , A., N Ü R N B E R G E R , H . , R U T T L O F F , H . , K A L B , H . und B E H N K E , U . , 1979b. Verfahren zur Behandlung von Cerealienstärke. DDR-Wirtschaftspatent 139 361. T Ä U F E L , A., R U T T L O F F , H . , SCHLOBACH, C. R . , GABOR, R . und B E H N K E , U . , 1 9 7 8 . Einfluß der a Amylase auf die Proteasewirkung in kleberhaltigen Substraten. Nahrung, 22, 819. T O N T A N A , A., B O C C U , E. and V E R O N E S E , F. M., 1976. Effect of EDTA on the conformational stability of thermolysin. I n : Z U B E R , H . (Editor), Enzymes and Proteins from Thermophilic Microorganisms; Structure and Function, p. 55. Proceedings of the International Symposium Zürich, 1975. Birkhäuser Verlag, Basel and Stuttgart. W E A V E R , L . H . , K E S T E R , W . R . , T E N E Y C K , L . F. and M A T T H E W S , B. W . , 1 9 7 6 . The structure and stability of thermolysim. I n : Z U B E R , H . (Editor), Enzymes and Proteins from Thermophilic Microorganisms; Structure and Function, p. 31. Proceedings of the International Symposium Zürich, 1975. Birkhäuser Verlag, Basel and Stuttgart. Mailing address: Dr. U . B E H N K E , Zentralinstitut für Ernährung der Akademie der Wissenschaften der D D R DDR-1505 Bergholz-Rehbrücke, ArthurScheunert- Allee 114/116

Zeitschrift f ü r Allgemeine Mikrobiologie

22

8

1982

521-527

(Department of Botany, Assiut University, Assiut, Egypt)

Seasonal fluctuations of freshwater fungi in River Nile (Egypt) FARIDA T . E L - H I S S Y , A . H . MOUBASHER a n d M . A . E L - N A G D Y

(Eingegangen am 16.

2.1982)

The seasonal fluctuations of aquatic fungi were studied over 24 water sample collected monthly between May 1979 and April 1981 from the River Nile near Assiut (about 375 km, south from Cairo, Egypt). The richest periods in aquatic fungal genera and species were September—December 1979, January—March, October—December 1980, and January—April 1981. These periods represent low or moderate temperature months. The poorest periods were May—August 1979, April, May, July, August and September 1980 which are almost summer months. Twenty-nine species and one variety of Olpidiopsis saprolegniae which belong to ten genera of aquatic fungi were collected during this investigation. Three genera were of high seasonal occurrence (19—24 months) namely: Achlya, Saprolegnia and Dictyuchus. Three other genera were of moderate seasonal occurrence (6 — 11 months) namely: Isoachlya, Leptolegnia and Pythium. The rest of genera were of low or rare seasonal occurrence. Seasonal f l u c t u a t i o n s of aquatic freshwater fungi were f o l l o w e d b y COKER (1923), MILOVTSOVA (1935), WATERHOTJSE (1942), B O C K (1956), D A Y A L a n d T A N DON (1962) a n d R O B E R T S (1963). T h e y all reported seasonal periodicity in t h e c o m p o s i t i o n of t h e fungal flora of freshwater. I n E g y p t , little a t t e n t i o n has been g i v e n t o t h e s t u d y of f u n g a l e c o l o g y of freshw a t e r fungi. E L - H I S S Y (1974) in a preliminary s t u d y , isolated 22 species belonging t o 15 genera of a q u a t i c fungi a n d 33 species belonging t o 17 genera of terrestrial fungi f r o m t h e R i v e r N i l e near Assiut. T h e present i n v e s t i g a t i o n is a n i n t e n s i v e s t u d y c o n c e n t r a t i n g o n seasonal f l u c t u a tions of a q u a t i c fungi of t h e R i v e r N i l e in E g y p t .

F O R B E S (1935a, b),

Materials

and

methods

Water samples (5 liter each) were collected monthly from the surface water of the Nile near Assiut during the period May 1979 to April 1981. The time required for bringing the samples from the River Nile to the laboratory was about one hour. Then the organic matter content and the total soluble salts were determined. p H values and temperature of the water were recorded. For the recovery of aquatic fungi from the water samples, aliquots of 100 ml of water were introduced into each of six sterile Petridishes (15 cm in diameter). Three sterilized hemp-seeds and three sterilized halves of Zea mays grains (EL-HISSY 1974) were put in each Petri-dish as baiting substances and left in natural light and room temperature. After 24 hours, the colonized seeds and grains were transferred to Petri-dishes containing sterile filtered Nile water to which penicillin (2000 units/1 water) (ROBERTS 1963) was added, to depress bacterial growth. The dishes were then incubated at 22 °C for 4—6 weeks during which time the aquatic fungi which colonized the seeds and grains were examined weekly. After each examination the colonized seeds and grains were transferred into sterile Petri-dishes containing sterile filtered Nile water. The recovered aquatic fungi were purified on glucose-peptone (GP) agar medium according to WILLOUGHB Y a n d PICKERING ( 1 9 7 7 ) .

The purified genera and species were identified according to the following references: COKEB

( 1 9 2 3 ) , FITZPATRICK ( 1 9 3 0 ) , JOHNSON ( 1 9 5 6 ) , SCOTT ( 1 9 6 1 ) , SEYMOUR ( 1 9 7 0 ) , WATERHOUSE ( 1 9 6 7 ,

1968). In addition the identification of some of these aquatic fungi was checked by the help of Dr. WILLOUGHBY (Ferry-House-Windermere Laboratory, Freshwater Biological Association, England).

522

F . T . E L - H I S S y , A . H . MOUBASHER and M . A . E L - N A G D Y

•jdy

+ +

I

I

I +

•J-EJ^

+ +

I

I

I +

•QAJ

+ + I + I +

+ 1

•UUP

+ + + 1 1 +

I I

tao

I I I +

+ +

I +

+

I + +

I

I + + +

I I

I

I

I +

I I

I

I

I

I I

I

I I

I + I + I +

I +

II

I +

I +

I

+

I

I

1 +

1 +

1

+

1

I I

1 +

1

I

I

I

+

•09Q

C

3 T3

'

A 0

N

•^OO

>

•dag

S

•3NY

¿3

XJNP CS £

9UTIP

a

3 ^ w 00 A 2 O — •FT

•jdy

&

O < ° I

•QAJ

Tj o .13 » ^

m

i l l £5 =8 O ojji tí.SP ® ft o c i». h & üo Ss Ph o ? ^ tí ^ ® W§ O > •31® ft © oo © © © © © l•Sl Sa d« © a

o

00

o

tí . s o (M H 13 O

ce

X

3C3 ®

£

T3 i n e OJ ti cS

O

O

M

N

Ó

O

O

O

^

^

' Ph

á n d

H

H

^

H

d

r

o

o

o

o

o

o

IO

9

o~Hp

' '5

S u E K A N E

O

O

I i z u k a

H .

