Acta Biotechnologica: Volume 9, Number 3 1989 [Reprint 2021 ed.] 9783112581841, 9783112581834

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Ada Blotectnologica || ^ A ^

Journal of microbial, biochemical and bioanalogous technology

Akademie-Verlag Berlin ISSN 0138-4988 Acta Biotechnol., Berlin 9 (1989) 3, 201-298

Volume 9 • 1989 • Number 3

Instructions to Authors

1. Only original papers that have not been published previously will be accepted. Manuscripts may 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 content 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 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 (DDR-7050 Leipzig, Permoserstr. 15, Dr. Dimter) as soon as possible.

Acta BiltKllQlBliCfl Journal of microbial, biochemical and bioanalogous technology

Volume 9 1989

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

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

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

Managing Editor:

L. Dimter, Leipzig

Number 3

AKADEMIE-VERLAG

• BERLIN

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

Acta Biotechnologica Herausgeber: Institut für Biotechnologie der AdW der DDR, Permoserstr. 15, DDR - 7050 Leipzig (Prof. Dr. Manfred Ringpfeil) und VEB Chemieanlagenbaukombinat Leipzig—Grimma, Bahnhofsstr. 3 - 5 , DDR-7240 Grimma, (Dipl.-Ing. Günter Vetterlein). Verlag: Akademie-Verlag Berlin, Leipziger Str. 3 - 4 , P F 1233, DDR-1086 Berlin; Fernruf: 2236201 und 2236229; Telex-Nr. 114420; Bank: Staatsbank der DDR, Berlin, Konto-Nr.: 6836-26-20712. Redaktion: Dr. Lothar Dimter (Chefredakteur), Martina Bechstedt, Käthe Geyler Permoserstr. 15, DDR-7050 Leipzig; Tel. 2392255 Veröffentlicht unter der Lizenznummer 1671 des Presseamtes beim Vorsitzenden des Ministerrates der Deutschen Demokratischen Republik. Gesamtherstellung: VEB Druckhaus „Maxim Gorki", DDR-7400 Altenburg. Erscheinungsweise: Die Zeitschrift „Acta Biotechnologica" erscheint jährlich in einem Band mit 6 Heften. Bezugspreis eines Bandes 198,— DM zuzüglich Versandspesen; Preis je Heft 33,— DM. Der gültige Jahresbezügspreis für die DDR ist der Postzeitungsliste zu entnehmen. Bestellnummer dieses Heftes: 1094/9/3. 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. © 1989 by Akademie-Verlag Berlin. Printed in the German Democratic Republic. AN (EDV) 18520 03000

Acta Biotechnol. 9 (1989) 3,203—209

Akademie-Verlag Berlin

Dynamics of Redox Potential in Bacterial Cultures Growing on Media Containing Different Sources of Carbon, Energy and Nitrogen OKTYABRSKY, O . N . , SMIRNOVA, G . V .

Department of Ecology and Genetics of Microorganisms Institute of Plant and Animal Ecology Ural Department of USSR Sciences Academy Perm, 614600, Lenin 11, USSR

Summary Two kinds of redox-potential changes were observed in batch cultures of E. coli, B. subtilis and B. megaterium with intensive aeration and pH maintenance at constant level: i) a gradual decrease of the redox potential during continual bacterial growth as a result of interactions between platinum electrode and cell surface; ii) the redox jumps in the generation of which the soluble redox substances take part under the conditions of different transitional processes (exhaustion of the sources of carbon, energy or nitrogen, metabolism switching from one source to another and so on). The redox monitoring may be useful for cultivation control in these situations.

Introduction Combining modern methods and improving the existent methods for controlling and regulating bacterial populations are required for biotechnology development. Redox potential (Eh) monitoring is one of these methods being useful for bacterial cultivation [1,2].

The main factors determining the culture's Eh value are the changes in oxygen partial pressure and pH if aerobic and facultative anaerobic bacteria grow at low aeration and without p H maintenance [1—3]. Redox-active substances excreted by the cells are supposed to be a factor which affects the bacterial culture's Eh [1], However, the nature of these substances is not fully understood. In recent years it was shown t h a t during redox-potential monitoring of bacterial cultures growing in modern fermenters with automatic stabilization of pH and high p 0 2 level maintenance, Eh changes were recorded which could not be related to pH and p 0 2 changes. These Eh changes were the leaps of Eh to the more negative values and were observed in transitional processes caused by glucose and ammonium exhaustion in the medium (E. coli, B. subtilis, S. marcescens), during heat shock of growing cells (E. coli) and under conditions of cell division in synchroneous cultures of B. subtilis [4—6]. In the present paper we show that the list of situations causing such characteristic Eh changes, which were not related to p 0 2 and pH, may be significantly extended. Materials and Methods The strains of E. coli K-12; A cya 854 (F~, trp, pur, A cya 854, str) adenylate cyclase deficient; AN 120 (F", arg, E 3 , thi, mtl, xyl, str 804, unc A 401) ATPase deficient; their parental strain AN 180 (F arg, E 3 , thi, mtl, xyl, str 804) (the last three were obtained 1*

204

Acta Biotechnol. 9 (1989) 3

from B O L S H A K O V A T. N., Gamalea Institute); AS-1 with high permeability of cell envelope (the strain was received from G R I N I U S L . L . , Vilnus University); Bacillus subtilis VKM-428 and Bacillus megaterium VKM-517 obtained from All-Union collection of microorganisms were used. Bacteria for inoculation were grown in a thermostat at 36 °C using the M-9 minimal medium with 2 g/-l glucose. The cultural medium for E. coli A cya was supplemented with 1 % tripton. The cultural medium for E. coli AN 120 and AN 180 additionally contained 100 mg arginine and 5 mg thiamine. Fed-batch-culture experiments were carried out in fermenters ANKUM-2 at 36°C (rate of rotation: 500 rpm, culture volume: 11). Bacteria E. coli A cya were cultivated in M-9 medium supplemented with 100 mg tryptophane, 100 mg adenine and necessary quantity of glucose. Cultures of E. coli AN 120 and AN 180 were grown in M-9 medium with 100 mg arginine and 5 mg thiamine and necessary quantities of glucose. Other bacteria were grown in M-9 medium with the carbon and energy source being added. The necessary pH was maintained by automatic addition of 1 N. NaOH. Aeration was provided so that the oxygen pressure (p0 2 ) was not lower than 70%. p 0 2 was measured by means of a platinum membrane electrode. The redox potential in the culture was measured by a smooth platinum electrode and reference silver chloride electrode. The role of the cell surface in redox-potential changes was investigated in the way described by M A T S U N A G A with some variations [7], where Eh was simultaneously measured by two pairs of calibrated electrodes. The measuring electrode of the first pair was separated from the medium by a membrane filter "Sympor" (0.3 (j.m) or by a cellophane membrane, for the other pair the measuring electrode was opened and contacted both with the cells and the medium. The electrodes were calibrated by adding cysteine, changing pH and barbotaging argon. I t was shown that both electrodes responded in the same way to cysteine addition, pH and oxygen pressure changes. However, after cysteine addition the hew steady state was achieved with the closed electrode 2—4 min later than with the open one. All parameters (pH, Eh, p0 2 , t°, optical density) were continually recorded. /9-Galactosidase activity was assayed in toluene-treated cells according to the method of E P S T E I N [8]. Results and Discussion In the first series of experiments glucose as a limiting substrate was added to the cultures of E. coli K-12 and B. subtilis in portions of 0.2—0.5 g/1 0.5—1 hour after growth cessation. The redox-potential measurement was carried out simultaneously with two platinum electrodes, one of them was opened and the other was separated from the medium by a membrane (see „Materials and Methods"). The potentials of these two electrodes were different under the conditions described (Fig. 1). If the redox-potential measurement was carried out by means of a closed electrode, the basic value of Eh was constant during all the growth periods. If Eh was recorded by an open electrode, the basic value lineally decreased to the negative region, and this deviation of the basic line from the initial level was proportional to the cell amount. Against the background of these changes other changes of Eh were observed which took place at bacterial-growth cessation caused by glucose exhaustion. They had the shape of potential jumps to more negative values and were recorded by both closed and open electrodes. The feature of these potential jumps was a small dependence of their amplitude (ziEh) on the cell amount in both E. coli and B. subtilis cultures. The amplitude of the Eh discontinuities measured by means of a closed electrode amounted to 46 i 4% (n = 21) and 60 ± 6% (n = 35) of the ¿1Eh measured by an open electrode for E. coli K-12 and B. subtilis, respectively. These differences in electrode readings described above cannot be explained by differences in pH and p0 2 on the two sides of the membrane filter separating

