Acta Biotechnologica: Volume 9, Number 1 1989 [Reprint 2021 ed.] 9783112581889, 9783112581872

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Acta Biotertniloglcg | |

Journal of microbial, biochemical and bioanalogous technology

Akademie-Verlag Berlin ISSN 0138-4988 Acta Biotechnol., Berlin 9 (1989) 1, 1—96

Volume 9 • 1989 • Number 1

Instructions to Authors

Í. Only original papers that have not been published previously will be accepted. Manuscripts may be submitted in English, German or Russian (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 in English and the title in English. 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. Diniter, Permoserstr. 15, D D R - 7 0 5 0 Leipzig) as soon as possible.

Acta MMlAliCt

Journal of microbial, biochemical and bioanalogous technology

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—Grimma by M. Ringpfeil, Berlin and G. Vetterlein, Leipzig

Editorial Board : 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 . Skryabin, Moscow M. A. Urrutia, Habana

1989

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

Number 1

Managing Editor:

L. Dimter, Leipzig

Volume 9

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

•

B E R L I N

"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 book-shop for foreign languages literature or to the competent news-distributing agency; — in the FEG and Berlin (West): to a book-shop 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 Akademie-Verlag 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, Bahnhofstr. 3—5, DDR-7240 Grimma (Dipl.-Ing. Günter Vetterlein). Verlag: Akademie-Verlag Berlin, Leipziger Straße 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 Jahresbezugspreis für die DDR ist der Postzeitungsliste zu entnehmen. Bestellnummer dieses Heftes: 1094/9/1. 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) 1, 3 - 8

Zur biotechnologischen Relevanz von Regulationsvorgängen des mikrobiellen Sekundärstoffwechsels BORMANN, E . J .

Zentralinstitut für Mikrobiologie und experimentelle Therapie Akademie der Wissenschaften der D D R , Beutenbergstr. 11, Jena, 6900 D D R Vortrag anläßlich der 8. Vortragstagung der GATM in Neubrandenburg, Januar 1988.

Summary Although the regulation of microbial secondary metabolism belongs to the important objects of actual investigations the results and knowledges are used rather poorly in industrial fermentations. This situation arises from the discrepancy between the economically driven development of the know how as soon as possible contrary to the more slow progress of profound examination of the complicated network of secondary metabolism. Nevertheless some well known principles of metabolic regulation, e.g. catabolite repression or resistance against products or metabolites are considered in fermentation processes by means of suitable substrate application as well as improvement of high yield strains. B o t h aspects are discussed with respect to examples of fermentations of ^-lactams, polyketides and glycopeptides.

Einleitung Obgleich der mikrobielle Sekundärstoffwechsel zu den intensiv bearbeiteten Forschungsrichtungen gehört, finden Resultate und Vorstellungen in nur relativ wenigen bekannten Fällen ihre unmittelbare Anwendung in industriellen Hochleistungsverfahren. Ursache hierfür ist zum einen der ökonomische Zwang, möglichst zügig zu einem effizienten und originellen „Know how" zu gelangen, bevor eine zeitaufwendige und gründliche Durcharbeitung des speziellen Objektes in biochemisch-regulativer Hinsicht erfolgen kann. Zum anderen lehrt der Umgang mit biologischem Hochleistungsmaterial, daß kaum zu verallgemeinernde Erfahrungen genutzt werden können, da die Entstehungsweise und die Stoffwechselleistung des jeweils speziellen Objektes stets an biochemische Besonderheiten geknüpft sind. Überdies beeinträchtigen patentrechtliche Informationsbarrieren den Informationsaustausch entscheidend, zumal die in der wissenschaftlichen Öffentlichkeit zugänglichen Ergebnisse und Verallgemeinerungen zumeist an ökonomisch nicht mehr attraktiven Stämmen gewonnen wurden. Insofern kann eine Gegenüberstellung von Literaturkenntnissen und biotechnologischen Gegebenheiten auf dem Gebiet des Sekundärstoffwechsels zu einer realistischen Beurteilung der Sachlage beitragen. Biochemie und Biotechnologie Unabhängig von der im Einzelfall zu betrachtenden Sekundärstoffbildung wird jeder diskontinuierliche oder semikontinuierliche Fermentationsprozeß — als gegenwärtig noch vorherrschende industrielle Prozeßauslegung bei der Sekundärmetabolitgewinnung 1*

4

Acta Biotechnol. 9 (1989) 1

Tab. 1. Regulative Aspekte im Sekundärmetabolismus Biogenetische Gruppe

Phänomen

Biochemische Aspekte

Zuordnung

Aminoglycoside

Produktresistenz

Intermediat- und Produktphosphorylierung Adenylierung Acetylierung Permeabilitätsänderung cAMP, ppGpp A-Faktor C-Faktor Sekmet-ZellwandSynthesekonkurrenz

Modelle Enzymatik

Präkursorpool aus Primärmetabolism. TCC, Pyruvatkinase Pyruvatdehydrogen. Citratbildung PEP-Carboxylase Adenylatlevel PhosphatasenRepression, Derepression Anhydrotetracyclinoxygenase Kompartimentierung Metabolittransport Exoenzyme Yalinsynthese Alanindehydrogenase Bistabilität cAMP

Stammvergleich

Differenzierung

Acetat-Polymalo- Entwicklungsphasen nat-Gruppe

Phosphatkontrolle

C-KatabolitRepression N-KatabolitRepression Differenzierung Aminosäurederivate

C-Katabolitrepression P-Katabolitrepression N-Katabolitrepression Stimulierung (Methionin) P-Kontrolle

Anwendung

Selektion

Modelle Populationsschrittmacher?

Substratfeeding (Lipide, C-Substrate) Modelle zur Enzymatik

Selektion

Substratfeeding NH 4 -Deponie? Modelle Modell Substratfeeding Enzymatik

Sekmet-Enzyme Präkursor-pool Sekmet-Enzyme

Modell

Präkursor-pool

Modell

Reverse Transsulfurierung Phosphatasen und Substrataufschluß

Modell

Modell

Selektion P-feeding P-Deponie

Shikimat-Deriv.

Produkthemmung

Sekmet-Enzyme Permeabilität

Mevalonat-Deriv.

Induktion

Präkursoreffekt Modell Kompartimentierung

BORMANN,

E. J., Microbial Secondary Metabolism

5

— durch die Einflußgrößen der Zellkonzentration (als Trockenmasse X), der spezifischen Produktbildungsrate g prod und der produktiven Zeit tp bestimmt. Für die Penicillinherstellung beispielsweise geht es darum, das Integral (1) zu optimieren [1]. Dabei kann es in der Prozeßgestaltung gleichermaßen zur Veränderung der Trockenmassekonzentration über die Wachstumsrate oder den Ertragskoeffizienten unter Beibehalt von qp über einen optimal dauernden Zeitraum kommen oder aber um die Veränderung von qp bzw. tp bei vorher ermittelter optimaler Zellkonzentration. Während für die Beeinflussung des Wachstumsprozesses generelle Gesichtspunkte der Substratbilanzierung berücksichtigt werden können [2], bedarf es bei der Manipulation der beiden anderen Einflußgrößen zumeist spezifischerer Kenntnisse hinsichtlich der Regulation der betreffenden Produktsynthese, die sich auf die Verlaufskurve der retardierenden Wachstumsrate und vor allem auf die Substratdeponie oder gezielte Zuführung beziehen. Der Sekundärstoffwechsel gilt als besonders empfindlicher Bereich. Wie Tab. 1 zeigt, wurden in den wichtigen biogenetischen Gruppen [3] zahlreiche regulative Zusammenhänge nachgewiesen, die z. T. bis auf die molekularbiologische Ebene verfolgt werden konnten. Man darf annehmen, daß die gentechnischen Fortschritte bezüglich des mikrobiellen Sekundärstoffwechsels [4] in absehbarer Zeit hierzu einen beträchtlichen Informationsschub liefern werden. Dennoch beschränkt sich die Anzähl der biotechnologisch relevanten Befunde auf relativ wenige Beispiele, wobei neben der Anwendung in der Prozeßauslegung die Selektion unter Einbeziehung biochemisch begründbarer Strategien im Vordergrund steht [5]. Für den Fermentationsprozeß selbst, namentlich an bewährten Verfahren, werden Substratdosierungen zur Präkursornachlieferung oder Vermeidung inhibitorischer Effekte hauptsächlich verwendet, während in der Stammentwicklung die Resistenz gegen Metabolite, Endprodukte oder ausgewählte Sioxen eine Rolle spielt. Dem Wunsch, möglichst bald auch genetisch manipulierte Sekundärstoffbildner in die Hand zu bekommen, die eine erhöhte Leistung aufweisen, stehen bislang noch die polygenische Determinanz sowie die Kompliziertheit der Kontrollmechanismen in diesem Stoffwechselbereich entgegen. Bei der Selektion von Leistungsstämmen nach biochemisch begründbaren Strategien wurde allerdings die Erkenntnis gewonnen, daß zwar beabsichtigte Genotypen hinsichtlich des als wichtig erachteten Merkmals erhalten werden, jedoch in nur geringem Maße die „gezielte Veränderung" auf die Erhöhung der Produktsynthese durchschlägt (Tab. 2, [6]). Demzufolge finden sich in eingesetzten Hochleistungsstämmen nur teilweise die Eigenschaften wieder, die aufgrund biochemischer Kenntnisse als Voraussetzungen für die Leistungserhöhung erwartet wurden (Tab. 3, [7]). Die neuerdings unter Einsatz der „resting cell"-Technik gewonnenen Einsichten etwa in die Enzymatik der /}-Lactam-Biosynthese [8] haben diese Nichtdeterminiertheit des Sekundärstoffwechsels insofern erhellt, als sich gezeigt hat, daß analoge Enzyme ganz unterschiedlich auf Katabolitrepression auslösende Komponenten reagieren (Tab4, [8]). J a , nicht einmal die /S-Lactamstruktur als gemeinsames Grundprinzip entsteht auf biosynthetisch einheitlichem Wege. Bei der Analyse der Tetracyclinbiosynthese konnte der seit langem bekannte PhosphatInhibitionseffekt zwar der Sensibilität der Anhydrotetracyclinoxygenase als dem wesentlichsten Kontrollenzym zugeordnet werden [9], ohne jedoch bei Phosphatresistenten immer auch erhöhte Produktbildung anzutreffen [10]. Aus eigenen Arbeiten zur Optimierung der Nourseothricinproduktion, einem Ergotropicum der Streptothricine, ergab sich die Erfahrung, daß gleichsam biochemisch inerte Veränderungen im Genotyp zu beträchtlicher Leistungssteigerung beitragen können. So erwies sich ein gegen 0,5 M NaCl resistenter Stamm zunächst im konventio-