«O N

h

CO CO O i

S

O

io o

oo oo o

H H CO

tí tä ho

CO S o w

o

eo s i

S

O

«

(

t.

o

i

» o . fe S

« Co Co « ¡¿ S s s ös u

ÖS ÖS o

tx ÖS i co O O

i S S O o o ^ K ' • e -S «s ^ O rfá o o I § û

s v

s

ii

ÖS ÖS

g « « " O co o œ oo in co © oo t o © o © e i © © ( N © © © o ¿ © © - ^ i © i í i - H pH CO IO (M i-l pH (M pH

© co >oco co •4 1 COco lO 00 r-l IO 05 pH © © © io io THIO cpH œ pH pH pH pH pHio pH pH p H

s «0 eo «o S S S I § evo s S co w o e • •S l ' Ss e.« ^ g. Ä s -o S 0O O Oo o ' « "e - Se 1 § § . § . § § S £-3 S b » i« gO . ®s s s s s w S S » » « œ w ^ ^ «» §à J» Os s Sfc. i» fe. "e ET-o o ô s» 1 CO oo co 1 - 5 S Î 3 . | j l §i 5i?gg g § 3e -o S S § 2's. e « « H U

'2 S rfS ^ 2 S S Ç u u S s s s e e QQOQCQCQGqcqoqoqgqgqGQOQOQGQGQGQGQCQGQOQGQGQOQCQGQCQCQÖQQQCQOQ

579

580

M . SUEKANE a n d H . IIZUKA

molar concentration, 0.8 M glucose, 0.05 M phosphate buffer (pH 7.5), 0.1 M MgS0 4 , 0.01 M COCL and 2 ml of crude enzyme solution, and was 4 ml in volume. After incubation for 3 hours at 60 °C, the reaction was stopped by withdrawing a 0.5 ml aliquot and mixing this with 4.5 ml of 0.5 M percholic acid solution. After centrifugation and 100 times dilution, the content of D-fructose was determined by the cysteine-carbazole method modified by MAKSHALL and Kooi (1957). Fructose was identified by the thin-layer chromatography method of STAHL and KALTENBACH (1961). Protein determination was carried out by the method of LOWBY et al. (1951). Enzyme assay: The assay of glucose isomerase was carried out according to the procedure described previously (SUEKANE et al. 1978). Induced mutation: Spores of Streptomyces olivochromogenes (from xylose-starch agar slant) were suspended in a sterilized 0.85% saline solution. The suspension was filtered aseptically using Toyo Eoshi filter paper No. 2. The number of spores per ml of this suspension was counted by the direct microscopic method. Then the suspension was diluted with the 0.85% sterilized saline so t h a t the final concentration was below 10 spores per ml. 10 ml of the suspension was then poured into a sterile Petri dish and irradiated with ultraviolet rays. The plate was shaken gently at a distance of 40 cm from the TJV lamp (SANKYO DENKI 15 W) in a sterilized box for a given period. After the irradiation the suspension was diluted so as to give 20—50 colonies when 0.1 ml of the sample was spread evenly over a xylose-starch agar plate. The plate was incubated a t 30 °C for 2 days. The colonies on each plate were transferred to xylose-starch agar slants. The enzymatic activities of these UV-irradiated strains were assayed after culturing in the xylose-starch liquid medium for 2 days. The crude intracellular enzyme solution was obtained as described above. Culturing media and procedures: The culture of Streptomyces olivochromogenes 5-17-3, which was obtained by induced mutation described in this report, was chosen based on the superior yield of glucose isomerase. The inoculum preparation and enzyme production were carried out in a SAKAGUCHI-flask at 28 °C for 2 days with shaking on a reciprocal shaker. Simplifying the X-S starch which had been used for the screening, the following media were used at the time the studies contained in this report were initiated: Medium for culture maintenance: Xylose 1 (w/v%); Yeast extract 0.1, Beef extract 0.1, Bacto tryptone 0.2, CoCL • 6 H 2 0 0.024, Agar 2, p H 7.0. Medium for inoculum preparation: Corn steep liquor (CSL) 4 (w/v%), Xylose 0,5, Corn starch 0.5, MgS0 4 • 7 H 2 0 0.05, CoCl2 • 6 H 2 0 0.024, p H 7.1. After the mycelia had proliferated in the above medium, the suspension was used to inoculate the enzyme production medium. The volume of the inoculum was 5% of the volume of the enzyme production medium. Enzyme production medium and procedures: CSL 4 (w/v%), Xylose 1, Corn starch 1, MgS0 4 • 7 H 2 0 0.05, CoCL • 6 H 2 0 0.024, Dow corning antifoam FG-10 0.1. A 500-ml SAKAGUCHI flask containing 50 ml of the inoculum medium was inoculated from a stock culture which had been grown on an agar slant of the maintenance medium. The flask, after inoculation, was then incubated a t 30 °C on a reciprocal shaker for 2 days. I t was then used for inoculating a series of enzyme production flasks at a 5 % inoculum level. The enzyme production flasks, which were generally run in duplicate, were placed on the reciprocal shaker a t 30 °C for 2 days. The intracellular glucose isomerase activities of the culture broths were determined according to the procedure described as above. Results S c r e e n i n g of c u l t u r a l

strains

T h e results o b t a i n e d w i t h t h e 63 strains of Streptomyces are s h o w n in Table 1. Glucose isomerase was f o u n d in a wide range of Streptomyces species. T h e intracellular e n z y m e s of S. olivochromogenes 1 2 — 5 a n d S.venezuelae 17 — 1 showed t h e highest f r u c t o s e conversion rates (38.9% and 38.3%). A t t e n t i o n w a s also f o c u s e d o n t h e app a r e n t extracellular glucose isomerase a c t i v i t i e s of various strains of Streptomyces. T a b l e 1 lists a f e w strains w h o s e culture filtrates indicated a c t i v i t i e s significantly greater t h a n t h e range of e x p e r i m e n t a l error. I t was f o u n d t h a t S. venezuelae 1 7 — 1 h a d t h e highest apparent extracellular glucose isomerase a c t i v i t y . Induced

mutation

The survival curve o b t a i n e d b y t h e usual p l a t e count m e t h o d after UV-irradiation is s h o w n in Fig. 1.

