Acta Biotechnologica: Volume 10, Number 3 1990 [Reprint 2021 ed.] 9783112582008, 9783112581995

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Acta ßlotecmnluica •

Volume 10 • 1990 • Number 3

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

Akademie-Verlag Berlin ISSN 0138-4988 Acta Biotechnol., Berlin 10 (1990) 3, 213-316

Instructions to Authors

1. Only original papers that have not been published previously will be accepted. Manuscripts should be submitted in English (in duplicate). The name of the institute (with the full address) from which the manuscript originates should be stated below the name(s) of the author(s). The authors are responsible for the contents of their contributions. 2. Original papers should not exceed 20 typewritten pages (double spaced), including references, tables and figures; short original communications may contain a maximum of six typewritten pages. 3. Each paper should be preceded by a summary. 4. Latin names of species as well as passages to be printed in italics for greater emphasis should be marked by a waving line. Please use only units and symbols of the Si-system. 5. Tables may be used to shorten the text or to make it more comprehensible. They should be numbered consecutively throughout the text and be supplied with a brief heading. They should not appear in the text, but should be written on separate sheets. 6. The numbers and sizes of illustrations should be limited to a minimum, they should be numbered consecutively and be quoted on separate sheets. Line drawings, including graphs and diagrams, should be drawn in black ink. Half-tone illustrations should be presented as white glossy prints. Figure legends are to be typed in sequence on a separate sheet. The back of each sheet should bear the name(s) of the author(s). 7. References listed at the end of the contribution should contain only works quoted in the text. They should be numbered in the order in which they are first mentioned in the text. Please give surnames and initials of all authors, the name of the journal abbreviated according to "Chemical Abstracts — List of Periodicals", volume number, year of publication, issue number or month, first page number. Books are to be cited with full title, edition, volume number, page number, place of publication, publisher and year of publication. 8. Notes to the text may be presented as footnotes on the same page. 9. 50 offprints are free of charge. Additional ones may be ordered on payment. 10. The author will receive two galley proofs for correction. They are to be returned to the managing editor (Dr. Dimter, Permoserstr. 15, Leipzig, 7050-DDR) as soon as possible.

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Acta Biotechnol., Berlin 10 (1990) 3

Acta Blattdmaiaaica Journal of Biotechnology in Industry, Agriculture, Medicine, and Environmental Protection

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

Volume 10

Managing Editor: L. Dimter, Leipzig

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

1990 Number 3

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

B E R L I N

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

"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 und Prof. Dr. Dieter Pöhland, Institut für Biotechnologie der AdW der DDR Permoserstr. 15, DDR-7050 Leipzig. Verlag: Akademie-Verlag Berlin, Leipziger Straße 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 Lizenznummer 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. Der gültige Jahresbezugspreis für die DDR ist der Postzeitungsliste zu entnehmen. Bestellnummer dieses Heftes: 1094/10/3. 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 03000

Acta Biotechnol. 10 (1990) 3, 2 1 5 - 2 2 4

Akademie-Verlag Berlin

Modelling of the Calculation of the Power Input for Aerated Single- and Multistage Impellers with special Respect to Scale-up MÖCKEL, H . - O . 1 , W O L L E S C H E N S K Y , E . 2 , D R E W A S , S . 2 , R A H N E R , H . - J . 3

1 2 3

VEB KCA Dresden 8012 Dresden PSF 184, G.D.R. VEB Jenapharm, 6900 Jena PSF 150 G.D.R. VEB Chemieanlagenbau Erfurt-Rudisleben 5211 Rudisleben PSF 213/214, G.D.R.

Summary For both laboratory and industrial scale fermenters the stirred fermenter is often being used. Especially for the design procedure of industrial fermenters with variable speed drive it is important to know the exact power characteristics for single and multiple impellers. The calculation procedure used for estimating the gassed power input gives the possibility to calculate the gassed power drawn by at maximum 3 impellers in vessels up to 3.5 m in diameter in aerated water. For calculation one only needs known parameters. This power input is valid for low and medium viscous fermentation broths too because especially after the addition of antifoam agents the properties of the liquid phase are then comparable to water as a coalescing liquid. It is possible to calculate the gassed power drawn by each impeller of a multiple system. This calculation procedure is applicable to different fermentation broths where in the range of low aeration rates the.resultb differ from those for water.

Introduction In spite of the development of new types of fermenters nowadays the stirred fermenter holds a dominating position in the field of fermentation, too. This is valid for both laboratory and industrial scale fermenters. Especially for the design procedure of industrial fermenters with variable speed drive it is important to know the dependence of the power characteristics on the aeration rate for single and multiple impellers. At the operating point with maximum speed the stirrer(s) have to draw the nominal power of the drive to get the maximal possible power input. Previous Work A large number of publications concerning the calculation of the power input under gassed conditions underline the importance of these questions. Ref. [1] contains an extensive comparison of literature data. The main aim of the article is to demonstrate, that the calculation procedure proposed in [2] and [3] allows to correlate the literature data and can be used for multistage stirrers, too. While for a single stirrer could be included measurements from a 500 m 3 stirred fermenter with a vessel diameter of ^ = 7.0 m, for the two and three stirrer systems only data up to a vessel Size of 3 m 3 or dy = 0.9 m were available. Therefore the transferability to industrial apparatuses is not confirmed finally. 1*

Acta Biotechnol. 10 (1990) 3

216 Problem

F o r t h e design of the drive of industrial scale stirred fermenters calculation correlations are necessary which consider the influences of — — — —

vessel diameter d1 diameter ratio d2jd1 number of stirrers z superficial gas velocity vg

and give t h e possibility to use only known parameters. Experiments All experiments for measurement of power input under gaSSed conditions were carried out in geometrically similar Stirred fermenters of industrial scale designed according t o F i g . 1. T h e liquid media were water or different fermentation broths gassed with air. W e measured t h e electrical power drawn b y the DC-drive P e l , the number of revolutions of the Stirrer n, the aeration rate V G and t h e air inlet parameters pqe and Under technical Scale conditions it is necessary t o measure t h e electrical power input because there is no possibility to measure the torque Separately. I n this ease the power of idle motion is to be determined Several times, i.e. with an e m p t y vessel. T h e n the net power input of t h e stirrer is Pn = m

• [U • J -

(u • j)lAle]

(1)

Fig. 1. Dimensions of the fermenters and measured data, valid for all measurements

Vg,tgE,PgE Air inlet

¿1 [m]

h^m [m]

Ah^rn [m]

Z

i

1.6

0.66

1.0

1 2 3

0.84 1.44 2.06

3.2

1.1

1.76

1 2 3

0.5 1.03 1.63

3.47

1.2

2.2

1 2 3

0.9 1.42 2.3

MÖCKEL, H.-O., WoLLESCHENSKY, E . et al., Aerated Single- and Multistage Impellers

217

From measurements with and without aeration for a given fermenter the ratio Po

(2)

\iJTdJ

is plotted where the relation (3)

g • di

exists. Power Input for the System Water-Air According to Ref. [1] the equation (4)

Po

1+ A

Ìf~di

has been derived. The constant A depends on the number of stirrers with 1 stirrer: A = 750 2 stirrers: A = 490 3 stirrers: A = 375 I t is to be shown that equation (4) is valid for industrial scale stirred fermenters, too, and first of all for reasons of the uniform medium measurements in the water-air system are compared. Fig. 2 to 4 show the results of measurements from technical scale fer1.0 r

o?

^ 0.5

10-3

10-2 Vg fiRÌ

Fig. 2. Power input for a gassed stirrer, water-air, one stirrer d1 = 0.4—7.0 m [1]

Symb. dt

d2jdx

hjd1

x O A

0.414 0.394 0.4

0.9 0.844 0.5

[m]

3.47 1.6 3.4

VL

[m*] 26.0 2.4 10.0

Acta Biotechnol. 10 (1990) 3

218 1.0 r

0

i 10-3

5

i 2

i 5

i 10-2

I 2

3.47 1.6 3.2

X O A

5

KMi

VL [m»]

0.414 1.42 0.394 1.47 0.4 1,05

43.4 4.5 24.0

H*

Symb. d1 [m]

to&4

Fig. 3. Power input for gassed stirrers, water-air, two stirrers = 0.4 m [1]

10-3

2

5

10-2

2

Vg^df Fig. 4. Power input for gassed stirrers, water-air, three stirrers dx = 0.4 m [1] Symb. d1 [m]

d2jd1

Klh

X

3.47 0.414 2.3

+ o

0.5 0.5

0.4 0.25

•

1.6

0.369 2.06

A

3.2

0.4

2.3 2.3 1.63

[m3] 70.0 0.22 0.22 6.43 39.0

MÖCKEL,

H.-O.,

WOLLESCHENSKY,

E. et al., Aerated Single- and Multistage Impellers

219

menters [4], [5] in comparison to former measurements in laboratory fermenters. The figures demonstrate that the measured data are with 80- "90% within the variance of the laboratory measurements. From this follows that the applied procedure of modelling the power input under aeration offers the possibility to correlate the power characteristics over a wide range of fermenter sizes if they are geometrically similar. The calculation model gives the advantage to Start the calculation with known or given parameters. All experiments were made with 4 coils as baffling. The results can be used for other types of baffles, too, if the influence of different baffles is considered in the calculation ofP0Power-Input for the System Fermentation Broth-Air For verifying the applicability of equation (4) for the System fermentation broth-air measurements were carried out during the fermentations of Lysin, Penicillin and Oxytetracyclin in fermenters with one or three stirrers. In comparison to water the liquid properties of a fermentation broth are very different and the coalescence is especially influenced. Therefore the relations found differ from the relations for water where there is a dependence on the number of Stirrers. Fig. 5 and 6 show the power input for one stirrer in an aerated Penicillin or Lysin broth respectively. A significant influence of liquid properties on power-characteristics is to find out for both broths only if vgl]!g • dx < 3 • 10~3.

Fig. 5. Power input for a gassed stirrer, Penicillin broth-airone stirrer, d t = 0.4 m, h0jd1 = 1.0 [1] Symb.

o

• o 0 X

•

+ T

d2/d1

Time

Baffles

M

0.40 0.35 0.35 0.30

125 125 150 150

4 blades

0.40 0.35 0.30 0.30

168 188 168 188

4 tubes

220

Acta Biotechnol. 10 (1990) 3

/Or

0.°

^ 0.5

10-3

10-^ \/gdi

Fig. 6. Power input for a gassed stirrer, Lysin broth-air, one stirrer dx = 0.4 m, = 1.0 [1] Symb.

o

d1 [m]

VL [m 3 ]

0.4

0.4

0.045 35 45 56 70 72

1.4

0.4

2.2

0.4

0.4

0.045 15

V A •

® + •

Time* [h]

35 70

* age of fermentation

In this iange the hold-up within the stirrer and the gas cavities behind the stirrer blades are mainly determined by the gas recirculation from the zones above and below the stirrer. In these zones the hold-up depends on the coalescence properties of the liquid. The tendency of gas bubbles to coalesce is reduced or completely cancelled which results in a higher gas recirculation rate. At higher sparging the influence of the fed air dominates and the found relations agree with characteristics for water. This is the range of technical interest. According to Ref. [7] for water, too, the ratio of recirculated gas to fed gas reduces with increasing Sparging and the behavioui can be explained phenomenologically. If three stirrers are used the changed liquid properties of a fermentation broth are more distinct. Because the upper stirrers are mainly influenced by gas recirculation their percentage on whole power input is reduced if the hold-up increases. Therefore the reduction of power input is more significant than for one stirrer. Fig. 7 and 8 show measurements for antibiotic fermentation broths and Lysin fermentation broth respectively for three stirrers. In this case the influence of the liquid properties is to notice for v • d1 < 0.01. It is impossible to find an exact connection with mean or rather local hold-up because the closed construction of the fermenters does not allow to measure these data. Additio-

M ö c k e l , H.-O., W o l l e s c h e n s k y , E. et al., Aerated Single- and Multistage Impellers

0\ 5

i

i

10-3

i

2

i

5

221

i

10-2 2

JL_ i/

Protein-Kinase

Phosphorylase-Kinase-P

Phosphorylase-P

> Gluc„-i

(a)

+Gluc-6-P

Abb. 2. Regulation der Aktivität von Phosphorylase mittels cAMP zur Phosphorylierung von Stärke

243

KLUDAS, K.-H., Biosynthese für die Impfstoffproduktion

scheinlich auch Peptide als Mediatoren eine besondere Rolle. Im Medium, das frei von Peptiden ist, findet keine Entnahme von Stärke statt. Wir vermuten, daß die Rolle des Adrenalins und Glucagons, einem Polypeptid aus 29 Aminosäuren, zur Aktivierung der Adenylatzyklase auch niedermolekulare Peptide mit einer bestimmten Aminosäuresequenz übernehmen können. Es könnten dies die Peptide His-Ser oder Asp-Ser sein, die im Trypton enthalten sind (Abb. 2). Physiologische Bedingungen zur optimalen Bildung immunogener Stoffe durch Bordetella bronchiseptica Die Ausbildung der erforderlichen immunbiologischen Parameter durch bronchiseptica: HL-Toxin: Fimbrien: Kapsel: Hämagglutination: jö-Hämolyse: Trypsin:

Bordetella

40 dlm/1 x 1010 Zellen 12 im ELMI nachweisbar 70% 1 : 2048 ( + + + ) bei 1 x 1010 Zellen/ml positiv 1 y.g/1 x 1011 Zellen

sind bei geeigneten Stämmen nur bei hoher Belüftungsrate (p02 = > 60%) möglich. Nur unter diesen Bedingungen werden genügend freie und gebundene Aminosäuren aufgenommen. Bei einem p0 2 -Gehalt von 60% werden durch 1 x 1010 Zellen/ml 2,098 g freie und 3,200 g gebundene Aminosäuren pro 1 Kulturbouillon benötigt. Da Fimbrien-, Kapsel- und HL-ToxinSynthese von einer optimalen Peptidkonzentration abhängig sind, muß die ProteinaSeaktivität durch Zusatz von Glucose so gesteuert werden, daß in der Kulturbouillon eine Trypsin-Aktivität von 0,5 [Lg/5 X 1010 Zellen/ml vorhanden ist. Sinkt der p0 2 -Gehalt auf 30% ab, erfolgt nur eine Aufnahme von 0,769 g freien und 0,064 g gebundenen Aminosäuren. Bei Sauerstoffreduzierung stehen also weniger Aminosäuren und Peptide zur DeSaminierung und Gluconeogenese zur Verfügung. Unter diesen Bedingungen sinkt die HL-Toxinsynthese und der Hämagglutinationstiter. Neben einem hohen Sauerstoffangebot ist es erforderlich, daß das Angebot an Leucin 1100 mg/1, an Tyrosin 320 mg/1 und an Phenylalanin 750 mg/1 nicht überschreitet. Wildstämme von Bordetella bronchiseptica können keine aus der Spaltung von Stärke entstehende GlucoSe an das Medium abgegeben. Bordetella-bronchiseptica-Stämme benötigen zur Vermehrung ein Vielfaches ihres Gewichtes an Aminosäuren. Eingegangen: 3. 1. 1989 Überarbeitet: 28. 2. 1989

Literatur [1] KIELSTEIN, P . , EKLER, W . : Mh. Vet. Med. J e n a 4 8 (1988), 87. [ 2 ] W E I D E , H . , PACA, [3]

lag, 1987, 96. B E M I S , D . a.,

J.,

KNORRE, W . A .

GREISEN, H . A . , A P P E L ,

— In: Biotechnologie, Jena, V E B Gustav Fischer Ver-

M.

J . G.: J .

Clin. Microbiol.

6 (1977), 471.

[4] KASUGA, T., NAKASE, Y . , KAWAHIRA, M . : P a t e n t s c h r i f t 2 2 1 2 2 7 7 v o m 14. 8. 1 9 7 2 . [5] ENDO, M., TAKEZAWA, T., NAKASE, Y . : Microbiol. Immunol. 2 4 (1980), 95.

Acta Biotechnol. 10 (1990) 3, 244

Akademie-Verlag Berlin

Book Review I. A . F . O P DEN

KAMP

Membrane Biogenesis NATO ASI Series H.: Cell Biology. Nr. 16 Berlin: Springer-Verlag, 1988. 477 S., 160 Abb., D M 2 3 8 , - , ISBN 3-540-18566-6

Der Sammelband „Membrane Biogenesis" enthält die Vorträge einer im Herbst 1987 unter dem Titel „New Perspectives in the Dynamics of Assembly of Biomembranes" durchgeführten Tagung. Die 31 Beiträge behandeln in einem ersten Abschnitt biophysikalische Aspekte der Phospholipidstruktur in Doppelschichtmodellmembranen (4 Artikel). Mit der Rolle der Plasmamembranproteine bei pathologischen Veränderungen in Erythrozyten, Endothelzellen der Aorta während der Erythropoese und im Herzmuskel beschäftigen sich 4 Beiträge. Den ausgewogensten Abschnitt des Buches Stellen die Originalmitteilungen zur Thematik „Proteintransport und Insertion in Membranen" dar, in denen sowohl pro- wie eukaryontische Modelle diskutiert werden (11 Beiträge). Schließlich müssen zwei aktuelle Artikel über die Azylierung von Proteinen erwähnt werden, die erst in den letzten Jahren als Proteinmodifikation mit möglicher Bedeutung für Transport- und Erkennungsfunktionen von Membranproteinen erkannt worden ist. Insgesamt ist der Band für Leser zu empfehlen, die sich mit einzelnen Aspekten der Themenkreise vertieft beschäftigen wollen. Zellbiologen, Biochemiker und Biophysiker sind besonders angesprochen. Gentechnische Annäherungen an membranbiologische Fragestellungen werden lediglich in zwei Beiträgen erwähnt. R.

GROSSE

Acta Biotechnol. 10 (1990) 3, 2 4 5 - 2 5 1

Akademie-Verlag Berlin

Comparison of Mitotic Activity and Growth in two Long Term Callus Cultures of Matricaria recutita L. CELLABOVA, E . , RYCHLOVA, M . , SEIDELOVA, A . , HONCAKIV, R .

Department of Special Biology Faculty of Science P. J . Saf&rik University Mänesova 23, 041 54 KoSice, C/24, Czechoslovakia

Summary Callus cultures of two tetraploid plants of Matricaria recutita L. were initiated from leaves and cultured on LINSMAIER-SKOOG culture medium supplemented with 0.01 mg • L-1 IAA or 3.00 mg • L-1 2,4-D for three years. The relationship between mitotic activity and growth of calli was studied. The time a t which mitotic activity began in both cultures was different. I n the callus culture cultivated on the medium with IAA the maximum mitotic activity was observed on the 14th to 17th day of subculture. I n the one cultivated on the medium with 2,4-D, the increase of mitotic activity was observed earlier and corresponded with the beginning of exponential growth. I n addition of total mitotic activity individual mitotic phases have also been evaluated. Most mitotically active cells in both cultures were in prophase. Their courses were similar to those of total mitotic activity. Metaphase and telophase stages were present in low frequency. Anaphase occured rarely. The functional dependences of mitotic activity and growth were characterized by appropriate mathematical models.