6

Acta Biotechnol. 9 (1989) i Tab. 2. Effektivität von „gezielter" Selektion bei Cephalosporin produzierenden Stämmen Resistenz gegen

Analoges von

Anzahl von Resistenten

Anzahl von Überproduzenten

Selenocystein Allylglycin Norvalin D, L-ABS S-2-AEC Selenomethionin Trifluoromethionin -Methylserin Trifluoroleucin Selenoethionin 2-DesoxygIucose Acriflavin Nystatin Amphotericin Kampfer Acenaphthen Quecksilber-IIchlorid Kupfer-II-chlorid Kaliumchromat Phenylmercuriacetat

Cystein Cystein Valin Valin Lysin Methionin Methionin Serin Leucin Methionin Glukose DNA-Effekt Membraneffekt Membraneffekt Ploidieeffekt Ploidieeffekt Komplexmetall

45 25 27 151 16 153 55 25 35 87 48 32 157 55 31 91 37

0 0 0 1 1 8 2 1 1 6 1 1 3 0 1 1 1

0 0 0 0,66 6,2 5,2 3,6 4 3 7' 2,1 3,1 2 0 3,1 1,1 2,7

Komplexmetall Komplexmetall Komplexmetall

37 125 55

0 1 0

0 0,8 0

Tab. 3. Merkmale von Leistungsstämmen von Pénicillium

%

chrysogenum

Phänomen

Biochemischer Aspekt

Sachverhalt

Lysinausschüttung

Verminderter Lysinfeed-back der Homocitratsynthase

Befund ohne weitere Leistungssteigerung

Polyenresistenz

Permeabilitätsänderung über Membraneffekte Penicillinausfluß ?

Fungicidin- bzw. Nystatinresistenz ohne weitere Überproduktion

Resistenz gegen Mitoseblocker, Schwermetalle, Schwefelanaloge, Aminosäureanaloge

Ploidieänderung

Gieringe Erhöhung der Leistung ohne systematische Zuordnung

PhenylacetatToleranz Fe 2 +-Sensibilität Penicillinacylaseaktivität erhöht, Acyltransferaseaktivität erhöht, Produkthemmung vermindert

Komplexbildung mit Penicillin Präkursornachschub

Enzymaktivitäten im Sekundärmetabolismus

Befunde in Leistungsstämmen ohne Kenntnis der Ursachen

BOBMANN, E. J., Microbial Secondary Metabolism.

7

Tab. 4. Regulationsphänomene an Enzymen der /J-Lactambiosynthese Mikroorg.

ACV-Synthetase

Zyklase

P. chrys.

R: Glukose Lysin

R: Lysin I: G-6-P

Str. lact.

R: Glukose

S: Glukose

R: Glukose NH 4 ' I: G-6-P FDP

Str. clav.

R: NH 4 -

R: NH 4 I: P 0 4 "

R: NH 4 P04"

(R :) Glukose

R: Glukose NH4I: G-6-P FDP

C. acremon.

Expandase

Epimerase

R: NH 4

R: Repression, I: Inhibition, S: Stimulation

nellen batch-Verfahren als leistungsstärker und in seiner Eignung für cyclische fed-batchRegime bzw. spezielle Phosphatdosierungsstrategien als überaus erfolgreich [11, 12]. In den hier genannten wenigen Beispielen industrieller Sekundärstoffproduktionen gelang die Realisierung von Produktkonzentrationen bis zum lOOOfachen des Wildstammniveaus stets durch die Gewinnung eines potenten Stammaterials auf empirisch systematischem Wege, dessen biosynthetisches Leistungsvermögen durch die Korrespondenz von angemessener Technologie und sorgfältig beachteten Substratverhältnissen — einschließlich spezieller Sterilisationseffekte — ausgeschöpft wurde. War es bei der Penicillin-G-Fermentation die diffizile Regelung der pH-p0 2 -Glukose-Relation im Verein mit bilanzierter Präkursordosierung, Wachstumsführung oder TemperaturprofilSteuerung [7], so standen bei der Oxytetracyclinherstellung die cyclischen fed-batchVerfahren [13] bzw. die Lipiddosierung [14] im Vordergrund. Im Falle der Nourseothricinfermentation wurde unter Beachtung ausreichender C- und N-Versorgung die Einstellung eines geeigneten Phosphatlevels durch Phosphatdeponie oder -Dosierungsprofil realisiert [15]. Daß auch an nichttradionellen Produktsynthesen eine bis zu lOfache Leistungssteigerung innerhalb von 2 Jahren auf 10 g Produkt/1 aufgrund der genannten Gesichtspunkte gelingt, zeigte neuerdings das Beispiel des Thiostreptons [16]. Schlußbemerkungen Die dargestellten Beispiele sollten demonstrieren, daß der mit der industriellen Sekundärstoffgewinnung befaßte Biotechnologe in der Fermentation zwar biochemisches Wissensgut verwenden kann, jedoch aus der Literatur keine generelle „Anleitung zum Handeln" in bezug auf bekannt gewordenen Regulationsmechanismen erwarten kann. Bislang hat jeder besondere Leistungsstamm, neben gewissen Übereinstimmungen, etwa hinsichtlich Phosphatstoffwechsel oder C-Katabolirepression, entscheidende „eigene" biochemische Charakteristika von zumeist unbekannter Natur. Dennoch wären Skepsis über die Beachtung von Grundlagenergebnissen an Modellobjekten oder leistungsschwächeren Selektanten oder aber Ignoranz gegenüber dem „akademischen" Wissensfortschritt unangebracht, da das Verfolgen der Entwicklung den Blick für entsprechende Phänomene am konkreten Objekt schärft und zumindest die Kenntnis analoger Biosynthesen das Auffinden ansprechbarer Stoffwechselzusammenhänge erleichtert.

8

Acta Biotechnol. 9 (1989) 1

Dieses biochemisch orientierte Wissen muß allerdings einhergehen mit entsprechenden Kenntnissen zur Fermentationstechnologie wie auch zu Fragen der Substratbilanzierung und Substratformierung im technischen Prozeß. Bis zu dem Zeitpunkt, da auch mit Hilfe der Genetik vollkommen durchschaubar fermentiert wird, mögen die nicht ganz ernsthaft gemeinten Formulierungen noch Gültigkeit besitzen: Know how -+- no why = Biotechnologie; no how + know why = Biochemie. Eingegangen: 19. 2. 1988

Literatur [1] SWARTZ, R. W.: Penicillins, in: Mou YOUNG, M.: Comprehensive Biotechnology, Vol.3, Pergamon Press N. Y. (1985), 7. [2] BABEL, W., MÜLLER, R. H.: Appl. Microbiol. Biotechnol. 22 (1985), 201. [3] MALIK, V. S.: A d v . Appl. Microbiol. 28 (1982), 27. [4] PEPERDY, J . F . , ILLING, G . T . : i n

Symp.

Soc. Gen.