581

Glucose isomerase production by Streptomyces

Fig. 1 Survival curve Table 2 Selected mutants of UV-irradiated 8. olivochromogenes Culture strain No.

Glucose isomerase activity (u/ml)

Dry cell weight (g/1)

Specific activity (u/g-cell)

Parent 1-1 1-8 1-32 3-13 3-36 4-25 5-17 5-46

1.95 2.10 2.11 2.07 2.10 2.36 2.13 3.33 2.05

9.0 11.1 11.7 8.9 11.1 9.4 10,7 9.9 10.0

216 189 180 233 189 251 199 336 205

Slants were prepared from approximately 50 colonies of survivors of 60-second UV exposure. About 300 colonies were transferred to the xylose-starch agar slants and then their enzyme activities were determined. Apparent mutants which showed higher activities than the parent strain are listed in Table 2. The most active strain (5 — 17) produced 1.7 times the enzymatic activity of the parent strain. The enzymatic activities of cells from five simple colonies isolated from strain 5—17 were determined and are shown in Table 3. Strain 5—17 — 3 showed the highest activ-

582

M. StrEKANE a n d H . IIZTJKA

Table 3 Enzymatic activities of monocolony isolates Culture strain No.

Glucose isomerase activity (u/ml)

Dry cell weight (g/1)

Specific activity (u/g-cells)

5-17 5-17-1 5-17-2 5-17-3 5-17-4 5-17-5 5-17-6

3.33 3.29 3.25 3.70 2.53 3.47 3.15

9.9 8.0 9.6 9.6 8.0 8.8 9.2

336 411 339 385 316 394 342

ity (3.7 units/ml). This corresponds to 1.9 times the parent strain's activity. Optimum culturing conditions for enzyme production Initial enzyme yield The yield of enzyme initially obtained in the media (using the procedure outlined above) was 2.4 and 3.2 units/ml from duplicate flasks, giving an average of 2.8 units/ml. Effect of yeast extract on enzyme production The first medium change to produce an increased yield of glucose isomerase was the addition of yeast extract ( K Y O K U T O CO., Ltd). This ingredient added to the medium at a 0.5% concentration produced a yield of 3.7 units/ml of glucose isomerase. It was also found in the same experiment that increasing the level of xylose in the medium containing yeast extract to 2 % instead of 1 % increased the yield of glucose isomerase enzyme to 4.1 units/ml. Later evaluation demonstrated that a 0.25% concentration of yeast extract produced optimum yields with 2% xylose. Thus, the updated basal medium had the following composition: CSL 4 (w/v%), Xylose 2, Corn starch 1, MgS0 4 • 7H 2 0 0.05, CoCl2 • 6H 2 0 0.024, yeast extract 0.25. Effect of glycine on enzyme production With the basal medium just outlined, the effect of glycine addition was studied. Table 4 shows that the 0.1 or 0.25% glycine additions increased the enzyme yield, but that more than the 0.5% glycine addition decreased the enzyme yield. Table 4 Effect of glycine on glucose isomerase production Medium

Glucose isomerase activity (u/ml)

Basal (control)

Basal -f 0.5% glycine Basal + 1.0% glycine

Specific activity (u/g-cells)

8.9

384

15.5

280

15.1

271

17.1

151

12.0

198

(3.42)(b)

Basal + 0.1% glycine Basal + 0.25% glycine

Dry cell(a) weight (g/1)

I : S

I I M )

til (4.65) (5.04) (4.98) (4.67) (3.86)

(a)

Dry cell weight (g/1) 9.4 10.7 8.8

9.9 10.0 10.0 9.5 9.7 9.6 9.4 9.4 9.2

(a) Average (b) Not included in average Basal medium: CSL 4 (w/v%), 15 DE Starch Hydrolyzate 1, Xylose 2, Glycine 0.25, MgS0 4 • 7 H . 0 0.05, pH 7.1

584

M. S i t e r a n e a n d H . I i z u k a

the other is the effect of dilution which sometimes occurs in fermentation tanks where direct steam injection is used for sterilization. As m a y be observed in Table 7, the optimum concentration of the medium is over 100%, however, there is a substantial drop in enzyme production between 105% and 110%. Table 7 Effect of varied total medium concentration on glucose isomerase production Medium concentration Basal at 80% of normal concentration Basal at 85% of normal concentration Basal at 90% of normal concentration Basal at 95% of normal concentration Basal at 100+ of normal concentration Basal at 105% of normal concentration Basal at 110% of normal concentration

Glucose isomerase activity (jx/ml) Î32 4.90 4.72 4.71 4.65 4.75 5.50 4.83 4.87 5.55 5.17 4.65 4.88

(4.20(a) (4.81) (4.68) (5.13) (4.85) (5.36) (4.77)

Dry cell weight (g/1) 9.4 9.0 9.1 8.3 8.5 8.7 9.5 9.6 9.8

10.1 10.0

10.7 9.0 9.2

(a) Average Basal Medium : CSL 4 (w/v %), 15 DB Starch hydrolyzate 1, Xylose 2, Glycine 0.25, MgSO„ 0.05, CoCl2 • 6 ILO 0.024, pH 7.1 Table 8 Effect of varied concentrations of CSL on glucose isomerase production CSL in medium (w/v %) 4 3.75 3.5 3.25 3 2.75 2.5 2.25 2

Glucose isomerase activity (u/ml) 5.70 5.96 6.60 6.50 6.17 6.90 5.50 5.82 5.20 5.05 4.77 5.15 4.45 4.72 4.54 4.17 4.35

(5.83) (a) (6.34) (5.70) (5.51) (4.91) (4.80) (4.63) (4.26)

Dry cell weight (g/1) 11.2 10.0 10.3 9.8 9.9 9.6 9.4 9.1 8.9 8.3 8.4 7.4 7.6 8.4 7.3 7.3 7.6

Specific activity (u/g-cells) 509 595 641 663 623 615 585 640 584 608 568 696 585 637 622 571 572

