Acta Biotechnologica: Volume 10, Number 4 1990 [Reprint 2021 ed.] 9783112582022, 9783112582015

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ACD I BlotecfeRQluica •

Volume 10 • 1990 • Number4

Journal of Biotechnology in Industry, Agriculture, Health Care, and Environmental Protection

Akademie-Verlag Berlin ISSN 0138-4988 Acta Biotechnol., Berlin 10 (1990) 4, 317-384

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Ada BiotediiMgia Journal of Biotechnology in Industry, Agriculture, Health Care, and Environmental Protection

Volume 10

Edited at the Institute of Biotechnology of the Academy of Sciences of the G.D.R.; Leipzig by M. Ringpfeil, Berlin and D. Pohland, Leipzig

Editorial Board: R. v. Baehr, Berlin A. A. Bajev, Moscow M. E. Beker, Riga S. Eukui, Kyoto P. P. Gray, Kensington I. Y . Hamdan, Kuwait G. Hamer, Zurich L. Herrera, Havana J. Hollo, Budapest

Managing Editor: L. Dimter, Leipzig

1990 Number 4

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

M. V. Ivanov, Moscow D. Meyer, Potsdam A. Moser, Graz P. 0. Okonkwo, Enugu G. Pasternak, Berlin W . Scheler, Berlin R. Schulze, Halle B. Sikyta, Prague G. Vetterlein, Leipzig

B E R L I N

"Acta Biotechnologica" publishes original papers, short communications, reports and reviews from biotechnology in industry, agriculture, health care and environmental protection. The journal is to promote the establishment of biotechnology as a new and integrated scientific field. The technological character of the journal is guaranteed by the fact that papers on microbiology, biochemistry, chemistry and physics must clearly have technological relevance.

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Acta Biotechnologica Herausgeber: Prof. Dr. Manfred Ringpfeil, Akademie der Wissenschaften der DDR Robert-Rössle-Str. 10, DDR -1115 Berlin-Buch. Prof. Dr. Dieter Pöhland, Institut für Biotechnologie der AdW der DDR Permoserstr. 15, DDR - 7050 Leipzig. Verlag: Akademie-Verlag Berlin, Leipziger Str. 3 - 4 , P F 1233, DDR-1086 Berlin; Stellv. Cheflektor Zeitschriften: Armin Beck, Redakteur der Abt. Zeitschriften: Cornelia Wanka; Fernruf: 22362 29 und 2236295; Telex-Nr. 114420; Bank: Staatsbank der DDR, Berlin, Konto-Nr.: 6836-26-20712. Redaktion: Dr. Lothar Dimter (Chefredakteur), Martina Bechstedt, Käthe Geyler, Permoserstr. 15, DDR-7050 Leipzig; Tel. 2392255. Veröffentlicht unter der Registriernummer 1671. Gesamtherstellung: VEB Druckhaus „Maxim Gorki", DDR-7400 Altenburg. Erscheinungsweise: Die Zeitschrift „Acta Biotechnologica" erscheint jährlich in einem Band mit 6 Heften. Bezugspreis eines Bandes 210,— DM zuzüglich Versandspesen; Preis je Heft 35,— DM. Bestellnummer dieses Heftes: 1094/10/4. Urheberrecht: Alle Rechte vorbehalten, insbesondere der Übersetzung. Kein Teil dieser Zeitschrift darf in irgendeiner Form — durch Photokopie, Mikrofilm oder irgendein anderes Verfahren — ohne schriftliche Genehmigung des Verlages reproduziert werden. — All rights reserved (including those of translation into foreign languages). No part of this issue may be reproduced in any form, by photoprint, microfilm or any other means, without written permission from the publishers. © 1990 by Akademie-Verlag Berlin. Printed in the German Democratic Republic. AN (EDV) 18520

Acta Biotechnol. 10 (1990) 4, 319—327

Akademie-Verlag Berlin

Acid Phosphatases Purified from Industrial Waste Mycelium of Aspergillus niger Used to Produce Citric Acid 2YLA, K .