Microbiol., ed. b y

GREENWOOD, D . ,

O'GRADY, F., Cambridge Univ. Press (1985), 283. [5] GRAPE, U.: Z. Allg. Mikrobiol. 21 (1981), 373. [6] CHANG, L. T., ELANDER, R. P.: Dev. Ind. Microbiol. 20 (1978), 367. [7] HERSBACH, G . , J . H . , VAN DER BEEK, C. P . , VAN D I J K , P . M. W . : i n : VANDAMME, E . J . :

Biotechnology of Industrial Antibiotics, M. Dekker Inc., N. Y., Basel (1984), 45. [8] GRÄFE, U . , BORMANN, E . J . : B i o l . Z b . 1 0 6 (1987), 3 3 . [9] ERBAN, V., TRILISENKO, L . V . , NOVOTNA, J . , BEHAL, V . , KULAEV, I . S., HOSTALEK, Z . : F o l i a

Microbiol. 82 (1987), 411. [10] KADIR, O . A . , BLUMAUROVA, M . , NOVOTNA, J . , BEHAL, V., VANEK, Z . : P r o s . I n t . S y m p . o n

Actinomycetes, Debrecen (1985), 314. [11] WEIDE, H., PACA, J . , KNORRE, W . A.: Biotechnologie. V E B G. Fischer Verlag J e n a (1987), 241.

[12] MÜLLER, P. J., OZEGOWSKI, J . H.: Zbl. Bakt. Hyg. A 260 (1985), 15. [13] BOSNJAK, M . , STROJ, A . , CTTRCIC, M . , ADAMOVIC, V . , GLUNCIC, Z., BRAVAR, D . : B i o e n g . X X V I I (1985), 3 9 8 .

Biotechnol.

[14] ETTLER, P . : A c t a Biotechnol. 7 (1987), 3.

[15] BOCKER, H.: DP 249 713 A 1 (1987). [16] SUZUKI, T., YAMANE, T., SHIMIZU, S.: Appl. Microbiol. Biotechnol. 25 (1987), 526.

Acta Biotechnol. 9 (1989) 1, 9 - 1 6

Enrichment of Soybean Milk with Calcium PRABHARAKSA, C h . , OLEK, A . C., STEINKRAUS, K .

H.1

Institute of Food Science, Cornell University Geneva, New York 14456

Summary Calcium in soymilk was increased to t h a t of human milk by the addition of 0.2% calcium lactate. I t was, however, impossible to raise the calcium content of soymilk to t h a t of cow's milk. The maximum amount of calcium lactate t h a t could be added to soymilk without coagulation of the milk protein was found to be 0.45% which resulted in about 75 mg of Ca/100 ml of soymilk. This amount was approximately 60% of the calcium in cow's milk. Sodium citrate had to be added in combination with calcium lactate to reinforce colloidal stability of the milk. The ratio of Ca to P in the milk containing added 0.45% calcium lactate was comparable to t h a t of cow's milk and a t the amount normally consumed daily-by infants and children it seemed to meet the requirements of Ca and P.

In developing countries, the problem of protein deficiency is very critical, especially for post-weaning infants and young children. In these areas, the dairy industry is inadequate to provide milk as a protein source. The use of imitation milks as a protein supplement appears to be necessary. Soybean milks may play a significant nutritional role in these countries in addition to their economic advantage over cow's milk and dried skim milk [1]. Imitation milk, as defined by the United States Federal Filled Milk Act, is a non-dairy product resembling cow's milk in appearance, flavor and nutritive value. Imitation milk may be produced from vegetable protein such as soy protein with vegetable fat added. It may also be in the form of milk in which no milk-derived ingredients except sodium caseinate are used. Either soy protein or sodium caseinate when used as a protein source at high concentration can adversely affect flavor of the milk [2]. Attempts have been made to improve the quality of imitation milks in terms of flavor and nutritive quality. When compared to the nutritional value of cow's milk, many nondairy imitation milks show pronounced deficiencies in protein, amino acids, essential elements and vitamins, making non-dairy imitation milks nutritionally less desirable in developing countries than in developed countries as noted by K O S I K O W S K I [ 3 , 4 ] . A major nutritional deficiency in soymilk is its low mineral content compared to that of cow's milk. Proteins and major minerals such as calcium, phosphorus and iron are present in cow's milk in considerably greater concentrations than in human milk [5]. In order to supply the mineral requirements of infants or pre-school children fed with soymilk, such nutrients should be supplemented. To whom correspondence should be addressed.

10

Acta Biotechnol. 9 (1989) 1

In addition to nutritive value, palatability of the milk is very important. The characteristic beany flavor of soymilk has hindered acceptance of the product and has caused problems for commercial companies attempting to develop commercially acceptable products [6]. STEINKRATTS [7,8] developed processes patented by Cornell University in which the beany flavor of soybeans is removed by extraction of the crude soy lipids including phospholipids. Such soymilks can simulate the flavor of cow's milk. The purpose of this study was to formulate soymilks with nutritive values similar to that of human and cow's milk by supplementing soymilks with calcium salts. This would then allow soymilks to meet the Recommended Dietary Allowances for calcium established by the Committee on Dietary Allowances Food and Nutrition Board [9].

Materials and Methods Soybeans of the Harosoy variety with initial moisture about 10% were used throughout this study. The gross composition of the soybeans is given in Tab. 1. Tab. 1. Proximate composition of whole soybean (Harosoy) Component

Composition [%] dry weight

Protein (N x 6.25) Fat 1 Ash N-free extract 2

42.08 19.97 4.86 33.09

1 2

Ether-extractable fat Calculated by difference

Effect of added calcium salts on viscosity and stability of soymilk. Calcium salts used to raise the Ca1 + content of soymilk were calcium sulfate and calcium lactate. Soymilks were prepared by soaking 100 g of soybeans in 300 ml of 0.048 N NaOH at 50 °C for 2 h in a water bath. After soaking, the hydrated beans were drained and boiling water was added to obtain a ratio of 1 : 8 (dry beans: water, w/v). The mixture was ground for 5 min in a WAKING blender. The resulting slurry was immediately filtered through 4 layers of cheesecloth, followed by filtration through a milk filtering pad (Agway #87-0564, 8 G-l) in a BTTCHNER funnel under reduced pressure to remove insoluble residue. Two percent sucrose was added and the milk was divided into portions. Calcium sulfate and calcium lactate at vario.us concentrations (0.05, 0.075, 0.1, and 0.125%, w/v) were added to individual portions. The milks were sterilized at 121 °C for 15 min. The viscosity in centipoise [cp] of all resulting milks were determined with a viscometer (OSTWALD-FOLIN, C34, size 200) at 30 °C. Effect of sodium citrate on viscosity of soymilk fortified with calcium lactate Soymilk with 0.125% calcium lactate added was prepared as described. Sodium citrate was added to provide concentrations of 0.2,0.3,0.4,0.5,0.6, and 0.7% (w/v) in the milks. The milks were autoclaved at 121 °C for 15 min. The viscosity of the milks was determined.

11

PRABHARAKSA, Ch., OLEK, A. C. et al., Soybean Milk

Effect of homogenization

on viscosity of soymilk fortified with calcium

lactate

Soymilk was prepared as described. The filtered milk was homogenized (MANTON-

GAULIN homogenizer type 15M 8BA-SMD) at 8000 psi, double pass. Two percent su-

crose was added and the milk was divided into 4 portions. Increasing concentrations of calcium lactate, 0.125, 0.15, 0.175, and 0.2% (w/v), were added individually to portions. The milks were mixed and autoclaved at 121 °C for 15 min. Viscosities of the resulting milks were determined. Effect of calcium lactate and sodium citrate added to soymilk before or after

homogenization

Two sets of experiments were performed in this study: — Addition of calcium lactate and sodium citrate to the milks before homogenization. — Addition of calcium lactate before homogenization and sodium citrate after homogenization.