(a) Average Basal Medium: 15 DE Starch hydrolyzate 1 (w/v %), Xylose 2, MgS0 4 • 7 H 4 0 0.05, CoCl2 • 6 H 2 0 0.024, Glycine 0.25, pH 7.1

585

Glucose isomerase production by Streptomyces

Effect of varied concentrations of CSL in medium on enzyme production An inoculum was prepared using a glycine-containing inoculum medium for an experiment designed to investigate the effect of various concentrations of CSL in the enzyme production medium. Table 8 shows the results. Based on these results, the enzyme production medium now contains 3.6% CSL instead of 4 % . Effect of ammonium salts on enzyme production As previously demonstrated (Table 5), the addition of ammonium acetate to the enzyme production medium depressed the yield of glucose isomerase to less than 1 unit/ml. Other ammonium salts, however, acted as potential sources of nitrogen and enhanced the yield of glucose isomerase. Table 9 presents the results obtained. All of the ammonium salts tested exhibited some enhancement of glucose isomerase production at some level of use. Ammonium nitrate appeared to be somewhat superior to the others. Effect on enzyme production of the use of glycine and ammonium nitrate in culture maintenance and inoculum preparation The effect of separate addition of glycine and ammonium nitrate to the enzyme production medium was enhancement of glucose isomerase production. To see if a Table 9 Effect of ammonium salts on glucose isomerase production Ammonium salts in medium

Glucose isomerase activity (u/ml)

Dry cell weight (g/1)

Specific activity (u/g-cells)

Basal (control)

4.9 4.9

9.1 8.9

539 550

(NH 4 ) 2 S0 4

5.7 6.6 5.7 6.25 5.9 6.2

8.7 9.6 8.75 8.45 8.5 9.3

655 688 652 740 694 667

0.1% 0.25% 0.5%

NH 4 N0 3

0.1% 0.25% 0.5% 1.0%

(NH 4 ) 2 HP0 4 0 . 1 % 0.25% 0-5% 1.0% Ammonium 0 . 1 % citrate 0.25% 0.5% 1.0%

10.7 10.1 9.6 9.0 10.1 10.2 8.3 8.5

654 684 745 739 618 614 699 689

5.9 8.6 5.6 9.1 5.05 8.6 5.0 8.2 8.0 3.9 4.15 7.9 Toxic — No cell groth

686 615 587 610 488 526

5.6 9.3 5.6 9.5 6.5 10.3 6.5 9.9 Toxic — No cell growth Toxic — No cell growth

602 590 631 656

7.0 6.9 7.15 6.65 6.25 6.25 5.8 5.8

Basal medium: CSL 4 (w/v %), Xylose 2, Com starch 1, MgS0 4 • 7 H„0 0.5, CoCl2 • 6 H„0 0.024, pH 7.1

586

M . SUEKANE a n d H . IIZUKA

great enhancement of yield could be obtained, their use in culture maintenance and inoculum development was investigated. Accordingly the following experiment was conducted : A culture was maintained on the basal slant medium supplemented with 0.25% glycine. Another culture was maintained on the same basal medium with the addition of ammonium nitrate. Thus there were three cultures used in this experiment: 1) regular culture, 2) culture grown in the presence of glycine, and 3) culture grown in the presence of ammonium nitrate. These three cultures were then used to inoculate three different inoculum media: 1) the basal inoculum medium, 2) the basal inoculum medium plus 0.25% glycine, and 3) the basal inoculum medium plus 0.25% ammonium nitrate. Each inoculum was then used to initiate culturing in three different enzyme production media having the following compositions : 1) CSL 3.6 (w/v %), Xylose 2.0, Corn starch 1.0, MgS0 4 • 7 H 2 0 0.05, CoCl2 • 6 H 2 0 0.024. 2) Basal plus 0.25% glycine. 3) Basal plus 0.25% ammonium nitrate. The pH of each medium was adjusted to 7.1. Table 10 shows the results obtained. Some stimulation occurred in the enzyme production using the basal medium when glycine was used in the culture maintenance and inoculum preparation stages. The specific activity is increased even though in some cases there was an increase in cell growth. Based on this observation, it may be stated while glycine and ammonium nitrate may increase the growth, the increase in enzyme yield is not in correlation with the growth since the enzyme production is enhanced to an even greater degree, as seen in the increase in enzyme yield per gram of cells. Effect on enzyme production of ammonium nitrate alone and in combination with glycine An experiment was designed to compare the effect on enzyme production of glycine and ammonium nitrate alone and in combination at various concentrations. Table 10 Effect on enzyme production of the use of glycine and ammonium nitrate in culture maintenance and inoculum preparation Medium composition Enzyme production Basal

Slant and inoculum Basal Basal + glycine Basal +

Basal + Glycine

NH4NOO

Basal Basal + glycine Basal +

Basal + N H 4 N 0 3

NH4N03

Basal Basal + glycine Basal +

NH4N03

Glucose isomerase activity (u/ml)

Dry cell weight (g/1)

Specific activity (u/g-cells)

2.75 3.90 4.86 4.76 4.63 3.38 6.34 4.46 6.37 6.71 5.39 5.62 6.94 7.55 7.55 8.10 7.45 7.73

6.1 7.8 7.6 7.6 7.8 6.7 11.6 9.8 10.6 10.3 10.3 10.9 9.0 9.6 9.0 9.4 9.0 9.5

450 500 640 640 593 504 546 556 601 652 524 515 772 786 839 862 825 814

587

Glucose isomerase production by Streptomyces

Table 11 shows the results with respect to cell mass, glucose isomerase activity and specific activity. These glucose isomerase activity and cell mass results are summarized in Table 7. Combinations of glycine and ammonium nitrate produced a higher cell mass than either compound alone. However, the enzyme yield did not increase proportionally with the growth. Conversely, based on this results, it appears that ammonium nitrate was the superior compound with respect to the yield and specific activity of the glucose isomerase enzyme. Optimum medium composition Based on the above-mentioned observations and with an eye to the cost, the optimum medium composition was determined as follows: Table 11 Effect on glucose isomerase production of NH 4 N0 3 alone and in combination with glycine Medium co mposition Glycine (%)

NH 4 NO 3 (%)