Introduction The growth of callus cultures includes the processes of mitotic division, increase in cell volume and/or differentiation. The cell proliferation is significantly influenced by the composition of culture medium and the presence of plant growth substances in appropriate concentrations [1 — 3]. Growth and differentiation processes including mitotic activity have been studied in several species which have been cultured in various culture systems. Most of the cells start to divide after a 20 to 24 h lag phase. The first cell division is usually synchronous [4]. Literature data characterizing mitotic activity in later stages of callus development are fragmentary. Only the values of total mitotic activity due to given culture conditions are shown [5—7]. The mitotic activity of callus cultures, especially during long term cultivation, should be influenced by polyploidy and karyological and chromosomal instability [5 — 8]. In this paper results are presented of a study of the relationship between mitotic activity and growth of Matricaria recutita L. callus cultures.

3

Acta Biotechnol. 10 (1990) 3

246

Acta Biotechnol. 10 (1990) 3

Material and Methods We used three years old callus cultures of Matricaria recutita cultivated on Linsmaier-Skoog culture medium [9]. Primary cultures were derived from leaf explants which were obtained from two tetraploid plants (2n = 36). Callus culture K2-81-7 was cultivated on RM medium with 0.01 mg • 1 _1 3-indoleacetic acid (IAA) and the second culture Kl-107 was cultivated on RM medium supplemented with 3.0 mg • l - 1 2,4-dichlorophenoxyacetic acid (2,4-D). The cultures were grown under fluorescent tubes of 2500 lux illuminance, 16/8 h photoperiod, 26/22 °C temperature and 70% relative humidity. The calli were subcultured monthly. Callus growth was measured by increases in fresh and dry weight over a 49 day period. The samples were dried to a constant weight at 105°C for 3 h. The mitotic activity of the cultures was studied over a 30 day period. The samples for cytological analysis were taken from mitotically active parts of callus tissue in 2 to 4 day intervals. They were fixed in ethanol/glacial acetic acid (3 : 1) for 18 h at 4°C and then hydrolyzed in IN HCl at 60°C for 8 min. Squashed preparations were obtained by a cellophane method [10] and stained with Giemsa solution diluted by Sorensen phosphate buffer (pH 7.0) 1: 9. Stained preparations were dehydrated in acetone and xylene and mounted in solacryl (a mixture of butylmetacrylate and methylmetacrylate in xylene). The mitotic activity and frequency of individual mitotic phases were evaluated in eight squashed preparations for each time interval. The average values of mitotic activity and mitotic phases are given as the percentage of the total number of approximately 10000 evaluated cells. Fresh and dry weight increases and the functional dependence of mitotic activity were statistically evaluated by regression and correlation analysis [11]. The formal mathematical relationship of these variables as they relate to the length of culture interval has been determined. The correlation index Iy • x has been used as the measure of the tightness of this relationship. The determination index R 2 in individual equations indicates the percentage of the total variance which is reproduced by given linear and nonlinear model. According to BakytovX et al. [12], the values R2 = 0.8 testify to the satisfactory explanation of the equations. Increase in fresh weight in relation to the lenght of subculture is expressed in percentage (initial weight 100%). The increase in dry weight during subculture was evaluated based on the absolute values of dry weight and on its percentage of fresh weight.

Results and Discussion The growth of callus cultures is expressed by increases in fresh and dry matter (Fig. 1). The growth of callus culture K2-81-7 was characterized by a 7 day lag phase. Exponential growth continued until the 28th day of Subculture. The values of dry matter expressed as a percent of fresh weight decreased after the 15th day of subculture. Percentage expression of fresh weight increases in reference to the length of the culture interval leads to the linear relationship which is expressed by the equation of the quadratic regression y = 1145.476a; - 117.881a:2 - 740.997 {R2 = 0.929, Iy • x = 0.936). In this expression, i.e. if we consider the initial weight of inoculum, the production of fresh cells proceeds at about the same rate from the 7th until 28th day of subculture. A similar pattern exists in the relation between the absolute values of dry matter and the length of culture interval, which is characterized by the equation of the quadratic parabola y = 81.207« - 9.125a;2 - 13.628 (E2 = 0.900, Iy • x = 0.908). The suitable mathematical model for the dependence of dry matter percentage to the length of culture interval is expressed by the equation of the regression y = a + b^x + b2x. Its estimate is the Sample regression equation y = 4.478 — 1.099a; + 0.009a;. Jt2 = 0.967 indicates that the latter estimate is better than the former. Iy • x = 0.805. According to this expression the percentage of dry weight decreased at a constant rate after the 7th day of subculture.

CELLABOVA, E., RYCHLOVA, M. et al., Mitotic Activity and Growth

247

28 35 Time Id 1 > /

Increase in fresh

weight

>Increase in dry weight / Duration of maximal mitotic

s*

K1-107

activity

K2-81-7"

2

U

21

28 35 Time Tdl

42

49

Pig. 1. Growth curves of Matricaria recutita callus cultures

The course of mitotic activity in both Matricaria recutita callus cultures during the subculture is characterized by the average mitotic index. The mitotic activity of K2-81-7 callus culture during the subculture is characterized by a one peak curve (Fig. 2). During the first eight days of subculture the frequency of mitosis slowly increased. After this period the mitotic activity increased significantly and the highest values of mitotic index were registered between day 14 and 17. The 14th day of subculture corresponds with the 7th day after the beginning of the- exponential growth phase. The duration of maximum mitotic activity was about 4 days. During the following days the frequency of mitoses started to decrease despite the prolonged exponential growth period. In this period the values of mitotic activity were about 1 to 1.5%. The course of mitotic activity in reference to the length of subculture is different from that of the growth characteristics. The dependence should be expressed by appropriate continuous functions. Until the 14th day the mitotic activity is characterized by the equation of the linear regression y = 1.385 + 2.492a; {R2 = 0.819, Iy • x = 0.854). 3*

248

Acta Biotechnol. 10 (1990) 3 Total Prophase Metaphase Telophase

5

~î—I 1

10

20 Time

Cdl

Fig. 2. Course of mitotic activity and mitotic phases in K2-81-7 callus cultures

In the interval between the 16th and 20th day of Subculture the course of mitotic activity should be expressed using the regression polynomial of the 3rd degree: y = 129.61 - 5.96« + 0.0052a;2 + 0.0020s 3

(R2 = 0.997, Iy x = 0.990).

Between the 21st and 28th day of Subculture the frequency of mitoses decreased. The course of the growth curve of callus culture Kl-107 is similar to that of K2-81-7. After the 21st day of Subculture the absolute fresh weight increases were lower but the dependence of the fresh and dry weight increases in reference to the length of subculture was similar. The dependence of fresh matter in % is expressed by the equation of the regression y = 486.762a; - 48.381a;2 - 83.286 (R2 = 0.950, Iy • x = 0.908). Fresh and dry weight of calli decreased slightly after the 35th day of subculture. The dry matter increases in absolute values in relation to the length of subculture are characterized by the equation of the regression y = 6.928 + 76.540a; — 8.467a;2 (R2 = 0.912, Iy • x = 0.918). For the dependence of percentage values of the dry matter on + b2x does not represent the length of subculture in Kl-107 the equation y = a + an appropriate mathematical model (R2 = 0.600). The mitotic activity in this culture shows an asymetrical one-peak curve (Fig. 3). The increase of mitotic activity started earlier. On the second day after transferring the calli to fresh medium, the mitotic activity was about 2% and a significant increase continued during the following few days. The highest mitotic activity was found on the 8th day and continued for 4 to 5 days. During further cultivation the mitotic activity decreased significantly. On the 21st day the values of mitotic activity were about 1% and remained at this level until the end of culture interval. The beginning of maximal mitotic activity correlates with the onset of exponential growth phase. Fresh weight of callus culture increased until the 28th day of subculture (Fig. 1), since the mitotic activity at that time significantly decreased. After the 28th day the callus culture was in stationary phase which was characterized by approximately 1% mitotic activity. The maximal mitotic activity was observed very early. Because of the small number of independent variables it cannot be expressed by a regression function. The curve started to decrease before the 14th day of subculture. The regression polynomial of the 3rd degree does not explain the course of mitotic activity between the 8th and 28th day of

CBLLABOVA,

E.,

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M. et al., Mitotic Activity and Growth

249

Total Prophase —

Metaphase Telophase

2K Vj o o

S

1 0

10

20 Time

Cdl

Fig. 3. Course of mitotic activity and mitotic phases in Kl-107 callus cultures

cultivation satisfactorily (y = 125.140 - 12.960a; + 0.581a;2 - 0.010a;3; E2 = 0.720, Iy • x = 0.899). The percentage portion of individual mitotic phases was evaluated in addition to the total mitotic activity in both cultures (Fig. 2, 3). This evaluation shows that in both cultures the prophases were prevalent (about 60%). The course of prophase in the subculture was very similar to that of total mitotic activity. Metaphases and telophases were present in very low frequency. Normal bipolar anaphases were present very rarely or were not observed at all. The frequency and morphology of prophases in callus culture K2-81-7 appeared to be normal (Fig. 2), but the metaphaSe morphology was atypical and similar to colchicine metaphases. The presence of atypical metaphase stages in various levels of ploidy (2n to 16w) (Fig. 4) and the occurence of aneuploid metaphases indicated injury which influenced the onset of the later stages.

Time

Cdl

Pig. 4. Distribution of polyploid methaphases in K2-81-7 callus cultures during subculture

250

Acta Biotechnol. 10 (1990) 3

The callus culture K2-81-7 was characterized by significant genome heterogeneity. Polyploidization was due to restitution mitoses. According to the quantitative evaluation of the frequency of individual levels of ploidy it is evident that the spontaneous polyploidization occured between the 8th and 21st day of subculture. The beginning of the occurence of restitution mitoses occured at the same time as the culture began the exponential growth phase. The highest frequency of restitution mitoses corresponded with the highest mitotic activity (Fig. 4). The karyological and genome alterations observed in callus culture suggested a dysfunction of the microtubular apparatus in callus cells. Because the nuclei were well stained it was possible to identify their morphological structure. Callus culture K2-81-7 was genetically heterogeneous and consisted of meristematic cells located mainly on the surficial regions of callus and of parenchymatous cells of various size and shape. We did not observe any organization of the cells into tissues or differentiation of organ primordia. In the callus culture Kl-107 prophase was also the prevalent mitotic phase which was detectable in mitosis. Metaphase and telophase occured less frequently and anaphases were observed very sporadically (Fig. 3). The analysis of the structure of nuclei showed variable numbers of chromocenters and an increase in the size of nuclei. Such alterations are typical for endomitosis. In contrast to the previous sample, most of chromosomes in metaphaSe cells were found in an equatorial position. For this reason it was not possible to count them exactly and to get information about the ploidy levels present in the culture. The morphology of this culture was heterogenous. The typical characteristics were cell pairs and irregular cell clusters. The texture of this culture was very friable. Studying the mitotic activity of plant cells cultured in vitro enables one to investigate the mitotic process in individual growth phases of cell and callus cultures [8]. The results obtained from the study of the first stages of callus development in Helianthus tuberosus indicate the early beginning of the exponential growth phaSe with a high degree of Synchronization of cell division [4, 13, 14]. Long-term cultivated callus cultures are often characterized as being very heterogenous cell systems depending on the pre-existing conditions and exogenous factors as well [1, 15]. In our experiment we have studied the mitotic activity in two longterm cultivated callus cultures of Matricaria recutita. The course of mitotic activity was characterized by a one-peak curve and was Specific for each culture. The average fresh weight of the K2-81-7 callus culture cultivated on RM medium with the phytohormone IAA increased 19.41-fold at the end of exponential growth. The highest dry matter content was observed on the 7th day of Subculture. The ascent of the frequency of mitoses was slow and reached a maximum on the 14th to 17th day when the dry matter content started to decrease significantly. The callus culture Kl-107 was cultivated on the baSal medium supplemented with 2,4-D. The Stimulative effect of 2,4-D, especially in higher concentrations, is widely used as a promoting factor for the initiation and growth of calli in primary as well as long-term cultures [16,17]. Nevertheless, we observed only 11.12-fold increase in average fresh weight at the end of exponential growth phase but the maximal values of mitotic index were observed between the 7th and 12th day. The early beginning of maximum mitotic activity caused its aSymetrical course. In this culture the period of maximal mitotic activity coincided with the period in which dry matter content was maximal. The increase in fresh weight expressed as a percentage of the initial weight in both cultures is characterized by the equation of the quadratic regression which prove about the Same onset of the individual growth phases. The differences in the relation between dry matter content and culture interval indicate

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251

that the fresh weight increase in callus culture K2-81-7 was not caused by cell division but rather by cell growth and cell vacuolization. The effect of length of culture interval on the course of mitotic activity is different from that of growth parameters because of the different initiation of mitotic activity. • From the cytological point of view the callus culture K2-81-7 was characterized by genome instability. The polyploidization was due to the presence of restitution mitoses retarded in metaphase. The restitution mitoses in callus and cell cultures are well-known from many experiments [5 — 7, 18]. The high frequency of polyploidy and aneuploidy and the occurence of mixoploid calli may be caused by plant growth substances and nutrient composition of the culture medium [19, 20]. An important role is also played by the genotype of the original plant [21]. The quantitative evaluation of the individual ploidy levels in K2-81-7 callus culture showed the beginning of polyploidization at the 8th day of subculture which correlated well with the values of mitotic activity. The high frequency of mitosis in Kl-107 callus culture may have been effected by the presence of 2,4-D. The position of metaphase chromosomes was similar to that in normal plant cells in situ. Acknowledgement The authors wish to thank Dr. Russell WEIGEL, Florida Institute of Technology, U.S.A. for reviewing the English manuscript and Mrs. Eva HUBOCIKA and Mrs. Terezia MEDVECOVA for excellent technical assistance. Received January 20, 1989

References [1] CIONINI, P . G., BENNICI, A., D'AMATO, F . : P r o t o p l a s m a 9 6 (1978), 101. [ 2 ] LANDA, Z., NOVAK, F . J . , OPATRNY, Z., LANDOVA, B . , PETRU, E . — I n : T h e use of tissue

culture in genetics and plant breeding. Praha, Academia, 1980.

[3] PATAU, K . , DAS, N. K . : Chromosoma 1 1 (1961), 553. [ 4 ] YEOMAN, M. N., EVANS, P . K . : Ann. B o t . 8 1 (1967), 3 2 3 . [ 5 ] DOLE2EL, J . , NOVAK, F . J . : J . P l a n t Physiol. 1 1 8 (1985), 4 2 1 .

[6] KNOSCHE, R.: Biol. Zbl. 100 (1981), 55.

[7] KNOSCHE, R., GUNTHER, G.: Biol. Zbl. 99 (1980), 311.

[8] FROLOVA, L . V., SHAMINA, Z. B . : Cytology ( U S S R ) 2 2 (1978), 5 5 1 .

[9] LINSMAIER, E. M., SKOOG, F.: Physiol. Plant. 18 (1965), 100.

[10] MURIN, A.: Stain Technol. 85 (1960), 351. [11] ECKSCHLAGER, K., HORSAK, I., KODEJS, Z. — In: The evaluation of analytical results and

methods. Praha, SNTL, 1980.

[12] BAKYTOVA, H., URGON, M., KONTSEKOVA, O. — I n : T h e fundamentels of statistics. B r a t i -

slava, Alfa, 1975.

[13] YEOMAN, M. N., DYER, A. F . , ROBERTSON, A. I . : Ann. B o t . 2 9 (1965), 265.

[14] YEOMAN, M. N., EVANS, P. K., NAIK, G. G.: Nature (London) 209 (1966), 1115. [15] NOVAK, F . J . , VYSKOT, B . : Z. Pfl. 7 5 (1975), 6 2 .

[16] KARTHA, K. K. — In: Cryopreservation of plant cells and organs. Boca Raton, CRC Press, 1985. [17] ONDREJ, M. — In: Cytogenetics and molecular genetics of plants. Praha, CSAV, 1985. [ 1 8 ] DOBEL, P . : Biol. Zb. 1 0 2 (1983), 4 3 1 .

[19] BAYLISS, M. Y. — In: VASIL, I. K. (Ed.) Perspectives in plant cell tissue culture. Int. Rev. Cytol. Suppl. IIA, 1980, 113. [ 2 0 ] N n z E K i , M., KITA, F . , TAKAHASHI, M. E . : J a p . J . Breed. 2 8 (1978), 3 0 4 . [ 2 1 ] WERSTJHN, G., DATKE, U . : Biol- Zbl. 1 0 2 (1983), 5 5 1 .

Acta Biotechnol. 10 (1990) 3, 252

Akademie-Verlag Berlin

Book Review A. EBNEB

Verzeichnis bibliographischer Datensammlungen und Datenbasen zu Grundlagen- und Anwendungsgebieten der Biotechnologie (Directory of Bibliographie Data Bases in Biotechnology) Berlin: Akad. Wiss. DDR, WissenSchftl. Inform.-Zentrum, 1989. 110 pp.

The results of an analysis of the current situation regarding bibliographic biotechnology information in the GDR have been compiled in a directory. The aim of this survey is to inform on bibliographic data bases in the field of biotechnology that are available in the GDR. This directory is the second issue of the "Biotechnology Information Service of the GDR" published by the Scientific Information Centre of the Academy of Sciences of the GDR. Together with the "Directory of Factographic Data Bases in Biotechnology", the first issue of the "Biotechnology Information Service of the GDR", this directory gives an overview on data bases relevant to basic and applied fields of biotechnology and available in the GDR. These directories are obtained from : Scientific Information Centre of the Academy of Sciences of the GDR, Schiffbauerdamm 19, DDR-1040 Berlin E . POETZSCH

Acta Biotechnol. 10 (1990) 3, 2 5 3 - 2 6 0

Akademie-Verlag Berlin

Anwendung von Methoden der Mustererkennung bei mikrobiell hergestellten Trockenproduktchargen N O E T Z E L , R . , SCHULZ, P .

Akademie der Landwirtschaftswissenschaften der DDR Institut für Biotechnologie Potsdam Abteilung Eberswalde Lichterfelder Wassertorbrücke Eberswalde -Finow 1300, DDR

Summary The application of methods ol Pattern Recognition makes it possible to classify dehydrated SCPproducts which were produced under various conditions of production by a low number of signs. These mathematical methods not only allow to draw technological conclusions but also to decide on correctness of the way to produce suitable SCP-products for feeding of monogastric animal species. There are a large number of possibilities to use these methods of Pattern Recognition in the field of biotechnological research.