The milk was prepared by soaking the soybeans in 0.5% NaHC0 3 ( 1 : 3), w/v) at 50 °C for 2 h. The beans were drained and blanched for 4 min in fresh 0 . 5 % NaHC0 3 solution (1 : 3, w/v, original dry beans to solution). The weight of solution absorbed was noted. Distilled water (temperature higher than 90 °C) was added to the beans (1 : 8, w/v, dry beans to water) in the WARING blender. Grinding and filtering steps were performed as previously described. In each set, filtered soymilk was divided into 12 portions. Concentrations of calcium lactate (0.2, 0.3, 0.4 and 0.5% w/v) and sodium citrate (0.0, 0.2 and 0.4% w/v) were added to the soymilk in various combinations. All the milk samples were homogenized at 8000 psi, double pass and then were sterilized for 15 min at 121 °C. The soymilks were observed for precipitates and general appearance. Viscosities were also determined. Determination of the maximum concentration of calcium lactate that couM be added to soymilk without precipitation of soy protein Soymilk was prepared by soaking and blanching soybeans in 0.5% NaHC0 3 as described. Two percent sucrose and 0.45% calcium lactate were added. The milk was homogenized at 8000 psi, double pass. Three concentrations of sodium citrate (0.2, 0.3, and 0.4%) were added to the homogenized milk. The milks were sterilized at 121 °C for 15 min and the viscosities of resulting milks were determined. Effect of heat sterilization in combination with addition of sodium citrate on stability of soymilk The milk was prepared as described. Two percent sucrose was added to the filtered soymilk. The milk was divided into 2 parts, one with and one without 0.45% calcium lactate added and then was homogenized at 8000 psi, double pass. Various concentrations (0, 0.1, 0.2, 0.3, and 0.4%, w/v) of sodium citrate were added to each part. Each milk sample was mixed well and the viscosities of all samples were measured before sterilization. All milk samples were then sterilized at 121 °C for 15 min, cooled to 30 °C and again the viscosities were determined. Elemental

analysis

Calcium present in soymilk prepared by the addition of 0.45% (w/v) calcium lactate was determined by the method of AOAC [10], sections 7.096—7.098a and 7.102a. One gram of dried, defatted soymilk sample was digested using 30 ml conc. H N 0 3 and 10 ml 7 0 %

12

Acta Biotechnol. 9 (1989) 1

perchloric acid on a hot plate. The solution was cooled, 50 ml of water were added and the mixture was boiled to remove N0 2 fumes and filtered. Appropriate dilution was made with 0.1 N HC1 to bring sample solution into the analytical range of the Perkin-Elmer 305B Atomic Absorption Spectrophotometer. Results and Discussion

Effect of added calcium salts on viscosity and stability of soymilk Soymilks fortified with calcium sulfate were found to be very viscous. The viscosity increased greatly (4.86 to 25.3 cp) as the concentration of calcium sulfate increased from 0 to 0.125% (Fig- 1). Also, the milk coagulated after standing at room temperature for a very short'period of time (24 h). Therefore, no further attempt was made to use calcium sulfate as the source of calcium in soymilk. 26

2 0•

0.025 0.050 0.075 0.100 0.125 %

Fig. 1. Changes in viscosity of soymilk containing various concentrations of added calcium salts

Calcium salt in soymilk[w/v]

Added calcium lactate also raised the viscosity of soymilk but the increase was much less than that of calcium sulfate (Fig. 1). The milk did not coagulate and the flavor of milks was acceptable. However, the milk with 0.125% calcium lactate added was rather thick and the viscosity (10.1 cp) was about 7 times of that of cow's milk (1.55 cp). Furthermore, 0.125% of calcium lactate added provides only 16 mg of Ca + + /100 ml of soymilk which is substantially less than that in cow's milk. Effect of sodium citrate on viscosity of soymilk fortified with calcium lactate The viscosity of soymilk decreased as the concentration of sodium citrate increased (Fig. 2). I t was found that sodium citrate also improved the flavor of soymilk. The beany flavor was less detectable in the soymilks with sodium citrate added than it was in the control without added sodium citrate. There was not much difference between the flavors of milks with 0.2% citrate and higher. The color of soymilks with high concentrations of citrate was darker. This was quite obvious in the milks containing more than 0.4% sodium citrate. Viscosity decreased less rapidly as concentrations of sodium citrate higher than 0.5% were added to the milks.

13

P r a b h a r a k s a , Ch., O l e k , A. C. et al., Soybean Milk

Fig. 2. Effect of sodium citrate on viscosity of soymilk containing 0.125% calcium lactate

Effect of homogenization on viscosity of soymilk fortified with calcium lactate

Homogenization had a remarkable effect on the viscosity of soymilk containing added calcium lactate. The viscosity decreased about 60% in homogenized soymilk (2.55 cp) from t h e control soymilk without homogenization (6.42 cp). Both milks contained 0.125% calcium lactate and 0.2% sodium citrate. The higher the concentration of calcium lactate in the range of 0.15, 0.175, and 0.2%, added to homogenized milk, the higher the viscosity of the milk (Fig. 3). However, the viscosity of the milk containing 0.2% calcium lactate was only 3.77 cp which was still less t h a n the control soymilk without added calcium lactate and without homogenization (4.54 cp). At a concentration of 0.25% calcium lactate, the milk coagulated after sterilization. This phenomenon also occurred when higher concentrations of sodium citrate (0.3 and 0.4%) were added. Therefore, other factors besides homogenization and concentration of sodium citrate are involved. coagulated

Fig. 3. Effect of homogenization on viscosity of soymilk containing different amounts of calcium lactate and 0.2% sodium citrate 0.05

0.10

%

Caticium

0.15

0.20 0.25

laciaie

[w/v]

Effect of calcium lactate and sodium citrate added to soymilk before or after homogenization

I n the first experiment, calcium lactate and sodium citrate were added to the milk before homogenization. I t was found t h a t the milks containing 0.3% calcium lactate and higher coagulated either before or after sterilization regardless of the amount of sodium citrate added (Tab. 2).

14

Acta Biotechnol. 9 (1989) 1 Tab. 2. The viscosity [cp] of soymilk fortified with calcium lactate and sodium citrate before and after homogenization Sodium citrate

r o/ 0/ J1 L

0.2 0.3 0.4

Calcium lactate [%] before homogenization

after homogenization

0.2

0.3

0.4

0.5

0.2

0.3

0.4

0.5

3.80 3.20 3.50

* * *

* » *

+ + +

3.10 2.98 2.80

* 3.35 3.27

* 3.35 3.42

+ + +

* Coagulated after sterilization Coagulated before sterilization

+

I t was concluded that when calcium lactate and sodium citrate were added together before homogenization, they possibly interacted, decreasing the availability of sodium citrate to stabilize the soymilk emulsion and the milk protein precipitated because of the high concentration of calcium lactate added. When calcium lactate was added before homogenization and sodium citrate was added after, the effect of sodium citrate on the stability of the milk was evident (Tab. 2). Soymilks without sodium citrate added coagulated except for the one with lowest concentration of calcium lactate (0.2%). At 0.5% calcium lactate, the milk coagulated right after homogenization. This indicated that 0.5% calcium lactate was too high to maintain stability in the milk. The viscosities of the milks at the same level of calcium lactate but with different concentrations of sodium citrate (0.2 and 0.4%) did not differ much. Determination of the maximum concentration of calcium lactate that could be added to soymilk without precipitation of soy protein Calcium lactate could be added to soymilk up to 0.45% without coagulation when 0.4% sodium citrate was added in combination. The viscosities of soymilk containing 0.45% calcium lactate and 0.2,0.3, a)nd 0.4% added sodium citrate were 3.96, 3.79, and 3.80 cp, respectively. However, the milks containing 0.2 and 0.3% sodium citrate precipitated slightly after standing at room temperature for a few days. Therefore, it would appear that to prevent coagulation of the milk containing 0.45% calcium lactate, 0.4% sodium citrate must be added. At this level, the flavor and color of the milk were still acceptable. Effect of heat sterilization in combination with addition of sodium citrate on stability of soymilk Sterilization also had a remarkable effect on viscosity of soymilk containing added calcium lactate. The viscosity of the milk decreased about 3 to 4 times after sterilization (Tab. 3). As observed earlier, the milk without sodium citrate added coagulated after sterilization. With a small amount of sodium citrate added (0.1%), the milk did not coagulate but after standing overnight at room temperature, slight precipitation was observed. At higher concentrations of sodium citrate (0.2% and higher), which were beyond the level required t o prevent coagulation of the milk, there was not much effect on the viscosity of the milk (Tab. 3). In the milks without calcium lactate added, sterilization had little effect on viscosity. There was a slight decrease in viscosity of the milks before sterilization when higher con-

PRABHARAKSA,

Ch.,

OLEK, A .

C. et al., Soybean Milk

15

Tab. 3. Viscosities of the milks with and without added calcium lactate before and after sterilization Sodium citrate [%]

0 0.1 0.2 0.3 0.4

Viscosity [cp] with 0.45% lactate

without 0.45% lactate

sterilization before

sterilization before

16.07 12.49 11.93 11.18 8.98

after *

3.50 3.00 3.12 3.01

2.83 2.80 2.77 2.73 2.67

after 2.63 2.67 2.68 2.71 2.35

* Coagulated after sterilization.

centrations of sodium citrate were added. However, the viscosities of the milks after sterilization were relatively constant at different concentrations of sodium citrate and were just slightly less than those before sterilization Elemental

analysis.