0.0

0.0

0.1

0.0

0.2

0.0

0.3

0.0

0.4

0.0

0.5

0.0

0.0

0.1

0.0

0.2

0.0

0.3

0.0

0.4

0.0

0.5

0.1

0.1

0.2

0.1

0.3

0.1

0.4

0.1

0.5

0.1

0.1

0.2

0.2

0.2

0.3

0.2

0.4

0.2

Glucose isomerase activity (u/ml)

Dry cell weight (g/1)

2.25 3.32 4.04 3.91 4.38 4.74 5.02 4.72 3.50 4.80 5.18 3.94 5.75 5.69 6.16 6.14 6.74 7.56 6.10 5.84 6.5 5.25 5.48 5.48 5.96 6.20 4.84 5.59 5.59 5.59 3.53 3.18 5.95 6.29 5.35 5.33 4.58 4.01 3.70 4.03

7.4 7.8 8.5 8.2 9.5 8.2 10.5 10.4 12.0 11.7 10.9 11.0 9.6 10.2 8.9 8.4 9.6 9.8 8.1 8.4 7.8 8.1 8.2 8.6 11.3 11.1 10.3 11.7 11.3 13.4 10.5 10.5 10.0 10.3 9.9 11.5 11.4 15.2 12.3 10.9

Specific activity (u/g-cells) 341 426 475 477 461 578 478 454 292 410 475 358 599 558 692 731 702 771 753 695 833 648 668 637 527 559 470 478 495 417 336 303 595 611 540 463 402 264 301 370

M. S u e k a n e and H . I i z u k a

588

Table 12 Effect of NH4NO3 and glycine on glucose isomerase yield N H 4 N 0 3 added (%)

Glycine added ( % ) 0

0 0.1 0.2 0.3 0.4 0.5 0 0.1 0.2 0.3 0.4 0.5 — : Not determined.

2.79 5.73 6.15 7.15 5.97 5.88 7.6 9.9 8.7 9.7 8.3 8.0

0.1

0.2

0.3

Glucose isomerase activity (u/ml) 3.98 4.51 4.86 6.08 5.48 5.22 5.34 6.12 4.30

0.4

0.5

4.15 5.59 3.84

4.56 3.36

—

—

—

—

—

—

—

—

—

—

8.5 8.4 10.2 —

D r y cell weight (g/1) 10.5 8.9 11.2 11.0 10.7 13.3 —

—

—

—

11.9 12.4 11.6

11.0 10.5 —

—

—

—

—

—

—

—

—

—

—

—

CSL 3.6 (w/v %), Xylose 2.0, Corn starch 1.0, NH 4 N0 3 0.2, Glycine 0.1, MgS0 4 • 7 H 2 0 0.05, Antifoam PPG 20000 1 dwp. Cobalt chloride was deleted from the view point of food safety. Using this enzyme production medium, 6.5 — 7.6 units/ml of glucose isomerase was obtained. Discussion Since T s u m u r a and S a t o ( 1 9 6 5 ) first reported that Streptomyces phaeochromogenes produces glucose isomerase, various species of Streptomyces have been reported to possess glucose isomerase activity. In the present experiment, glucose isomerase was also found to be distributed in a wide range of species of Streptomyces. No relationship was found between the taxonomical position and glucose-isomerizing activity. The activity seemed to be found on a strain-by-strain basis, as is said to be true the production of antibiotics. Some strains produced glucose isomerase both intra- and extracellularly. A true extracellular glucose isomerase has not been reported to date among actinomycetes. The extracellular glucose isomerase reported here was probably released from the cell by autolysis and was not a true extracellular enzyme.,Chen et al. (1979) recently reported that Streptomyces flavogriseus produced extracellular glucose isomerase togegether with intracellular glucose isomerase. They also concluded that the extracellular glucose isomerase was probably released by autolysis. No remarkable increase in enzyme yield was obtained, but strain 5—17—3 showed about two times the specific activity of the parent strain. Strain 5—17—3 could be referred to as a hyper-mutant. Furthermore, mutant strain 5—17—3 was found to be sufficiently stable to reproducibly yield 3.3—3.5 units of glucose isomerase per ml. Medium studies are usually conducted by addition, at varied concentrations, of compounds which are known to be metabolically active with respect to the microorganisms being investigated. When a compound is found to produce a desirable effect, then réévaluation of all of the ingredients in the medium becomes necessary. By this procedure, optimization of the medium composition to maximize the glucose isomerase yield was attained. The addition of ammonium nitrate and glycine were found to effect the enzyme production, but the mechanism has yet to be elucidated.

Glucose isomerase production b y

589

Streptomyces

References P., A N D E R S O N , A . W . a n d H A N , Y . W . , 1 9 7 9 . Production of glucose isomerase by Streptomyces flavogriseus. Appl. Environ. Microbiol., 37, 3 2 4 — 3 3 1 .

CHEN, W .

LOWBY, 0 . H . , ROSEBROUGH, N . J . , PARR, A . L . a n d RANDALL, J . R . , 1 9 5 1 . P r o t e i n m e a s u r e m e n t s

with t h e Folin-phenol reagent. J . biol. Chemistry, 193, 265—275. MARSHALL, R. 0 . a n d Kooi, E. R., 1957." Enzymatic conversion of D-glucose to D-fructose. Science, 125, 6 4 8 - 6 4 9 . STAHL, E . u n d

KALTENBACH, U . , 1961.

Dunnschichtchromatographie.

VI.

Spurenanalyse

von

Zuckergemischen auf Kieselgar G-Schichten. J . Chromatogr. 5, 351 —355. S U E K A N E , M . , T A M U R A , M . and T O M I M U R A , C., 1 9 7 8 . Physico-chemical a n d enzymatic properties of purified glucose isomerases f r o m Streptomyces olivochromogenes and Bacillus stearothermophilus. Agric. Biol. Chem., 4 2 , 9 0 9 - 9 1 7 . S U E K A N E , M. a n d I I Z U K A , H . , 1 9 8 1 . Enzymes of glucose isomerization in various microorganisms. Z. Allg. Microbiol., 21, 4 5 7 - 4 6 8 . TAKASAKI, Y., 1966. Studies on sugar-isomerizing enzyme. Production a n d utilization of glucose isomerase from Streptomyces sp. Agric. Biol. Chem., 30, 1247 — 1253. T S U M U R A , N. and S A T O , T . , 1 9 6 5 . Enzymatic conversion of D-glucose to D-fructose. V I . Properties of t h e enzyme f r o m Streptomyces phaeochromogenes. Agric. Biol. Chem., 29, 1129 — 1134. Mailing address:

40 7, Allg. Mikrobiol , B d . 21, H

8

Dr. M. SUEKANE 502 Mitsui-Kamimaezu Haitsu 17—35 Fujimi-cho Naka-ku Nagoya-shi Aichi, 460 J a p a n

Zeitschrift für Allgemeine Mikrobiologie

Kurze

22

8

1982

591-594

Originalmitteilung

(Akademie der Wissenschaften der DDR, Forschungszentrum für Molekularbiologie und Medizin, Zentralinstitut für Mikrobiologie und experimentelle Therapie, Jena, Direktor: Prof. Dr. rer. nat. habil. U. T A U B E N E C K )

Effect of short-chain alcohols on production of NADP-glycohydrolase by Streptomyces griseus U . GRÄFE

(Eingegangen

am 5.