Einführung Zur zusätzlichen Gewinnung von Eiweißfutterätoffen aus einheimischen Rohstoffen und Nebenprodukten wurden Fermentations versuche durchgeführt. Die Beurteilung der Produktmuster aus diesen Versuchen erfolgte hinsichtlich ihrer wertbestimmenden Inhaltsstoffe und auch möglicher Schadstoffe. Ferner wurde in Stickstoffbilanzversuchen an Ratten geprüft, ob das Produktmuster als eine potentielle Eiweißquelle für die Ernährung monogastrider Tierarten geeignet ist. Die Ergebnisse zeigten, daß einzelne Chargen aussichtsreich schienen, andere aber völlig ungeeignet waren. Aus diesem Grunde wurde zu geschlossenen, unsterilen Fermentationsverfahren übergegangen, um die Vorteile hinsichtlich wählbarer konstanter Milieubedingungen, Prozeßkontrolle sowie kontinuierlicher Kultivierung zu nutzen und die Ergebnisse damit reproduzierbar zu machen. Ein schwieriges Problem dabei ist die Einordnung von nach unterschiedlichen HerStellungSvarianten gewonnenen Trockenproduktchargen in eine eng umgrenzte Produktgruppe, um eine hinreichend genaue Aussage Sowohl im Sinne der Einordnung als auch des technologisch begründbaren Rückschlusses zu ermöglichen. Eine Sehr leistungsfähige Gruppe von mathematischen Verfahren zur Lösung derartiger Probleme sind die Methoden der Mustererkennung (Pattern Recognition). Wenn davon ausgegangen wird, daß jedes gewonnene mikrobielle Trockenprodukt ein Objekt darstellt, welches durch die Ergebnisse der chemischen Analyse bzw. Ergebnisse der Fütterungsversuche an Ratten ein bestimmtes Merkmalsmuster besitzt, so können diese Objekte nach ihren Mustern eingeordnet werden. Nach H E N E I O K u. a. [ 1 ] wird davon ausgegangen, daß verschiedene KlaSSen von Objekten Sich in verschiedenen Klassen von Objektmustern widerspiegeln und die Erkennung von Objektklassen bzw. -kategorien somit auf die Erkennung von Musterklassen zurückgeführt werden kann..

254

Acta Biotechnol. 10 (1990) 3

Damit wird die Existenz eines Zusammenhangs zwischen den direkter Beobachtung leicht zugänglichen Merkmalen und dem Merkmal, zu einer bestimmten Objektklasse zu gehören, unterstellt. Die Stärke der Mustererkennungsverfahren besteht darin, daß dieser möglicherweise Sehr komplizierte Zusammenhang Selbst nicht bekannt zu sein braucht, Sein Vorhandensein jedoch zu einer Vorhersage der eigentlich interessierenden Objekteigenschaft aus eigentlich nicht interessierenden, jedoch leicht beobachtbaren Eigenschaften, dem Objektmuster, genutzt wird. Beispiele der Anwendung der Methoden der Mustererkennung sind aus der analytischen Chemie [2] und aus der Wasserwirtschaft [3] bekannt.

Mathematische Grundlagen Aufstellung

der

Datensätze

Zielgrößen der durchgeführten Mustererkennungsanalysen waren: a) Aussagen zur Einordnung der mikrobiellen Trockenprodukte in Gruppen mit großen Gemeinsamkeiten b) Rückschlüsse zum Einfluß von HerStellungävarianten auf die Produktqualität. Für die unter a) zu erreichende Zielgröße wurden 18 Trockenproduktchargen, die nach unterschiedlichen technologischen Varianten produziert wurden, aus einem Untersuchungszeitraum zwischen 1979 und 1986 ausgewählt. Die Charakterisierung der Produkte erfolgte nach qualitativ gleichen Merkmalen. Es wurden drei Datensätze aufgestellt und verrechnet: — Datensatz D l : 36 Objekte (Summe der Objekte aus den Datensätzen D2 und D3) Bei den Objekten 19 bis 36 handelt es sich um die Produkte aus dem dritten Datensatz. — Datensatz D2: 18 Objekte (Merkmale: Rohprotein-, Rohasche- und Lysingehalt, Stickstoffeinnahme, Stickstoffbilanz, Lebendmassezunahme, biologische Wertigkeit, scheinbare Verdaulichkeit). — Datensatz D3: 18 Objekte (Merkmale: wie D2, ergänzt wurde hier die Futterration der Ratten durch eine Methioninzugabe in Höhe von 3% des Rohproteins). Der Datensatz D2 Stellt Somit den eigentlich relevanten Datensatz dar. Beim Datensatz D3 wurde durch die Methioninzugabe eine Modifizierung vorgenommen, um durch eine Pärallelrechnung der Datensätze eine Bestätigung der Ergebnisse des zweiten Datensatzes zu erhalten oder bei größeren Abweichungen Anhaltspunkte für eine Neuformierung der betrachteten Merkmale zu gewinnen. Um den Punkt b) abarbeiten zu können, war die Aufstellung eines „technologischen" Datensatzes erforderlich. Dabei handelt es sich um eine zusammenfassende Kennzeichnung der Herstellungsbedingungen für die 18 Trockenproduktchargen. Um Doppeldarstellungen zu vermeiden, sind Angaben zur Art und Weise der Herstellung ausgewählter Trockenproduktchargen den zusammengefaßten Ergebnissen der Mustererkennungsverfahren in Tab. 1 zu entnehmen. Folgende Faktoren wurden berücksichtigt: 1. Herkunft des Substrates (Orte: A — Eberswalde, B — Leipzig, C — Dresden) 2. Vorbehandlung des Substrates — Belüftung — Abtrennung der Feststoffe durch Zentrifugation

NOETZEL, R., SCHULZ, P., Mustererkennung bei Trockenproduktchargen

y

255

3. Fermentationsbedingungen — Einsatz einer Starterkultur bei dei Fermentation — Temperatur (30 °C und 50 °C) — Fermentortyp (Druckstrahler, offenes Ortbetonbecken mit Saugkreisel) — Biomasserückführung — Arbeitsvolumen (501, > 50001) 4. Aufarbeitung des Produkts — Konzentration der Biomasse (Zentrifugation, Flotation) — Produktwaschung durch Säure 5. Trocknungsart — Vakuumgefriertrocknung — Sprühtrocknung — Trockenschrank — Walzentrocknung. Methoden der Mustererkennung Aus der Literatur sind verschiedene Verfahren der Mustererkennung bekannt. Man kann die Verfahren in folgende drei Gruppen einteilen: 1. Display-Verfahren (Hauptkomponentenanalyse, Nonlinear Mapping) 2. hierarchische Verfahren der Mustererkennung (Average-, Single- und Complete Linkage) 3. nichthierarchische Verfahren der Mustererkennung (z.B. Potentialverfahren CLUPO T) Aus jeder dieser drei Gruppen wurde ein Verfahren der Mustererkennung ausgewählt, um Unsicherheiten bei der Darstellung und Interpretation der Ergebnisse auszuschließen und Erfahrungen beim Umgang mit derartigen Methoden hinsichtlich der formulierten Zielgrößen zu sammeln. Zur Anwendung kamen die Hauptkomponentenanalyse und die Mustererkennungsverfahren Average Linkage und CLUPOT. Die Darstellung der Ergebnisse erfolgte grafisch in Displayform bei der Hauptkomponentenanalyse bzw. in Form von Dendrogrammen bei Average Linkage und CLUPOT. Die für die Analyse notwendigen Rechnerprogramme wurden in der Programmiersprache BASIC von H E N E I O N u . a . (1985-•• 1987) erarbeitet. Genaue mathematische Erläuterungen können bei H E N R I O N U. a. [4] nachgelesen werden. Als Rechner fanden ein Personalcomputer PC 1715 und ein Homecomputer Sinclair ZX spectrum + Anwendung. Ergebnisse und Diskussion Hauptkomponentenanalyse

der drei Datensätze

Bei der Auswertung einer Hauptkomponentenanalyse tritt durch die Reduzierung der Darstellungsdimensionen ein Informationsverlust auf. Obwohl dieser Verlust durch eine linearkombinatorische Berechnung der reduzierten Abbildungsdimensionen minimiert wird, kommt es insbesondere bei einer größeren Anzahl von Merkmalen zu beachtenswerten Informationsverlusten. So betrug bei der Verrechnung des zweiten Datensatzes der Varianzanteil der ersten beiden Hauptkomponenten 70%. Der Anteil der dritten Hauptkomponente lag bei 14%. I n diesem Fall wäre eine dreidimensionale Darstellung

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dem zweidimensionalen Display vorzuziehen. Aus Gründen der Anschaulichkeit des Flächendisplays wurde darauf verzichtet. Deutlich läßt sich eine Klassifizierung der 18 Trockenproduktchargen in fünf Gruppen erkennen (Abb. 1). Die Chargen 16 und 17, Produkte aus vor- und nachbehandlungslosen Fermentationsvarianten, ordneten Sich nachfolgend in die Gruppe I I des dargestellten Displays Haupkomponentenanalyse ein. Eine weitere Zuordnung des Objektes 13 erfolgte nicht. Die Verrechnung des ersten Datensatzes mit seinen 36 Objekten bestätigte im wesentlichen die Gruppierungen der einzelnen Trockenproduktchargen. Der Varianzanteil der ersten beiden Hauptkomponenten betrug 72%. Auf die dritte Hauptkomponente entfielen 11%.

Abb. 1. Display der Objektgruppierung des Datensatzes D2 (18 TrockenproduktChargen aus dem Untersuchungszeitraum 1979---1986) im Ergebnis der Hauptkomponentenanalyse

I m Verlauf der weiteren Berechnungen, die an dieser Stelle aber nicht im vollen Umfang wiedergegeben werden können, änderte sich die Gruppenzugehörigkeit einzelner Objekte. So ist die spätere Zuordnung der Objekte 4 und 12 (zwei Trockenproduktchargen aus Fermentationen in Druckstrahlfermentern) in die große Gruppe der Produktchargen 1, 2, 3, 5, 6, 7, 14 und 18, die ebenfalls unter geschlossenen, unsterilen Fermentationsbedingungen hergestellt wurden, als Zeichen f ü r bestehende Gemeinsamkeiten zu werten. Clusteranaly.se nach dem Average-Linlcage- Verfahren Als Verfahren zur Mustererkennung oder als Clusteranalyseverfahren werden Methoden bezeichnet, die Objekte mit gruppentypischen Merkmalen einander zuordnen. Diese durch gemeinsame typische Merkmale gekennzeichneten Gruppen werden Cluster genannt. Dabei ist es das Ziel der Clusterung Objekte so zu gruppieren, daß Sie innerhalb der Gruppe ähnliche, zwischen den Gruppen aber möglichst unterschiedliche Muster aufweisen. Hierarchische Clusterungen erzeugen eine Folge von Partitionen P x bis P n . Eine Partition ist hierbei eine Menge von paarweise disjunkten Teilmengen. Ausgehend von der kleinsten Partition P1 wird durch die Zusammenfassung zweier Objekte die nächstgröbere Partition P 2 erreicht. Nach n — 1 Durchrechnungen wird so schrittweise die Gesamtmenge erreicht.

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Das Average Linkage ist somit ein agglomerativ verlaufendes Verfahren mit der im speziellen Fall erfolgten Berechnung der mittleren Euklidischen Abstände der Objekt voneinander. Das Dendrogramm des zweiten Datensatzes ist in der Abb. 2 dargestellt. Die Auswertung wurde so vorgenommen, daß die Clusterbildung auf zwei Ähnlichkeitsniveaus erfolgte. E s k a n n eingeschätzt werden, daß die Bildung der Einzelcluster recht gleichmäßig die gesamte Breite der Ähnlichkeitsverteilung erfaßte. Dabei sollten die

1

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Abb. 2. Dendrogramm der Ergebnisse einer hierarchischen Clusterung der Objekte des Datensatzes D2 nach dem Average-Linkage-Verfahren

Ähnlichkeitsgrenzen als fließend angesehen werden, obwohl die Objekte sich einzeln zuordnen lassen. Auffällig bei den folgenden Verrechnungen war der'Wechsel des Objektes 4 in Verbindung mit dem Objekt 12 und wiederum von Objekt 12 mit dem Objekt 18. Wird in diesem Zusammenhang noch einmal die Hauptkomponentenanalyse betrachtet, so zeigt sich, daß die Objekte 18 und 4 auch dort zum Objekt 12 passen. Da die im Datensatz D 3 dargestellten Trockenproduktchargen bei der Verfütterung an R a t t e n durch eine Methioninzugabe aufgewertet wurden, wird deutlich, daß das Objekt 4 ohne Methioninzugabe zwischen zwei Qualitäten steht und mit der Zugabe in eine Gruppe springt, die als stabiles Cluster bezeichnet werden k a n n sowie aussichtsreiche Trockenproduktchargen repräsentiert.

Clusteranalyse nach dem

GLUPOT-Verfahren

Bei den nichthierarchischen Verfahren wird im Gegensatz zu den hierarchischen Clusterungsverfahren f ü r jedes Ähnlichkeitsniveau eine völlige Neuberechnung vorgenommen. Daraus ergeben sich Überschneidungen mit Clustern auf kleineren Berechnungsniveaus. Demzufolge ist es möglich, daß Objekte Cluster verlassen und zu schon bestehenden zugeordnet werden. Die Ermittlung von „springenden" Objekten und die damit verbundene Feststellung nichtstabiler Cluster ist ein Vorteil der nichthierarchischen Verfahren gegenüber den hierarchischen. Aus den stabilen Clustern lassen sich ebenfalls Dendrogramme aufstellen.

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Acta Biotechnol. 10 (1990) 3

Die Berechnungen ergaben, daß im ersten und dritten Datensatz keine „springenden" Objekte vorhanden waren, im zweiten Datensatz aber Schwankungen auftraten. Diese liegen besonders im'Bereich der größeren Ähnlichkeitskennzahlen. Als Yergleichsebene diente auch hier ein gleichmäßiges Ähnlichkeitsniveau bei allen drei Datensätzen zur CluSterfestlegung (Abb. 3). Das Klassifizierungsergebnis des zweiten Datensatzes ordnet sich aber in die Reihe der Ergebnisse der HauptkomponentenanalySe und des AverageLinkage-Verfahrens zur Mustererkennung ein. 2

18 16 17

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11 10 15 13 9

Abb. 3. Dendrogramm der Ergebnisse einer nichthierarchischen CluSterung der Objekte des Datensatzes D2 und dem CLUPOT-Verfahren

Einschätzung der Ergebnisse Die Tab. 1 macht deutlich, daß die betrachteten 18 Trockenproduktchargen durch die angewendeten Mustererkennungsverfahren in zwei große Gruppen aufgeteilt wurden. In der ersten Gruppe finden sich alle Produkte mit gesonderter Behandlung wie Vorbehandlung' (Belüftung, Zentrifugation), Fermentation mit Starterkultur, thermophile Fermentation, Fermentation mit Druckstrahlfermentoren und Säurewäsche als Nachbehandlung wieder. In der zweiten Gruppe vereinen sich solche Varianten wie die Fermentation in offenen Becken, vor- und nachbehandlungslose Technologie und auch augenscheinlich weniger geeignete Trocknungsverfahren (Walzentrocknung unter atmosphärischen Bedingungen, Trocknung im Trockenschrank, Sprühtrocknung). Aus den Ergebnissen der Klassifizierung kann weiter abgeleitet werden, daß eine Vorbehandlung generell Einfluß auf eine höhere Qualität der mikrobiellen Trockenproduktchargen ausübt. Dabei konnte nicht geklärt werden, ob Belüftung oder Feststoffentlastung oder beide gemeinsam die Produktqualität positiv beeinflußt haben. Deutlich wird der Einfluß bei der Verwendung einer Starterkultur, bei der thermophilen Fermentation und der Sauren Wäsche der Produkte, wobei additive Effekte bei gleichzeitiger Anwendung der genannten Verfahren nicht geklärt werden konnten. Die Verwendung eines einheitlichen Fermentortyps bei der Herstellung der Produktchargen ist als entscheidender Beitrag für eine Klassifizierung im Hinblick auf die Reproduzierbarkeit des Ergebnisses anzusehen.

Noetzel,

E.,

Schulz,

P., Mustererkennung bei Trockenproduktchargen I addnjQ

H

fe fe ^fePfe fe -P P fe P bciS tí '0) © © © © © © ï tí o

260

Acta Biotechnol. 10 (1990) 3

Die Vakuumgefriertrocknung ließ keine Beeinflussung der Produktqualität erkennen. Zusammenfassend kann gesagt werden, daß mit den dargestellten Verfahren zur Mustererkennung bei unterschiedlichen mikrobiellen Trockenproduktchargen mathematische Methoden Anwendung fanden, die einen wesentlichen Beitrag zur qualitativen Beurteilung angewendeter Herstellungsvarianten leisten und damit die Möglichkeit bieten, gezielt technologische Schlußfolgerungen ziehen zu können. Mittels dieser Methoden kann zur Entscheidungsfindung über die Richtigkeit eines eingeschlagenen Weges zur Gewinnung geeigneter Trockenprodukte wesentlich beigetragen werden. Für die Anwendung der methematischen Methoden zur Mustererkennung ergeben sich große Anwendungsmöglichkeiten im Bereich der biotechnologischen Forschung. Eingegangen: 21. 7. 1988 Überarbeitet: 8. 12. 1988

Literatur [1] HENRIOST, A.: Zeitschrift für Chemie 27 (1987), 56. [ 2 ] HENRION, G . : C h e m i s c h e T e c h n i k 3 8 ( 1 9 8 6 ) , 5 2 5 . [ 3 ] HEININOER, P . : C h e m i s c h e T e c h n i k 3 8 ( 1 9 8 6 ) , 4 0 0 .

[4] HENRION, G.: Beispiele zur Datenanalyse mit BASIC-Programmen. Berlin, Verlag der Wissenschaften, 1988. [5] ANDERBEBG, M. R.: Cluster Analysis for Applications, Academic Press, New York, 1973. [6] DEICHSEL, G. U. TRAMPISCH, H. J.: Clusteranalyse und Diskriminanzanalyse, G. Fischer Verlag, Stuttgart 1985.