Oktyabrsky, O. N., Smienoya, G. V., Redox Potential in Bacterial Cultures

205

Fig. 1. Redox potential dynamics in fed-batch culture of E. coli K-12 1 — optical density (D), 2 — Eh measured by open electrode, 3 — Eh measured by membrane filter closed electrode. Arrows denote the moments of glucose addition.

the ecletrode from the medium. The cell-Surface interaction with the open platinum electrode is the most probable reason for different electrode readings. Under the condition of glucose exhaustion, the Eh-leap parameters for E. coli K-12 were dependent on the ion composition of the medium. A K + -concentration change from 10~2 to 10" 5 with the replacement of K + by an equimolar quantity of N a + did not influence the amplitude of the potential jump. A decrease of K + concentration in the medium to 2 • 10" 6 M caused a Eh leap, lowering the amplitude to a 5th—7th, which was followed by a sharp decrease in growth rate. Eh gradually increased if glucose was added, however, bacteria did not resume their growth simultaneously, as is possible at a high K + concentration, but only 15 min after glucose addition (Fig. 2). In transitional processes caused by glucose exhaustion in E. coli K-12 cultures the Eh leap disappeared at a S0 4 2 - concentration in the medium below 10 - 4 M. A S 0 4 2 - limit simultaneously leads to a decrease of both the rates of bacterial growth and respiration. After S0 4 2 " addition to the medium in the form of Na 2 S0 4 or MgS0 4 solutions, the bacteria were capable of generating Eh leaps again due to glucose exhaustion. A decrease of Ca 2+ and Na + concentrations down to trace amounts did not affect both the Eh-leap profile and amplitude. Different strains of E. coli (K-12, M-17, AN 120, AN 180, A cya, AS-1) were investigated in order to test their ability for Eh-leaps generation. All of the strains listed here, with the exception of A cya, were able to generate reversible leaps of Eh to more negative values at glucose exhaustion. In a culture of E. coli A cya the open platinum electrode recorded two types of Eh changes: gradual decrease of Eh which was proportional to the cell amount during growth, and characteristic redox-potential changes under both conditions of glucose exhaustion and addition (Fig. 3). However, after glucose exhaustion in E. coli A cya a higher increase of Eh (10—30 mV) was observed in contrast to an Eh decrease, which was typical for other strains. This increase of E h was accompanied by a fall of the bacterial growth rate and by the rise of p 0 2 due to the decrease of oxygen consumption. Acidification was replaced by alkalization after the exhaustion of glucose. Glucose addition caused the acceleration of bacterial growth and the rapid decrease of

206

Acta Biotechnol. » (1989) 3

_i OS

Time [hi

i 10

i 15

Fig. 2. Growth of E. coli K-12 in fed-batch culture at different K + concentrations in the medium 1 — concentration of biomass (X) and 3 — Eh at 2 • lO"2 M K+; 2 — X and 4 - Eh at 2 • 10-« M K+. The growth of cells ceased due to glucose exhaustion and resumed after glucose addition to the culture at the moments denoted by arrows.

Time [h] Fig. 3. The transitional process under glucose exhaustion in E. coli A cya i — optical density (D), 2 — Eh measured by open electrode in E. coli A cya culture, 3 — Eh measured by open electrode in E. coli K-12 culture, 4 — p0 2 . The cells of E. coli K-12 and A cya were grown in M-9 medium with tryptophane (100 mg/1) and adenine (100 mg/1). The moments of glucose addition are indicated by arrows.

O k t y a b r s k y , O. N., Smtbnova, G. V., Redox Potential in Bacterial Cultures

207

E h to negative values. An analysis of kinetics, size and direction of E h and p 0 2 changes showed t h a t p 0 2 changes might contribute to Eh change. The bacterium E. coli A cya has a low activity of adenylate cyclase, a key enzyme in cAMP synthesis. The cAMP system takes p a r t in the regulation of carbon- and energysources metabolism and of membrane-related functions, including bioenergetic ones [9]. We propose that the inversion of the redox-potential leap in E. coli A cya under the condition of glucose exhaustion may be caused by disturbances of bioenergetic functions. Eh leaps at growth cessation were also observed during growth of E. coli K-12 with lactose and glycerol and during B. megaterium growth with glucose if these substrates were the sole sources of carbon and energy.

Time [hi

Fig. 4. The transitional process with diauxic growth of E. coli K-12 1 — optical density (D), 2 — pH, 3 — Eh measured by open electrode, 4 — p0 2 , 5 — /?-galactosidase activity. The arrow denotes the moment of glucose and lactose mixture addition.

Eh. changes in E. coli K-12 under the condition of diauxia are shown in Fig. 4. I n these experiments a mixture of glucose and lactose was added to a bacterial culture nonadapted to lactose, and then a typical diauxic growth was observed. Four phases of growth were observed: i) growth with glucose, ii) 25 —40 min pause, iii) growth with lactose, iv) cessation of growth due to lactose exhaustion. Two E h leaps have been recorded with amplitudes of about 125 mV, each of them was followed by growth cessation after glucose and lactose exhaustion, respectively. The time period during which the first E h leap occured agreed with the pause when a delay of bacterial growth and an increase of /5-galactosidase activity up to the level sufficient for growth with lactose were observed. A diauxia pause was absent, and growth continued until both substrates were consumed if a mixture of glucose and lactose was added to a lactose-adapted E. coli culture. Accordingly, only one E h leap was observed in this case. If several carbon and energy sources, for example, glucose, lactose, acetate, etc. were available in the medium

208

Acta Bicitechnol. 8 (1989) 3

simultaneously, a successive generation of Eh leaps was recorded whenever the next substrate was exhausted. These Eh changes were not directly connected with p0 2 and pH changes in the medium. In all experiments described above bacteria were cultivated in the M-9 medium with NH4C1(1 g/1) as nitrogen source, and the carbon and energy s6urces (glucose, lactose, glycerol, etc.) were the limiting components of the medium. In Fig. 5 the Eh values and other parameters of E. coli K-12 grown in the M-9 medium with two nitrogen sources (NH4C1 and histidine) are demonstrated. The concentrations of limiting components were chosen in such a way that at first NH4C1 and then glucose were exhausted. After ammonium exhaustion the E. coli cells continued their growth with glucose and histidine without any pause.

Time [rnin] Fig. 5. The transitional processes in E. coli K-12 under condition of a successive exhaustion of ammonium and glucose in the medium Cultural medium contained the M-9 components with addition of histidine, NH4C1 and glucose. Details are given in the legend to the Fig. 4.

The bacterial growth rate fell visibly, the acidification of the medium was stopped, and oxygen consumption decreased sharply. The leap of Eh measured by means of an open platinum electrode was a feature of transitional processes. The Eh amplitude amounted to 50—110 mV with a mean value of 70 i 4 mV, the duration of the decreasing phase was 15—30 min. Further growth of E. coli with glucose and histidine was performed at a lower steady state of the redox potential. If NH4C1 solution was added to the medium at any moment after NH 4 + exhaustion, all culture parameters, including Eh, recovered their significance for the growth with ammonium rapidly (dotted line). If E. coli was grown with histidine as the sole nitrogen source, the reaction of the open platinum electrode to growth cessation at glucose exhaustion was contrary to that observed during growth with ammoriium: Eh increased at growth cessation and decreased at its resumption. Every change of culture parameters demonstrated in Fig. 5 was not directly connected with pH and p0 2 changes. During the growth of aerobe and facultatively anaerobe bacteria a low aeration and without pH stabilization the changes of oxygen pressure and pH in the culture are so