2.1982)

Short-chain alcohols as e.g. methanol and ethanol were found to suppress both the formation of NADP-glycohydrolase by S. griseus and the excretion of enzyme into the medium. The results suggest that the generation of enzyme represents a compartmentized process in which membrane structure is involved.

The enzyme NADP-glycohydrolase (E.C. 3.2.2.6) has been proposed to be involved in the regulation of cyto-differentiation of Streptomyces griseus as a coordinative signal (VORONINA et al. 1978, G R A F E et al. 1981). This particular role may be due to the interference of the phosphoadenosine-diphosphoribose (the product of the NADPglycohydrolase) with some NADH-dependent enzymes of the pentose and citrate cycles, respectively (VORONINA et al. 1978, G R A F E et al. 1980). On the other hand, competitive inhibition by this metabolite of the NADP-dependent isocitrate dehydrogenase in S. griseus can be expected to disturb the whole metabolism in such a manner that the organism would not grow on carbohydrates without concomitant accumulation of citrate. This is clearly not the case and, by contrast to the expectation, 2-oxoglutarate accumulation can be observed with fermentations of S. griseus (BORMANN 1966). This suggests that both the NADP-glycohydrolase and its product phosphoadenosine-diphosphoribose are separated from the isocitrate dehydrogenase due to compartmentation. Support to this contention was provided by the finding that in S. griseus H P both NADP-glycohydrolase and NAD-glycohydrolase (E.C. 3.2.2.5) are secreted into the medium (GRAFE et al. 1981). In order to check whether these enzymes can be formed and/or excreted by membrane-bound systems (RAMALEY

1979) we studied the effect of methanol and ethanol on their production and excretion by 8. griseus HP, since short-chain alcohols are known for their capacity to interfere

with the formation and the function of microbial cytoplasmic membranes (INGRAM

1974,

GRAFE

et al. 1979,

RIGOMIR

et al. 1980).

Strain and conditions of cultivation: 8. griseus H P was propagated as it has been described earlier ( G R A F E et al. 1981). All cultivations were performed in 500 ml shaking flasks (240 r.p.m., 5 cm stroke, 25 °C) containing 80 ml of media composed as follows (g • l - 1 ) : Medium A : maize starch, 30; D,L-alanine, 3 ; NH 4 N0 2 , 3; NaCl ,2.5; CaCO,, 3; K H 2 P 0 4 , 0.5; pH 6.2. Medium B : D-glucose 25 (sterilized separately); L-glutamic acid, 1; D,L-alanine, 1; D,L-aspartic acid, 1; (NH 4 ) 2 S0 4 , 3; K H 2 P 0 4 , 0.5; NaCl, 2.5; FeCl 3 , 0.005; MgCl2 • 6 H 2 0 , 3; MnS0 4 • 2 H ? 0 , 0.02; CaC0 3 , 1; CaCL, 1; pH 6.2. By use of this media, 48 h inocula were prepared starting with agar slant cultures. Each 3 ml of appropriate culture samples were used to inoculate the main cultures on the same medium. Methanol or ethanol were added at zero time. 40*

592

U . GRÄFE

Assays: NADP-glycohydrolase was measured either in cell-free extracts of disrupted mycelia or in culture liquids according to ZATMAN et al. (1953) as it was described in a preceding paper (GRAFE et al. 1981). Mycelium extracts were prepared by sonication of freshly harvested mycelia (90 sec, 0 °C, Tris buffer, pH 7.4, 0.1 M, LABSONIC 1510, Braun, Melsungen, F . E . G . ) and centrifugation of the cell debris at 2 3 0 0 0 g (15 min). Dry weights were estimated gravimetrically, and protein was measured by means of LOWRY'S method.

Fig. 1 shows the effect of increasing concentrations of methanol and ethanol on the activity of NADP-glycohydrolase in cell-free mycelium extracts of 8. griseus HP during cultivation on the medium A. Beginning with a subtoxic concentration (1 — 2%) the methanol had a profound suppressory effect on the level of enzyme. The same behaviour was observed in the presence of ethanol (0.5 — 1%). When NAD was used as the coenzyme instead of NADP, adequate alterations of enzyme activities were measured. Similarly, during growth of the strain on soluble medium B, the de novo formation of mycelial NADP-glycohydrolase and its secretion into the medium was suppressed strongly in the presence of methanol (or ethanol, not shown here). In addition to short-chain alcohols, other detergent-like compounds as e. g. sodium oleate and

Fig. 1. Effect of methanol and ethanol in the medium (zero time addition) on the activity of NADPglycohydrolase in cell-free mycelium extracts of S. griseus H P during growth on medium A. (enzyme activity in ¡¿moles • m i n - 1 - m g - 1 of extracted mycelial protein). O control, no additions, methanol added: 1 % (v), 2 % (&), 3 % (•), 4 % (•), 5 % (•), ethanol added: 0 , 5 % (T), 1 % (A)

Production of NADP-glycohydrolase by Streptomyces

593

s e v e r a l T w e e n - e m u l g a t o r s were established to alter t h e p a t t e r n of e n z y m e p r o d u c t i o n b y S. griseus H P . T h e a b o v e results m a y be interpreted in t e r m s of t h e f o r m a t i o n of N A D P - g l y c o h y d r o l a s e (and/or N A D - g l y c o h y d r o l a s e ) w i t h i n m e m b r a n e - a s s o c i a t e d biochemical c o m p a r t m e n t s ( R A M A L A Y 1 9 7 9 ) . I t appears likely, t h a t alterations of m e m b r a n e structure i n d u c e d either b y m u t a t i o n ( G R À F E et al. 1 9 8 1 ) or b y changes in