The maximum amount of calcium lactate t h a t could be added without coagulation of the milk protein was found to be 0.45% (w/v). The total calcium content of whole Harosoy soybeans was 234.22 mg/100 g soybeans. The soymilk control (no calcium lactate added) contained 20.3 mg Ca/100 ml. Soymilk prepared with 0.45% calcium lactate (57.6 mg Ca/100 ml) had an average Ca level of 74.7 mg/100 ml. The recovery of Ca was 95.5%. The amount of Ca found in soymilk without added calcium lactate (20 mg/100 ml) was about one-sixth of the Ca in cow's milk (120—125 mg/100 ml) and slightly more t h a n a half of Ca in human milk (34 mg/100 ml). The addition of 0.45% calcium lactate (57.6 mg Ca/100 ml) made the calcium content in soymilk twice as much as t h a t of human milk and about 60% of Ca in cow's milk. I t is unlikely t h a t the calcium content can be increased to the level found in cow's milk since coagulation of soymilk protein occurred when the higher concentrations of calcium lactate were added. The ability of infants to absorb Ca from human milk is assumed t o be about 60% of intake on the average and the calcium requirement for the infant is assumed to be Tab. 4. Recommended Daily Dietary Allowances of Ca for infants and children1'2 Age [year] from up to

Ca [mg]

0.0-0.5 0.5-1.0 1.0-3.0 4.0-6.0

360 540 800 800

1 2

The allowances are intended to provide for individual variations among most normal persons as they live in the United States under usual environment stresses. Recommended Dietary Allowances [9] RDA 1980

16

Acta Biotechnol. 9 (1989) 1

270 mg/day, an amount supplied by 800 ml of human milk as reported by FOMON [5]. He also stated that the infant not fed human milk should consume 500 mg Ca/day. Therefore, about 700 ml of soymilk with 0.45% calcium lactate added would be adequate to supply the Ca requirement of infants. The Recommended Daily Dietary Allowances (RDA) of calcium for infants and children are shown in Tab. 4. Received March 10, 1988

References [1] STEINKRAUS, K . H . : Appl. N u t r . 4 (1976), 49. [2] KOSIKOWSKI, F. V.: Milk F d . Techn. 31 (1968), 174. [3] KOSIKOWSKI, F. V.: J . Dairy Sci. 52 (1969), 756. [ 4 ] K O S I K O W S K I , F . V . : Food Sci. 3 6 ( 1 9 7 1 ) , 1 0 2 1 . [ 5 ] F O M O N , S . J . : I n f a n t Nutrition, Philadelphia a n d L o n d o n : W . B . S A U N D E R S C O . , 1 9 6 7 . [ 6 ] W I L K E N S , W . F . , H A C K L E R , L . R . : Cereal Chem. 4 6 ( 1 9 6 9 ) , 3 9 1 . [7] STEINKRAUS, K . H . : Method for defattering soybean meal. U.S. P a t e n t 3, 721, 569 issued March 20, 1973. [8] STEINKRAUS, K . H . : Process for producing d e f a t t e d a n d debittered soybean meal. U.S. P a t e n t 4, 496, 599 issued J a n . 29, 1985. [9] Recommended D i e t a r y Allowances 9th ed. National A c a d e m y of Sciences, Washington, D.C. 1980. [10] Association of Official Agricultural Chemists, Official Methods of Analysis, 14th ed. (WILLIAM, 5. ed.) AOAC, Washington, D. C. 1984.

Acta Biotechnol. » (1989) 1, 17-23

A Reduced Specific fithanol-Forming Performance of Yeast at High Biomass Concentrations as a Result of a Changed Ethanol-Tolerance Behaviour of the Cells under Condition of Limitation I. A Theoretical Treatment RICHTEB, K .

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

Summary The loss of fermentative activity of yeast cells, observed in continuous fermentation experiments at increasing biomass concentration is explained by the assumption that the ethanol-tolerance behaviour of the microorganisms changes if a growth-stabilizing factor limitation is present. A mathematical specification of the relationships existing in this context is given and an improved steady-state productivity model of ethanol production is derived.

Introduction In continuous fermentation experiments carried out with the yeast Saccharomyces cerevisiae Sc 5 in an ideally mixed high-flow rate fermenter significant differences were found between the obtained productivities of ethanol formation and the values expected from theoretical considerations [1, 2]. Apparently the microorganisms could not use their potential power of fermentation if the ethanol concentration in the medium exceeded the critical value P1 = 42.0 g/1. In these cases the used steady-state productivity model v x

p

•fldeal —

»

fU (I;

. d 1 + -5x

was not fitted by the experimentally attained productivities. Mostly the latter were less t h a n the model values. A good correspondence was observed at alcohol concentrations P < Pl only. The loss of activity was found to be a unique function of t h e biomass concentration x and the hydraulic dilution rate D. Empirically so a corrected equation could be derived, which reflects the overall relationships between the real productivities and the quantities x and D in a sufficient degree: ^real

=

. 1 +

M>( l - e " * « + W ) .

d D

(2)

X

From equation (2) we can clearly notice that the specific activity of the cells decreases with increasing biomass concentration. Similar effects were also reported on by other authors [3—5], but without giving a detailed explanation for it. 2

Acta Biotechnol. 9 (1989) 1

18

Acta Biotechnol. 9 (1989) 1

I t seems that a limitation was the cause for the observed loss of fermentative power in the case investigated by us. Under condition of steady-state in high-flow rate fermenters the at the time maximum possible metabolic performance is permanently demanded from the microorganisms. Therefore a limitation must affect the specific growth rate in a particular degree in such a situation. Since a deficiency of substrate and anorganic ions, respectively, could be excluded in all experiments, it had to be assumed that an urgent shortness of essential growth factors (vitamins, amino-acids, unsaturated f a t t y acids etc.) lay before in the medium. This supposition was confirmed by the fact t h a t a significant enhancement of the specific fermentative activity of the cells could be observed after adding extra doses of yeast extract. Consequently, as an explanation for the loss of activity found in the experiments mentioned above a change of the ethanol-tolerance behaviour of the microorganisms as result of a limitation is obliged to discuss. The aim of this paper is to demonstrate the fundamental relationships existing in this context and to connect them with the phenomenon of the decrease of the metabolic power of yeast cells with increasing biomass concentrations. Materials and Methods The point of contact for the theoretical considerations made in this paper are the results of continuous fermentation experiments carried out in a high-flow rate fermenter by using the yeast Saccharomyces cerevisiae Sc 5. The fermenter applied in this research was a 1.01-CSTR equipped with a membrane module in the bypass. I n the whole reactor system the liquid was ideally mixed. A more detailed description of the used fermenter is given in [2], I n all of the interpretations attempted by us on this field steady-state values was taken into consideration only. The specific growth rate and the specific ethanol formation rate refer to the portion of active cells in the fermenter liquid. Theoretical Considerations The starting-point for the following reflections is an ethanol-forming yeast, the inhibitory behaviour of which is characterized by the linear functions ¡i = ¡j.o — a'{P — P x ) ,

(3)

v = v0 — aP.

(4)

Such relationships were found for the yeast Saccharomyces cerevisiae Sc 5 in the case of a limitation-free fermentation [6, 7]. Whereas the ethanol formation is inhibited over the whole ethanol-concentration interval 0 < P s j P " , the hindrance of growth begins not till exceeding the inhibitory threshold concentration P j . A diagram of the both functions is shown in Fig. 1. The equation (4) can be modified by applying the known equation of Lxjedeking and P i b e t [8] V

=

OijU

+

(5)

as follows : 0 < P r g P 1i > (

(6)

" = («ijMo + A>) — a ( P — P i ) , for

P ^ P ^ P ' ,

(?)

v=

P ' ^ P ^ P " .

(8)

* = K " o + A>) - aP, -a(P

-P'),

for for

RICHTER K., Ethanol-Tolerance Behaviour

Pi Ethanol concentration

19

P" P [g/Il

Fig. 1. A schematic diagram of the both inhibitory functions of growth and ethanol formation of a yeast having a linear ethanol-tolerance characteristic including an inhibitory threshold for growth under condition of a limitation-free fermentation

Accordingly the specific ethanol formation rate is regarded as the sum of a growthlinked term g ml- 1 ]

Growth [gl-1]

Lysine [gl-1]

Thiamine HCl

0.10 1.00 10.00 100.00

2.01 2.12 2.14 2.26

ab bcde cdef f

1.87 1.94 2.19 1.53

bed def g a

Riboflavine

0.10 1.00 10.00 100.00

2.00 2.01 2.01 1.98

ab ab ab a

1.83 1.83 1.82 1.80

be be be b

Ca-pantothenate

0.10 1.00 10.00 100.00

2.00 2.02 2.03 2.03

ab abc abed abed

1.84 1.84 1.84 1.84

bed bed bed bed

Pyridoxine HCl

0.10 1.00 10.00 100.00

2.04 2.15 2.20 2.14

abed def ef cdef

1.85 1.98 2.04 1.87

be ef f bed

0.01 0.10 1.00 10.00

1.98 2.18 1.97 1.97

a ef a a

2.02 1.91 1.83 1.51

f cde be a

Biotin

Control

No vitamin added

1.98 a

2Fa

1.81 be

Values in a column followed by the same letter are not significantly different at the 5 % level as determined by DUNCAN'S multiple range test.