OKTYABRSKY,

O. N.,

SMIRNOVA,

G. V., Redox Potential in Bacterial Cultures

209

considerable t h a t E h values recorded by the system with metal electrodes are connected to a marked degree with the changes of these parameters [2]. I n these situations E h is useful as a parameter for the control of bacterial growth, but indirectly it reflects the oxygen concentration in the culture. Our studies showed t h a t under intensive aeration and at a constant p H level a system for E h measuring with an open platinum electrode recorded two types of potential changes. The changes of the first type observed during continual growth were expressed in the form of a gradual Eh decrease proportional to the cell amount. They are the result of the interaction between electrode and cell surface. The changes of the second type in the form of Eh leaps were observed under transitional processes and are caused b y the action of both the cell surface and the solute redox substances on the electrode. The results of our research together with previously known facts [4—6, 10, 11] permit the presentation of an enlarged list of situations in which investigated bacteria generate E h leaps not connected with p H and p 0 2 changes: the exhaustion of the sources of carbon, energy and nitrogen, metabolism switching from one source to another, the influence of substances changing the intracellular p H , heat shock, and cell division in synchroneous cultures. Probably the E h leap during lysozyme action observed b y HEW I T T in Micrococcus lysodeikticus may be attributed to this list [3]. W h a t is the nature of the substances and intracellular processes causing the redox jumps under the conditions of transitional processes? I t has been shown in ouj- experiments t h a t the E h jumps in transitional processes caused by glucose and ammonium exhaustion in the medium were connected with the increase in the amount of accessible S H groups in the medium and on the cell surface. W e propose t h a t the intracellular p H decrease is the immediate reason for these changes [12]. As a whole, data about redox potential dynamics in intensively aerated cultures demonstrate t h a t E h may be a sensitive indicator of the physiological state of bacteria. This enhances the informational value of E h monitoring and opens wider possibilities for its application in biotechnology. Received April 21, 1988 Revised June 20, 1988

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]

RABOTNOVA, I. L.: The Role of Physical Chemical Conditions (pH and rH2) in Life Activity of Microorganisms. Moskow: Edit. U S S R Sci. Acad., 1957, p. 145. J A C O B , H. E.: Redox Potential — In: Methods in Microbiology. London: Academic Press, 1970. Vol. 2, p. 91-123. H E W I T T , L. F.: Oxidation-Reduction Potentials in Bacteriology and Biochemistry. Edinburgh: Livingstone ltd., 1950, p. 215. OKTYABRSKY, O . N., SMIRNOVA, G. V., Z E L E N I N , E. N.: Biophysica 2 8 (1984) 5, 831. OKTYABRSKY, O . N., PSHENICHNOV, R. A.: Microbiologiya 5 1 (1982) 3, 515. OKTYABRSKY, O . N . , SMIRNOVA, G . V.: Biophysica 3 1 (1986) 3, 459. MATSUNAGA, T., K A R U B E , I., S U Z U K I , S . : Appl. Environ. Microbiol. 37 (1979) 1, 117. E P S T E I N , W.: J. Mol. Biol. 8 0 (1967) 3, 529. D I L L S , S . S . , DOBROGOSZ, W. J.: J. Bacteriol. 1 8 1 (1977) 3, 854. K A L I M I , G . , H E N R I Q U E S , B. M. A., P E R E I R A , L.: Indian J. Exp. Biol. 1 9 (1981) 9, 808. OKTYABRSKY, O . N., SMIRNOVA, G. V. — In: Thesis of Reports of IVth All-Union Conference "Control Cultivation of Microorganisms". Puchshino, 1986, p. 140. OKTYABRSKY, O . N . , SMIRNOVA, G . V . : BIOCHIMIYA, in press.

Act» Biotechnol. 9 (1989) 3, 210

Akademie-Verlag Berlin

Book Review Daniel

AMMAN

Ion-Selective Microelectrodes Principles, Design and Applications Berlin, Heidelberg, New York, Tokyo: Springer-Verlag, 1986. 346 pp., 153 fig., 42 tab., DM 1 5 8 , - , ISBN 3-540-16222-4 In Anbetracht der Tatsache, daß Fortschritte im biotechnologischen und medizinischen Bereich einen Erkenntniszuwachs auf zellulärer Ebene bedingen, stellen ionenselektive Mikroelektroden (ISM) einen experimentellen Schlüssel für den Zugang insbesondere intrazellulärer Transportvorgänge dar. Das vorliegende Buch widerspiegelt umfassend sowohl theoretisch als auch praktisch die Thematik ionenselektiver Mikroelektroden und sich abzeichnende neue Entwicklungen dieser Technik. Aufgrund seiner langjährigen Erfahrungen geht der Autor bis ins Detail der Arbeitsweise ionenselektiver Elektroden allgemein und der von ISM speziell, den auftretenden Schwierigkeiten und Fehlermöglichkeiten beim Messen mit ionenselektiven Elektroden nach. So zerstört er z. B. die Legende von der "tailored" ISM, ohne deshalb auf gesicherte theoretische Erkenntnisse, die eine Suche nach ionenselektiven Substanzen erleichtern, zu verzichten, zumal diese Erkenntnisse auch Bedeutung für die Einschätzung der Zellkontamination durch die aus den ISM austretenden Substanzen besitzen. Weiterhin werden beispielsweise ausführlich Unterschiede zwischen Diffusions- und Tip-Potential herausgearbeitet oder es wird eingehend auf die Komplexität vor allem biologischer Meßmedien und ihre Konsequenzen für die Meßergebnisse eingegangen. Das Buch ist logisch aufgebaut und übersichtlich gegliedert. Nach der systematischen Klärung theoretischer Grundlagen wie Struktur und Eigenschaften ionenselektiver Substanzen, Zusammensetzung und Optimierung ionenselektiver Membranen, Potentialbildung an ionenselektiven Membranen, Selektivitätsfaktor, Einzelionenaktivitäten u. a. m. wird auf die Herstellung verschiedener Formen von ISM eingegangen, wobei die modernen Verfahren bevorzugt behandelt und Vor- und Nachteile der verschiedenen Elektrodenausführungen erläutert werden. Auf die häufig recht komplizierten älteren Technologien wird durch zahlreiche Literaturangaben verwiesen. Besondere Aufmerksamkeit widmet der Autor den Flüssigmembran-ionenselektiven Mikroelektroden auf der Basis neutraler Carrier, die eingebettet in weitere Kapitel bezüglich Theorie, praktischen Aufbau und ihre Anwendung ausführlich behandelt werden. Dabei wird auf Messungen in lebenden Zellen eingegangen, daß Ausmaß der mechanischen Zerstörung der Zelle und der chemischen Veränderung des Zellinhaltes durch die ISM untersucht und die Rolle des intrazellulären Wassers mit seiner besonderen Struktur sowie der Ionenstatus in der Zelle werden diskutiert. Hier beschränken sich die Betrachtungen nicht auf Messungen mit ISM allein, sondern es werden auch die Bedeutung der Meßwerte zur Klärung solcher Probleme wie das der Membrantheorien von P F E F F E R und L I N G herausgestellt und weitere zur Messung zellulärer Vorgänge angewandte Techniken wie Verteilung schwacher Säuren und Basen, optische Methoden (Biolumineszenz, Verwendung von Photoproteinen) und NMR-Spektroskopie gegenübergestellt. Ein spezielles Kapitel behandelt die Ermittlung der Konzentrationen bzw. Aktivitäten von H + , Li + , Na+, K + , Mg2+ und Ca2+ und im abschließenden Kapitel werden ethische Probleme von Experimenten an lebenden Organismen betrachtet, wobei sich der Autor nachdrücklich zu seiner Verantwortung gegenüber dem Lebewesen, an dem experimentiert wird, bekennt. Sehr schön die zusammenfassenden Bemerkungen am Schluß jedes Kapitels und hilfreich die zahlreichen Abbildungen. Hier allerdings sind manche Diagramme für den mit potentiometrischer Arbeitsweise nicht Vertrauten nur wenig informativ. Hervorzuheben ist unbedingt die äußerst gründliche Literaturarbeit. Obwohl das Buch vorwiegend für den in der Elektrophysiologie Tätigen geeignet erscheint und überall dort, wo Forschungen auf zellulärer Ebene betrieben werden, nicht fehlen sollte, ist es darüber hinaus für alle jene von Nutzen, die mit ionenselektiven Elektroden oder Mikroelektroden arbeiten, da die erschöpfend behandelten fundamentalen Probleme allerorts die gleichen sind. B . GRÜNDIG C. KRABISCH

Acta Biotechnol. 9 (1989) 3, 211-217

Akademie-Verlag Berlin

Characterization of the Course of D-Glucitol Biotransformation by Strains Gluconobacter oxydans PELECHOVÄ, STAN£K, J . 1

2 3

J.1,

KULHANEK,

M.2,

GRAMANOVA,

I.1,

KREJÖF, J . 1 ,

HEEMANKOVI,

V.3,

jr.3

Department of Fermentation Chemistry and Technology Institute of Chemical Technology 166 28 Prague, ÖSSR Research Institute for Pharmacy and Biochemistry 130 60 Prague, ÖSSR Laboratory of Monosaccharides Institute of Chemical Technology 166 28 Prague, ÖSSR

Summary Production strains Glwonobaeter oxydans used in manufacture of L-sorbose from commercial D-glucitol were evaluated by HPLC determination of D-glucitol, L-sorbose, D-fructose, D-mannnitol, D-arabinitol, D-xylulose, and D-iAreo-2,5-hexodiulose in the course of fermentation. Serious differences were observed among the strains apparently identical by standard methods.