Academy of Agriculture in Cracow Department of Biotechnology 31-425 Cracow, 29-Listopada Ave., 46. Poland

Summary The waste mycelium of Aspergillus niger 'Z' used to produce citric acid was recognized as a rich source of intracellular acid phosphatases ( E . C . 3 . 1 . 3 . 2 . ) . Three forms of enzyme were isolated and purified 5 0 — 1 5 0 fold according to a procedure involving ammonium sulphate fractionation, Sephadex G - 2 0 0 gel chromatography, and DEAE-cellulose ion-exchange chromatography. The cytoplasmic form ( 8 0 % of the whole activity) was found to have molecular weight 2 3 0 0 0 0 , 'MICHAELIS' constant 2 . 4 m l (when measured with pNPP), optimal temperature 6 0 ° C , pH optimum 1.8 — 3.0 and contained 3 0 % of carbohydrate in the molecule. This form was able to hydrolyse pNPP, beta-glycerophosphate, inorganic pyrophosphate, glucose-6-phosphate, phytate compounds and phosvitin, but did not hydrolyse beta-casein nor lecithin. One of two forms connected with the cell walls shown similar properties, whereas the other had molecular weight of 9 5 0 0 , 'MICHAELIS' constant 1 . 4 M M , pH optimum 5 . 0 and was much less thermostable.

Introduction Several e n z y m e activities were mentioned t o be present in t h e industrial waste mycelia a f t e r citric acid fermentation [1 — 3]. I n such a mycelium of Aspergillus niger 'Z' pectinases, proteases, cellulases, as well as glucanases h a v e been found b y G A L A S e t al. [2], who suggested p r o d u c t i o n of these enzymes preparation for t h e fruit-juice industry. I n t h e previous s t u d y [4] it was found out t h a t a n crude acid phosphatase originated f r o m this mycelium was able t o hydrolyse n a t u r a l p h y t a t e compounds, w h a t m a y be of i m p o r t a n c e in food technology and nutrition. T h e aim of this work was t o purify enzyme in order t o learn some properties of Aspergillus niger acid phosphatase — a n a c t i v i t y , which is not well established up t o now.

Material and Methods Assays The activity of acid phosphatase was measured in 0.1 M acetate buffer pH 4.5 using 5.5 mM solution of p-nitrophenyl-phosphate (pNPP) as a substrate. The final volume of the incubated mixture was 1.05 ccm. After 15 minutes of incubation at 40°C the reaction was stopped by the addition of 1*