lysine production. Pyridoxine at 10 [¿.g m l - 1 significantly stimulated growth and lysine production but was less prominent than thiamine or biotin. Biotin and thiamine for maximal lysine production was 0.1 ¡xg m l - 1 and 10 [ig ml - 1 . Above this concentrations of biotin or thiamine resulting in maximal stimulation of growth but lysine production was less. Higher concentrations of pyridoxine and riboflavine similarly inhibited lysine production. Thus with optimal concentration of biotin and thiamine an increase of 11 % and 21%, respectively was obtained over the control. Effect of trace elements: Since trace elements affect growth and lysine yield by microorganisms [5, 12], it was thought desirable to test the effect of some of the trace elements. Five trace salts, more or less arbitarily chosen for this purpose, were Cu 2+ , Mn 2 + , Ni 2 + ,

SEN,

S. K . , C H A T T E R J E E , S.

P., Lysine Production

65

Zn 2+ and V 0 3 2 - . From the Tab. 2 it is evident that of the five trace salts tested growth and lysine production was stimulated by Mn 2+ and Zn 2 + . However, growth and lysine yield was significantly less if Ni 2+ or VO, 2 - were added. The optimal concentrations for Mn 2+ , Zn 2 + , Cu 2+ were of 1.0 ¡xg ml - 1 , 1.0 ¡j.g m l - 1 and 0.1 fig m l - 1 respectively, however, in the case of Ni 2+ even at 0.1 ¡Ag m l - 1 was inhibitory. At concentrations 5.0(j.gml _ 1 , 5.0 ¡xg m l - 1 and 1.0 y.g ml - 1 in case of Mn 2+ , Zn 2 + and Cu 2+ respectively both growth and lysine yield were lowered. Interestingly, lysine production increased significantly with the addition of these salts where growth was maximum and decreased where growth was inhibited. Tab. 2. Effect of trace elements on growth and lysine production by M. varians Salts

Concentration [(xg l" 1 ]

Growth [gi-1]

Lysine [gl-1]

MnCl2

0.1 1.0 5.0 10.0

2.05 2.23 2.21 1.96

ghij jk jk fgh

2.01 2.12 2.09 1.86

ijklm lmno klmno ghi

ZnCl 2

0.1 1.0 5.0 10.0

2.17 2.32 2.22 2.09

ijk k jk hij

2.14 2.21 2.17 2.02

mno o o jklmn

CuClj

0.1 1.0 5.0 10.0

2.06 1.96 1.91 1.63

ghij fgh fgh de

1.94 1.89 1.76 1.63

hijk ghij fg ef

NiCl2

0.1 1.0 5.0 10.0

1.88 1.51 1.27 1.01

fg cd b a

1.86 1.45 1.19 1.03

ghi cd b a

Na2V03

0.1 1.0 5.0 10.0

2.01 1.81 1.59 1.41

ghi ef cd be

1.97 1.79 1.54 1.37

ijkl gh de e

Control

No salt

1.98 fgh

2Fa

1.88 ghij

Values in a column followed by the same letter are not significantly different at the 5 % level as determined by DUNCAN'S multiple range test

Discussion Effect of vitamins on lysine accumulation has been studied by S H I G A T O [8] in Micrococcus glutamicus, by A R E S H K I N A et al. [9] in Brevibacterium, C H A T T E R J E E and B A N E R J E E [10] in Bacillus megaterium and C H A T T E R J E E and C H A T T E R J E E [11] in Micrococcus luteus. K I N O S H I T A [1] and S H I G A T O [8] reported that all lysine producing strains of M. glutamics required biotin. Similarly, A R E S H K I N A et al. [9] found that biotin was essential for lysine accumulation by Brevibacterium sp. The experiment on the effect of vitamins on growth and lysine production indicated that though the organism is not totally dependent on the vitamins, nevertheless, biotin and thiamine stimulated lysine accumulation. 5

Acta Biotechnol. 9 (1989) 1

Acta Biotechnol. » (1989) 1

66

The exact role of biotin in amino acid producing microorganisms is not definite. While T A N A K A et al. [ 1 3 ] hold the view that biotin functions by limiting growth and thus carbon and nitrogen sources are rather available for formation of amino acids than for the synthesis of cell matter. In other authors [ 1 4 — 1 6 ] opinion low biotin concentration makes the bacterial cells more permeable allowing a higher leaching out of amino acid into the surrounding medium. The results obtained by C H A T T E R J E E and B A N E R J E E [ 1 0 ] in case of Bacillus megaterium and B. coagulans support neither of these two hypotheses. In studying the effect of manganese, zinc, nickel, copper and vanadate on growth and lysine yield, it was observed that except nickel and vanadate the other three metal ions had promoting effect to varying extent. In individual case the effect of the ions tested on growth and lysine yield was almost parallel. From this experiment it can be deduced that the trace elements used play no direct role but they have some influence on growth and lysine yield in this organism. Among the more important factors influencing growth and quantitative yield of metabolites are the mineral constituents of the medium. [17] W E I N B E R G [ 1 8 ] studied the role of trace elements in microorganisms and observed that zinc was one of the key metals for certain microorganisms. Growth promoting effect of zinc in several species of Streptomyces was noted by H E I N and L E C H E V A L I E R [ 1 9 ] . B A N E R J E E and N A N D I [ 5 ] observed the stimulation of growth of a strain of Streptomyces by zinc, copper, and manganese. However, K A J I W A K A et al. [ 2 0 ] reported that the addition of more than 1 [xg ml - 1 of Cu a+ the medium improved the yield of lysine by Brevibacterium and Micrococcus. Inhibition of lysine yield by Ni 2+ may be due to the inhibition of Di-amino-pimelic-decarboxylase activity by the metal ion in M. glutamicus [21]. Regarding the role of trace element in microorganisms M A R T I N and M C D E N I E L [ 2 2 ] suggested that the metal ions probably acted as activator of the enzymes involved in synthetic steps of metabolites. The actual mechanism of stimulation or inhibition of. growth and lysine production observed with trace elements is still to be worked out [12] Acknowledgement Authors are grateful to the UGC, India for financial assistance during this period of investigation. We also thankfully acknowledge the help extended by Mrs. Dr. Manika C H A T T E R J E E and Dr. Subrata RAY. Received November 9, 1987 Revised January 25, 1988

References [1] K I N O S H I T A , S.: Adv. Appl. Microbiol. 1 (1959), 201. [2] S E N , S . K., C H A T T E R J E E , M., C H A T T E R J E E , S . P.: Biol. Bull. India 4 (1982), 131. [3] S E N , S . K., C H A T T E R J E E , M., C H A T T E R J E E , S . P.: Microbiol. Espanola. 36 (1983), 15. [4] M A K U L A , R., F I N N E R T Y , W . R. : J . Bacteriol. 95 (1968), 2108. [5] B A N E R J E E , A. K., N A N D I , P.: Trans. Bose. Res. Inst. 27 (1964), 87. [ 6 ] W O R K , E.: Methods Enzymol. 5 ( 1 9 6 2 ) , 8 6 4 . [7] D U N C A N , D . B . : Biom. 11 (1955), 1. [8] S H I G A T O , M.: Nippon Nogei Kagaku Kaishi. 36 (1962), 809. [9] ARESHKINA, L . Y . , BAKER, N . E . , BUKIN, V. N., XARKLINS, R . , KLYUEVA, M. N . ,

L.

Prikl. Biokhim. Microbiol. 1 (1965), 396. [ 1 0 ] C H A T T E R J E E , S., B A N E R J E E , A . K . : Indian J . Exp. Biol. 1 1 ( 1 9 7 3 ) , 4 4 6 . [11] C H A T T E R J E E , M., C H A T T E R J E E , S. P.: Acta Biotechnol. 2 (1982), 287. [12] S E N , S . K., C H A T T E R J E E , S . P.: Acta Biotechnol. 5 (1985), 215. S., LIEPIN, G . :

KTTTSEVA,

SEN, S. K . , CHATTERJEE, S. P., Lysine Production

67

[13] TANAKA, K . , IWASAKI, H., KINOSHITA, S. : Nippon Nogei K a g a k u Kaishi. 34 (1960), 593. [14] SHIIO, I., OTSUKA, S., TAKABASHI, M . : J. Biochem. 51 (1962), 56. [15] VELDKAMP, H., BERG, Z., ZEVENHUIZEN, D. P . T . M. : Antonie van Leeuwenhoek J. Microbiol. Serol. 29 (1963), 35. [16] OTSUKA, S., MIYAJIMA, R., SHIIO, I . : J. Gen. Appi. Microbiol. 11 (1965), 285. [17] WAKSMAN, S. A . : A d v . Appi. Microbiol. 11 (1962), 1. [18] WEINBERG, E. D . : A d v . Microbiol. Physiol. 4 (1970), 1. [19] HEIN, A . H., LECHEVALIER, H. A . : Mycologia. 48 (1956), 628. [20] KAJIWARA, T., KINOSHITA, K . , VOSHINAGA, P., OKUMIJRA, S.: Chem. Abstr. 76 (1971), 2555c. [21] WELWARD, L „ FUSKA, J., SRANKA, K . , HANO, A . : Chem. Abstr. 77 (1971), 60073a. [22] MARTIN, J. F „ MCDANIEL, L . E . : A d v . Appi. Microbiol. 21 (1977), 1.