Acta Biotechnol. 10 (1990) 3, 261—269

Akademie-Verlag Berlin

Purification and Immobilization of the Inducible Form of Extracellular Laccase of the Fungus Trametes versicolor ROGALSKI, J . 1 , DAWIDOWICZ, A . L . 2 , LEONOWICZ, A . 1 *

1

2

M. Curie Sklodowska-University Department of Biochemistry M. Curie-Sklodowska Sq. 3, 20-031 Lublin, POLAND M. Curie-Sklodowska University Department of Chemical Physics M. Curie-Sklodowska Sq. 3, 20-031 Lublin, POLAND

Summary The inducible form of extracellular laccase (EC 1.10.3.2) produced by Trametes versicolor culture in a 10 1 glass fermenter was isolated and purified by ion-exchange chromatography on the DEAESephadex A-50 column and by affinity chromatography on a syringyl-CPG column. The enzyme was further immobilized on functionalized porous glass. The paper also discusses some properties of immobilized and free enzyme, respectively. Introduction Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) is a multiple enzyme inducible in fungi b y various phenolic compounds [1—4], cycloheximide [5] or 2,5 xylidine [6]. I t has been shown that the enzyme occurs both in inducible and constitutive forms [1, 3, 4, 7 — 10] and t h a t the inducible forms are much more active towards t h e phenolic compounds toxic for fungi [1, 4, 9, 11]. The aim of the presented work was isolation and m a x i m u m purification of the inducible form of extracellular laccase from Trametes versicolor, also its immobilization, and comparison of the activity and some properties of the immobilized enzyme with those of a free one. Materials and Methods Enzyme Source and

Activity

The strains Trametes versicolor (L. ex Fr) Pil No 7, Fomes fomentarius (Linnaeus) Pries No 94, Fomes annosus (Fries) Cooke No 89 and Rhizoctonia praticola (Saksena ex Vaartaja) No 99 coming from our collection were maintained at 4°C on the agar slants [1]. The laccase activity was detected colorimetrically with syringaldazine as a substrate [12], The activity was calculated in the international units from the following formula: A =

DA*, . J_„ 5 x F x 10" 225 nkat/1 ES x Dt xv

and the specific activity was expressed by the relationship: ^spec = nkat/p * To whom correspondence should be addressed. 4

Acta Biotechnol. 10 (1990) 3

262

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where DA = the increase in absorbance at 525 nm, V = the total reaction volume (ml),

ES = 65000 (the molecular adsorption coefficient according to HARBIN and OBST (13]), Dt = the

reaction time in seconds, v = the enzyme volume (ml), p = mg protein. The activity of immobilized laccase was assayed by a polorographic analysis with a CLARK oxygen electrode provided with a linear TZ 4100 recorder (Praha, Czechoslovakia). In this one the incubation mixture contained in 0.1 M citrate-phosphate buffer 0.6 FXM of phenolic substrate and such a quantity of immobilized enzyme, which was necessary to utilize 1 ¡¿mol of oxygen per minute at optimum pH with syringaldazine as a substrate. The activity was expressed in nmoles of oxygen consumed per minute by each substrate. Culture Conditions Liquid LINDEBEBG medium [14] was dispensed in 300 ml ERLENMAYER flasks (100 ml each) and sterilized at 0.075 MPa for 30 min. The medium was inoculated and the submerged cultures were run at 28 °C on a rotary shaker at 220 rpm. After 7days of growth (time fo]ind as optimal) the cultures were transferred as inoculum (10% of total volume) into a 10 1 glass-fermenter filled with 6 1 of the LINDEBERG medium sterilized as above. The aerated (6 1 air min -1 ) and stirred (350—400 rpm) fermenter culture was run 11 days at 28 °C. Occasionally the antifoam B emulsion (SIGMA, St. Louis, Missouri, USA) was used for breaking the foam. To Stimulate the production of laccase, 2,5-xylidine (SIGMA) in 50% ethanol was added as an inducer to the concentration of 0.2 MU. Isolation of Proteins In order to remove the mycelium from the growing medium the suspension from reactor was filtered through the MIRACLOTH quick filtration material (Chicopee Mills, Inc., New York, USA; distributed by CALBIOCHEM). For the best concentrating the filtrate proteins three ways were chosen: first precipitation by various agents such as ammonium sulphate in various saturation ranges, ethanol, acetone or a mixture of ethanol and 3% acetone, second-rotary evaporation at + 48 °C and third-ultrafiltration by using a TCF-10 cell and a UM-10 membrane (AMICON, Oosterhout, Holland). The content of protein was determined according to LOWBY et al. [15]. The protein in the fractions of the column effluents was measured by light absorbance at 280 nm. Preparation of CPQ Support The controlled porous glass was prepared from Vycor glass (composed of 7 mol% Na 2 0, 23 mol% B 2 O a and 70 mol% Si0 2 ) by thermal treatment and a leaching process described elsewhere [16—17]. Support Activation Glass supports were activated by y-aminopropyltriethoxysilane (y-APTES) [18—19]. In order to shift the reaction equilibrium in the direction of APTES-CPG formation and to obtain the maximum coverage density, the ethanol formed simultaneously during the reaction was removed from the reaction mixture according to the method of ROGALSKI and DAWIDOWTCZ [20]. The activated APTES-CPG support was further used both for affinity chromatography of laccase (after coupling with a ligand) and for enzyme immobilization (after activation with glutaraldehyde). Coupling of Idgand for Affinity Chromatography The bonding procedure of the laccase substrate (syringylaldehyde) to activated APTES-CPG was as follows: 5 g of APTES-CPG were mixed at 20°C on a rotary shaker with 100 mg of syringylaldehyde and 40 ml water [21]. The constant pH level (pH 10) was maintained by successive addition of 1 M NaOH. The SHIFF'S bases formed were reduced according to the method of RYAN and FORTELL [22]. To saturate the primary amino groups on the support surface the last procedure was. repeated three times and the final product was washed with 100 ml of 0.2 M acetic acid and subsequently with 100 ml of 0.1 M phosphate buffer pH 5.2.

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ROGALSKI, J . , DAWIDOWICZ, A. L . e t al., Extracellular Laccase

Purification of Laccase The culture filtrate containing laccase activity was saturated with ammonium sulphate a t the saturation range 0—0.8 and centrifuged (15 min, 15000 X g). The precipitat was dissolved in 0.01 M phosphate buffer p H 6.0 and desalted on a Sephadex G-50 column. The fractions showing the highest laccase activity were applied onto a DEAE-Sephadex A-50 column (5 X 25 cm), equilibrated with 0.1 M phosphate buffer, pH 6.0. The enzyme preparation was eluted from the column with a linear concentration gradient of 0.1—0.5 M phosphate buffer, pH 6.0. The fractions containing the inducible form of laccase were detected according to LEONOWICZ et al. [11] and pooled, dialysed against 0.1 M phosphate buffer pH 6.0 and applied onto a syringyl-CPG column (0.7 X 3 cm) equilibrated with the same buffer. The fractions bonded with the carrier were removed by using 0.5 M (NH 4 ) 2 S0 4 . These fractions were further desalated on a Sephadex G-50 column and kept at -f 4°C or lyophylized.

Polyacrylamide-gel Electrophoresis Electrophoresis was performed in the TRIS/borate system, p H 8.45, protein bands were visualized with Coomassie Blue, and laccase, after adjustment of pH in the gels to 4.5, by the reaction with p-phenylenediamine according to LEONOWICZ et al. [11].

Immobilization of Laccase The enzyme was coupled to APTES-CPG using glutaraldehyde (SIGMA) according to the method by LAPPI et al. [19]. In this case 5 mg of laccase protein and 5 g of support were used. Finally the preparate contained ca. 1 milligram of protein per gram glass.

Results and Discussion In order to select the best producer of extracellular laccase some strains from our Department collection known from their ability to laccase formation were grown in shaken cultures for 10 days and then the laccase activity was stimulated with 2,5-xylidine. The results of laccase production by these fungi and the enzyme inducibility by 2,5-xylidine are shown in Tab. 1. As it can be seen Trametes versicolor among other investigated fungi is the best producer of extracellular laccase and also in this fungus laccaSe is stimulated by 2.5-xylidine to the largest extent. For this reason Trametes versicolor was subjected to further investigations. The optimal period after which the culture of this fungus ought to be induced is 8 —10 days. Then mycelium reached its Stationary growing phase and glucose level lowered below 30% of its initial amount (Fig. 1). The culture filtrate prepared at this time served as a source of laccase for further investigations. At the beginning of the next step, the methods of protein isolation and concentration from the culture fluid were applied. Laccase was precipitated either,by ammonium Tab. 1. Extracellular laccase activity of the selected strains growing on LINDEBEBG medium After 10 day shaken culture (for details see Materials and Methods). Fungal strains

Trametes versicolor Fomes fomentarius Fomes annosus Rhizoctonia praticola, 4*

Laccase activity [U/ml]

p H optimum for

Non induced culture

2,5 xylidine

laccase aotlvlt y

5.1 0.9 1.1 0.3

15.2 6.9 9.8 0.3

4.0 4.3 4.6 7.2

264

Acta Biotechnol. 10 (1990) 3

Fig. 1. The fermenter culture of Trametes versicolor. Conditions as described in Materials and Methods. 1 mycelium growth, 2 glucose content in the culture, 3 laccase activity, 4 pH

sulphate or by organic solvents (see Materials and Methods) in various rations to the culture fluid volume or it was concentrated by ultrafiltration. The results presented in Tab. 2 show that the ammonium sulphate saturation (0—0.8) is the best way both for concentrating protein and laccase activity. Thus this method was used as preliminary step of laccase purification (see Tab. 3). The inducible form of Trametes versicolor laccase obtained from the precipitated proteins (after desalting on the Sephadex G-50 column and ion exchange chromatography on the DEAE-Sephadex column as described earlier by LEONOWICZ et al. [11]) was finally highly purified by affinity chromatography on the syringyl-CPG column. In the latter case, 0.1 M phosphate buffer pH 6 as a Starting eluent was employed. The laccase was removed from the column by 0.5 M ammonium sulphate Solution in starting buffer (Fig. 2) according to the procedure described elsewhere [4]. The fractions exhibiting laccase activity were pooled and desalted on the Sephadex G-25 column. In order to check the homogeneity of the obtained enzyme fractions, they were controlled electrophoretically according to ref. [11]. As it can be seen from Fig. 3, the enzyme preparation is not to Split off at the conditions of disc electrophoresis (a single protein band exhibiting laccase activity). It is very stable, especially after lyophilization. Storing for a year at — 20 °C does not cause any loss of activity. The results obtained in the successive steps of the purification are given in Tab. 3. As it can be seen laccase has been purified about 400-fold; Such a high degree of purification of extracellular laccase from Trametes versicolor had not been achieved previously. Affinity chromatography for purification of laccase from Neurospora crassa was first applied by FROEHNER and ERIKSSON [5]. In this case the ConA-Sepharose column was

Rogalski, J., Dawidowicz, A.

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L. et al., Extracellular Lacease

Tab. 2. Thé influence of various methods of precipitation and concentration of Trametes versicolor culture fluid on laccase activity Method

Protein in relation to the initial content [ % ]

Laccase activity [U/ml]

Laccase activity in relation to the initial content [ % ]

Precipitation by ethanol in concentration: 0.25 25% 33% 0.16 50% 0.05

39.0 24.0 7.2

14.5 11.8 6.9

27.2 22.0 13.0

Precipitation by mixture of ethanol and 3 % acetone in concentration: 0.36 25% 33% 0.19 0.10 50%

58.0 38.5 18.7

28.8 19.9 14.5

58.1 46.4 33.7

Precipitation by acetone in concentration: 0.47 25% 33% 0.20 0.10 50%

61.0 42.5 18.7

25.5 22.8 18.7

36.6 60.1 43.3

Precipitation by ammoniumsulphate in the Saturation range: 0 -0.4 0.4-0.8 0.8-1.0

0.25 0.57 0.29

15.5 64.5 18.3

17.0 56.0 3.0

13.1 77.4 2.3

Ultrafiltration a t : 1° 11° Evaporation Lyophylization* Crude culture fluid

0.01 0.9 0.78 1.06 0.08

10.4 86.5 97.5 99.0 100.0

0.2 74.0 10.0 68.0 6.5

2.2 85.0 29.4 75.9 100.0

Protein concentration [mg/ml]

* The data obtained after dissolving of maximum lyophylized preparate in 1 ml of water. Tab. 3. Isolation and purification of the inducible extracellular laccase form from Trametes versicolor Purification step

Total protein [mg]

Specific activity [U/mg protein]

Culture fluid Ammonium sulphate in range 0—0.8 DEAE-Sephadex A-50 column chromatography Syringyl-CPG column chromatography

1100.0

0.9

1.0

100.0

520.0

1.9

2.2

94.6

1.5

97.5

112.1

17.1

0.1

350.8

402.0

3.3

Purification [-fold]

Yield [%]

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Acta Biotechnol. 10 (1990) 3

20 Fraction

30 number

Fig. 2. The affinity chromatography of laccase on the syringyl-CPG column. The experimental conditions were the same as described in the text. • absorbance, •— laccase activity A

B

^

Fig. 3. Polyacrylamide gel electrophoresis of the purified laccase preparation. Gel electrophoresis was performed by the procedure as described in Materials and Methods. A protein, B laccase activity

used and the degree of the final enzyme purification was about 100-fold. Rather poor results could be explained by a non-specific complexing of laccase with concanavalin through a-glycosidic groups of the carbohydrate component of the laccase protein. Since, in addition to laccase, many other glycoproteins occur in fungi, this procedure results only in separation of glycoproteins from other proteins. Much better results were obtained in our laboratory, when for the first time the ligand — syringylaldehyde specific for laccase was attached to the polysaccharide matrix [4]. In that case, the syririgylAH-Sepharose 4 B column was used for purification of constitutive and inducible forms of Pholiota mutabilis intracellular laccase. In the present work glass matrix used as a support for laccase-specific ligand guarantees not only high purification of the enzyme, but also reproducibility of the affinity column which is very important from both biotechnological and economical points of view.

ROGALSKI,

J.,

DAWIDOWICZ,

A. L. et al., Extracellular Laccase

267

The laccase preparation obtained after affinity chromatography was further immobilized on y-APTES-CPG support. As a result of the bonding procedure 90.6% of protein and 89.2% of laccase activity were coupled to the Support. Fig. 4 shows the pH and temperature profiles for free and immobilized enzymes. I t can be Seen, that there exists a small shift of optimum pH for the immobilized enzyme toward less acidic region compared to the free enzyme (from pH 4.0 to 4.3). Such shifts (explained by partitioning protons in the active centre of the enzyme affected by ionized active groups of the Support [23]) had been recorded previously for various immobilized enzymes [24], The immobilized enzyme is characterized by higher Stability in pH range 3.0—7.0. The temperature optima for immobilized and native forms are similar and equal to 55 °C. Besides, the immobilized enzyme is a little more thermally Stable in the temperature range + 4 to + 6 5 °C.

Pig. 4. Effect of pH (A) and temperature (B) on the activity of the purified laccase preparations The experimental conditions as described in the text. ^ laccase immobilized, • native enzyme

The effects of the Storage time at various temperatures on the activity of free and immobilized forms of laccase are presented in Tab. 4. The results point to higher stability of the immobilized preparation. Even at 30 °C about 9 % of the enzyme initial activity is preserved after 1 year storage. However, the most convenient storage temperature for the immobilized preparation is + 4 ° C . Under these conditions the immobilized form preserved about 100% of its activity after 2 years. The influence of the enzyme Storage at —20 °C is almost the same for the immobilized and native forms. About 10% drop of activity is observed in both caSeS just after first freezing. That suggests the significant influence of freezing and thawing on the activity of both forms. The data in Tab. 5 refer to the activity losses of the immobilized and native laccases after successive freezing and thawing. The destructive role of freezing and thawing processes is particularly oovious in the case of immobilized laccase. After 10-time repeated operations, the final product exhibits about 10% of its initial activity. Probably it is due to breaking of the peptide chains in the immobilized enzyme chemically bonded with the Support surface. High strains occur in these chains during water-ice phase transition as a result of different thermal expansion coefficients. Fungal laccase occuring commonly in wood-degrading species is involved into important natural processes; e.g. lignin degradation [25] and humuS synthesis [26]. Especially active in these processes is the inducible form of enzyme [11]. LaccaSe has also various

268

Acta Biotechnol. 10 (1990) 3 Tab. 4. The stability of purified laccase preparations preserved at various temperatures Time of preservation [days]

Laccase free [% of activity after preservation at °C]

Laccase immobilized [% of activity after preservation at °C]

-20

+4

+30

-20

+4

+30

0 1 12 22 34 42 63 180 360 720

100.0 92.3 92.2 91.8 91.8 91.2 91.0 91.0 89.9 89.9

100.0 100.0 100 0 100.0 87.9 76.6 51.2 40.2 0.1 0.0

100.0 81.7 38.3 11.7 1.6 0.2 0.0 0.0 0.0 0.0

100.0 90.2 90.0 90.0 90.0 89.9 89.9 89.9 89.9 89.8

100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0

100.0 100.0 100.0 100.0 80.2 65.9 55.4 45.2 9.1 0.0

Tab. 5. The influence of freezing and thawing (in the temperature range —20 °C to + 2 0 °C) on the activity of laccase preparations Number of freezing and thawing ° processes

Laccase activity remained [%] • 77T " free immobilized

0 1 2 3 4 5 6 7 8 9 10

100.0 91.0 89.2 79.6 72.6 70.0 68.2 64.5 50.1 46.2 39.2

100.0 89.9 89.9 76.3 70.0 62.8 54.6 41.7 36.2 24.1 11.8

applications, among them the ability to detoxify the aquatic waters seems to be important [27]. The enzyme isolated from Trametes versicolor by various authors has been previously immobilized by binding to various supports, such aS ConcanavalinA-SepharoSe [5], agar, polyacrylamide and methacrylate gels [28], polyacrylonitrile [29] or celite [30]. These supports however, are not suitable for large Scale operations due to their chemical and mechanical instability and low resistance against a microbial attack. Controlled porosity glasses used in this work are much more stable and resistant as support itself and in addition, they can be sterilized and regenerated [24]. Consequently, we have demonstrated a convenient method for relatively high purification and immobilization of the inducible form of Trametes versicolor extracellular laccase. In both cases we have used the same, home-made and very cheep glass beads of controlled porosity. High activity and considerable Stability seem to lend hope to the possible future usage of the enzyme preparation for detoxification of the environment polluted by phenolic derivatives of natural and industrial origins as well aS the transformation of xenobiotics to humic materials.

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L. et al., Extracellular Lacease

Acknowledgements The authors wish to thank Miss Grazyna MOBAWSKA for her skillful technical assistance. This work was supported by the Polish Scientific Projets Nos. CPBR 3.13.2.1.18, CPBP 04.11 2.33 and CPBP 04.02/2.2. Received September 29, 1988 Revised April 7, 1989

References [ 1 ] BOLLAG, J.-M., LEONOWICZ, A.: Appl. [ 2 ] G I G I , O . , MARBACH, I . , M A Y E R , A . M . :

Environ. Microbiol. 4 8 ( 1 9 8 4 ) , 8 4 9 . Phytochemistry 1 9 ( 1 9 8 0 ) , 2 2 7 3 . [3] HAABS, A., HÜTTEEMANN, A.: Arch. Microbiol. 184 (1983), 309. [4] LEONOWICZ, A., MALINOWSKA, M . : Acta Biochim. Polon. 29 (1982),"219. [5] FROEHNEB, S. C., ERIKSSON, K . - E . : J . B a c t e r i o l . 1 2 0 (1974), 450. [6] [7] [8] [9] [10] [11]

FAHBAEITS, G . , REINHAMMAB, B . : Acta Chem. Scand. 2 1 ( 1 9 6 7 ) , 2 3 6 7 . LEONOWICZ, A., TBOJANOWSKI, J . : Acta Biochim. Polon. 2 2 ( 1 9 7 5 ) , 2 9 1 . LEONOWICZ, A . , TBOJANOWSKI, J . : MICBOBIOS 1 8 ( 1 9 7 5 ) , 1 6 7 . LEONOWICZ, A., TBOJANOWSKI, J . : Acta Biochim. Polon. 2 5 ( 1 9 7 8 ) , 1 4 7 . LEONOWICZ, A . , TBOJANOWSKI, J . , NOWAK, G . : Microbios 6 ( 1 9 7 2 ) , 2 3 . LEONOWICZ, A., TBOJANOWSKI, J . , OBLICZ, B.: Acta Biochim. Polon. 2 5 ( 1 9 7 8 ) , 3 6 9 .