320

Acta Biotechnol. 10 (1990) 4

5 com 40 MM NaOH. The amount of liberated p-nitrophenol was determined spectrophotometrically at 405 nm. One unit of enzyme activity [U] was defined as 1 (¿mole of p-nitrophenol liberated per minute under the above conditions. The concentration of protein was determined by means of LOWRY'S method [5] using bovine serum albumin as a standard. Carbohydrate content was assayed by the method of DUBOIS [6]. Strain The strain 'Z' of Aspergillus niger (owned by Citric Acid Factory at Zgierz) was grown under normal industrial conditions in surface culture on molasses rich medium. Preparation of Crude Extracts When the fermentation process was completed (190 h) the mycelium was separated from the medium, washed With water, dried and milled on a laboratory grinder to the powder form, then stored at 4°C. A weighed portion of mycelium powder was mixed with cold acetic acid solution (pH 3.0), homogenized and centrifuged. Supernatant obtained in this way was used as a source of cytoplasmic enzyme. The cell walls debris were washed five times with buffer, then incubated overnight in the presence of 1.6M NaCl in 0.1 MTris-HCl buffer pH 7.2 and'Triton X-100'. The contents of incubation vessels was centrifuged and served as a source of these enzyme forms which are connected to cell wallls. Purification of Enzyme Ammonium sulphate precipitation was the first step of purification procedure. In the case of cytoplasmic form, precipitate showing enzyme activity was collected between 50 and 100% of full saturation. This was slightly modified when activity from cell walls was under preparation: Crude extract was saturated to the level of 80%, and precipitated proteins were discarded. The supernatant contained an enzymatically active fraction, which was then dialysed overnight at 2 °C against several changes of 0.1 M Tris-HCl buffer pH 7.2 and finally precipitated in 72% ethanol. The next chromatographic techniques of purification procedure were performed for all enzyme fractions in the same way using ISCO equipment for liquid chromatography (Dialagrad Model 382 programmed gradient pump, absorbance monitor UA-5 with Type 6 optical unit and fractions collector type 328). For gel permeation chromatography the column K 16/60 was filled with Sephadex G-200. A sample of enzyme solution from the previous step was applied and the elution was carried out with the aid of 10 mM Tris-HCl buffer. The fractions which showed acid phosphatase activity were collected, concentrated and finally equilibrated with 10 mM acetate buffer pH 4.5. Ion-exchange chromatography was the last step of purification. For this purpose the column 18 X 80 mm was filled with 'WHATMAN DE 11' DEAE-cellulose, and the following elution conditions were chosen: 20 mM acetate buffer pH 4.5 was pumped through the column at the flow rate of 30 ccm/h since all non-adsorbed proteins are washed out. Then, a linear gradient of ionic strength was applied using 1 M NaCl to the final concentration of 0.5 M. This was followed by two hours' washing with starting buffer. The fractions which acid phosphatase activity had shown were collected within 'peaks', concentrated by 'polyethylene glycol 20000', and lyophilized. Characterization of Enzyme The pH optimum of all enzyme forms was examined using citrate-HCl buffer, in the range of pH from 1.6 to 4.8, and acetate one from pH 4.6 to 5.6. These buffers were also applied for pH stability determination, when samples of enzyme protein were incubated with pNPP at different pH for 3 h at 55 °C. The optimum of temperature was tested in temperature range from 30 to 80 °C with glucose-6phosphate as a substrate.

¿YLA,

321

K., Acid Phosphatases

Thermostability determination was performed during 15 minutes' incubation at 30—80°C at optimal pH value for each enzyme forms, or at pH 4.5. In order to estimate M I C H A E L I S - M E N T E N ' S constant 0.86 to 220 mM solutions of pNPP were incubated with enzyme for 15 minutes at 40 °C. Molecular weight of enzyme fractions was established by means of gel chromatography on Sephadex G-200, as described above. The following molecular weight markers were applied: gammaglobulin: 67000, globulin FR I I : 176000, and yeast fructofuranosidase: 270000. The inhibitory effect of different substances on acid phosphatase forms activity was tested by short preincubation of enzyme protein solution with an inhibitor solution at room temperature, and then pNPP was added and phosphatase activity was measured in the common way. Substrate specificity of cytoplasmic form was established by incubation of 5.5 mM solution of a phosphate containing compound with 2 U of acid phosphatase for one hour at 40°C and pH 4.5. The reaction was terminated by the addition of 5 ccm of 5% TCA. The liquid obtained after filtration was subjected directly for inorganic phosphorus determination by the L O W B Y and L O P E Z method [7].

Results The cytoplasmic form of Aspergillus niger acid phosphatase from the waste mycelium was purified 50-fold, as assigned to crude extract (Tab. 1). Tab. 1. Purification of cytoplasmic acid phosphatase Total enzyme [U]

Yield [%]

Protein concent. [mg/cm3]

Total protein [mg]

Crude extract

3.57

514

21

3025

5.90

100

Ammonium sulphate fractionation

9.75

78

287

2300

29.50

76

Sephadex G-200 chromatography

0.39

10

50

1361

136.00

45

DEAE-cellulose chromatography

0.12

35

768

304.00

25

2.53

Enzyme activity [U/cm 3 ]

Specific activity [U/mg]