Introduction L-Sorbose, as an intermediate of the classical R E I C H S T E I N synthesis of vitamin C, is manufactured in increasingly large amounts by fermentative dehydrogenation of Dglucitol with acetic bacteria, the method invented by P E L O U Z E [1] and B E R T R A N D [2, 3]. Nowadays, the selected strains of Gluconobacter oxydans [4—6] are used exclusively, but even in these the dehydrogenation is accompanied by the production of side metabolites, mainly D-fructose and D--2,5-hexodiulose; 3 D-xylulose; 4 D-fructose; 5 D-mannitol; 6 D-arabinitol; 7 1-butanol; 8 D-glucitol

[min]

fractometer (Meopta, Czechoslovakia); the linear relationship between concentration of water solutions of D-glucitol and the refractive index [16] is valid even for high concentrations, the refractive indexes of water solutions of D-glucitol and L-sorbose are practically identical. In paper chromatography, phenol saturated with water was used for elution, the detection was made with periodate or with ammonia silver nitrate solution. The pH values were measured on a pH-meter PO 201/2 (RADELKIS, Hungary), dissolved oxygen with a probe connected with a D. O. Analyzer (NEW BRUNSWICK SCIENTIFIC, USA). Biomass was determined on Synpor filters No. 5 (KAVAIJER, Czechoslovakia) or nephelometrically on a spectrophotometer UNICAM SP 800 (England) at 600 nm. The concentration of the live cells was determined by a four-day cultivation on agar medium.

214

Acta Biotechnol. 8 (1989) 3

Results and Discussion Thé production strains of Gluconobacter oxydans were preliminary evaluated in flask cultivations by standard analytical methods (sugar dry matter, determination of reducing substances, paper chromatography), the resulting degrees of conversion of D-glucitol to L-sorbose are given in Tab. 1. Liquid chromatography was then used to follow in detail the course of cultivation of the best, apparently equivalent strains S 232, S 664, and CCM 2356. The data obtained (Tab. 2—4) clearly show the serious differences among these strains, which were almost identical according to standard methods. Tab. 2. Changes in saccharide concentration in the course of conversion of commercial D-glucitol to L-sorbose by Gluconobacter oxydans S 232 Time D-GIucitol

L-Sorbose

D-Fructose

D-Mannitol

D-Arabinitol

D-Xylulose

[h]

[g/1]

[g/1]

[g/1]

[g/1]

[g/1]

[g/1]

D-threoTotal 2,5-hexodiulose [g/1] [g/1]

0 2 4 6 8 10 12 14 16 18 20 22 24 28

172.3 170.3 166.2 159.7 151.5 137.5 124.8 106.1 91.2 76.5 60.1 48.2 38.6 27.5

23.9 26.8 30.6 39.6 47.5 55.6 70.0 89.7 106.1 119.1 135.2 146.0 152.8 163.4

0.38 0.46 0.55 0.63 0.82 1.09 1.39 1.80 2.20 2.48 2.81 3.10 3.30 3.80

3.5 3.2 3.1 3.0 2.8 ' 2.7 2.6 2.4 2.3 2.0 1.8 1.5 1.3 1.0

0.50 0.42 0.34 0.30 0.28 0.23 0.21 0.20 0.20 0.19 0.19 0.18 0.17 0.16

0.10 0.18 0.27 0.38 0.32 0.36 0.37 0.37 0.38 0.39 0.40 0.40 0.45 0.45

0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.3 0.4 0.6 0.8 1.1

200.88 201.56 201.26 203.81 203.42 197.68 200.47 200.77 202.58 200.96 200.90 199.98 209.12 197.41

Tab. 3. Changes in saccharide concentration in the course of conversion of commercial D-glucitol to L-sorbose by Gluconobacter oxydans S 664 Time D-Glucitol

L-Sorbose

D-Fructose

D-Mannitol

D-Arabinitol

D-Xylulose

[h]

[g/1]

[g/1]

[g/1]

[g/1]

[g/1]

[g/1]

D-threo- Total 2,5-hexodiulose [g/1] [g/1]

0 2 4 6 8 10 12 14 16 18 20 22 24

177.0 175.5 173.0 167.9 155.8 146.8 125.7 102.1 76.0 51.0 38.3 27.2 22.1

16.6 20.1 21.2 25.7 37.1 48.4 66.9 92.5 118.7 139.2 154.5 162.8 170.5

0.8 0.9 1.0 1.3 1.5 1.7 2.2 2.8 3.5 4.2 4.8 5.3 5.5

3.5 3.5 3.5 3.3 3.3 3.1 2.7 2.5 2.2 1.7 1.7 1.3 1.2

0.58 0.58 0.57 0.57 0.54 0.50 0.45 0.44 0.41 0.33 0.22 0.13 0.11

0.07 0.07 0.07 0.08 0.10 0.14 0.18 0.21 0.25 0.34 0.44 0.52 0.54

0.9 1.1 1.2 1.2 1.3 1.5 1.7 2.0 2.0 2.5 3.0 4.0 5.0

199.55 201.75 200.54 200.05 199.61 202.14 200.83 201.65 203.06 199.27 202.96 201.25 204.95

215

PELECHOVA, J . , KULHANEK, M. et al., D-GIucitol Biotransformation

Tab. 4. Changes in saccharide concentration in the course of conversion of commercial D-glucitol to L-sorbose by Qlvamobacter oxydans COM 2356 Tinje D-Glucitol

L-Sorbose

D-Fructose

D-Mannitol

D-Arabinitol

D-Xylulose

[h]

[g/I]

[g/1]

[g/1]

[g/1]

[g/1]

[g/1]

~D-threo- Total 2,5-hexodiulose [g/1] [g/1]

0 3 6 9 12 15 18 21 24

170.0 165.5 148.2 126.0 104.4 59.0 23.4 19.4 13.9

30.2 36.9 53.9 75.1 94.0 139.3 165.6 169.2 173.9

0.4 0.5 0.8 2.1 3,7 4.9 5.1 5.5 6.0

3.6 3.6 3.5 2.4 1.3 0.1 0.0 0.0 0.0

0.5 0.5 0.5 0.4 0.3 0.3 0.2 0.2 0.1

0.1 0.1 0.1 0.2 0.3 0.3 0.4 . 0.4 0.5

0.1 0.1 0.4 0.5 0.7 1.4 2.2 3.0 4.1

204.3 206.6 206.9 206.7 204.7 205.3 196.9 197.7 198.5

In all cases (Tab. 2—4), the course of D-glucitol decrease, like the increase of L-sorbose had a similar shape. D-Mannitol present in the medium was dehydrogenated to D-fructose slightly more quickly than D-glucitol, especially with the strain COM 2356, where it was used up already in the early stages of cultivation. The increase of D-fructose concentration, however, was larger than calculated from the decrease of D-mannitol, it increased also in the final stage of conversion when the concentration of D-mannitol equaled zero. In the case of strain S 664, the amount of D-fructose was even twice as large as the" theoretical amount formed from D-mannitol. The relationship between the concentration of this excess D-fructose and that of D-

o

CH2-CH-~~^CH-CH2 S V \ /

0

0

I

0

-0-SI-~-NH-CH2-CH-~~-CH-CH2 1 1 \ / 0 OH 0

IB

HO-CH2

,

A——0

0 1

SI 3 - 0 - S I — N H - C H 2 - C H - ~ ~ - C H - C H 2

"

1

0

1

1

\ s

OH

+

lx

nu

l,À\9 HO \I

h

I 0 SI

i

§-O-SI—NH-CH2-CH~~-CH-CH2-O-CH2

l

0 1

1/ OH OH

I

OH

I

OH

1—-A

0

-

I 0 SI

$-0-SI—•-NH-CH2-CH-~~CH-CH2-0-

i-

I 1

0

I

OH

I

OH 1C

Fig. 1. Schematic presentation of chemical reactions occuring during modification of C P G by y-APTES (Pig. 1 A ) ; of y - A P T E S - C P G by bioxirane (Fig 1 B ) , and the reaction occuring during glucose binding to e p o x y - A P T E S - C P G (Fig 1 C ) .