5*

Acta Biotechnol. 9 (1989) 1, 68

B o o k Review

F.

SEBBA

Foams and Biliquid Foams-Aphrons Chister, New York, Brisbane, Toronto, Singapore: J o h n Wiley & Sons, 1987, 236 S., L 29.50, I S B N 0-471-91685-4

Schäume treten im Zusammenhang mit Mehrphasensystemen in den verschiedensten Bereichen der Technik auf. Sie können erwünscht bzw. gezielt erzeugt werden oder auch unerwünscht sein, wie dies in Abwasseranlagen und Bioreaktoren der Fall ist. Schaumprobleme sind sehr komplex, insbesondere dort, wo Tenside während des Prozesses entstehen können, wie z. B. bei Fermentationsprozessen. F ü r die Lösung solcher Schaumprobleme ist in erster Linie die Kenntnis der Vorgänge a n der Phasengrenzfläche unerläßlich. Diesem Ziel, der Vermittlung dieser Kenntnisse und der Erläuterung der d a m i t zusammenhängenden Phänomene, ist dieses Buch gewidmet. Bs stellt eine gute Einführung f ü r einen breiten Interessentenkreis dar, weil es auf theoretische Betrachtungen die umfangreiche mathematische Kenntnisse erfordern würden, bewußt verzichtet, d a f ü r aber alle in der Praxis auftretenden Effekte eingehend beschreibt. Der Schaumbegriff bleibt, wie im Titel zum Ausdruck k o m m t , nicht nur auf Gas-Flüssigkeits-Systeme beschränkt, sondern wird auf flüssig-flüssig bis hin zu fest-flüssig-Systemen ausgedehnt. Der Anwendung in der Praxis sind 3 Kapitel gewidmet, die sich auf Gasblasen, kolloidale Gasdispersionen und flüssig-flüssig-Dispersionen beziehen. Dabei liegt gleichfalls der Schwerpunkt auf der Darstellung der auftretenden Prozesse a n der Phasengrenze wie z. B. bei der Solventextraktion, der Öl-Sand-Trennung oder der Flotation, weniger auf technische Einzelheiten der Verfahren, wie mechanische Schaumzerstörungseinrichtungen, Dispergatoren u. ä. Der Charakter und das Anliegen des Buches wird besonders deutlich durch die beiden letzten Kapitel „Cancer" und „Painting on W a t e r " . Der Autor versucht darin Parallelen zwischen dem Verhalten von Tumor- und Metastasenbildung und den Erscheinungsformen, die in Öl-Tensid-Wassersystemen auftreten, zu ziehen. Mit „Painting on W a t e r " wird gezeigt, daß sich die Spreitungsbilder von ölschichten auf Wasser in Verbindung mit entsprechenden Beleuchtungstechniken g u t f ü r die Entwicklung dekorativer Muster eignen. Diese sehr weitgefaßte Darstellung der Vorgänge und Erscheinungen an Phasengrenzen und Schäumen läßt das Buch zu einer recht allgemeinverständlichen und anregenden Lektüre f ü r jeden werden, der nach einer leichtverständlichen Einführung sucht. Der im Titel enthaltene und in der Literatur noch wenig gebräuchliche Ausdruck „ A p h r o n " bezeichnet die kleinste Struktureinheit des Schaumes. W . STEPHAN

Acta Biotechnol. 9 (1989) 1, 6 9 - 77

Characterization of Biotechnological Processes and Products Using High-Performance Liquid Chromatography (HPLC) IY. SEC, HIC, and IEC Separations of Proteins GEY, M., Thiem, M., GRUEL, H. Academy of Sciences of the G.D.R. Institute of Biotechnology, PermoserstraBe 15, Leipzig, 7050 G.D.R.

Summary Proteins are separated by means of size-exclusion (SEC), hydrophobic-interaction (HIC) and ionexchange chromatography (IEC). Analytical and semipreparative HPLC glass columns are the basis of the chromatographic analyses. Using a short (100 x 3.8 mm i.d.) column packed with Si 200 Polyol standard proteins of different molecular weights (ovalbumin, MW = 43000; chymotrypsinogen A, MW = 25000; ribonuclease, MW = 13700 and contrycal, MW = 6512) could be distinguished. Basic proteins (e.g., chymotrypsinogen A, cytochrome C) are separated on aluminium oxide (LiChrosorb Alox T) by cation-exchange chromatography. Correlations between the retention times of proteins and their isoelectric points or the buffer concentration of the mobile phase are investigated. Furthermore, two examples of liquid chromatographic purification procedures for enzymes of biotechnological interest are demonstrated. One enzyme extract (thermostable protease) is separated by hydrophobic-interaction chromatography, another one (/3-galactosidase from a thermophilic microorganism) is purified on a weakly basic anion-exchange resin (pore size: 130nm) based on a styrene-divinylbenzene copolymer.

Introduction Currently, the HPLC analysis of proteins is an area of rapid development. Whereas separations and purifications of biopolymers lasting several hours or days were typical of the past, today chromatographic analyses are achievable in less than one hour or in only a few minutes. The most widely used separation systems for proteins are size-exclusion chromatography, SEC [1—5], hydrophobic-interaction chromatography, HIC [6—8] and reversed-phase chromatography, RPC [9], ion-exchange chromatography, IEC [10—14] and affinity chromatography, AC. Glass columns are important in bioanalytical research and especially for biopolymer separations because of their inertness and transparency. We already reported applications of short (50 or 100 X 3.8 mm i.d.) and efficient HPLC glass columns e.g. in sugar [15—17] and organic acid [18] analyses. Recently, we discribed a cartridge with semipreparative glass columns, e.g. 150 X 8 mm i.d., [19]. In the present study we prepared and tested different glass columns for protein analysis.

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Acta Biotechnol. 9 (1989) 1

They were packed with Si 200 Polyol (SEC), Butyl = Si 300 Polyol (HIC), LiChrosorb Alox T (IEC) and a weakly basic anion-exchange resin (IEC). Also some separation examples of enzymes — a thermostable protease and a /?-galactosidase from a thermophilic microorganism — using these glass columns will be given. Material and Methods HPLC systems The liquid chromatograph (Knauer, F.R.G.) included two 750/04 pumps, 750/36 decilinear programmer, 72.00 UV/fluorescence monitor, TY recorder, Rheodyne 7010 injector (Rheodyne, CA, U.S.A.) and a further high-pressure pump from a 1304 liquid chromatograph (U.S.S.R.) for flow rates smaller than 0.1 ml/min. Stationary

phases

SEC: Si 200 Polyol, 3 ¡xm, pore size: 20 nm HIC: Butyl = Si 300 Polyol, 5 [xm, pore size: 30 nm (Serva, F.R.G.) IEC: LiChrosorb Alox T, 5 ¡xm, pore size: 6 nm (Merck, F.R.G.) and a weakly basic anion-exchange resin based on a styrene-divinylbenzene copolymer, 10—20 ¡im, pore size: 130 nm (CKB Bitterfeld, G.D.R.) Mobile phases, chemicals Na 2 HP0 4 , NaOH, NaCl, HC1, propanol-2, (NH 4 ) 2 S0 4 (Laborchemie Apolda, G.D.R.) Packing procedures of HPLC glass columns Glass columns were packed as described previously [15, 16]. For preparing columns with Si 200 Polyol and Butyl = Si 300 Polyol a second, already filled HPLC column was connected after the glass column to reduce a sudden pressure increase that may cause sharing forces, which are able to destroy the packing material. Semipreparative glass columns were filled with ion-exchange resin as reported in [19]. Standard proteins Ovalbumin, ribonuclease, bovine serum albumin (Pharmacia Fine Chemicals, Sweden); chymotrypsinogen A, myoglobin, cytochrome C, contrycal (Serva, F.R.G.) Thermostable protease An enzyme preparation was obtained from the culture filtrate of novel thermophilic Bacillus strain (I) by ammoniumsulfate precipitation, dialysis and acetone fractionation. Enzyme activity The proteolytic activity was measured with the chromogenic substrate azocasein (Serva, F.R.G.). The reaction mixture contained 0.1 M tris-acetate-buffer (pH = 8.0), azocasein at a concentration of 1 % and a suitable amount of enzyme. This solution was incubated at 70 °C for 20 minutes. The reaction was stopped with one volume part of trichloro acetic acid (TCA). After centrifugation 0.25 ml of TCA supernatant was pipetted into 1.25 ml of 0.25 N NaOH. The absorbance of this solution at 440 nm was proportional to the proteolytic activity.