Purification step

The gel permeation chromatography was found to be a very effective stage of purification procedure, which let a large ballast of low-molecular weight proteins and peptides be removed and allowed to have the enzyme activity in a single, but not well-distinguished peak (data not shown). In the course of the ion-exchange chromatography on DEAE-cellulose the enzymatically active proteins were found in one main peak, which was eluted from the column at NaCl concentration of 0.2 M (Fig. 1.). Within this fraction (named 'i') three minor peaks are to be seen. The cytoplasmic enzyme may be therefore supposed to exist in multiple molecular forms. The fraction eluted from the column at 0.3 M NaCl had specific activity ca 45 U/mg only, and therefore was not investigated any more. The activity connected to the cell walls (20% of total acid phosphatase found in mycelium) was separated into two fractions with the aid of gel chromatography (Fig. 2.). These fractions were chromatographed on DEAE-cellulose independently. In the case of fraction having higher molecular weight the enzymatically active proteins were eluted at 0.2 M NaCl (fraction 'bi'), whereas the second fraction ('bii') was not adsorbed in the column at pH 4,5, and was eluted before the gradient of ionic strength had been applied. Chromatogram of the 'bii' fraction is shown on Fig. 3.

322

Acta Biotechnol. 10 (1990) 4

Elution

time ChJ

Fig. 1. Chromatogram of cytoplasmic acid phosphatase from Aspergillus niger used for citric acid production (DEAE-cellulose column)

-O 80 - o 0.8 0 00 CM

1 60 hi

0.6

i5 4 0 x 20

O.k 0.2

4 5 Elution time

6 Lhl

Pig. 2. Sephadex G-200 chromatogram of acid phosphatases bound to the cell walls of Aspergillus niger (Detail) 0.050 r °

3 4

A. lipoferum strain NRC 8670

5 «

A. brasilense strain NRC 5677

7 S

A. brasilense strain NRC 1771

• • • • •

c3 60 V

M

O O

15

m -e ® "R o

M O o

"o w>

a ci H > M

o .H a •S 3 fc ^ *H Ml ® fl H fi

£ ir1

^

a

'„•k o a

cd d -S bC

.PH O "S O

O Jg£ £P £ o a.

50

56

79.4

703

43.0

112.9

+12.9

+11.4

+24.3

9.1

100

32

70.4

692

42.3

101.7

+11.3

+9.8

+21.1

8.9

50

14

52.9

565

34.5

93.6

+12.1

+10.0

+22.1

8.9

100

11

48.4

562

34.4

86.1

+11.0

+8.8

+19.8

8.8

50

40

63.7

633

38.7

100.6

+12.1

+10.6

+22.7

9.1

100

20

60.7

622

38.0

97.6

+11.2

+9.4

+20.6

8.8

50

89

95.2

792

48.4

120.2

+13.5

+12.2

+25.7

9.1

100

71

79.6

713

43.6

111.6

+12.2

+11.1

+23.3

9.0

2 ® OS lipoferum strain NRC 1244

N ^ bo

£ S

1 2

«

G

£

Initial most probable number: Initial total nitrogen: Initial organic carbon: Initial pH: Incubation temperature:

+L J-

l à

£ 1

w

PH

4 x 103 cells/ml Zero mgl -1 1636 mgl -1 6.8 30°C

• Different amounts of semisolid N 2 malate medium were taken in different sets of ERLENMEYER flasks having identical capacity (300 ml)

growth conditions. As a general trend, there were considerable variations in the amounts of the total mineral nitrogen and the nitrogen gains among the different strains under either condition of cultivation. For example, in A. lipoferum strains 1244 and 8670, (the total mineral nitrogen & the nitrogen gains) were 33.6 & 106.9 mgl -1 and 28.2 & 69.9 mgl -1 after 20 days for 50/300 ml growth condition, while in the case of 100/300 ml growth condition the corresponding figures were 30.2 8c 91.9 mgl -1 and 25.9 & 59.5 mgl -1 after the same period. On the other hand, in A. brasilense strains 1771and5677, (the total mineral nitrogen & the nitrogen gains) were 35.2 & 120.6 mgl -1 and 31.4 & 78.6 mgl -1 after 20 days for 50/300 ml growth condition, while in the case of 100/300 ml growth condition the corresponding figures were 31.9 & 92.9 mgl -1 and 27.6 & 68.5 mgl -1 after the same period. Worth of notice, the amounts of nitrogen fixed is usually linked to cell proliferation, which confirms the findings of J E N S E N [12] and S H A W K Y [13]. The DL-malic acid content gradually decreased with time. The organic carbon consumed within twenty days for A. lipoferum strains 1244 and 8670 was 55.1 & 39.8% and 49.5 & 36.9% under 50/300 ml and 100/300 ml growth conditions, respectively, while in the case of A. brasilense strains 1771 and 5677, the corresponding figures were 58.3 & 41.6%