Temperature,

Optimization

In order to optimize temperature, the reactions were carried out as follows. Samples of 1 g suction-dried APTES-CPG were mixed with 1 ml of oxirane and 5 ml 0.1 M Britten ROBINSON'S buffer pH 1 0 . 0 . The samples were then brought to different temperatures [4-4; + 2 8 ; + 4 5 ; +70°C) and mixed on the shaker over 24 h. As before the labelled glucose was bound to the prepared materials.

278

Acta Bioteehnol. 9 (1989) 3

Reaction-Time Optimization The optimal reaction time was found by shaking 1 g of suction-dried APTES-CPG mixed with 1 ml of dioxirane and 5 ml 0.1 M Britten R O B I N S O N ' S buffer p H 10.0 for different periods of time (12; 24; 48 h) at 28°C. Successive steps were the same as before. Oxirane-Amount

Optimization

1 g portions of suction-dried APTES-CPG were mixed with 0.5; 1; or 2 ml of oxirane and such amounts of 0 . 1 M Britten R O B I N S O N ' S buffer pH 1 0 . 0 to reach the final mixture volume 6 ml. The reaction was carried out over 24 h at 28 °C. The obtained material was then subjected to the reaction with the labelled glucose. I n all cases described above the amount of the labelled glucose bound with the prepared sorbents (epoxy APTES-CPG) was treated as the measure of oxirane amount bound to the support surface. In order to bind the labelled glucose with the obtained sorbents, the following procedure was applied: 1 g of prepared epoxy-APTES-CPG was placed into the mixture of 1 kBq 14C [U] glucose (8.5 GBq/mmol; Amersham) and 100 mg of non-labelled glucose. The next steps of these reactions were the same as in case of the ligand coupling procedure (see below). Ligand-Coupling Procedure The coupling procedure of ligands was accomplished by adding 1 g of the reactant dissolved in 5 ml of 0.1 M Britten R O B I N S O N ' S buffer p H 10.0. Glucose, fructose, lactose, galactose and amino acids as alanine, phenylalanine, serine, tyrosine and asparagine were used as reactants. The conditions of the reaction were as follows: time = 12 h, temperature = 28 °C. In all cases the reactions were stopped by washing the gel on a glass-filter funnel with 100 ml of water, 50 ml of monoethanolamine and 100 ml of water. Glucose-Bonding Procedure Glucose is one of the most popular monosaccharides. I t can be also treated as a substrate of some enzymes for example glucose oxidase (EC 1.1.3.4), glucose dehydrogenase (EC 1.1.1.47), hexokinase (EC 2.7.1.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), glucose-6-phosphatase (EC 3.1.3.9), glucose-l-phosphatase (EC 3.1.3.10), glucose-6phosphate isomerase (EC 5.3.1.9), glucose phosphomutase (EC 2.7.5.1). As this ligand is useful in many cases, the glucose bonding procedure was optimized. As a support, epoxy-APTES-CPG obtained according to the above described method was used. During the optimization synthesis, the labelled glucose was applied. pH Optimization 1 g samples of suction dried epoxy-APTES-CPG were mixed with 5 ml of 0.1 M Britten R O B I N S O N ' S buffer p H 8—12 which contained 1 0 0 mg of non-labelled glucose and 1 kBq 14 C [U] glucose. The reactions were carried out at 28 °C for 24 h at 150 rpm. The reaction was stopped as before by the washing procedure (see Ligand Coupling Procedure). Reaction Time and Temperature Optimizations The optimal conditions of the reaction time and temperature were found similarly to. the optimal p H conditions. The reactions were carried out at constant p H 10.0, and constant temperature + 2 8 °C over different periods of time (1; 3; 6; 12; 24; 48) or at constant p H 10.0 and constant time 24 h at different temperatures ( + 4 ; + 2 8 ; + 4 5 ; + 7 0 °C). The radioactivity of the synthetized materials was treated as a measure of the bond glucose amount.

ROGALSKI, J . , DAWIDOWICZ, A. L., B i n d i n g of Glucose

279

Methods Surface-Area

Measurements

The specific surface area was determined from nitrogen thermal desorption measurements (BET method) [21]. For this purpose, nitrogen sorptomat (Sorptomatic 1806 — Carbo Erba, Milan, Italy) was employed. Pore Volume and Pore-Diameter

Measurements

The mean pore diameter and porosity (pore volume) of the prepared CPG were calculated from the porosimetric data [22]. The porosimetric investigation were made by means of the mercury porosimeter Type 1500 ( C A R B O . E R B A , Milan, Italy). Determination of Oxirane Groups In order to find out the end of the washing procedure, the epoxy groups were determined in supernatant after washing. STTNDBEBG and PORATH [19] method was employed for this purpose. Measurements of Radioactivity The radioactivity bound into the matrix was measured according to K B I T C H E V S K Y and MAT.TTOTRA [23] using Aquasol 2 (NEN, England) scintillator and Isocap 300 (Nuclear Chicago, USA) scintillation counter. Besults and Discussion The controlled porous glass used in the investigations was characterized by specific surface area $ B E T = 71.8 m 2 /g and mean pore diameter D = 71 nm. These pores are sufficiently large to allow for the entrance of the globular protein molecules of the molecular weight between 0.13—30 Mdaltons [24], The reaction of chemical bonding in the experiments is hypothetically illustrated in Fig. 1 A, B, C. The reaction presented in Fig. 1A is known very well from literature [25—26] and it is not the subject of this paper. Fig. 2 shows the influence of different parameters on the amount of bound epoxy modifier with APTES-CPG (in agreement with I B reaction). I t should be reminded that the amount of bound epoxy agent was estimated on the basis of 14C glucose radioactivity level connected to epoxy-APTES-CPG in further stage under the constant conditions. However, this value is not quite real but there is no doubt radioactivity changes a^e in this case proportional to the change of chemically bound oxirane. Besides, this solution is relatively easy and precise. I t seems to be extremely useful if one takes into account the problems of the epoxy-group determination on support surface and errors which can occur during analysis especially at low coverage density. As it can be seen from Fig. 2 A the binding reaction of epoxy agent with CPG covered by aminophase runs at p H = 11.0 most effectively. The further increase of p H causes the radical drop of oxirane bound with the surface. Probably it results from hydrolysis of siliceous-CPG structure in spite of APTES layer which can also play a protecting role. The destructive role of high concentration of O H - ions (pH = 10.0) in the reaction of oxirane chemical binding is also illustrated by Fig. 2B presenting the influence of reaction time on the amount of the bound epoxy agent. The significant decrease of bound oxirane amount is observed during the increase of the reaction time from 24 to 48 h. Twenty four hours of the reaction time as follows from Fig. 2B is the most optimal.

280

Acta Biotechnol. 9 (1989) 3

0.5

10

Oxirane amount [ml/g APfES-CPO

20 J

4

28

45

Femperoture

70 [°C]

Fig. 2. Oxirane content expressed as an amount of bound glucose as a function of pH (Pig 2 A); reaction time (Fig. 2 B ) ; bioxirane amount (Fig. 2C) and temperature (Fig. 2D).

In Fig. 2C the relationship between the amounts of bound epoxy compounds and the employed substrate is presented. As results from this plot, the most adequate amount of oxirane in relation to the APTES-CPG is 1 cm 3 of oxirane per 1 g APTES-CPG. The double increase of epoxy agent amount does not lead to the increase of bound modifier but causes some decrease. On the one hand it can result from the experimental error but on the other hand it confirms the saturation of free amine groups in APTES-CPG surface and/or the reaction among epoxy groups on the forming support surface. According to Fig. 2D, the discussed reaction l b is the most effective at higher temperature. The 80% increase of bound oxirane is observed during the temperature changes from + 4 to + 2 8 °C. The further temperature increase (from + 2 8 to + 4 5 °C) caused only the 8 % increase. As mentioned in the introduction, one of the purpose of the paper was to find out the optimal conditions of glucose binding to synthetized support (epoxy-APTESCPG). For this reason, a numerous reactions of glucose binding under various conditions of pH, temperature and time were carried out. In the experiments, the support with constant coverage density by epoxyrAPTES radicals was applied. As results from Fig. 3 A, the influence of temperature in this case is different from that during coupling of the oxirane to APTES-CPG. Diminution of bound glucose is observed with the temperature increase. The temperature change from + 4 to + 2 8 °C leads to the 5 0 % drop of glucose contents coupled with the support. Considering the influence of hydroxyl-ion concentration on the reaction the maximum at pH = 9.0 is seen (Fig. 3 B ) . The pH increase causing drop of the bound glucose probably results from the hydrolysis reaction. At pH lower than 9.0 pH units, the surface glucose concentration is not significantly different. I t can be connected with lower reactivity of the epoxy groups at pH lower than 9.0. According to Fig. 3C, the optimal time of the reaction is between 6—12 h. The 9 % decrease of the bound glucose is observed after 24 h in relation to the amount coupled at optimal time. Taking into consideration the data from Fig. 2 it can be said that the