[12] LEONOWICZ, A., GBZYWNOWICZ, K.: Enzyme. Microb. Technol. 8 (1981), 55. [ 1 3 ] H A B K I N , J . M . , OBST, J . R . : Experientia 2 9 ( 1 9 7 3 ) , 3 8 1 . [ 1 4 ] L I N D E B E B G , G . , HOLM, G . : Physiol. Plant. 5 ( 1 9 5 2 ) , 1 0 0 . [15] LOWRY, O. H . , ROSEBBOFGH, N . J . , PARK, A . L . , RANDALL, R . J . : 1 9 8 (1951), 265. [ 1 6 ] H A L L E R , W . J . : J . Chem. PhyS. 4 2 ( 1 9 6 5 ) , 8 6 6 . [ 1 7 ] DAWIDOWICZ, A. L . , WAKSMTTNDZKI, A., D E B Y L O , A.: Chem. Anal. 2 4 ( 1 9 7 9 ) , 8 1 1 . [ 1 8 ] ROBINSON, P . J . , D U N N I L L , P . , L I L L Y , M . D . : Biochim. Biophys. Acta 2 4 2 ( 1 9 7 1 ) , 6 5 9 .

[19] LAPPI, D . A . , STOLZENBACH, F . E . , KAPLAN, N . O., KAMEN, M . D . : B i o c h e m . B i o p h y s . R e s .

Commun. 69 (1976), 878. [20] DAWIDOWICZ, A. L., ROGALSKI, J . : Polish Patent, P-270379 (1988). [ 2 1 ] PASZCZYI&SKI, A . , TBOJANOWSKI, J . : Microbios 1 8 ( 1 9 7 7 ) , 1 1 1 . [22] R Y A N , E . , F O B T E L L , P. P.: F E B S Lett. 28 (1972), 73. [23] TBEVAN, M. D.: Immobilized Enzymes: An Introduction and Application in Biotechnology, John Wiley and Sons, Chichester—New York—Brisbane—Toronto, 1980. [ 2 4 ] ROGALSKI, J . , SZCZODBAK, J . , DAWIDOWICZ, A . , ILCZUK, Z . , LEONOWICZ, A . : E n z y m e M i c r o b .

Technol. 7 (1985), 395. [25]

SZKLABZ, G . , LEONOWICZ, A.: Phytochemistry 25 (1986), 2537. [ 2 6 ] LEONOWICZ, A . , BOLLAG, J . - M . : Soil Biol. Biochem. 1 9 ( 1 9 8 7 ) , 2 3 7 . [27] BOLLAG, J.-M.: Aquatic and Terrestrial Humic Materials, CHBISTMAN,

R. F. and

GJESSING,

E. T., Ann Arbor Science The Butterworth Group, 1983, p. 127. [28] YABOPOLOV, A. I., MALOVIK, V.: Zh. Anal. Khim. 38 (1983), 503. [ 2 9 ] GONCHABOVA, N . A . ,

GAVRILOVA, V . P . , SHAMOLINA, 1 . 1 . , LOBOVA, A . B . , •'VOLF, L . A . : 2 1 ( 1 9 8 5 ) , 330. [ 3 0 ] SHTJTTLEWOBTH, K . L . , BOLLAG, J.-M.: Enzyme. Microb. Technol. 8 ( 1 9 8 6 ) , 1 7 1 .

Prikl. Biokhim. Mikrobiol.

Acta Biotechnol. 10 (1990) 3, 270

Akademie-Verlag Berlin

Book Review E.-L. WrNisrACKER, R . HTJBER (Ed.)

Protein Structure and Protein Engineering 39th Mosbacher Colloquium Berlin, Heidelberg, New York, London, Paris, Tokyo: Springer-Verlag 1988. 131 pp., DM 7 8 , - , ISBN 3-540-50394

In the last years many symposia and conferences dealt with problems of protein engineering and protein structure research. Also the 39th Mosbacher Colloquium was attended to this research field. The organizing commitee was successful to win important scientists from all over the world. They represented in their papers the most recent research results of their groups. The given talks touch nearly all problems of this research field, e.g. — — — —

application of protein engineering analysis and determination of protein structure stability and flexibility of proteins thermodynamic aspects of hydrophobic interactions, folding dynamics, and energy and entropy changes during conformational changes of proteins — molecular backround of enzyme catalysis — design of new proteins, especially of biologically active peptides and synthetic antibodies -r computer techniques in protein research and data handling.

The now available bock contains all contributions of this colloquium. The book is mainly directed to specialists. But it is also recommended to other interested researchers and advanced students which want to get informations on recent results and new research directions in this field. In no scientific library it should be missed. A . STEXJDEL

Acta Biotechnol. 10 (1990) 3, 271—275

Akademie-Verlag Berlin

Wirkung eines mikrobiellen Enzympräparates mit zellwandlytischen Eigenschaften auf Saccharomyces cerevisiae MENZEL, G . 1 , SCHÖPS, K . 1 , K L E I N , G . 1 , H Ä F N E B , B . 2

1 2

Ingenieurhochschule Kothen, Sektion Biotechnologie/Lebensmitteltechriik Bernburger Straße 5 2 - 5 7 , Kothen, 4370, D D R V E Forschungszentrum Biotechnologie Berlin Alt-Stralau 62, Berlin, 1017, D D R

Summary The effect of an enzyme preparation of Streptomyces spec. K T 3 was tested in view of its cell wall lytic activity on cells of Saccharomyces cerevisiae. According to electron micrographs under the influence of the enzyme preparation protoplasts b u t no spheroplasts were formed. The yield of protoplasts amounted dependent on the enzyme charge to 75—99 per cent, most of the protoplasts were released after 60 minutes. Cells of 12 strains of Saccharomyces cerevisiae were able to be protoplasted and stabilized immediately thereafter by mannitol (0.8 M) or KCl (0.6 M). The presence of stabilizers already in the enzyme-containing medium inhibited, however, protoplasting considerably. The stabilized protoplasts could be stored for six days a t 4 °C without any noticeable changes.

Einführung Biotechnologische Verfahren mit Mikroorganismen setzen leistungsstarke Stämme voraus. Es ist deshalb eine vorrangige Aufgabe, die Leistungsparameter bestimmter Stämme zu verbessern oder neue Produktionöstämme zu schaffen, die hohe Biomasse- bzw. Produktausbeuten gewährleisten. Um dieses Ziel zu erreichen, bedient man sich verschiedener Methoden, eine davon ist die in den letzten Jahren mehrfach angewandte Methode der Genomzusammenführung auf parasexuellem Wege durch induzierte Fusion von Protoplasten selektierter Stämme (u. a. FEBENCZY [ 1 ] , ALFÖLDI [ 2 ] , MACH [ 3 ] ) . Sehr häufig wurden zur Protoplastierung der Magendarmsaft von Helix pomatia oder daraus hergestellte Enzyme eingesetzt ( E D D Y U. WILLIAMSON [4], PEBEBDY [ 5 ] , D I A TEWA et al. [ 6 ] , W E B E R [ 7 ] u. a.). Eine weitere Möglichkeit, Protoplasten zu gewinnen, ergibt sich durch Anwendung zellwandlytischer mikrobieller Enzyme (s. Kuo u. YAMAMOTO [8] sowie POPOV u. KONSTANTINOVA [9]). Wir untersuchten die Wirkung eines Streptomyceten-Enzympräparates auf die Protoplastenbildung verschiedener Stämme von Saccharomyces cerevisiae.

Material und Methoden Hefestämme: Saccharomyces cerevisiae Meyen ex Hansen (1883) Kolin, Schönebeck, Pardubice, Trenöin, 2yrard6w, f, 12, erhalten vom V E B Gärungschemie Dessau, außerdem Weißburgunder F, Riesling F, Müller-Thurgau F, Silvaner F, Traminer F , isoliert aus Traubenpreßmost.

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Medien und Chemikalien: 1. Nährmedium: Hefeextrakt-Pepton-Glucose-Medium (YEPG), zur Verfestigung Agar, 2% 2. Phosphat-Citrat-Puffer nach Mc IIVAINE 3. Tris-EDTA-Lösung, 10 mM 4 . 2-Mercaptoethanol-Lösung: 2 (JTL in 5 0 ml Tris-EDTA-Lösung ( 1 . — 4 . nach W E B E R [7]) 5. Enzympräparat (VE Forschungszentrum Biotechnologie Berlin): Enzymkomplex aus 1,3-/9Glucanasen und Proteasen mit für die Zellwandlyse vorteilhaftem Serinproteaseanteil, Produktionsstamm: Streptomyces spec. KT3, Einsatzform: Lyophilisat bzw. Flüssigkonzentrat* 6. Osmotische Stabilisatoren: Mannitol, 0,8, 0,6, 0,4 M: Sorbitol, 1,4 M; KCl, 2,0, 0,8, 0,6 M.

Protoplastierung der Hefezellen Die Gewinnung der Protoplasten erfolgte in Anlehnung an die von W E B E R [7] beschriebene Methode. Von einer Vorkultur auf YEPG-Agar wurden die Hefen mit physiologischer NaCl-Lösung abgeschwemmt und in 100 ml YEPG-Flüssigmedium bei 30 °C unter Schütteln 15 h inkubiert. 5 ml dieser Suspension wurden erneut in 100 ml YEPG-Flüssigmedium bei gleichen Bedingungen 3 h kultiviert, danach wurden die Zellen abzentrifugiert und zweimal mit Aq. dest. gewaschen. Die gesamte Hefemenge wurde 10 min mit 2-Mercaptoethanollösung behandelt, anschließend zentrifugiert und wiederum zweimal gewaschen. Von der jeweiligen Enzymcharge wurden meist 1 oder 2 mg Protein in 10 ml Phosphat-Citrat-Puffer aufgenommen bzw. 1 bis 2 ml Flüssigkonzentrat mit Puffer ad 10 ml aufgefüllt. Zu diesem Gemisch wurde ein Teil der vorbehandelten Zellen gegeben und bei 30 °C im ERLENMEYERkolben auf dem Horizontalschüttler der zellwandlytischen Wirkung des Enzympräparates ausgesetzt. In Abständen von 10 min erfolgte die mikroskopische Kontrolle des Zellzustandes bzw. die Bestimmung der Protoplastenausbeute in der Zählkammer nach THOMA. Die abzentrifugierten Protoplasten wurden in 0,8 M Mannitol- oder 0,6 M KC1Lösung bei 4°C aufbewahrt.

Präparation zur elektronenmikroskopischen Untersuchung der Hefezellen Um den Prozeß der Zellwandlyse und der Protoplasterifreisetzung stadienweise verfolgen zu können, wurde die Einwirkungszeit der Enzymlösung variiert. Jeweils 2 ml der Zellsuspension wurden zentrifugiert, mit 0,6 M KCl-Lösung gewaschen, wieder zentrifugiert und 24 h bei 4°C mit Glutardialdehyd, 3,6%, in KCl-haltigem Phosphatpuffer, pH 7,3, fixiert. Nach zweimaligem Waschen mit diesem Puffer wurde das Sediment 5—6 h bei Zimmertemperatur durch l%ige phosphatgepufferte 0 s 0 4 -Lösung nachfixiert und anschließend dreimal mit Phosphatpuffer gewaschen, danach stufenweise durch Aceton entwässert. Nach dem Intermedium Propylenoxid wurde in Araldit eingebettet. Die Ultradünnschnitte (Ultracut von REICHERT/JUNG) wurden durch Bleicitrat (REYNOLDS [10]) nachkontrastiert. Zur Aufnahme der Zellen diente das TransmiSsionselektronenmikroskop D2 (VEB Carl Zeiss Jena).

Ergebnisse und Schlußfolgerung Über lytische Aktivitäten von Streptomyces-Arten bzw. -Stämmen liegen einige Berichte vor, u. a. von S K U J I N S et al. [11], SUGIMOTO [12], K u o und YAMAMOTO [8], R A D E MÄCHEK und R E U T E K [13], BILLICH et al. [14]. Wir untersuchten an praxisrelevanten Hefestämmen die Wirksamkeit eigener Präparationen aus Streptomyces spec. KT3. Alle 12 getesteten Stämme von Saccharomyces cerevisiae waren zur ProtoplaStenfreisetzung fähig. Der Beginn der Protoplastierung wurde mittels Lichtmikroskop (ohne und mit * Daten zur näheren biochemischen Charakterisierung des Enzympräparates liegen im VE Forschungszentrum Biotechnologie Berlin vor.

MENZEL, G . , SCHÖPS, K . U. a . , E n z y m p r ä p a r a t e m i t z e l l w a n d l y t i s c h e n E i g e n s c h a f t e n

273

Phasenkontrasteinrichtung) an einem Vorwölben der Zellbegrenzung erkennbar. Mit fortschreitender Enzymwirkung wurde die Protrusion größer, bis schließlich der Protoplast je nach verwendeter Enzymcharge im Verlaufe von 20 — 90 min aus der Zelle schlüpfte und sich abkugelte. Wurde bereits der Enzymlösung zur Erhaltung der austretenden Protoplasten ein Stabilisator in Form von KCl, Mannitol oder Sorbitol zugesetzt, so zeigten die damit behandelten Hefezellen meist nur Ansätze zur Protoplastierung; das Schlüpfen der Protoplasten erfolgte jedoch nicht. Zu ähnlichen Ergebnissen gelangten auch A B N O L D und G A B B I S O N [ 1 5 ] bei der Verwendung von Schnecken-/?-glucanase. Des100

0

20

i0

60 80 t [min]

100 120

Abb. 1. Freisetzung von Protoplasten unter der Einwirkung des zellwandlytischen Enzympräparates von Streptomyces spec. K T 3 auf Saecharomyces cerevisiae Kolin

halb wurden die stabilisierenden Verbindungen erst nach der Freisetzung der Protoplasten von uns zugegeben. Am geeignetsten erwiesen sich KCl, 0,6 M, und Mannitol, 0,8 M. Die Ausbeute an Protoplasten schwankte in Abhängigkeit von der Zusammensetzung des Enzympräparates zwischen 75 und 9 9 % (Abb. 1). Um zu entscheiden, ob durch Einwirkung des Äire^omyces-Präparates tatsächlich Protoplasten und keine Sphäroplasten gebildet werden, untersuchten wir die Hefezellen auch elektronenmikroskopisch. Ausgangsmaterial, Stadien der Protoplastierung und deren Ergebnis zeigen die Abbildungen 2 —12. Die Protoplasten wurden ohne anhaftende 2

"ü

Abb. 2—4. Ausgangsmaterial für die Protoplastierung: Zellen von Saccharomyces cerevisiae

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Abb. 5—8. Stadien der Protoplastierung: Abb. 5, Erste lytische Erscheinungen an der Zellwand Abb. 6. Beginnender Austritt des Protoplasten nach Öffnung der Zellwand Abb. 7. Weiterer Austritt des Protoplasten bei stark vergrößerter Öffnung der Zellwand Abb. 8. Ablösen des Protoplasten von der Zellwand

Abb. 9—12. Ergebnis der Protoplastiernng: Abb. Abb. Abb. Abb.

9. Protoplast 10. Begrenzung des Protoplasten durch die Cytoplasmamembran (Ausschnitt) 11. Zellwand nach Ausschlüpfen des Protoplasten 12. Zerfall der Zellwand in Bruchstücke

MENZEL,

G.,

SCHÖPS, K . U.

a., Enzympräparate mit zellwandlytischen Eigenschaften

275

Zellwandteile freigesetzt, sie waren also nur von der Cytoplasmamembran begrenzt (Abb. 9 u. 10). Zurück blieb die Zellwand, die nach längerer Enzymeinwirkung zerfiel (Abb. 11 u. 12). Die Lebensfähigkeit der Protoplasten wurde unter Verwendung von Methylenblau geprüft. 6 Tage nach der Aufbewahrung bei 4°C schienen die meisten Protoplasten noch lebensfähig zu sein, nur wenige waren lysiert. Darüber hinaus lieferten erste Versuche zur Fusogenität der Protoplasten Hinweise für die Intaktheit der Zellen. Die dargelegten Ergebnisse sind uns Anlaß, das Streptomyceten-Enzympräparat für die Herstellung von Saccharomyces cera>mae-Protoplasten im Rahmen biotechnologischer Zielstellungen positiv zu bewerten. Zeichenerklärung cm cw pb st v I 1

Cytoplasmamembran Zellwand periplasmatisches Körperchen Narbe Vakuole 1 um

Für zuverlässige technische Assistenz danken die Autoren Frau Sabine Helga ZLEGELGÄNSBERGER.

WINTERFELD

und Frau

Eingegangen: 9. 5. 1988 Überarbeitet: 28. 2. 1989

Literatur [1]

L. — I n : Genetics as a Tool in Microbiology. Eds.: CLOVER, S. W., H O P W O O D , D. A. Cambridge, 1981, 1. [2] ALFÖLDI, L. — I n : Basic Life Sciences, Vol. 19, Genetic Engineering of Microorganisms for Chemicals. Eds.: HOLLAENDER, A . , D E MOSS, R . D . , K A P L A N , S . , K O N I S K Y . ' J . , SAVAGE, D . , WOLFE, R. S. New York, London, 1982, 59. [3] MACH, F. — I n : Abh. Akad. Wias. DDR, Abt. Mathem., Naturwiss., Technik, N2; Biotechnologie, 2. Symp. soz. Länder, 2 . - 5 . 12. 1980, Leipzig. Hrsg.: RINGPFEIL, M. Berlin, 1982, 91. [ 4 ] E D D Y , A . A . , WILLIAMSON, D . H . : Nature 1 8 3 ( 1 9 5 9 ) , 1 1 0 1 . [5] PEBERDY, J . F.: Annu. Rev. Microbiol. 33 (1979), 21. FERENCZY,

[6] DIATEWA, M., VIARD, I . , STAHL, A. J . C.: Biochimie 6 3 (1981), 67.

[7] WEBER, II. : Zellbiologie und Genetik der Hefen. Berlin, 1982, 80ff. [8] Kuo, S.-C., YAMAMOTO, S. — In: Methods in Cell Biology, Vol. 11. Ed.: PRESCOTT, D.M. New York, San Francisco, London, 1 9 7 5 , 1 6 9 — 1 8 3 . [9] POPOV, B . K . , KONSTANTINOVA, R . D . : Zbl. Mikrobiol. 1 4 1 (1986), 217.

[10] REYNOLDS, E. S.: J . Cell Biol. 17 (1963), 208. [11] SKTTJINS, J . J . , POTGIETER, H . J . , ALEXANDER, M . : Arch. Biochem. Biophys. 111 (1965), 358.

[12] SUGIMOTO, H.: Agric. Biol. Chem. 31 (1967), 1111. [13] RADEMACHER, K . H., R E U T E R , G. : Z. Allg. Mikrobiol. 18 (1978), 107. [14] BILLICH, A., KELLER, U . , KLEINKAUF, H . , ZOCHER, R . : A p p i . Microbiol. B i o t e c h n o l . 2 8

(1988), 442. [15] ARNOLD, W . N . , GARRISON, R . G . : J . Bacteriol. 187 (1979), 1386.