SHAWKY, B. T., Dinitrogen Fixation

333

Tab. 2. Growth and nitrogen-fixing activity of different Species and strains of Azospirillum, grown in semisolid N2 malate medium (Incubation period : 10 days)

sI d I— O

I c5 g eh A.lipoferum

strain NRC 1244

3

A.lipoferum

5

6

A® .2

A ® .2

g O

S O

l ^ f I " i r I ? M >3 3 8 — =3 s^ S J .-ëa» spa» sf>c3 s a taÊ o â o è

2

4

d

2 1 X ^

* 1

1

r-i

ao o

5

& w w w g a

+ Jm* mi g a

55.1

118.5

810

49.5

69.9

651

10

59.5

50

25

100

g a

i i -S "3b ¿ a

^ w

+ 18.1

+ 15.5

+ 33.6

8.9

113.5

+ 16.6

+ 13.6

+ 30.2

8.9

39.8

107.4

+ 14.1

+ 14.1

+ 28.2

9.2

603

36.9

98.7

+ 12.9

+ 13.0

+25.9

8.9

78.6

681

41.6

115.4

+ 16.6

+ 14.8

+ 31.4

9.2

18

68.5

652

39.9

105.1

+ 14.2

+ 13.4

+27.6

9.0

50

178

120.6

953

58.3

126.5

+ 19.2

+ 16.0

+ 35.2

9.1

100

112

92.9

815

49.8

114.0

+ 16.8

+ 15.1

+ 31.9

9.0

£

£

| i,

character", oxygen-sensitive enzyme of preparations obtained from Clostridium. This conformational protection could operate in two ways. Either the oxygen-sensitive sites could be inaccessible to oxygen, or they could be stabilized by conformational features of the nitrogenase complex so that, though accessible to oxygen, they would be undamaged by it. (2) respiratory protection, whereby respiration functioned as an "oxygen-wasting system", which maintained a low redox potential (E h ) value within the cell, presumed to be necessary for nitrogen fixation, i.e., respiration not only performs its usual physiological function but also protects nitrogenase by scavenging oxygen from the neighbourhood of the nitrogen-fixing site. Accordingly, the microaerophilic character of dinitrogenfixing azospirilla means that these organisms have low Q0 2 values and hence can afford little respiratory protection. The previous results clearly emphasize that the available 0 2 is one of the most important factors affecting the outcome of N 2 -fixation by Azospirillum. The different ways of cultivation materially affect the amount of nitrogen fixed as well as of carbon consumption, which is reflected on the efficiency of N2-fixation by a particular Azospirillum strain. Consequently, it is recommended that, in recording the power of dinitrogen fixation of an organism, the method of cultivation be mentioned in addition to other factors influencing the N 2 -fixation process. Of particular note was the observation of the considerable rise in pH, which took place during azospirilla growth and could be attributed to the oxidation of the malate, i. e., it is capable of growing in alkali medium even up to pH 9.5. For this wide range of pH, Azospirillum, may have a nitrogen-fixing enzyme system different from that of other dinitrogen fixers. Further studies will be necessary to prove the validity of this assumption. 2*

336

Acta Biotechnol. 10 (1990) 4

Summing up, this research has led to the conclusion that the activity of dinitrogen fixation b y azospirilla is affected not only b y bacterial species properties, but also b y the strain and the culturing conditions employed. Received June 30, 1989

References [ 1 ] DÖBEREINER, J . , MABRIEL, I . E . , NERY, M . : C a n . J . M i c r o b i o l . 2 2 ( 1 9 7 6 ) , 1 4 6 4 .