Rogalski, J., DawidowICZ, A. L., Binding of Glucose

Temperature

_J

I

6

12

1

Time

I'Cl

L_

?4

281

Fig. (Fig (Fig oose

3. Influence of temperature 3A); pH (Fig 3B) and time 3C) on the amount of bound gluto prepared epoxy-APTES-CPG.

48 [h]

most optimal conditions of oxirane binding to APTES-CPG are as follows: pH = 10.5; temperature = + 2 8 °C; reaction time = 24 h and the amounts of substrates 1cm 3 oxirane/1 g APTES-CPG. On the base of Fig. 3: pH = 9.0, reaction time = 12 h and temperature = +4°C was assumed to be optimal for the synthesis of glucose with epoxy-APTES-CPG. In order to control the reproducibility of glucose-binding procedure with epoxy-APTESCPG surface, the series of 10 syntheses of glucose binding was carried out. The syntheses were performed under optimal conditions. From 10 syntheses it was found that the amount of bound glucose equals to 16.4 mg (A + 0.44 mg)/l g support (92 ¡¿mole; A ± 2 ¡j.mole/1 g of support). The analogous conditions of APTES-CPG activation by oxirane and binding of ligands to the obtained support were employed in the case of similar saccharide compounds and some amino acids. The data concerning these syntheses are given in Tab. 1. As it can be seen from Tab. 1 the similar binding degrees of the above mentioned substances with epoxy-APTES-CPG were obtained for monosaccharides galactose and fructose and for bisaccharide lactose which is made of one glucose molecule and one galactase molecule coupled by /?-l,4-glucoside bond. A little bit higher value of bound lactose in relation to galactose proves that the employed binding conditions are more advantageous for reaction of glucose with epoxy-APTES-CPG than for galactose with the same support. As lactose is composed of these two saccharides, both of them can be casually coupled with the support. The obtained results for lactose are a little lower than for glucose and a little higher than in the case of galactose. 6

Acta Biotechnol. 9 (1989) 3

282

Acta Biotechnol. 9 (1989) 3 Tab. 1. Amount of bound ligands to epoxy-APTES-CPG Isotopes used for binding

Supplier and specific activity of used isotopes

Amount of bound isotope explained as the amount of bound ligand [mg of ligands per 1 g of support]

D [U- U C] galactose [D-glucose 1-14C] lactose D [U-14C] fructose L [U-14C] phenylalanine L [U-14C] alanine L [U-14C] serine L [U-14C] tyrosine L [U-14C] asparagine

AMERSHAM; 1 . 8 7 - G B q / m m o l AMERSHAM; 1 . 8 5 - G B q / m m o l AMERSHAM; 5 . 5 6 - G B q / m m o l

14.4 A ± 0 . 4 3 15.9 A ± 0 . 4 5 12.4 A ± 0 . 4 4

AMERSHAM; 1 6 . 6 - G B q / m m o l

2.4A±0.11

AMERSHAM; 5 . 5 0 - G B q / m m o l

2.4A±0.12

AMERSHAM; 5 . 5 0 - G B q / m m o l AMERSHAM; 1 6 . 6 - G B q / m m o l AMERSHAM; 3 . 7 0 - G B q / m m o l

3.7 A ± 0 . 1 0 3.8 A ± 0 . 0 9 2.6 A ± 0.11

On the basis of amino-acid-binding data it can be said that amine groups show lower affinity for epoxy-APTES-CPG. The employed coupling conditions probably do not stabilize the bonds between epoxy groups and amine groups belonging to amino acids. It is also confirmed by 60% increase of bound amino acids which possess additionally the hydroxyl group (serine, tyrosine) in relation to their analogues which do not possess these groups (alanine, phenylalanine).

Conclusions 1) The results presented show that it is possible to synthetize siliceous supports with reactive epoxy groups on their surfaces not by direct method with application of proper silane modifier possessing epoxy groups on opposite ends, but by a doublestep reaction on the surface. 2) The synthetized support allows for coupling different compounds having free hydroxyl and amine groups (e.g. saccharides and amino acids). 3) As follows from the presented data the binding reaction should be optimized individually for each compound. 4) The reactions with compounds having hydroxyl groups run easier than in the case of substances with amine groups. Acknowledgement This work was Supported in part by the Polish Scientific Project PR

I 08

and CPBR.

3.13.2.1.18.

Received April 15, 1988 Revised June 20, 1988

References [ 1 ] MIEDZIAK, I . , WAKSMTTNDZKI, A . : Wiadomosci Chemiczne 88 ( 1 9 8 4 ) , 1 4 7 . [ 2 ] WISEMAN, A . : Handbook of Enzyme Biotechnology. Ed. : WISEMAN, A . New

Sydney, Toronto, 1975, p. 243.

York, London,

ROGALSKI, J . , DAWIDOWICZ, A. L., Binding of Glucose

283

[3] SOLOMON, B . : A d v a n c e s in Biochemical Engineering. E d s . : T. K . GHOSE, A. FIECHTEB,

N. BLAKEBBOUGH) Berlin, Heidelberg, New York: Springer-Verlag, 1978, p. 131. [4] SANDERSON, C. J . , WILSON, D. V.: I m m u n o l . , 20 (1971), 1061. [ 5 ] ZABORSKY, O . R . , OGLETREE, J . : B i o c h e m . B i o p h y s . R e s . C o m m u n . 6 1 ( 1 9 7 4 ) , 2 1 0 . [ 6 ] PORATH, J . , AXEN, R . , ERNBACK, S . : N a t u r e 2 1 5 ( 1 9 6 7 ) , 1 4 9 1 .

[7] LOWE, C. R., DEAN, P. D. G. : Affinity Chromatography. London, New York, Sydney, Toronto: J . Wiley and Sons, 1974, p. 206. [8] ibid. p. 250. [9] VRETBLAD, P.: Biochim. Biophys. Acta 434 (1976), 169. [10] [11] [12] [13]

LANDT, M . , BOLTZ, S. C., BUTLER, L . G . : B i o c h e m i s t r y 17 ( 1 9 7 8 ) , 9 1 5 . SIMONS, P . C., VANDER JAGT, D . L . : A n a l . B i o c h e m . 8 2 ( 1 9 7 7 ) , 3 3 4 . E D Y , V . G . , BILLIAU, A . , d e SOMER, P . : J . B i o l . C h e m . 2 5 2 ( 1 9 7 7 ) , 5 9 3 4 . LÖNNERDAL, B . , CARLSSON, J . , PORATH, J . : F E B S L e t t . 7 5 ( 1 9 7 7 ) , 8 9 .

[14] SNYDER, L. R., KIRKLAND, J . J . : Introduction to Modern Liquid Chromatography. New York, Chichester, Brisbane, Toronto: J . Wiley and Sons, 1979, p. 488. [15] DAWIDOWICZ, A. L., WAKSMUNDZKI, A.: Wspólczesne kierunki w teorii i praktyce chromatograficznej. Wroclaw: Ossolineum, 1976, p. 29. [16] HALLER, W. J . : J . Chem. P h y s . 42 (1965), 686. [ 1 7 ] DAWIDOWICZ, A . L . , WAKSMTXNDZKI, A . , DERYLÓ, A . : C h e m . A n a l . 2 4 ( 1 9 7 9 ) , 8 1 1 . [ 1 8 ] DAWIDOWICZ, A . L „ ROGALSKI, J . : P L - P 2 7 0 3 7 9 . [ 1 9 ] SUNDBERG, L . , PORATH, J . : J . C h r o m a t o g r . 9 0 ( 1 9 7 4 ) , 8 7 .

[20] KLYSZEJKO-STEFANOWICZ, L.: Cwiczenia Z Biochemii. Warszawa: PWN, 1972, p. 713. [21] DAWIDOWICZ, A. L., P i x r s , S., NAZIMEK, D.: J. Anal. Appi. Pyrol. 10 (1986), 59. [22] DAWIDOWICZ, A. L., PIKUS, S.: Appi. Surface Sci. 17 (1983), 45. [ 2 3 ] KRITCHEVSKY, D . , MALHOTRA, S . : J . C h r o m a t o g r . 5 2 ( 1 9 7 0 ) , 4 9 8 .

