Acta Biotechnologica: Volume 8, Number 2 1988 [Reprint 2021 ed.] 9783112581766, 9783112581759

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Acta ßioiecliioligicfl Volume 8'1988 Number 2

Journal of microbial, biochemical and bioanalogous technology

Akademie-Verlag Berlin ISSN 0 1 3 8 - 4 9 8 8 Acta Biotechnol., Berlin 8 (1988) 2, 1 1 3 - 2 0 8

Instructions to Authors

1. 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. Dimter, Permoserstr. 15, Leipzig, 7 0 5 0 - D D R ) as soon as possible.

Acta Biitedniloiia 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 Konibinat of Chemical Plant Construction Leipzig—Grimma

M. Ringpfeil, Berlin and G. Vetterlein, Leipzig

Editorial Board:

1988

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

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

Number 2

Managing Editor:

L. Dimter, Leipzig

Volume 8

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 FRG and Berlin (West): to a book-shop or to the wholesale distributing agency Kunst und Wissen, Erich Bieber OHG, Wilhelmstr. 4—6, D-7000 Stuttgart 1; — 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 (Redaktion), 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 192,— DM zuzüglich Versandspesen; Preis je Heft 32.— DM. Der gültige Jahresbezugspreis für die DDR ist der Postzeitungsliste zu entnehmen. Bestellnummer dieses Heftes: 1094/8/2. 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. © 1988 by Akademie-Verlag Berlin. Printed in the German Democratic Republic. AN (EDV) 18520 03000

Acta Biotechnol. 8 (1988) 2, 115—123

Growth Kinetics of Top and Bottom Cultures of Saccharomyces spp. in a Chemostat Using Sugarbeet Molasses A L E X A N D E R , D . B . , ZAJXC, J . E . , J O N E S , L . P .

Department of Biological Sciences The University of Texas at E l Paso, Texas 79968

Summary Growth kinetics were evaluated for three yeast strains of the genus Saccharomyces. Two topfloating strains, SP 115 and SP 116 and one flocculant yeast SP 104 were analyzed in pure and mixed cultures in 1-liter continuous fermentation experiments in a chemostat. Growth was monitored for 72 h at 30 °C in a medium containing sugarbeet molasses and 1.0 g/liter each of NH 4 H 2 P0 4 and urea. SF 115 and SF 116 were found to have lower fi m i x values of 0.290 and 0.296 h - 1 , respectively, than SP 104, which had a /KH0CTb nOflBefleHHH noTOKOB nHTaTenbHOH c p e ^ H c oSteMHoii CKopooTbio Qi. MaTeMaraHecKoe onncaHne annapaTa COCTOHT H3 coBOKynHOCTH ypaBHeHHft MaTepnajibHoro 6ajiaHca, cooTHoineHHft AJIH pac^eTa rHflpoflHHaMHiecKHx H MaccooSMeHHux

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Acta Biotechnol. 8 (1988) 2, 1 5 9 - 1 6 7

Zum Lichteinfluß auf Fermentorkulturen von Nigella damascena L. SCHMATJDER, H . - P . * 1 , DÖBEL, P . * 2 * Während der Fermentorexperimente waren die Autoren Mitarbeiter des Institutes für Biochemie der Pflanzen der Akademie der Wissenschaften der DDR, Weinberg 3, Halle, DDR-4050 1 Friedrich-Schiller-Universität Jena, Sektion Biologie, WB Technische Mikrobiologie, Neugasse 23, Jena, 6900 DDR 2 Akademie der Wissenschaften der DDR Institut für Biotechnologie Permoserstraße 15, Leipzig, 7050 DDR

Summary: The growth behaviour of a Nigella damascena cell line cultivated in airlift fermenters in darkness as well as in ligth was investigated. Considerable variations were observed with regard to the morphology of the cells, the consumption of substrate and the content of yellow pigments. The UV absorption spectra of the yellow pigments show some similarities to the spectra known for thymoquinone.