[2] DAY, J . M.: Appl. Environ. Microbiol. 34 (1977), 640. [ 3 ] NEYRA, C. A . , DÖBEREINER, J . : A d v . A g r o n . 2 9 ( 1 9 7 7 ) , 1.

[4] BALDANI, V. L . D., DÖBEREINER, J . : Soil Biol. Biochem. 12 (1980), 434. [ 5 ] VAN BERKUM, P . , BOHLOOL, B . B . : M i c r o b i o l . R e v . 4 4 ( 1 9 8 0 ) , 4 9 1 . [ 6 ] PATRIQUIN, D . C., DÖBEREINER, J . , J A I N , D . K . : C a n . J . M i c r o b i o l . 2 9 ( 1 9 8 3 ) , 9 0 0 .

[7] SHAWKY, B. T.: Zbl. Mikrobiol. 144 (1989), 581. [ 8 ] COCHRAN, W . G . : B i o m . 6 ( 1 9 5 0 ) , 1 0 5 .

[9] JACKSON, M. L.: Soil Chemical Analysis. London 1959. [10] Amer. Publ. Health Assoc. and Amer. Wt. Wks. Ass. (A.P.H.A.): Standard Methods for the Examination of Water, Sewage, and Industrial Wastes, 10th ed., New York 1955. [11] DÖBEREINER, J., DAY, J . M.: I n : Symposium on nitrogen fixation. (Eds. NEWTON, W. E., NYHAN, C. J.), Washington States University Press, PULLMAN, W. A. (1976), pp. 518—538. [12] JENSEN, H. L.: Bacterid. Rev. 18 (1954), 195. [13] SHAWKY, B. T.: Zbl. Mikrobiol. 137 (1982), 445. [ 1 4 ] ABD-EL-MALEK, Y . , HOSNY, I . , SHAWKY, B . T . : Z b l . B a k t e r i o l . I I 1 3 4 ( 1 9 7 9 ) , 3 9 0 .

[15] SHAWKY, B. T.: Zbl. Mikrobiol. 137 (1982), 453. [16] [17] [18] [19] [20]

SHAWKY, B . T . , GHALI, Y . , AHMED, P . A . , KAHIL, T . : Z b l . M i k r o b i o l . 1 4 2 ( 1 9 8 7 ) , 4 4 1 . DALTON, H . , POSTGATE, J . R . : J . G e n . M i c r o b i o l . 5 4 ( 1 9 6 9 ) , 4 6 3 . DALTON, H . , POSTGATE, J . R . : J . G e n . M i c r o b i o l . 6 6 ( 1 9 6 9 ) , 3 0 7 . POSTGATE, J . R . : N a t u r e 2 2 6 ( 1 9 7 0 ) , 2 5 . POSTGATE, J . R . : J . A p p l . B a c t e r i d . 3 7 ( 1 9 7 4 ) , 1 8 5 .

[21] MULDER, E . G., BROTONEGORO, S . : I n : T h e biology of nitrogen f i x a t i o n . (Ed. QUISFEL, A.),

North-Holland Publishing Company, Amsterdam. (1974), pp. 37-85. [22] DÖBEREINER, J . : IN: The biology of nitrogen fixation. (Ed. QUISPEL, A.), North-Holland Publishing Company, Amsterdam. (1974), pp. 86 — 120. [23] MORRIS, J . G.: Adv. Microbial. Physiol. 12 (1975), 169.