Acta Biotechnol. 10 (1990) 3, 276

Akademie-Verlag Berlin

Book Review B . WITTMJLNN-LIEBOLD ( E d . )

Methods in Protein Sequence Analysis Berlin, Heidelberg, New York, London, Paris, Tokyo: Springer-Vertag, 1989. 201 Abb., 575 pp., DM 1 9 5 , - , ISBN 3-540-19433-9

The book contains the most important contributions of the Seventh International Conference on "Methods in Protein Sequence Analysis", held in July 1988 in Berlin. The aim of the conference was to summarize the current situation in protein structure research. I n twelve chapters different problems of this research are discussed, e.g. new methods for amino acid analysis N- and C-terminal sequence analysis protein and peptide purification crystallization of proteins and cell organelles design of sequencers and sophisticated equipment for structural analysis of peptides and proteins — theoretical aspects of protein folding — gene technology for protein characterization and the design of new proteins. — — — — —

The content of the now available anthology of this meeting demonstrates the rather rapid progress in molecular biology in our days. Especially the new methodical developments made it possible to broaden our current knowledge on proteins and their structure. Some new research trends are also indicated. The book is mainly directed to specialists. But it is no question that it will also find a large interest among scientist of different branches of natural sciences. The acquisition of this anthology is recommended for scientific libraries especially. A . STEUDEL

Acta Biotechnol. 10 (1990) 3, 277—282

Akademie-Verlag Berlin

Amorphous-Cellulose Dispersing Activity of Cellobiohydrolase I of Trichoderma reesei Suggestion for an Interpretation of the Cx-Effect HEITZ, H . - J . , WITTE, K . , WARTENBERG,

A.*

Fachrichtung Mikrobiologie, Universität des Saarlandes, Universität — Bau 2, D-6600 Saarbrücken, F.R.G.

Summary Two isoenzymes of cellobiohydrolase I (CBHI/1 and CBHI/2) of Trichoderma reesei purified from Celluclast™ are Shown to cause a new mode of reaction which is exhibited additionally to the known exo-glucanase reaction of CBHI: Dispersion of amorphous cellulose solution is increased by CBHI/1 and CBHI/2 to 49 and 64 p.c., respectively, before decompositions of substrate as induced by the same enzymes become detectable. Amorphous-cellulose dispersing activity of CBHI is reduced by cellobiose which is known to be an inhibitor of CBHI. Amorphous-cellulose dispersing activity was also observed of a CBHII isoenzyme (CBHII/2), an endo-glucanase and complete Celluclast, however, to lesser' extents (17, 12, 35 p.c., respectively). Observations with a phase-contrast microscope determined an increase of dispersion of particles of amorphous cellulose induced by cellulases of Celluclast. Decompositions of amorphous cellulose run almost linearly and quite exponentially when induced by CBHI and CBHII, respectively, allowing differentiation between exo-glucanase and endo-glucanase activities of these cellulases. Abbreviations: CBH, l,4-/?-D-glucan cellobiohydrolase (EC 3.2.1.91) or exo-glucanase; EG, 1,4(1,3; l,4)-/?-D-glucan 4-glucanohydrolase or endo-glucanase; CMC, carboxymethylcellulose; MU-, 4-methyI-umbelliferyl-; Introduction Nearly four decades ago R E E S E , S I U and LEVXNSON [ 1 ] proposed the Cj/Cx-concept for interpreting solubilization of native cellulose as induced by fungal cellulases especially by the cellulases of Trichoderma reesei (at that time named Trichoderma viride). (^-activity means the capability of a cellulaSe system to attack crystalline cellulose; C x -activity, however, stands for an attack on CMC in a random manner resulting in a rapid decrease of chain length of cellulose and in a low increase of reducing group concentration. Today the finding [2] is generally accepted that C 1 -action is related with the action of the cellobiohydrolase component of the cellulaSe System, despite the fact that no one has Successfully measured this different action separately. This study shows the hydrolytic activity of C B H I of Trichoderma reesei to be associated with a cellulose dispersing activity supposed to be a ^ - a c t i v i t y and having its effects Separately from the hydrolytic action of the enzyme, i.e. the cleavage of cellobiosyl units from the non-reducing ends of cellulose molecules [3, 4]. * To whom correspondence should be addressed. 5

Acta Biotechnol. 10 (1990) 3

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Materials and Methods Freeze-dried preparations of two isoenzymes of CBHI, two isoenzymes of CBHII and one endoglucanase were yielded by fractionation of Celluclast™ [4], a commercial cellulase preparation which was kindly provided by NOVO INDUSTRI (Bagsvaerd, Denmark). Aspergillus niger cellulase was a commercial preparation delivered by SERVA (Heidelberg, F.R.G.). Celluclast cellulases were used in final concentrations of 62.5 ¡xg/ml of incubation mixture, with A. niger cellulase in a concentration of 125 fig/ml. The incubation mixture contained 108 [ig/ml of amorphous cellulose being equivalent to 32 Nephelometric Turbidity Units [NTU], provided t h a t turbidity was not increased by a change of dispersion of cellulose which is the subject of this report. The methods used for chromatographic fractionation of the enzymes, for preparation of amorphous cellulose and for measuring turbidity of amorphous cellulose solutions were given previously [4]. The degree of accuracy of turbidity measurements was in the range of 1 NTU. The measuring points forming the curves of Fig. 1 and 2 are mean values in each case resulting from 400 single values.

50 CBHI/2 • CBH1/1 Peak 1.3.5 Control A. niger

CBHÏÏ/1

-5

•"• CBH n/2 Incubation

Celluclast

12 Measuring

time

Cminl

Fig. 1. Decompositions of amorphous cellulose recorded by nephelometric measurements as catalyzed by cellulases purified from Celluclast. Control measurements were performed with non-fractionated purified Celluclast and with Aspergillus niger cellulase. A basic control experiment was executed with an incubation mixture without enzyme. Incubation was started in each experiment 24 sec. before the first measuring cycle began. Enzyme concentration was adjusted to 62.5 fig/ml in each experiment, except that of A. niger cellulase which was 125 (ig/ml.

Results and Discussion Turbidity of an enzyme free solution of amorphous cellulose (pH 4.8) has been shown to remain constant over 1/2 h (Fig. 1). Additions to the incubation mixture of one of the cellulase fractions yielded from purified Celluclast were followed by different decreases of turbidity (Fig. 1) disclosing decomposition of cellulose. The kinetics of decreases of turbidity were strongly exponential in the cases of decompositions induced by CBHII/1, CBHII/2 and Celluclast, wheieas decreases of cellulose turbidity caused by CBHI/1

HEITZ, H . - J . , WITTE, K . e t al., Dispersing A c t i v i t y of Cellobiohydrolase I

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SO -

5» % •5

•

Cellobiose 0

•

1.9 6.0

t

• 19.0 mg/ml Control

Fig. 2 a

30 12 Measuring

time CminJ

50 Cellobiose 0

a s:

1.9 •

6.0

19.0 mg/ml

Control

30 12 Measuring

Fig. 2 b

time CminJ

Fig. 2. Decompositions of amorphous cellulose recorded by nephelometric measurements as catalyzed by CBHI/1 (a) and CBHI/2 (b) and being inhibited by cellobiose. The control experiment executed with a enzyme free incubation mixture was the same in Fig. 2a and 2b.

and CBHI/2 were nearly linear (Fig. 1). For comparison, control experiments were performed with an endoglucanase (peak 1.3.5 from the chromatographic fractionation of Celluclast [4]) and with Aspergillus niger cellulase, whereby both were shown to be active towards CMC [4] but failed to be active towards amorphous cellulose in the turbidity assay (Fig. 1). In the experiments with CBHI/1 and CBHI/2 just after addition of an enzyme to substrate, strong increases of turbidity (50 to 70 p.c.) occurred before decomposition of substrate could detected. This primary change of turbidity of cellulose Solution was obviously caused by an increase of dispersion of the particles of amorphous cellulose which by aggregation form clusters of different Sizes in the solution (Fig. 3). Purified Celluclast, CBHII/2 and endo-glucanase from Celluclast (peak 1.3.5) induced the primary increase of cellulose turbidity to a leSs extent compared with the effects induced by 5»

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Fig. 3. Phase contrast microscopy of particles of amorphous cellulose influenced by the buffer (0.01 M KH 2 P0 4 , pH 4.8) and by cellulases. a. Initial state of amorphous cellulose solution, b. Clusters of particles of amorphous cellulose formed by aggregation after mixing the initial solution of amorphous cellulose, with buffer solution, c. d. e. f. States of aggregation of amorphous cellulose after addition of CBHI/1, CBHII/1, complete Celluclast and Aspergillus niger cellulase, respectively. The photographs were taken 2 min. (b., c., d., e.) and 24 h (f) after incubation.

CBHI/1 and CBHI/2 (Fig. 1). In contrast, no primary turbidity increase could be detected in the experiments with CBHII/1 and with A. niger cellulase. The results of the experiments of Fig. 2 a and 2 b make evident that the primary increase of turbidity of amorphous cellulose is really induced by the cellulase used in the respective experiments of Fig. 1. Addition of cellobiose to the reaction mixture which contains CBHI/1 or CBHI/2 was followed by an inhibition of cellulose decomposition being in agreement with the well known inhibitory effect of cellobiose on cellobiohydrolase [2, 4, 5,6], At the same time cellobiose inhibited the increase of cellulose dispersion so that the primary increase of turbidity was retarded and could now be observed directly during the first minutes of measurement (Fig. 2a, 2b). Decomposition of amorphous cellulose (detectable by decrease of cellulose turbidity) and increase of dispersion of amorphous cellulose (detectable by an increase of turbidity) are obviously different reactions catalyzed by the Same cellobiohydrolase. This differentiation between the reactions is disclosed by the different kinetics of the reactions: Cellulose dispersion took place in the experiment which was not inhibited by cellobiose only within the first half minute of the experiment whereas decomposition of cellulose remained over the whole period of the experiment (12.4 min). Moreover, decomposition of amorphous cellulose was completely inhibited when cellobiose was used in the highest concentration whereas the increase of dispersion of cellulose was inhibited only in part (Fig. 2 a, 2 b). Fig. 3 gave proof of the interpretation of the results outlined in Fig. 1. Amorphous cellulose solution is represented by cellulose particles of an average size of 0.5 to 2 (im (Fig. 3 a). Mixing of this solution with buffer solution (pH 4.8) is followed by an aggregation of the particles to clusters (Fig. 3 b); the process is undone in part or completely by addi-

H e i t z , H.-J., W i t t b , K . et al., Dispersing Activity of Cellobiohydrolase I

281

tion of CBHI/1 (Fig. 3c), CBHI/2 or Celluclast (Fig. 3e). Additions of CBHII/1 (Fig. 3d) and A. niger cellulase (Fig. 3f) were not followed by an effect with regard to dispersion change. To Summarize for the present, the increase of cellulose turbidity as induced by CBHI/1 and CBHI/2 (Fig. 1) corresponds with dispersion of cellulose particles caused by the same enzyme (Fig. 3). CBHI/1 and CBHI/2 had been screened as exo-glucanaSeS due to their capability of decomposing MU-lactoside and their being inhibited with regard to MU-lactoSide decomposition by cellobiose [4]. The exo-glucanase feature of CBHI/1 and CBHI/2 were further confirmed by the kinetics of CMC decomposition which was nearly linear when plotted versus time [4]. This strongly contrasted with decomposition of CMC driven by endo-glucanases which run remarkably exponential course [4]. Decompositions of cellulosic materials driven linearly and exponentially by exoglucanases and endo-glucanases, respectively, resulted additionally from our present experiments using amorphous cellulose as a substrate (Fig. 1). The differences of CBHI and CBHII kinetics resulting from CMC decomposition experiments [4] correspond very well to kinetics resulting now for amorphous cellulose decompositions. This confirmeS the result [4] that the CBHII isoenzymes are endo-glucanaSeS corresponding to the respective finding reported previously [7]. This classification of CBHII/1 and CBHII/2 as endo-type cellulaSeS Still may contradict with their capability of decomposing MU-cellobioside [4] which is stated to be a substrate for exo-glucanases and (¡glucosidases [8, 9,10]. However, in other experiments only CBHI, not CBHII, had been found to be active towards MU-cellobioside [9]. If the deduction is right there must exist two different types of endo-glucanases which are specified to attack amorphous cellulose and CMC in the case of the first type and CMC alone in the case of the Second one, corresponding with respective conclusions [4, 7]. Differing from those results, CBHII was shown to be inactive towards CMC [7]. The Second and hitherto only known type of endo-glucanaSeS is represented in our experiments by the experiment with peak 1.3.5 cellulase prepared from purified Celluclast and with A. niger cellulase, both of which failed to attack amorphous cellulose to any extent. Amorphous-cellulose dispersing activity as detected in our experiments is mainly associated with the CBHI supporting the hypothesis that amorphous-cellulose dispersing activity is equivalent to Cj-activity of cellulaSeS. Corresponding with the literature cited in the preceding breaks our CBHI isoenzymes were real cellobiohydrolases: The nearly linear kinetics of CBHI, the final product being cellobiose and the isoelectric point (pi = 4.1) [4] prove this conclusion. Cellulose dispersion as Supplementally induced by the CBHI isoenzymes means an increase of degree of hydratation of cellulose molecules. Due to this increase of hydrate water content amorphous cellulose is rapidly decomposed by endo-type cellulases when additionally added, aS can be demonstrated [11] by the well known synergism between CBHI and CBHII [12], For the present, amorphouScellulose dispersing activity is resulting aS the capability of the CBHI isoenzymes to disperS amorphous cellulose independently of their reaction in decomposing substrate by cleaving cellobiosyl groups. One should consider that amorphous-cellulose dispersing activity was additionally associated with Celluclast endo-glucanase (peak 1.3.5) and with CBHII/2 though increases of turbidity induced by these enzymes were shown to be smaller compared to increases caused by the CBHI isoenzymes. Because amorphouscellulose dispersing activity is Strongest associated with CBHI, not with CBHII or conventional EGs, it seems not probable that this activity is equivalent to the "swelling factor" activity of cellulaSeS being exclusively associated with EGs [13, 14].

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Acknowledgements The authors are grateful to Mr. P. MEIERS for editing computer programs and to Mr. J. POCKLINGTON for proofreading the manuscript. Received January 20, 1989

References [1] REESE, E . T., Sitr, R . G. H., LEVINSON, H . S. : J . Bacteriol. 59 (1950), 485. [2] HALLIWELL, G., GRIFFIN, M. : B i o c h e m . J . 1 8 5 (1973), 587. [3] NUMMI, M., NIKTJ-PAAVOLA, M.-L., LAPPALAINEN, A., ENABI, T.-M., RATTNIO, V . : B i o c h e m . J . 2 1 5 (1983), 677. [4] WITTE, K . , HEITZ, H . - J . , WARTENBERG, A . : A c t a B i o t e c h n o l . 1 0 (1990), 41.

[5] PETTERSSON, L. G. : In Symposium on Enzymatic Hydrolysis of Cellulose, Aulanko, Finland (BAILEY, M., ENARI, T . M. & LINKO, M., eds) p p . 255, S I T R A , H e l s i n k i (1975). [6] VAN TILBETJBGH, H . , CLAEYSSENS, M . : F E B S L e t t . 187 (1985), 283.

[7] KYRIACOTJ, A., MACKENZIE, R . C., NEUFELD, J . R . : E n z y m e Microb. Technol. 9 (1987), 25. [8] HEPTINSTALL, J . , STEWART, J . C., SERAS, M. : E n z y m e Microb. Technol. 8 (1986), 70. [9] VAN TILBEURGH, H . , CLAEYSSENS, M., DE BRUYNE, C. K . : F E B S L e t t . 1 4 9 (1982), 152.

[10] WITTE, K., WARTENBERG, A.: Acta Biotechnol. 9 (1989), 179. [11] [12] [13] [14]

HEITZ, H . - J . , WITTE, K . , WARTENBERG, A . : u n p u b l i s h e d . FÄGERSTAM, L . G., PETTERSSON, L . G., F E B S L e t t . 1 1 9 (1980), 97. WOOD, T . M . : B i o c h e m . J . 1 0 9 (1968), 217. WOOD, T . M . : B i o c h e m . J . 1 1 5 (1969), 457.

Acta Biotechnol. 10 (1990) 3,283—292

Akademie-Verlag Berlin

The Controlled Porous Glass (CPG) with Reactive Epoxy Groups as Support for Affinity Chromatography II. Modified CPG as Support of Substrates or Coenzyme of Glucose Oxidase (GOD) for its Purification and Immobilization ROGALSKI, J . 1 , DAWXDOWICZ, A. 2 , FIEDUBEK, J . 3 , LEONOWICZ, A . 1 * 1

2

3

M. Curie-Sklodowska University Department of Biochemistry M. Curie-Sklodowska Sq. 3, 20-031 Lublin, Poland M. Curie-Sklodowska University Department of Chemical Physics M. Curie-Sklodowska Sq. 3, 20-031 Lublin, Poland M. Curie-Sklodowska University Department of Applied Microbiology M. Curie-Sklodowska Sq. 3, 20-031 Lublin, Poland

Summary Controlled porosity glasses (CPG) are materials very widely employed as supports for chromatography and biotechnology. In this paper CPG with epoxy groups have been applied as carrier of FAD, glucose and 2-deoxy-D-glucose. Glucose oxidase (GOD) was highly purified on these sorbents. The purified fraction of GOD was also immobilized on the activated CPG. The presented results suggest the higher stability of immobilized form of this enzyme in relation to the native enzyme.

Introduction Glucose oxidase (GOD, /J-D-glucose : 1-oxidoreductase EC 1.1.3.4) which catalyzes oxidation of glucose by molecular oxygen has come into notice as a very useful enzyme for its wide application, especially in food industry [1] and clinical analysis [2]. The enzyme was isolated directly from mycelia of Aspergillus niger [3] and Some Pénicillium, strains [2—3] and also from culture filtrate, e.g. from Pénicillium amagasakiense [4]. According to the data of ultracentrifugation and electrophoretic analysis the MR (molecular weight) of GOD from Aspergillus or Pénicillium is about 150000 D (daltons). The enzyme molecule contains FAD (2 moles per 1 mol of protein) [5] and carbohydrates (10 — 16%) as well [6—8]. O'MALLEY and WEAVES [9] have reported that chemical reduction of two disulfide bonds existing in the enzyme unit (by means of 2-mercaptoethanol) results in formation of the protomers with a MR of about 80000 D. The activity of Aspergillus niger glucose oxidase is generally specific for glucose but it also attacks 2-deoxy-D-glucose and other sugars [10 — 13] although with much lower yield (below 30% and 1% respectively, compared to glucose oxidation) [10]. On the contrary, glucose oxidase of Pénicillium purpurogenum is highly specific only for Dglucose and does not catalyze the oxidation of any other investigated sugars [14]. The raw preparations of glucose oxidase are usually contaminated by other enzymes such as catalase (EC 1.11.1.6), invertase (EC 3.2.1.26), amylase (EC 3.2.1.3) or galactose oxidase (EC 1.1.3.9) [15 — 16]. In order to purify such crude preparations, the fractio* To whom correspondence should be addressed.