[24] Operation Instruction CPG-10. Electro-Nucleonics, Inc., 368 Passaic Ave. Fairfield, N.Y. 07006. [25] ROBINSON, P. J., DUNNILL, P., LILLY, M. D.: Biochim. Biophys. Acta. 242 (1971), 659. [26] WILCHEK, J . : Methods in Enzymology. New York: Academic Press, 34 (1974), 59.

Acta Biotechnol. 9 (1989) 3, 284

Akademie-Verlag Berlin

Book Review Paul P R Ä V E , Merten Fritz WAGNER

SCHLINGMANN,

Wulf

CRUEGER,

Karl

ESSER,

Rudolph

THATTER,

Jahrbuch Biotechnologie, Band 2 1988/89 Wien, München: Carl Hanser Verlag, 1988. 620 S., 160 Abb., DM 6 8 , - , ISBN 3-466-15255-5

Mit dem Jahrbuch Biotechnologie soll im Laufe des Jahres eine Reihe entstehen, die wichtige wissenschaftliche, technische aber auch wirtschaftliche Themen, Entwicklungen und Daten zusammenfassend darstellt. So wurden auch für die vorliegende Ausgabe 2 — 1988/89 — wieder aktuelle Arbeitsgebiete der Biotechnologie ausgewählt, für die der jeweilige Stand der Technik von fachkundigen Autoren aufbereitet wurde. Mit seinen Übersichten über thematisch begrenzte Fachgebiete aus Forschung und Entwicklung, will das Jahrbuch dazu beitragen, die Informationsflut auf diesem Arbeitsgebiet sowohl für den Fachmann, als auch für den Einsteiger zu kanalisieren. So wird im Laufe der Zeit eine Enzyklopädie der Biotechnologie entstehen und fortgeschrieben werden, die einerseits dem Spezialisten raschen Einblick in die Randgebiete seines Arbeitsfeldes ermöglicht, andererseits dem Einsteiger den Zugang zum Erkenntnisstand sowie zu den wichtigsten aktuellen Themen erleichtert. Im Abschnitt „Praxis der Biotechnologie" werden in Fortführung der ersten Ausgabe des Jahrbuchs weitere neue Methoden der Laborpraxis beschrieben. Gerade auf einem so jungen, sich dynamisch entwickelnden Gebiet, wie der Biotechnologie, ist es geboten, erprobte Labormethoden möglichst rasch den Fachkollegen mitzuteilen. Nur allzuoft ist für diesen praktischen Aspekt in wissenschaftlichen Publikationen kein Platz. Hier will das Jahrbuch Biotechnologie eine Lücke schließen. Mit dem Teil „Informationen" zusammengestellten Fakten, Daten, Hinweisen und Vorschriften, soll ein Überblick über die Arbeitsbedingungen gegeben werden, die der Biotechnologe bei seiner Tätigkeit kennen und berücksichtigen muß. Ergänzend dazu finden sich in diesem Abschnitt auch Informationen aus dem Bereich Forschung/Lehre/Ausbildung, sowie Angaben über einige der auf diesem Gebiet tätigen Firmen.

Acta Biotechnol. 8 (1989) 3, 285—290

Akademie-Verlag Berlin

Anwendung der p02-Elektrode zur Gelöst-Sauerstoff-Bestimmung in organischen Flüssigkeiten W E I C H E R T , D . , K L A P P A C H , G . , SCHRÖTER, H .

Akademie der Wissenschaften der DDR Institut für Biotechnologie Permoserstraße 15, Leipzig 7050, DDR

Summary The method described allows the determination of dissolved oxygen in water-inmiscible organic liquids under normal pressure in a temperature range of 0°C to 80 °C depending on the boiling point of the organic material. The method' can be adapted to modified conditions. The method using the principles of 02-measurement by means of CLARK'S electrode offers an accuracy of 2—6%. The exact knowledge of dissolved molecular oxygen in organic liquids can be of interest for chemical or biotechnological processes.

Einleitung Die Kenntnis gelöster Mengen molekularen Sauerstoffs in flüssigen Medien ist für Wissenschaft und Technik von Interesse, weil dieser Sauerstoff noch in geringer Konzentration über eine beachtliche Reaktivität verfügt. Der für biologische Prozesse bedeutende Gelöst-0 2 wäßriger Lösungen ist nach zahlreichen Methoden bestimmt worden, wobei sich die Messungen mit der C L A R K S c h e n Elektrode durchgesetzt haben [1—3]. Diese Methode gestattet auch die Bestimmung gelösten Sauerstoffs in organischen Flüssigkeiten. Dieser Sauerstoff kann f ü r chemische oder biotechnologische Prozesse von Bedeutung sein, in denen Kohlenwasserstoffe, Öle, Monomere u. ä. eine Rolle spielen. Die nachfolgend beschriebene Methode zur Bestimmung von Gelöst-0 2 in organischen Flüssigkeiten erweitert die Möglichkeiten, die Rolle des molekularen Sauerstoffs in den genannten technischen Prozessen zu quantifizieren und dadurch exakter einschätzen zu können. Bestimmungsmethode Meßprinzip Das Meßprinzip beruht auf der Partialdruckmessung gelösten molekulare® Sauerstoffs mittels der membranbedeckten Ü L A R K s c h e n Elektrode nach der Gleichgewichtseinstellung in zwei nichtmischbaren Flüssigkeiten. Dazu wird eine bestimmte Menge sauerstoffgesättigten Wassers mit einer bekannten Menge sauerstofffreier organischer Flüssigkeit

286

Acta Biotechnol. 9 (1989) 3

in Kontakt gebracht und bei definierten Bedingungen durch Rühren der 02-Austausch bis zur Gleichgewichtseinstellung durchgeführt und dabei in der wäßrigen Phase mit einer in Wasser bei gleicher Temperatur calibrierten p0 2 -Elektrode die prozentuale Sättigung gemessen. Die für die Berechnung der Sauerstofflöslichkeit in der organischen Flüssigkeit erforderlichen Sauerstoffkonzentrationen 02-gesättigten Wassers sind für bestimmte Temperaturen und 02-Partialdrücke bekannt, so z. B. in einer Firmenschrift für ein j>02-Meter [3], aus der auszugsweise Werte in Tab. 1 zusammengefaßt wurden. Für die von der sauerstoffgesättigten wäßrigen Phase an die organische Phase abgegebenen Sauerstoffmenge m 0 , bei der sich eingestellten prozentualen Sättigung p 0 2 X gilt dann:

Daraus ergibt sich für die 02-Konzentration in der organischen Phase bei gleicher prozentualer Sättigung: (Vor. =

v m

°-

[mg 0 2 /kg]

' Or • SOr.

(2)

Die Kombination der Gleichungen' 1 und 2 gestattet die Berechnung der maximalen Sauerstofflöslichkeit in der organischen Flüssigkeit für die jeweilige Meßtemperatur ¿M und den jeweiligen 02-Partialdruck unter Meßbedingungen.

r

ü 2 A

[mg 0 2 /kg] [mg 0 2 /lJ

,

(3) ,4,

1

Es bedeuten: Co.w

= 0 2 -Konzentration in der Oa-gesättigten H 2 0-Phase unter Meßbedingungen i M und 2>O,-M (Werte siehe Tab. 1) [mg/kg] C 0 l _or. = Aktuelle bzw. maximale 0 2 -Konzentration in der organischen Phase unter Meßbedingungen [mg/kg] bzw. [mg/1] Fw = Volumen der wäßrigen Phase [1] F0r = Volumen der organischen Phase [1] p02X = Prozentuale 0 2 -Sättigung nach der Gleichgewichtseinstellung der Messung Öw> £?or. = Dichte der wäßrigen bzw. organischen Phase unter Meßbedingungen [kg/1]. Meßverfahren

Die in Prinzipskizze 1 dargestellte Meßapparatur bestand aus drei Glasgefäßen A, B und C, die mit einem Doppelmantel zur Thermostatierung, absperrbaren Zu- und Abführungen für Gase und Flüssigkeiten und einer Rührvorrichtung im Gefäß C ausgerüstet war. Alle Gefäße waren für die Volumenmessung graduiert. Die organische Phase wurde im Gefäß A mit Reinst-N2 und die wäßrige Phase im Gefäß B mittels Luft bis zur Sättigung begast, dabei wurde gleichzeitig die p0 2 -Elektrode im Gefäß B unter Meßbedingungen auf 100% Sättigung calibriert. Als Meßinstrument diente dabei das p0 2 -Meter M65 der Firma Metra Meß- und Frequenztechnik, Dresden — Radebeul [4], Anschließend wurde die j>02-Elektrode im vorher mit Reinst-N2 gespülten Gefäß C positioniert. Im Gefäß C wurden dann bei totaler Füllung definierte Volumina beider Phasen unter Rühren in Kontakt gebracht und in der wäßrigen Phase der sich einstellende Wert der prozentualen 0 2 -Sättigung gemessen.