I 1

2

3

4c/

1 2

3

id

1

2

3

id

Fig. 2. Influence of methanol on biomass concentration (a) (dry weight), extracellular activity of NADP-glycohydrolase (b) (in ¡¿moles • m i n - 1 • m g _ 1 of dry biomass per ml) and level of NADPglycohydrolase in cell-free mycelium extract ((xmoles • m i n - 1 • m g - 1 of extracted protein) during growth of 8. griseus on medium B. x control, no additions, methanol added: 1% (O), 2 % ( • ) , 3 % (A), 4 % (A), 5 % (v) t h e m e d i u m c o m p o s i t i o n can a f f e c t t h e p r o d u c t i o n of t h i s e n z y m e a n d its transport i n t o t h e m e d i u m . A t present, it remains a n u n a n s w e r e d question w h a t m a y be t h e true role of t h e extracellular N A D P - g l y c o h y d r o l a s e in t h e regulation of differentation. On t h e o t h e r h a n d , controlled release of t h e e n z y m e or its product from particular comp a r t m e n t s i n t o t h e cell m a y occur in d e p e n d e n c e o n lipid composition giving rise t o alterations of t h e m e t a b o l i c f l u x during differentiation.

A Thanks are due to Miss C.

WENTZKE

cknowledgements and G.

BERGTER

for technical assistance.

References BORMANN, E. J., 1966. Stoffwechselregulation und Brenztraubensäurestauung bei Antibiotikabildenden Streptomyceten. Arch. Mikrobiol, 53, 2 1 8 — 2 2 5 . GRÄFE, U . , BOBMANN, B . J . a n d TRUCKENBRODT, G . , 1 9 6 8 . C o n t r o l b y

phospho-adenosinediphos-

pho-ribose of NADP-dependent isocitrate dehydrogenase and 6-phosphogluconate dehydrogenase. Z. Allg. Mikrobiol., 20, 6 0 7 - 6 1 1 .

G R Ä F E , U . , R O T H , M . , CHRISTNER, A . a n d BOBMANN, E . J . , 1 9 8 1 . B i o c h e m i c a l c h a r a c t e r i s t i c s

of

non-differentiating mutants of Streptomyces griseus. I. Role of NADP-glycohydrolase in cell differentiation. Z. Allg. Mikrobiol., 21, 633—642. G R Ä F E , U . , SCHADE, W . , BOCKER, H . and THRUM, H . , 1979. Alcohol-induced switching-over of metabolic flux in Streptomyces noursei J A 3890b. Z. Allg. Mikrobiol., 19, 721—726. INGRAM, L. 0 . , 1974. Adaptation of membrane lipids to alcohols. J . Bacteriol., 125, 670—678. R A M A L E Y , R . F . , 1 9 7 9 . Molecular biology of extracellular enzymes. Adv. Appl. Microbiol., 2 5 , 37-55.

594

U . GRÄFE

RIGOMIR, D., BOHIN, J . P. and LXJBOCHINSKY, B., 1980. Effect of ethanol and methanol on lipid metabolism in Bacillus subtilis. J . Gen. Microbiol., 121, 139—149. VORONINA, 0 . 1 . , TOVABOVA, 1.1, and KHOKLHOV, A. S 1978. Studies on the A-factor induced inhibition of glucose-6-phosphat edehydrogenase in Actinomyces streptomycini. Bioorg. Khim., 4, 1538-1546.

ZATMAN, L. J., KAPLAN, N. 0 . and COLOWICK, S. P., 1953. Inhibition of spleen diphosphopyridine nucleotidase by nicotinamide, an exchange reaction. J . biol. Chemistry, 200,197—205. Mailing address:

Dr. U. GRÄFE Zentralinstitut für Mikrobiologie und und experimentelle Therapie der AdW DDR 6900 Jena, Beutenbergstr. 11

Zeitschrift für Allgemeine Mikrobiologie

Kurze

22

8

1982

595-599

Originalmitteilung

(Akademie der Wissenschaften der D D R , Forschungszentrum für Molekularbiologie und Medizin, Zentralinstitut für Mikrobiologie und experimentelle Therapie, Jena, Direktor: Prof. Dr. habil. U. T A U B E N E C K )

Effect of L-valine and L-isoleucine on fatty acid composition of Streptomyces hygroscopicus and S. griseus U . GRÄFE, M . BOTH a n d D . K R E B S

(Eingegangen

am 22.

3.1982)

The effects of L-valine and L-isoleucine on the composition of mycelial fatty acids were investigated during growth of differentiating parent strains of Streptomyces hygroscopicus and Streptomyces griseus as well as their non-differentiating derivatives ( A m y - strains) on a synthetic medium. B o t h in the Amy+ and A m y - strains, in the presence of L-valine, the portion of the isopalmitic acid (iC16:0) increased, but the addition of L-isoleucine led to an elevated level of the 12-methyltetradecanoic acid (aC15:0). The results suggest that the genetically determined alterations in the ratio of both fatty acids in the non-differentiating derivatives may be due to specific changes in the biosynthetic pathways of both amino acid precursors rather than due to changes of their catabolism.

Lipids from streptomycetes are known to contain methyl-branched iso and anteiso f a t t y acids (BATRAKOV and B E R G E L S O N 1978) whose ratio appears to play a crucial role in the regulation of membrane-associated functions (ARIMA et al. 1973, OKAZAKI et al. 1973,1974). Alterations in the composition of f a t t y acids may be due not only to changes of the environment (KOVALTCHUK et al. 1973) but may be caused also by genetic changes. Thus, the non-differentiating derivative CC1 (Amy~Tur~) of the turimycin-producing and aerial-mycelium-forming Streptomyces hygroscopicus JA6599 NG60—93 (Amy + Tur + ) has been reported to differ from the parent strain by the reduced portion of mycelial 12-methyltetradecanoic acid (aC15:0) during growth on a synthetic medium (GRÄFE et al. 1982a). Otherwise, the ratio of the 12-methyltetradecanoic acid (aC15:0) to isopalmitic acid (iC16:0) was elevated in the non-differentiating and non-streptomycin-producing derivatives (Amy~Str~) of Streptomyces griseus H P (Amy + Str + ) as compared with their ancestor (GRÄFE et al. 1982b). These results suggested t h a t differentiation in streptomycetes requires as a prerequisite t h a t the cells are capable of exactly regulating the composition of the above mycelial f a t t y acids even under partial nutrient limitation, e. g. in the absence of particular precursors of the f a t t y acids in the medium. Although both f a t t y acids (aC15:0 and iC16:0) are known to originate from isoleucine via 2-methylbutyryl coenzyme A and, respectively, from isobutyryl coenzyme A via valine, it seemed not clear from the above publications whether the biosynthesis of the amino acid precursors or their catabolism has been subject to genetic changes. If the degradation of both amino acids would play a major role in the regulation of f a t t y acid composition, it could be expected t h a t Amy + or A m y - strains would display a different response to feeding with L-valine and L-isoleucine. On the other hand, if changes in the biosynthesis of both amino acids would be responsible for the altered

596

U . GRÄFE, M . ROTH a n d D .