284

Acta Biotechnol. 10 (1990) 3

nation by means of 2.6 M ammonium sulphate at pH 5.0 can be used [17]. The other way consists in both ammonium sulphate precipitation (85% saturation) and subsequent dialysis for removing of inorganic salt [18]. The enzyme is purified in the next step by ion-exchange and size exclusion chromatography on the sorbents of Sephadex type [18-19]. Affinity chromatography, however, seems to be the most useful, cheap and quick method for purification of the enzyme. This technique allows the isolation of an interesting enzymatic protein from a very complex mixture due to complementary interaction between enzyme and specific for this enzyme ligand joined to sorbent. Usually, as the ligand substrates, cofactors or inhibitors of purified enzymes are employed. In the case of glucose oxidase, D-glucoSe, 2-deoxy-D-glucose or FAD can be used as the ligands. The first two are substrates for GOD and FAD is a cofactor which activates the enzyme during its catalytic reaction. All mentioned substances possess free hydroxyl groups. These groups can serve for chemical bonding of the ligands to the matrix. Usually it is realized by the reaction of hydroxyl groups with proper groups of the Support Surface. This paper deals with purification of glucose oxidase on sorbents composed of the mentioned ligands chemically bonded to the controlled porosity glass (CPG) [20]. Materials and Methods Organism and Enzyme Preparation The mutant Aspergillus niger G-13 selected earlier [21] from our collection was maintained a t + 4 ° C on malt agar slants. Both conditions of glucose oxidase production in fermenter cultures and the crude enzyme preparation were described earlier [10].

Determination of Enzyme Activity ctnd Protein Content GOD activity was determined in liquids directly according to the methods described elsewhere [22] but with our own modification as follows: the enzyme solution (1 ml) was added to the reaction mixture (2 ml) composed of 2 mg glucose, 0.2 mg o-dianisidine, 60 |_ig peroxidase (500 U/mg) and 0.8 ml 0 . 1 M M C I L V A I N E buffer p H 5.7. After 30 min incubation at 30 ° C the enzymatic reaction was stopped by adding 4 ml of 5 N HC1 and the absorbance at 525 nm was measured against a blank composed of the same mixture (but deprived of glucose and peroxidase) incubated under the same conditions. In order to determine activity of the immobilized enzyme 50 mg of the suctiondried preparate were used and the further steps were performed as described above. One unit of the enzyme was defined as the amount that oxidizes 1 [imol /f-D-glucose to D-gluconic acid min - 1 a t 37 °C and p H 5. The protein determination was carried out according to [23].

Preparation of Support The controlled porous glass was obtained from VYCOR glass (composed of 7 mol% Na 2 0, 23 mol% B 2 0 3 and 70 mol% Si0 2 ) by thermal treatment and a leaching process described elsewhere [24, 25],

Activation of the Support Glass supports were activated by y-aminopropyltrietoxy silane (APTES) according to the method described in [26]. As a result APTES-CPG was obtained. This material was then modified by means of bifunctional oxiranes (epoxy-APTES-CPG) [20, 27].

Coupling of Enzymes Coupling of the enzyme (5 mg of GOD protein) to APTES-CPG (5 g) was performed by means of glutaraldehyde as described elsewhere [28].

ROGAX.SK!, J., DAWIDOWICZ, A. et al., Support for Affinity Chromatography I I

285

Coupling of Ligands Coupling of the substrates and cofactor to activated CPG was performed as was described in [20]. Following this procedure 100 mg glucose or 100 mg deoxyglucose were added to the reaction mixture. In the case of FAD only 25 mg were used. Glucose concentration on the CPG surface was estimated as was described elsewhere [20] on the basis of radioactivity measurements of bonded 14 C[U]gIucose. Also the anthrone method was used for determination of glucose and deoxyglucose as well [29]. The difference between substance concentrations in the reaction mixture before and after the bonding reaction with epoxy-APTES-CPG was assumed to be the amounts of bonded glucose or deoxyglucose. FAD concentration was estimated spectrophotometrically at X = 260 nm employing the absorbance coefficient (e) = 38.6 10® at pH 9.0. The difference in FAD concentration in the reaction mixture before and after the bonding reaction was assumed to be the amount of cofactor bonded to epoxy-APTES-CPG. Coupling of Enzyme to the

Supports

Sorbents with bonded ligands (100 mg each) were placed in separate tubes containing 3 ml of 0.1 M MCILVAINE buffer [30] ranging from pH 4 to pH 8 with the step of 0.3 pH unit. To each tube, 100 ¡JLI (about 5 units) of GOD were introduced and mixtures while rotary shaking (150 rpm) were 5 min incubated and then centrifuged (5 min at 10000 g). Pellets were washed twice with the same buffer, centrifuged as before and GOD activity was measured in the supernatant and washings. Affinity

Chromatography

All prepared sorbents were packed into the column (30 X 8 mm). The samples containing on the average 5 mg of protein dissolved on 0.01 M MCILVAINE buffer pH = 6.0 were injected onto the column. As an eluent the same buffer was applied. The elution was reached by changing p H and ionic strength of buffer and by adding 0.7 M ammonium sulphate solution. The eluates were collected in 0.5 ml portions. Polyacrylamide

Oel

Electrophoresis

The cylindrical as well as slab gel electrophoreses were performed directly according to LAEMMLI [31]. In the first case 7.5% acrylamide run at 3 mA per tube, in the second 15% acrylamide in the presence of 0.1% sodium dodecyl sulphate (SDS) was applied employing 50 mA per plate (140 X 170 X 2 mm) [32—34]. The protein bands were stained for 0.5 h with 0.15% Coomasie Brillant Blue R-250 (SIGMA, St. Louis, Missouri) dissolved in the mixture of methanol, acetic acid, glycerol and water in the volume ratio of 16: 2 : 1: 23 and destained in the same mixture but in the ratio of 8 : 2 : 1 : 29. The activity was visualized after adjustment of pH in gels to 5.7 and the reaction with the mixture of o-dianisidin and peroxidase was applied for GOD activity determination (see Materials and Methods). For the determination of GOD molecular weight LMD Calibration Kit Proteins (PHARMACIA, Uppsala, Sweden) was used.

Results and Discussion The controlled porous glass used in these investigations was characterized by the specific surface area 200 °C zur EDA. Dafür wird eine geringe Menge an Dampf oder Heißwasser zur Aufwärmung der Hefesuspension vor der Separation auf ca. 80 °C benötigt.

295

PtroHNER, K,, HEMMANN, H.-G., Energetische Optimierung

Derzeitige Gehalt Hefetrockensubstanz gl kg 20-50

Technologien

Fermentation

Perspektivische

Technologien Kühlw max. 24'C

Kühlw. max 2t°C

Fermentation

max 2S°C

Kaltseparation mehrstufig

max 28 "C

Kaltseparation mehrstufig 90~150 °C

90-150

Eindampfung mehrstufig

Heißseparation

150-250

bis 210

Sprühtrocknung

Walzentrocknung

> 900

J_

J5Pz250

Sprühtrocknung

Extraktion Schwedt!

>900

>900

>900

Walzen trocknung >900

Extraktion (nur PCK

Schwedt)

>900

Pelletierung

bis 400

Wirbelschichttrocknung

>900

(nur PCK

Eindampfung FFEDAIRDEDA

£

Pelletierung

>900

Trocken futterhe fe

Trocken

futterhefe

Abb. 1. Futterhefeindustrie der DDR — derzeitige und mögliche perspektivische Technologien —

— Die klassischen Technologien der thermischen Aufarbeitung in Form von FF-EDA und RDEDA sind soweit optimiert, daß sie mit geringem Energiebedarf zur Verlustdeckung auskommen. Der einzusetzende Heizdampf wird im Temperaturbereich von 150°C bis 200°C benötigt. — Sollte sich die-Wirbelschichttrocknungstechnik durchsetzen, dann wird Dampf in einem höheren Temperaturbereich > 200°C benötigt. — Sollten sich weiterentwickelte Walzentrockner größerer Leistung mit anschließender Pelletierung durchsetzen, wird ein Heizmedium für die Direktbeheizung von > 150 °C benötigt.

Die Situation k a n n weiterhin auf Basis des absoluten Anfalls an Fermentorabwärme, des Bedarfes an Hilfsenergien und der Energieverluste in den einzelnen Stufen analysiert werden. I n Abb. 2 wird der Energiestrom f ü r die biotechnologische Umwandlung eines Modellsubstrates zu Futterbiomasse dargestellt. Es läßt sich aus Abb. 2 prinzipiell ableiten, daß das Wärmeangebot (mit niedrigem Temperaturniveau des Fermentors) den Wärmebedarf der eingesetzten Aufarbeitungsstufen weit übertrifft. Schließlich ist im Rahmen der energetischen Analyse auf die Abhängigkeit der Kühlkapazität der Sühleinrichtungen von der Tagestemperatur der Umgebungsluft hinzuweisen. Bekanntermaßen müßte in Futterhefewerken mit Kühltürmen in den Sommermonaten die umgewälzte Kühlwassermenge bis zum Mehrfachen des in Wintermonaten Normalen gesteigert werden. E s gibt in der D D R wenig Aussagen über Produktivitätsoder Qualitätsverluste in der Fermentation durch unzureichende Kühlkapazität in den Sommermonaten. 6*

Acta Biotechnol. 10 (1990) 3

296

N-Quelle

C-Quelle

Abb. 2. Futterbiomasseproduktion — Sankeydiagramm — (Werte in GJ/tTS)

S-g

?

.1- 900 g BTS/1.

Die letztere Teilanlage wurde auf Grund fehlender Versuchsergebnisse mit hochviskosen Biomassen nicht in die techn.-ökon. Berechnungen einbezogen, obwohl sich Vakuumwalzentrockner auch für den Einsatz von Wärmepumpen anbieten, wenngleich deren Einzelkapazitäten bisher sehr begrenzt sind. Das Wärmepumpenprinzip wird technisch folgendermaßen realisiert: Das als Arbeitsmittel eingesetzte Kältemittel R 12 entzieht dem als Verdampfer fungierenden Wärmeübertrager des Fermentors ca. 50% der Reaktionswärme bei einem Temperaturniveau von 35 °C und wird dabei auf 30 °C überhitzt. Im Verdichter erfolgt eine Erhöhung der Temperatur des Kältemittels bei 2 MPa auf 80 °C. Das Arbeitsmittel wird nunmehr bei einer Kondensationstemperatur von 75 °C sowohl zum Vorheizen des Fermentorablaufes, zum Beheizen der 1. Fallfilmeindampferstufe.sowie des Rotationsdünnschichteindampfers eingesetzt. Das Kältemittel-Kondensat verläßt die beheizten Systeme mit einer Temperatur von 45 °C und wird auf die Ausgangsparameter reduziert. Die eingesetzten Teilanlagen sind sämtlich in der DDR verfügbar: — Fermentoren aus dem Produktionssortiment des VEB Germania Karl-Marx-Stadt — Kältemittelverdichter vom VEB Kühlautomat Beflin Zum Einsatz gelangten die größten in der DDR verfügbaren Aggregate vom Typ S 3-2500, wobei Anpassungskonstruktionen notwendig wären. — FF- und RD-Eindampfanlagen vom VEB CAER Im Falle einer Beheizung bzw. Kühlung von Anlagen dieser Art mit dem o. a. Kältemittel wären ebenfalls Anpassungskonstruktionen notwendig.

Die Optimierung der Wärmepumpensysteme sowie der Vergleich mit einer konventionellen thermischen Aufarbeitung durch Serien-FF-EDA und RD-EDA erfolgte mit der Zielfunktion FA Min.! Die Elemente der Zielfunktion sind in Abb. 5 angegeben. Als weitere Optimierungskriterien wurden der Investaufwand sowie der Primärenergiebedarf herangezogen. Im Ergebnis der techn.-ökon-. Berechnung mit Einsatz von Elektroenergie zum Antrieb der Kältemittelverdichter konnte nachgewiesen werden, daß integrierte Wärmepumpensysteme der angeführten Art mit Elektroantrieb der Kältemittelverdichter mit konventionellen Eindampfsystemen nicht konkurrieren könneft. Obwohl der Primärenergiebedarf für sie Wärmepumpenschaltung um 25% fällt, steigen die InvestaufWendungen um ca. 50% an. Damit erhöhen sich die fondsbezogenen Aufwendungen um mindestens 7%. Der Grund für die Erhöhung der Kosten Hegt insbesondere in hohen Investaufwendungen für Kältemittelverdichter und Nachfolgeinvestitionen im Energiebereich. Die Größe der Verdichterleistung äb 500 kW/Verdichter rechtfertigen den dezentralen Einsatz von Dampfantrieben zum Antrieb der Kältemittelverdichter im Sinne der Wärme-Kraft-Kopplung. Diese Antriebsweisen setzen die Abnahme von Gegendruckdampf in einem zweiten Aufarbeitungsstrang bzw. in peripheren Bereichen voraus. Nach derartigen Schaltungen ist es möglich, die volkswirtschaftlichen Aufwendungen soweit zu minimieren, daß sie unter denen konventioneller Eindampfanlagen liegen. Hierbei

PÜOHNER, K., HEMMAUN, H.-G., Energetische Optimierung FA FA = = =

299

Min! SK + FZ EE + D + FW + A + Inst. + Sonst. K + FZ EE + D + FW + 23% IK /TM/a/

Legende: FA — Fondsbezogener oder gesellschaftlicher Aufwand SK — Selbstkosten FZ — Fondsbezogener Zuschlag EE — Kosten für Elektroenergie D — Kosten für Dampf A — Abschreibungen Inst — Instandhaltungsaufwand Sonst. K — Sonstige Kosten einschließlich Technol. Betriebsgemeinkosten und Betriebsleitungskosten IK

— Investaufwand

Abb. 5. Integrierter Wärmepumpenprozeß — Zielfunktion für technisch-ökonomische Optimierung —

sind noch nicht die ökonomischen Auswirkungen einer stabilen Kühlung einbezogen worden. Die gezeigten integrierten Wärmepumpensysteme haben einige Schwachstellen. Zum ersten kann die direkte Beaufschlagung von Kühler- und Verdampferflächen mit Kältemitteln angeführt werden, wobei der Eidbruch des Kältemittels in die Biosuspension in das Kalkül einbezogen werden muß. Andererseits sind jedoch zusätzliche Sicherheitskreisläufe aus verfahrenstechnischen Gründen undiskutabel. Zum zweiten wird der Einsatz von Kältemitteln der Art halogenierter Fluorchlorkohlenstoffe in neuester Zeit auch aus der Sicht der Emission dieser Produkte in die Erdatmosphäre gesehen. NOWOTNY [5] z. B. wies den Ernst der Situation nach, wenn durch Lecks oder Havarien größere Mengen dieser Kältemitteldämpfe emittieren können. Wir werden uns zwangsläufig in den nächsten Jahren mit Einsatz neuer Kältemittel auch für biotechnologische Verfahren beschäftigen müssen, wobei unterstellt werden muß, daß die Anforderungen der Hygiene, Sicherheit und des Umweltschutzes erfüllt werden können. In Auswertung dieser gezeigten Probleme haben wir eine Technologie konzipiert, die vollständig auf einen geschlossenen Linksprozeß mit Einsatz eines Kältemittels verzichten kann. In Abb. 6 wird ein derartiges Verfahren dargestellt. Es wurde als Verfahren der Verdampfungskühlung bezeichnet. Es zeichnet sich dadurch aus, daß der zur Abführung der Reaktionswärme erforderliche Wärmeübertrager wiederum als Verdampfer ausgebildet wird, wobei jedoch die Beheizung des Verdampfers mittels Kulturflüssigkeit erfolgt, die aus dem Fermentor entnommen und im Kreislauf geführt wird. Diese Kulturflüssigkeit gibt einen Teil der fühlbaren Wärme im Verdampfer an den Fermentorablauf ab, welcher hierbei unter Anlegung eines entsprechenden Vakuums verdampft. Auf diese Weise erfolgt sowohl die Fermentorkühlung ohne ein von dei1 Tagestemperatur abhängiges Kühlmedium und ohne ein Kältemittel als auch gleichzeitig die Aufkonzentrierung des Fermentorablaufes in einer ersten Stufe. Gekoppelte Fermentor-Eindampfsysteme der vorgestellten Art wurden noch nicht betrieben. Die Problematik liegt einerseits bei den Eindampfsystemen, die so arbeiten, daß die Temperatur des Heizmediums mindestens 5 grd über der Siedetemperatur des zu verdampfenden Stoffes liegt. Andererseits ist es erförderlich, daß die Volumenströme an Brüden nach dem Verdampfungskühler auf den für die nachfolgenden Eindampf-

300

Acta Biotechnol. 10 (1990) 3

S Hab'35MJIh 35 °C IWC^S'CI

Fall fil Fermentarablauf 25000 kglh *=S0gll t-35°C

Ts

meindampfung

Verdampfungskühler 31,

kPa

Fermentation

Rotai ionsdünnschichf eindampfung

VakuumwalzenrIrockner

Trockenprodukt >900g TSU

Abb. 6. Verdampfungskühlung zur thermischen Aufkonzentrierung von Biomassen

stufen notwendigen Druck und der entsprechenden Temperatur angehoben werden. Dazu mußten mehrstufige Verdichter der größten in der DDR verfügbaren Bauart und in mehreren parallelen Strängen eingesetzt werden. Im Ergebnis der ökonomischen Berechnungen konnte ermittelt werden, daß die Investaufwendungen für diese Kreiselverdichter mit Getriebe und konventionellem E-Antrieb alle Einsparungen am Fermentorsystem überdecken. > Selbst die optimale Variante zur Verdampfungskühlung mit Nutzung von 90% der Fermentorabwärme zur Eindampfung und Trocknung der Biomasse verursacht mehr als doppelt so hohe InvestaufWendungen wie der konventionelle Prozeß. Die laufenden Aufwendungen dieser Variante hegen ca. 50% höher als die Basisvariante, verursacht durch höhere investabhängige Kostenarten.

c> Q o>

250 I| 200

Verdampfungskühlung

>»-

^

konventionelle Kühlung und Eindampfung

0) 100 ,o> o 6 c

Ü]

35

40

45 Fermentor- rL0Q7 J • temperatur

Abb. 7. Verdampfungskühlung für Biomassen — Abhängigkeit der Investaufwendungen von der Fermentationstemperatur

PtrcHNER, K., HEMMANN, H.-G., Energetische Optimierung

301

Die Auswirkungen einer höheren Fermentationstemperatur bei einem möglichen Einsatz von thermophilen Hefen sind in den Abbildungen 7 und 8 angegeben. Es ergeben sich signifikante Einsparungen in den Aufwendungen für die einzusetzenden Kreiselverdichter, ohne daß jedoch die minimalen Kosten der Basisvariante erreicht werden. Es wird eingeschätzt, daß sich auch für den Antrieb von Kreiselverdichtern Dampfturbinen einsetzen lassen. Unter der Voraussetzung der Verwendung des Gegendruckdampfes bei Parallel- bzw. peripheren Anlagen lassen sich Einsparungen im Investbereich und in den Energiekosten erzielen, die gegenüber konventionellen Systemen lukrativ werden können. Für eine mögliche Realisierung sind jedoch noch umfangreiche theoretische als auch praktische Arbeiten notwendig. Aus allen Berechnungen ergab sich die Schlußfolgerung, nur soviel Brüden zu komprimieren, wie für die nachfolgende Verwertung im eigenen Prozeß notwendig ist und dazu bei möglichst niedriger Nutzwärmetemperatur.