Akademie-Verlag Berlin

Acta Biotechnol. 10 (1990) 4, 337 - 3 4 0

1-Acetyl-ß-carboline, a New Metabolite of Streptomyces kasugaensis PROKSA, B.1, UHBÎN, D.1, S T U R D Î K O V A ,

1 2

M . 2 , FTTSKA, J . 2

Institute of Chemistry, Slovak Academy of Sciences, CS-84238 Bratislava, Czechoslovakia Department of Biochemical Technology, Faculty of Chemistry, Slovak Technical University, CS-81237 Bratislava, Czechoslovakia

Summary 1 -Acetyl-/5-carboline, isolated as a metabolite of an actinomycete for the first time, was obtained from the culture filtrate of Streptomyces kasugaensis. The separated compound was identified by spectral means. Production of l-acetyl-/3-carboline was substantially rised after addition of equimolar amounts of DL-tryptophan and DL-alanine into the cultivation medium.

Introduction S o far three metabolites h a v e been isolated f r o m t h e cultivation m e d i u m of Streptomyces kasugaensis : hydrophylic k a s u g a m y c i n , structurally classified into aminoglycoside antibiotics and t w o lipophilic l,2-dithiolo/4,3-b/pyrrolo derivatives thiolutin and aureothricin [1], All t h e s e c o m p o u n d s reveal antifungal activities and are used in p l a n t protection. I n organic extract of t h e cultivation beer of 8. kasugaensis w e f o u n d a n e w m e t a bolite ; t h e isolation, structure elucidation as well as t h e proposed m e c h a n i s m of f o r m a t i o n of t h i s c o m p o u n d is presented in this paper.

Material and Methods Melting point was determined on a K O F L E R micro hot-stage, the I R spectrum was recorded with a P E R K I N E L M E R , model 983. The EI-MS was measured with a J E O L JMS 100 D apparatus a t 70 eV and 300 (¿A. The J H- and 13 C-NMR spectra of CDC13 and DMSO-d 6 solutions run with a BRTJKER AM-300 instrument operating a t 300 and 75 MHz, respectively, are relative to TMS; semiselective I N E P T experiment [2] was optimized for ° J c - H = Hz, evolution and refocusing intervals were set to 50 and 60 ms, respectively; the 90° *H pulse width was 10 ms. H P L C was proceeded with a column (150 X 3 mm) packed with S E P A R O N SGX C18 7 ¡xm (TESSEK, Praha), mobile phase methanol — water (60 : 40), flow rate 0.4 cm 3 • min - 1 , detector wavelength 254 nm. SILUFOL UV 254 in chloroform — methanol ( 9 : 1 ) and visualization a t 254 nm were used in TLC. Producing organism Streptomyces kasugaensis I F O 13851 was obtained from the Institute for Fermentation, Osaka, J a p a n . This train was maintained on B E N E T T agar (Catalogue ATCC medium No. 174).

Acta Biotechnol. 10 (1990) 4

338

Cultivation of S. kasugaensis Medium for the inoculum preparation consisted of (g • l - 1 ) : glucose 15, soybean meal 15, K H 2 P 0 4 1, MgS0 4 • 7 H 2 0 0.5, NaCl 3, CaC0 3 5, distilled water added to 1 1, pH 6.9. The medium was transferred in 100 ml portions to 500 ml flasks, sterilized (20 min at 120°C) and inoculated with S. kasugaensis. Cultivation time 72 h, temperature 28 °C. Production medium consisted of (g • 1 _ 1 ): glucose 15, soybean meal 12, K H 2 P 0 4 1, MgS0 4 • 7 H 2 0 0.5, NaCl 3, DL-tryptophan 0.08, DL-alanine 0.08, pH adjusted to 6.9. The sterilized medium was inoculated with 10% (v/v) of inoculum prepared. Cultivation time on rotary shaker (3.7 Hz, 28°C) was 144 h.

Isolation of l-Acetyl-fi-carboline (1) Cultivation broth (2 1) was filtered through a layer of microcrystalline cellulose, the filtrate was extracted 5-times with 200 ml of chloroform — 2-propanol ( 3 : 1), the combined extracts were dried over anhydrous sodium sulfate and the solvents were evaporated. The residue was chromatoTab. 1. 'H- and 13 C-NMR data of l-acetyl-/3-carboline (1) Position

13

C

*H ^ H - H (Hz)