KREBS

fatty acid spectra in the Amy" derivatives, both A m y + and Amy" strains should show the same adaptive alteration of fatty acid spectrum during oversupply with L-valine and L-isoleucine, respectively. These considerations prompted us to study the effect of exogenous valine and isoleucine on f a t t y acid composition of differentiating and non-differentiating strains of Streptomyces hygroscopicus and S. griseus. Organisms: Streptomyces hygroscopicus J A 6 5 9 9 N G 6 0 - 9 3 was obtained from the strain collection of the Zentralinstitut für Mikrobiologie und experimentelle Therapie, Jena. The non-differentiating derivative C C 1 has been described ( R O T H et al. 1 9 8 2 ) . Streptomyces griseus H P from the strain collection of V E B J E N A P H A R M , Jena, and the non-differentiating derivative LM 1 were reported elsewhere ( G R Ä F E et al. 1 9 8 1 ) . Medium: As complex agar medium, AL53 agar ( R O T H and N O A C K 1 9 8 1 ) was used for the propagation of the strains and the cultivation of emerged mycelia. The medium for submerged cultivations (SM) contained (g • l - 1 ): D-glucose, 25 (sterilized separately); D,L-alanine, 1; D,L-aspartic acid, 1; L-glutamic acid, 1; (NH 4 ) 2 S0 4 ,1; NaCl, 2.5; KBLP0 4 , 0.5; FeCl3, 0.005; MgCl2 0.02; CaC03, 1; CaCl? • 6 H 2 0, 2; pH 6.2. Growth conditions: Agar slant cultures were prepared as it has been described ( R O T H and N O ACK 1981, G R Ä F E et al. 1981). Spores or mycelia from agar slant cultures were spread on AL53 medium in Petri dishes in order to get laws of emerged mycelia. Incubation time was 8 d at 28 °C. All submerged cultivations were performed in 500-ml glass flasks containing 80 ml of medium on rotary shakers (240 r.p,m., 5 cm stroke). Precultures were inoculated with spores or mycelia from agar slant cultures and cultivated at 27 °C for 48 h. In each case 3 ml of precultures were transferred into 80 ml of medium SM. These cultures were shaken at 25 °C. Sterile solutions of L-valine or L-isoleucine were added at zero time. Extraction and analysis of fatty acids: Submerged mycelia from each 400 ml of cultures were collected by suction filtration. The lipid material was extracted by treatment with methanol/CHCl3 (1:2, v/v) for 48 h at room temperature ( K A T E S 1972). From the mycelium extracts the lipids were obtained by means of the common Folch procedure ( K A T E S 1972). They were treated with methanolic HCl (25 g HCl, 1_1, 48 h) in order to prepare the methyl esters of the fatty acids ( G R Ä F E et al. 1982a). Gas chromatography of fatty acid methyl esters was carried out with a gas Chromatograph model GCHF-18.3 (VEB CHROMATRON Berlin, G.D.R.) equipped with a flame ionization detector. Glass columns (3 m X 3 mm i.d.) were employed which were filled with 3% DEGS on Chromosorb G (80—100 mesh, S E R V A , Heidelberg, F.R.G.) and operated isothermically at 165 °C. The temperature of injector was 220 °C, that of the detector 200 °C. Nitrogen (35 ml • min -1 ) was the carrier gas. The identification of peaks was performed by comparison of their GC retention with authentic standards of fatty acid methyl esters ( S E R V A Heidelberg, F.R.G.). The calculations were based on peak areas. Shown in Table 1 is the effect of zero time additions of 1 % L-valine and 1 % L-isoleucine, respectively, to SM medium on the composition of mycelial f a t t y acids of Streptomyces hygroscopicus JA6599 N G 6 0 — 9 3 (ancestor strain, A m y + T u r + ) and its non-differentiating and non-turimycin-producing derivative CC1 (Amy~Tur~). As it has been reported ( G R Ä F E et al. 1982a), both strains differ by the altered ratio of the aC15:0 to iC16:0 f a t t y acids under these conditions which is indicated by a reduced level of the former fatty acid in the derivative CC1. In the presence of L-valine, the portion of the isopalmitic acid (iC16:0) rose strongly both in the A m y + and Amy~ strains. Concomitantly, the portion of the iC14:0 fatty acid increased while the level of the aC15:0 fatty acid declined. The latter fact may be due to feedback regulation of isoleucine biosynthesis by valine. Unexpectedly, L-valine reduced also the formation of the olefinic fatty acids. On the other hand, in the presence of L-isoleucine the level of the aC15:0 fatty acid rose markedly while the biosynthesis of the isopalmitic acid (iC16:0) was reduced. These results suggest that both valine and isoleucine can be used as carbon precursors for the biosynthesis of isopalmitic (iC16:0) and 12-methyltetradecanoic acids (aC15:0) both by the Amy+ strain NG60—93 and the non-differentiating mutant CC1. Similar results are summarized in Table 2. Zero time additions of L-valine and Lisoleucine to cultures of Streptomyces griseus H P (Amy + Str + ) and its mutant LM1

Effect of amino acids on «8 » S g O ¡3 ft ">i.2 g

Streptomyces

Tft-®io»o a a o t - M ® 05 ^^ SO O I I 00 NONhoÓM

vo vn oo o CO pI M © CO CÓ

p—IIOCO>0 — I I (N COCO COIM— I ItiC© OpH o CD

(35 CO OS 00 co i> iq oo oo e-* © id ni © io CO CO Tji CO © CO

o

oo ao Ti co >n i>co fhrttì; io 05 oó tu' oó ió t|Ì ai co' rii o> p^ ni >d