GEY, M., THIEM, M. et al., High-Performance Liquid Chromatography

71

/3-galactosidase An enzyme preparation was obtained from cell mass of a thermophilic Bacillus strain (II) after treatment with lysozyme and by ammonium sulfate precipitation, dialysis and acetone fractionation. Enzyme Activity: see [20]. Results and Discussion Size-Exclusion

Chromatography

(SEC)

Proteins are separated by size-exclusion chromatography because of their differences in molecular weight. The interactions of proteins with the stationary and mobile phases should be as small as possible. Large molecules that were not able to diffuse into the pores of the particles of the stationary phase are excluded and are eluted first together with the elution front (V { : intermediate particle volume). Smaller molecules pass through a more or less great part of the porous stationary phase and are eluted later. To find out the molecular weight a protein, it was separated by SEC with the elution volume (V e ), the separation column is to be calibrated by means of standard proteins of different molecular weights. One test protein should be excluded (V,), some other biomolecules should partly diffuse into the pores (F e ) and one standard protein should pass through the whole particle volume of the stationary phase ( F 0 : void volume). Plotting the logarithm of the molecular masses against the elution volume gives a straight line under ideal conditions. Based on this calibration curve the molecular weight of an unknown protein can be determined after measuring its elution volume. One premise is that the protein should elute within the pore volume (V p ) that is used for distinguishing proteins. For Vp the following equation is valid: VP = F 0 - F j In Fig. 1 a SEC separation of four standard proteins on a 100 mm glass column (i.d.: 3.8 mm) is demonstrated. The pore size of the stationary phase is 20 nm. Ovalbumin with a molecular weight of 43000 is excluded (F,) on this stationary phase. Contrycal (MW: 6512) elutes together with the void volume. This short SEC column is to be used for a "rough fractionation" of protein mixtures with molecular weights between approximately 5000 and 40000. Further molecular weight determinations, e.g. of jtf-galactosidase isolated from a thermophilic microorganism, by means of larger SEC columns (TSK G 3000 SW, 600 x 7.5 mm i.d.) have been described in detail elsewhere [21]. Hydrophobic-Interaction

Chromatography

(HIC)

Using reversed-phase chromatography with strongly hydrophobic stationary phases (e.g. R P 18) (larger) proteins can only be eluted by means of an organic modifier in the aqueous mobile phase. A disadvantage is that organic solvents may cause protein denaturation. In comparison to RPC the so-called hydrophobic-interaction chromatography is carried out on a stationary phase which is only weakly hydrophobic ( R P 3 or R P 4). The carbon content of such materials is approximately 10% of a R P 18 stationary phase [22]. In the first step of the HIC separation mechanism the proteins are fixed to the weakly hydrophobic matrix using a mobile phase with a high initial salt concentration (0.7 to 2 M). Reducing the salt amount by means of gradient elution with dilute buffers the hydrophobic interactions of the proteins with the stationary phase are also reduced and the proteins can elute from the column stepwise.

Acta Biotechnol. 9 (1989) 1 V1 M-

* +-

VE -

1

Time [mm] 1 1 1 1 1 2418 12 6 0 Fig. 1. Chromatogram of a test mixture 1: ovalbumin (MW: 43000), 2 : chymotrypsinogen A (MW: 25000), 3: ribonuclease (MW: 13700), 4 : contrycal (MW: 6512) glass column: 100 x 3.8 mm, stat. phase: Si 200 Polyol, 3 (xm, mob. phase: 0.05 M Na 2 HP0 4 /propanol-2 975: 25 v/v, pH = 7.5; flow rate: 0.02 ml/min, detection: UV, 254 nm, injection volume: 20 (jlI, concentration: 0.5 mg/ml for protein 1, 2, 4; 2.0 mg/ml for protein 3 I

I 70

1 60

1 50

1 AO

1 30

I 20

I 10

L 0

Fig. 2. Chromatogram of an extract of a thermostable protease 4 : protease activity glass column: 100 x 3.8 mm, stat. phase: Butyl = Si 300 Polyol, 5 [«n; mob. phase: A : 0.1 M Na 2 HP0 4 , 0.75 M (NH 4 ) 2 S0 4 , pH = 6.0; B : 0.1 M Na 2 HP0 4 , pH = 6.0; flow rate: 0.2 ml/min, pressure: 5. ..9 MPa, detection: UV, 254 nm, injection volume: 20 ¡xl

GEY, M„ THIEM, M. et al., High-Performance Liquid Chromatography

73

In Fig. 2 a chromatogram of a HIC separation of a thermostable protease on a 100 mm glass column (i.d.: 3.8 mm) packed with Butyl = Si 300 Polyol is shown. The protein precipitation on the separation column was carried out using a 0.75 M (NH 4 ) 2 S0 4 -buffer. For removing the proteins a 0.1 M phosphate buffer was used. The biological activity of the protease was determined in micropreparatively isolated fractions after the HIC separation procedure. The activity of the enzyme was conserved under the chromatographic conditions and the mean biological activity was measured in the protein peak 5. Ion-Exchange Chromatography (IEG) Besides ion exchanger based on silica gel mainly organic polymers (TSK-PW series or Mono Q, Mono S) are applied to liquid chromatographic protein separation. Aluminium oxide as a markedly cheaper stationary phase is an attractive alternative in some cases. Cation-exchange chromatography LAUBENT et al. [23] used aluminium oxide for protein separations because of its amphoteric character caused in aqueous solutions by the hydroxyl groups on the surface. Dependent on the pH value of the eluents aluminium oxide can be used as an anion or cation exchanger. Fig. 3 shows a cation-exchange separation on a 100 mm glass column (i.d.: 3.8 mm) filled with LiChrosorb Alox T. The elution proceeds in the order of increasing isoelectric point.

Time [minj 30

20

Fig. 3. Chromatogram of a test mixture i : bovine serum albumin (IP: 4.7 — 4.9), 2 : myoglobin ( I P : 6 . 8 - 7 . 3 ) , 3 : chymotrypsinogen A ( I P : 9.1) glass column: 100 x 3.8 mm, stat. phase: LiChrosorb Alox T, 5 ¡i.m; mob. phase: 0.2 M N a 2 H P 0 4 , pH = 8.0; flow rate: 0.1 ml/min, pressure: 2MPa, detection: UV, 254 nm, injection volume: 20 (1.1, concentration: 1 mg/ml for each protein

10

This is also shown in Fig. 4, where the dependence of the retention time on the isoelectric point is illustrated. Acid proteins, such as pepsin or bovine serum albumin, are not retained whereas proteins with isoelectric points greater than seven were separated. B y means of variations of buffer concentration (ionic strength), the retention time of basic proteins can be influenced (Fig. 5). An increase in ionic strength of the mobile phase is connected with a decrease of the retention time of the proteins. This effect is charac-

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Acta Biotechnol. 9 (1989) 1

chymo

pepsin

trypsinogen

myoglobin

aldolase

bovine serum albumin 10 IP 2 4 6 8 Fig. 4. Correlation between retention time (tR) and isoelectric point (IP) flow rate: 0.3 ml/min, further HPLC conditions as in Fig. 3

—i

1

1

1

1

01

0.2

0.3

OA

0.5

Fig. 5. Correlation between retention time (tR) and buffer concentration (c) • — bovine serum albumin chymotrypsinogen A x — cytochrome C flow rate: 0.4 ml/min, further HPLC conditions as in Fig. 3

[mot f1J

tenstic for the operating ion-exchange mechanism because higher concentrations of the counter-ions in the eluent are the reason for a quicker elution of the biomolecules. Acid proteins that are not retained on this cation exchanger did not show such a dependence in their retention behaviour (Fig. 5). The cowering of the i R -values of the proteins owing to higher buffer concentrations was exploited to elute proteins with "endless" retention time, e.g. cytochrome C, by gradient elution chromatography, as seen in Fig. 6. Also, by means of a flow gradient the analysis time could be further reduced. The separation of enzymes (thermostable protease and /J-galactosidase) on aluminium oxide was not achieved. Boths enzymes were eluted under these conditions together with the elution front.

GEY,

M.,

THIEM,

M. et al., High-Performance Liquid Chromatography

i 15

1 12

1 9

Time

1 6

[mmJ

1 3

75

1 0

Fig. 6. Chromatogram of a test mixture 1: bovine serum albumin, 2: myoglobin, 3: chymotrypsinogen A, 4: cytochrome C (IP: 10.1) mob. phase: A: 0.2 M Na 2 HP0 4 , pH = 8.0; B: 0.5 M NTa2HP04, pH = 8.0; flow rates: 0.1 ml/min for 6 min, then 0.5 ml/min; pressure: 2...5 MPa, concentration: 1 mg/ml, further HPLC conditions as in Fig. 3

Anion-Exchange

Chromatography

Anion-exchange separations of acid proteins on aluminium oxide were also impossible. Buffers of smaller pH values caused a drastic deterioration of peak height and shape (see Fig. 7). The positively charged surface of aluminium oxide may be the reason for the observed strong protein adsorption on the stationary phase. Organic polymers, on the other hand, are especially suitable for anion-exchange separations of proteins. We used a weakly basic anion-exchange material based on a styrene-