Verdampfungskühlung konventionelle Kühlung und Eindampfung

45

Fermentortemperatur

Abb. 8. Verdampfungskühlung für Biomassen — Abhängigkeit der fondsbezogenen Aufwendungen von der Fermentationstemperatur —

Eingegangen: 11. 11. 1988

Literatur [1] KATJKTTSCH: FCK Wolfen 1986, EG Mikrob. Eiweißfuttermittel. [2] Methodik zur Bestimmung der Effektivität beim Einsatz von Wärmepumpenanlagen in der mikrobiologischen Industrie RGW-Material 1987. [3] FRATSCHER : Kann aerobe Abwärme aus bioteehnologisehen Anlagen nutzbar gemacht werden? Merseburger Technologische Tage 1981. [4] ARRAS, M., SCHTJLZE, R.: Betrachtungen zur biotechnologischen Abwärmenutzung mittels Wärmetransformation Jahrestagung Verfahrenstechnik 1988, Kothen. [5] NOWOTNY, S.: Probleme, die sich aus der Emission von halogenierten Fluorchlorkohlenwasserstoffen für die Erdatmosphäre ergeben Ztschr. „Luft- und Kältetechnik" 3/88.

Acta Biotechnol. 10 (1990) 3, 302

Akademie-Verlag Berlin

Book Review R . MOOTANG

Genetische Erfindungen im gewerblichen Rechtsschutz Köln, Berlin, Bonn, München: Carl Heymanns Verlag KG, 1988 401 pp., DM 9 8 , - , ISBN 3-452-21273-4

Quite a number of studies have been published on the legal protection of industrial rights as far as techniques and findings in genetic engineering are concerned. This is not just another book on the shelves but a remarkable addition. R . MOUFANG compares European and U.S. patent law and regulations dealing with the protection of varieties to see if these efficiently promote new technologies. A detailed explanation of the scientific fundamentals is followed by a historical analysis of the German and American legal backgrounds. Further chapters deal with the concept of invention (technical character, reproducibility, discoveries), the meaning of exclusion clauses for ethical reasons as well, the application of protecion conditions to genetic inventions, the problem of disclosure, especially in connection with deposits, and finally the protective effect patents have on genetic inventions as well as special protection rights in plant genetics. All the relevant problem areas are so thoroughly treated t h a t it would be difficult to find anything similar in the German speaking world. Literary references and helpful foot notes are given with scientific precision. Clearly a must and of special benefit to those practising and studying patent law. But also a useful reference book for the natural scientist, and an indisputable pleasure to read for any other interested parties. Something few inaugural dissertations can claim for themselves. B . KONIECZNY

Acta Biotechnol. 10 (1990) 3, 3 0 3 - 3 0 5

Akademie-Verlag Berlin

Short Communications Verfahren zur Produktion von ColEl-verwandten Plasmiden N E U B A T J E B , P . , HOFMANN, K . , R I E T H D O R F , S . , H E C K E B , M .

Ernst-Moritz - Arndt-Universität Greifswald Sektion Biologie, Technische Mikrobiologie Jahnstraße 15 a, Greifswald, 2200 GDR Vortrag anläßlich der 4. Neubrandenburger Biotechnologietage 1988

Summary The plasmid ColEl and its derivatives are the most commonly used vectors in gene cloning experiments. A very often used method to obtain a sufficient amount of plasmid DNA is to treat exponential growing cells of Escherichia coli containing plasmids with chloramphenicol [2]. An amplification of these plasmids is also possible in Escherichia coli relA mutants if one of the necessary amino acid limits the growth [8]. On the basis of this system a fed-batch fermentation process was developed with a ten times higher yield of plasmid DNA than the method based on chloramphenicol.

ColEl-Plasmide gehören zu den derzeit am meisten benutzten Vektorsystemen. Da sie eine relativ geringe Größe sowie mehrere singuläre Schnittstellen für Restriktionsenzyme besitzen und außerdem in dem Bakterium E. coli vorkommen, sind sie für Klonierungsexperimente besonders geeignet. Für den breiten Einsatz dieser Plasmide ist ihre Fähigkeit zur Amplifikation in der Zelle bedeutend, da sie dadurch in ausreichender Menge verfügbar werden. Allgemein wird für die Amplifikation von ColEl-Plasmiden bei einer geringen Zelldichte während der logarithmischen Waehstumsphaäe Chloramphenicol in einer Konzentration von 10 bis 50 mg/1 zugesetzt [1, 2, 3, 4, 5]. Von H E C K E B et al. [6] wurde eine alternative Methode zur Plasmidamplifikation beschrieben, die auf der Tatsache basiert, daß nach Verbrauch einer Aminosäure im Kulturmedium in entsprechenden auxotrophen Mutanten von E. coli relA-Stämmen eine Anreicherung von ColEl-Plasmiden zu verzeichnen ist. Es wurde gezeigt, daß im E. coli relA-Stamm CP 79 [7] nach Aminosäureverbrauch das Plasmid pBR 322 um das fünf- bis achtfache angereichert wird [6]. Dies entspricht der AmplifikationSrate, die auch mit der Chloramphenicolmethode erreicht wird. Von R I E T H D O R F [5] konnte diese Methode auch für andere E. coli relA-Stämme (NF 162, CP 143) wie auch für verschiedene ColEl-ähnliche Plasmide bestätigt werden. Diese Variante zur Amplifikation von Plasmiden stellt Somit ein geeignetes Verfahren dar, mit dem Plamid-DNÄ vom ColEl-Typ in größeren Mengen billig produziert werden kann [8]. Diese Methode besitzt gegenüber der Amplifikation mit Chloramphenicol bestimmte Vorteile. So ist letztere nur bei den Plasmiden anwendbar, die keine Chloramphenicolresistenz tragen. Andererseits erreicht man, da das Antibiotikum während der logarithmischen Phase zugesetzt werden muß [4], nur geringe Zelldichten und somit

304

Acta Biotechnol. 10 (1990) 3

begrenzte Ausbeuten an Plaämid-DNA. Die Von H E C K E K et al. [6] beschriebene Variante erlaubt es außerdem, durch nachfolgende Aminosäurezugabe die hohe Gendosis in eine Überproduktion plasmidkodierter Proteine umzuwandeln, wie dies am Beispiel der /?-Glucanase von RIETHDORF [5] gezeigt wurde. Nach dem erfolgreichen Test der AmplifikationSmethode in einem Laborfermentor (LKB 1601 Ultroferm, L K B Bromma, Schweden) sollte die Raum-Zeit-Ausbeute des Verfahrens durch die Anwendung der Fed-batch-Kultivierung verbessert werden. Diese Optimierung wurde für zwei E. coli relA-Stämme (CP 79, CP 143) durchgeführt. Bei gleicher Höhe der Plasmidamplifikation im Batch- und im Fed-batch-Verfahren konnte durch die Erhöhung der Zelldichte eine zehnfache Steigerang der Raum-ZeitAusbeute erzielt werden (s. Tab. 1) [9], Tab. 1. Plasmidausbeuten von Batch- bzw. Fed-batch-Fermentationen der Stämme E. coli CP 79 (pBR 322) und E. coli CP 143 (pBR 322) Stamm

Batch-Fermentation

Fed-batch-Fermentation

ODboo

OD

Gehalt in mg/1

(ig/1 (OD 6 0 0 =

CP 79 (pBR 322) C P 1 4 3 (pBR 322)

3,0 3,0

2,2 3,6

Gehalt in mg/1

1)

750 1220

¡Jig/1 (OD500 =

23 31

20,3 47,8

1)

880 1550

Ein wichtiger Schritt für eine möglichst billige Verfahrensgestaltung war der Ersatz der teueren Aminosäurepräparate durch EiweißhydrolySate. Beim Einsatz von Casamino acids wurde dieselbe Plasmidamplifikationsrate erzielt wie bei der Supplementierung des Mediums mit den einzelnen Aminosäuren. Prinzipiell konnte gezeigt werden, daß auch andere EiweißhydrolySate (z. B. Hefeextrakt) eingesetzt werden können. Da für die gentechnische Verwendung der Plasmide eine Reinigung notwendig ist, wurden unter Nutzung der alkalischen SDS-Methode [2, 3] die Verluste an Plasmid-DNA bei den einzelnen Reinigungsstufen bestimmt (s. Tab. 2). Offensichtlich ist der entscheidende Schritt der Aufbereitung die SDS-Behandlung bzw. der wirkungsvolle Aufschluß der Zellen. Demgegenüber führt die Enteiweißung mit Phenol zu einem relativ Stabilen Verlust von ca. 18%. Eine Höchstreinigung mit der CsCl-Dichtegradienten-Zentrifugation bringt einen hohen Plasmidverlust mit sich und Tab. 2. Ausbeuten an p B R 322-DNA nach verschiedenen Reinigungsstufen Reinigungsstufe

Batch-Fermentation ((ig/1, OD 600 = 1) E. coli E. coli CP 79 CP 143 (pBR 322) (pBR 322)

Verlust des jeweiligen Schrittes L/oJ

Ausbeute vom AusgangsPlasmidgehalt L/oJ

1. Methode nach FRENKEL

700

1300

-

100

2. alkal. SDSMethode [2]

396

1100

29,5 ± 14

70 ±

15

3. Phenolextraktion [2]

311

950

17,6 ± 4

59 ±

15

4. CsCl-DichtegradientenZentrif ugation (2 X )

142

333

60 ± 5

23 ± 3

und BKEMER [ 4 ]

NEUBAUER,

P.,

H O F M A N N , K . U.

a., Produktion von Plasmiden

305

ist, da sie Sehr zeitaufwendig und kostenintensiv ist, nur sinnvoll, wenn eine entsprechende Reinheit unbedingt erhalten werden Soll. I m Ergebnis der Reinigung können ca. 20% der eingesetzten Plasmidmenge in hochreiner Form gewonnen werden. Durch den Einsatz anderer in der Literatur beschriebener Verfahren [z. B. 1 0 , 1 1 , 1 2 , 1 3 ] soll die Ausbeute bei der Reinigung noch erhöht werden. Mit dem beschriebenen Fed-batch-Verfahren ist es möglich, zehnfache Ausbeuten im Verhältnis zur herkömmlichen Batch-Kultivierung zu erzielen. Dies entspricht bei den durchgeführten Fermentationen einem Plasmidgehalt von 20 bis 50 mg/1 Kulturmedium. Nach Reinigung können unter Einbeziehung einer zweimaligen CsCl-DichtegradientenZentiifugation ca. 10 mg reiner PlaSmid-DNA/1 Kultur isoliert werden. Die erzielten Ausbeuten übertreffen um mindestens das Zehnfache die Werte, die mit der Chloramphenicolamplifikation erreicht werden. Im Vergleich zu den von F R E N K E L und B R E M E K [ 4 ] erzielten hohen Ausbeuten von 3 mg/1 Kulturvolumen mit einem E. coli B-Stamm Stellen sie immerhin eine mindestens siebenfache Steigerung der Ausbeute dar. Mit dieser Erhöhung der Raum-Zeit-Ausbeute konnte nachgewiesen werden, daß die Zellkonzentration von relA-Stämmen bei ausreichender NährstoffverSorgung in der logarithmischen Wachstumsphase und gesicherter Aminosäurelimitation zur Plasmidamplifikation für die Höhe des Plasmidgehaltes in der Zelle keine Bedeutung hat. Eine weitere Erhöhung der Zelldichte im Fermentationsmedium, die auf eine noch höhere Plasmidausbeute zielt, scheint unter Beachtung der Plasmidstabilität möglich. Eingegangen: 24. 10. 1988 Überarbeitet: 24. 1. 1989

Literatur [1] [2] [3]

D. B., H E L I N S K I , D. R.: J. Bacteriol. 1 1 0 (1972), 667. T., FRITSCH, E. F . , SAMBROOK, J. : Molecular cloning- a laboratory manual, Cold Spring Harbor Lab., New York, 1972. BIRNBOIM, H . C . , D O L Y , J . : Nucleic Acids Res. 7 ( 1 9 7 9 ) , 1 5 1 3 .

CLEWELL,

MANIATIS,

[4] FRENKEL, L., BREMER, H . : D N A 5 (1986), 539.

[5]

S. : Dissertation A, E.-M.-Arndt-Univ. Greifswald, 1988. Mol. Gen. Genet. 1 9 0 ( 1 9 8 3 ) , 3 5 5 . [7] FIEL, N., F R I E S E N , J. D.: J. Bacteriol. 95 (1968), 729. [8] HECKER, M . , MACH, F., SCHROETER, A., H O F A N N , K., VOLZKE, K. D. : DDR-Patent Nr. DD 239222 Al, WPC 12N/2782503 (1985). [ 9 ] H O F M A N N , K . , NETJBATJER, P . , RIETHDORF, S . , H E C K E R , M. : J . Basic Microbiol. ( 1 9 9 0 ) , im Druck. [10] T O W N S E N D , D. E., A S H D O W N , N., BOLTON, S., G R U B B , W. B . : Lett. Appi. Microbiol. 1 (1985), 87. [ 1 1 ] H U M P H R E Y S , G . O . , WILLSHAW, G . A . , A N D E R S O N , E . S . : Biochem. Biophys. Acta 8 8 8 RIETHDORF,

[ 6 ] H E C K E S , M . , SCHROETER, A . , MACH, F . :

(1975), 457.

J., ACHESON, C . M . , D H U R J A T I , P. : Biotechnol. Bioeng. M. A.: Anal. Biochem. 159 (1986), 280.

[ 1 2 ] COPPELLA, S .

[13]

HEDIGER,

2 9 (1987), 646.

Acta Biotechnol. 10 (1990) 3, 306

Akademie-Verlag Berlin

Book Review Lignocellulosic Materials (Edited by A.

FIECHTER)

Berlin: Akademie-Verlag, 1989. 148 pp., M 1 2 0 , - , ISBN 3-05-500662-3

The Volume 3 8 of the series "Advances in Biochemical Engineering/Biotechnology" (Ed. A. TEB) consists of following three contributions

FIECH-

— L . R . LYND

Production of Ethanol from Lignocellulosic Materials Using Thermophilic Bacteria — F . PARISI

Advances in Lignocellulosics Hydrolysis and in the Utilization of the Hydrolyzates — S t . MABSILI-LIBELLI

Modelling, Identification and Control of the Activated Sludge Process I n the first paper are considered resources and technological aspects of ethanol production from lignocellulosic substrates via thermophilic bacteria. Especially the biological treatment of solid wastes with concomitant ethanol production may become attractive in that solid wastes represent less expensive Substrates. Some features of thermophilic bacteria for this purpose are discussed in comparison to yeasts. This includes the advantages of pentose utilization and in situ cellulase production and cellulose utilization, and the disadvantages of low ethanol tolerance and low ethanol yield. Critical research areas are proposed for closing the gap between the potential of thermophilic bacteria and that which has been experimentally realized. The second paper is of great importance because the decision if acid or enzymatic hydrolysis of lignocellulosics will prevail over in the near future is still open. Different types of acid hydrolysis are described and compared. Some disadvantages and limitations of enzymatic hydrolysis are possibly neglected because of new hyperproductive strains of cellulolytic microorganisms and simultaneous hydrolysis and utilization of produced sugars. A survey of the present trends is given. The third paper is focussed on the dynamic aspects of the activated sludge treatment process and of its operational improvements. The three aspects of modelling, identification and control are reviewed. A simplified model for the pollutant biomass interaction and practical algorithms are discussed. G . KLAPPACH

Acta Biotechnol. 10 (1990) 3, 307—310

Akademie-Verlag Berlin

Ready-to-Use Microbiological Culturing Set Ö A D E R S K Y , I . , 1 K Y B A L , J . , 2 SLKYTA, B . 3

1 2 8

State Textile Research Institute, Radiation Technology Center CS - 66471 Veverska Bitygka Research Institute of Pharmacy and Biochemistry, Prague CS — 13060 Prague, Kourimski 17 Czechoslovak Academy of Sciences Institute of Microbiology, Prague CS - 14220 Prague, VidenskA 270

Summary A ready-to-use microbiological culturing set is described which contains a solid nutrient medium for single use. The set consists of a transparent circular dish with a flange along the upper rim, sterile solid medium, transparent lid with a grip, into which the dish loosely fits, and a multilayer covering foil which provides an air-tight sealing of the cover. The set can be used without any problems for 2.5 years after its fabrication, as verified by culturing different microorganisms on several solid nutrient media.

Introduction The cultivation of microorganisms on solid nutrient media in P E T R I dishes has formed an integral part of microbiological methodology for a long time. The formerly used glass dishes have been largely replaced by plastic ones which have the advantage of being marketed by the supplier in a sterile state. Their main shortcoming is that the nutrient media in P E T R I dishes can be stored only 1 to 2 weeks at a lowered temperature; another disadvantage is the possibility of contamination during transport and the drying up of the medium. These drawbacks have been alleviated by the suppliers in several ways. The P E T R I dishes may be coated by a plastic foil which prevents an accidental separation of the dish and the lid but does not preclude evaporation of moisture and drying up. Attachment of the dish to the lid by bonding agents or by adhesive tapes prolongs their storage life by several weeks at most. Coating of the dishes with a copolymer film is highly efficient but labourious and economically unsuitable.

Construction ol a Ready-to-Use Microbiological Culturing Set

We succeeded in eliminating the above shortcomings by constructing a ready-to-use culturing set. The configuration of the individual parts is illustrated in Fig. l a in a sectional view and in Fig. 1 b in a top view. A flat circular dish (1) with a flange (2) along its upper rim, containing a solid sterile nutrient medium (3) is covered by a transparent lid (4) with a grip (5). The shape of the dish with the flange and of the lid is such that, after adjustment, the dish fits loosely into the lid. Furthermore, when the set is opened

308

Acta Biotechnol. 10 (1990) 3 7

a

6

m

3

4 b

.7

5 r

v.

Fig. 1. Scheme of the ready-to-use microbiological culturing set: a : sectional view, b: top view

by peeling off the coating foil (6), the lid is lifted off the dish, the medium is inoculated and the lid is replaced back on the dish, there is a tight fit between the contact areas of the lid and the dish. The grip serves to facilitate the peeling off of the cover foil. The full line in Fig. l a and the hatched area in Fig. l b represent a peel weld (7) which tightly joins the multilayer covering foil with the circumferential area of the lid. To prevent damage to the loose unwelded part of the foil during the opening of the set before use caused by necessary plucking the peel weld is made to project in a gradually tapering protrusion from the circular contour into the grip. paper

layer

Fig. 2. Configuration of the multilayer covering foil

Both the dish and the lid are fabricated from a transparent unsoftened deep-drawing plastic 0.2—0.5 mm thick, such as unsoftened polyvinyl chloride suitable for blister packaging. The multilayer foil (Fig. 2) consists of a paper layer with a square weight of 45—100 g m _ a , a layer of polyethylene (PE) 0.03—0.09 mm thick and with a specific weight of 918—923 kg m~3, aluminium foil (Al) 0.01—0.04 mm thick and a peel layer. The peel layer consists of nitrocellulose or a copolymer such as ethylene-vinylcarbonic acid, ethylene-vinylacetate or vinyl chloride-vinyl acetate. Both the outer and the inner dish, as well as the coating material, were sterilized by irradiation by a gamma source 60Co (RCH, USSR) using a dose of 25 kGy. This dose was chosen because, unlike lower doses, it did not affect the growth of the microorganisms and ensured sufficient sterility of the irradiated material (Tab. 1). Testing the Set The set was tested by methods for assaying sterility and culturing efficiency (number of colonies appearing on the tested medium after a standard inoculation relative to that appearing in a freshly prepared control medium). These parameters were monitored at