WEICHERT, D., KLAPPACH,

287

G. U. a., Gelöst-Sauerstoff-Bestimmung

Tab. 1. Gelöst-0 2 -Konzentration in Wasser bei verschiedenen Temperaturen, Luftsättigung bei 760 mm Hg und einem 0 2 -Partialdruck von 160 mm Hg [3] Temperatur °C

Gelöst-0 2 -Konzentration [mg 0 2 /kg] Destilliertes Wasser

Wasser 5 g Salz/kg

Wasser 10 g Salz/kg

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

14,62 14,23 13,84 13,48 13,13 12,80 12,48 12,17 11,87 11,59 11,33 11,08 10,83 10,60 10,37 10,15 9,95 9,74 9,54 9,35 9,17 8,99 8,83 8,68 8,53 8,38 8,22 8,07 7,92 7,77 7,7 7,5 7,4 7,3 7,2 7,1 7,0 6,9 6,8 6,7 6,6

13,79 13,41 13,05 12,72 12,41 12,09 11,79 11,51 11,24 10,97 10,73 10,49 10,28 10,05 9,85 9,65 9,46 9,26 9,07 8,89 8,73 8,57 8,42 8,27 8,12 7,96 7,81 7,67 7,53 7,39 7,25

12,97 12,61 12,28 11,98 11,69 11,39 11,12 10,85 10,61 10,36 10,13 9,92 9,72 9,52 9,32 9,14 8,96 8,78 8,62 8,45 8,30 8,14 7,99 7,85 7,71 7,56 7,42 7,28 7,14 7,00 6,86

Als Beispiel soll die Bestimmung der maximalen Saueratofflöslichkeit in p-Xylol bei 16 °C und einem Barometerstand von 760 mm Hg, entsprechend einem 0 2 -Partialdruck von 160 mm Hg angeführt werden. Der Kontakt von 0,393 1 sauerstoffgesättigten Wassers mit 0.147 1 sauerstofffreiem p-Xylols im Gefäß C ergab bei 16 °C und einem Barometerstand von 768 mm Hg einen prozentualen 0 2 -Sättigungswert von 34,0% nach der Gleichgewichtseinstellung, gemessen mit einer Polycarbonatmembran bedeckten p0

Acta Biotechnol. 9 (1989) 3

288 Prinzipskihe

1

Abb. 1. Prinzipskizze der Meßanordnung

Elektrode der Firma Metra Meß- und Frequenztechnik, Dresden—Radebeul. Der C 0t wWert für 16 °C und 760 mm Hg Luftdruck (Tab. 1) betrug 9,95 mg 0 2 /kg H 2 0 . Mit Hilfe der Gleichung 3 wurde unter Berücksichtigung des Luftdruckes für p-Xylol bei 16°C und 760 mm Hg Luftdruck eine maximale Sauerstofflöslichkeit von _ =

9,95 • 0,393 • 0,9989 • 66,0 = 0,147.0,865-34,0

_Q 59 6 m g '

... °a/kg

bestimmt. In der Literatur [3] wurde ein Wert von 58,34 mg 0 2 /kg für die gleichen Bedingungen angegeben.

Ergebnisse und Diskussion Von 4 Kohlenwasserstoffen, die für biotechnologische Prozesse zur Gewinnung von mikrobiellem Protein von Interesse sein können, wurde die Gelöst-0 2 -Konzentration für Luftsättigung bei 30 °C und einem 0 2 -Partialdruck von 160 mm Hg bestimmt. Die Ergebnisse sind in der Tab. 2 zusammengefaßt worden. Die untersuchten Kohlenwasserstoffe wiesen im Vergleich zu Wasser eine 6—6,5fach höhere 0 2 -Löslichkeit auf. Bei den Messungen lag der mittlere prozentuale Fehler zwischen 0,5 und 6,6%. Für vergleichende Betrachtungen sind in der Tab. 3 die 0 2 -Löslichkeiten verschiedener organischer, nicht mit Wasser mischbarer Flüssigkeiten aufgeführt. Sie wurden der Literatur entnommen und wurden mit unterschiedlichen Methoden bestimmt.

W E I C H E S T , D . , KLAPPACH,

G. U. a., Gelöst-Sauerstoff-Bestimmung

289

Tab. 2. Gemessene Gelöst-02-Konzentration bei Luftsättigung, 30 °C und 160 mm Hg 0»-Partialdruck Untersuchte Flüssigkeit

Desorbat 2

Dieselkraftstoff 1

Dieselkraftstoff 2

Mepasin

Cetan

Messungen

Gelöst-02Konzentration [mg(yi]

Mittelwert Gelöst-02Konzentration [mg0 2 /l]

Mittlerer Fehler [mg 0 2 /l]

1 2 3 4

45,0 45,0 49,0 48,3

46,8

1,06

2,3

1 2 3 4 5

42,1 49,0 43,6 42,1 46,5

44,6

1,3

2,9

1 2 3 4 5 6 7

49,8 44,6 50,5 46,7 42,3 47,9 50,6

47,5

3,1

6,6

1 2 3

47,2 46,3 46,5

46,6

0,2

0,4

1 2 3 4 5

50,6 49,0 43,6 42,1 46,5

44,6

1,3

2,9

Tab. 3. 02-Löslichkeit in organischen Flüssigkeiten bei Luftsättigung [3] (160 mm Hg 0 2 -Partialdruck) Organische Flüssigkeit

Temperatur

n-Dodekan Isoparaffin Octan 1-Octen Benzol Toluol Xylol para Acetylentetrachlorid Tetrachlorkohlenstoff Nitrobenzol Eingegangen: 19. 5. 1988

Literaturzitat

[°C]

Gelöst-04Konzentration [mg 0 2 /l]

30 30 20 20 19 18 16

73 74 64 64 55,4 58,1 58,3

[5] [5] [6] [6] [3] [3] [3]

18

18,6

[3]

18 18

43,1 17,3

[3] [3]

[%]

Acta Biotechnol. 9 (1989) 3

290

Literatur [1] [2] [3] [4]

CLARK, L. C.: Trans. Am. Soc. Artif. Int. Org. 2 (1956), 41. CLABK, L. C.: Analysis Instrumentation 1964, 79 —82. BECKMAN Instructions 1675-A; Fieldlab TM-Oxygen Analyzer. Metra Meß- und Frequenztechnik, Dresden—Radebeul, DDR.

[ 5 ] SPRENGLER, G . , POHL, E . : B r e n n s t o f f c h e m i e 5 0 ( 1 9 6 9 ) , 2 7 6 .

[6] PETROCELLI, J . A., LICHTENSTEIN, D . H . : A n a l . C h e m . 8 1 (1959), 2017.

Acta Biotechnol. 9 (1989) 3, 2 9 1 - 2 9 3

Akademie-Verlag Berlin

Short Communications Lysine Production by Auxotrophic-Regulatory Mutants of Corynebacterium glutamicum PLACHY, J .

Research Institute of Antibiotics and Biotransformation 25263 Roztoky near Prague, Czechoslovakia

Summary A mutant of Corynebacterium glutamicum dependent on homoserine and resistant both to S-(2aminoethyl)-L-cysteine and lysine hydroxamate, cultivated under submerged conditions for 4 days in a medium containing sucrose, corn-steep and pea nut meal hydrolyzate, accumulated 33.5 g/1 lysine.

Introduction Auxotrophic-regulatory mutants are now widely employed for amino-acid production by fermentation. There has been isolated a mutant of Corynebacterium glutamicum resistant to S-(2-aminoethyl)-L-cysteine (AEC) and dependent on homoserine (Hse) producing relatively high amounts of lysine [1], T O S A K A et al. [2] have isolated mutants of Brevibacterium lactofermentum resistant to both AEC and