Einleitung Bei der Kultivierung pflanzlicher Zellen im Fermentor spielen eine ganze Reihe von Umweltfaktoren, in erster Linie mechanische Belastung, Belichtung, Belüftung und Nährmedienzusammensetzung, eine wichtige Rolle für die Ausprägung der gewünschten physiologischen und morphologischen Eigenschaften (vgl. die Übersichten [1—4]). Für einige chlorophyllfreie Kulturen wird angegeben, daß eine Belichtung die Produktbildung positiv beeinflussen kann [z. B. 5]. Andere Linien reagieren jedoch negativ auf eine Belichtung während der Fermentation und zeigen eine deutliche Abnahme der Zellteilungshäufigkeit, des Wachstums und anderer physiologischer Parameter, wie z. B. der Aktivitäten wichtiger Enzyme. Ein derartiges System ist die von S C H M A U D E R et al. [6] beschriebene feindisperse Kultur von Nigella damascena L. I n der vorliegenden Arbeit wird ein weiterer Fall von Lichtempfindlichkeit bei Nigella damascena-Fermentorkulturen beschrieben.

Material und Methoden Zellkulturtechnik Als Ausgangsmaterial für die Fermentorkulturen diente eine seit 1984 im 7-Tage-Umlegerhythmus in Dunkelheit gehaltene Schüttelkultur von Nigella damascena L. Dieser Zellstamm war aus Blütengewebe isoliert worden. Als Nährmedium kam ein modifiziertes Medium nach M U R A S H I G E und S K O O G (mit 2 mg/1 2,4-D statt IES, 2% Saccharose und ohne Glycin) zum Einsatz [7],

160

Acta Biotechnol. 8 (1988) 2

Bei der Fermentorkultivierung wurden die Airliftvarianten der Fermentoren des Typs L F 2 (CSSR/DDR) eingesetzt [8, 9]. Die Fermentoren waren mit 3,5 1 Medium gefüllt und wurden im Verhältnis 1:8 bis 1:10 beimpft sowie mit 1 vvm belüftet. Die Arbeitstemperatur bfetrug 28 °C. Das zur Beimpfung der Fermentoren verwendete Zellmaterial war 7 Tage im Dunkeln bei 28 °C auf Rundschüttelmaschinen vom Typ Fanal vorkultiviert worden.1 Die Belichtung der Fermentoren erfolgte mit Hilfe von 4 U-förmigen Leuchtstoffröhren (Typ LUn 40), die an der Außenseite der Fermentoren montiert waren, wobei eine Lichtmenge von 400—600 Lux angeboten wurde. Die Belichtung erfolgte im 12 Stunden Licht-Dunkel-Rhythmus. Analytik Das Trockengewicht wurde von aliquoten Mengen der Zellsuspension (5—15 ml) nach Abtrennung und gründlichem Waschen der Zellen durch Trocknen bis zur Gewichtskonstanz bei 90 °C ermittelt. Die Bestimmung des Gehaltes an anorganischem Phosphat, Ammonium- und Nitrat ionen erfolgte nach [10—12], Der Gehalt an Glucose bzw. Saccharose wurde mittels der Glucoseoxidase ([13], Fermognost®-Test des VEB Arzneimittelwerk Dresden) bestimmt. Zur Saccharosebestimmung wurden die Proben zwecks Spaltung des Disaccharides mit HCl behandelt. Aus der Differenz der ermittelten Glucoseanteile erfolgte die rechnerische Ermittlung des Saccharosegehaltes. Zur Isolierung des intrazellulär angereicherten Farbstoffes wurde die abfiltrierte Biomasse zunächst mit Sand zerrieben und anschließend mehrfach mit einem Gemisch aus Ethanol und 10%iger HCl (95:5 v/v) bzw. Ethanol extrahiert. Die Grob- und Feinreinigung erfolgte durch Dünnschichtchromatographie an Kieselgel PF 254 („MERCK") unter Verwendung der Laufmittelsysteme LM A: Ethanol: 10%ige HCl = 95:5 LM B: n-Butanol: Eisessig: Wasser = 80:20:20. Die Detektion erfolgte anhand der gelben Eigenfarbe sowie der UV-Fluoreszenzlöschung bei 254 nm. Zur Elution vom Kieselgel sowie als Lösungsmittel für die Bestimmung der UV-Absorption diente LM A. Die Spektren und der Farbstoffgehalt wurden mit dem Zweistrahl-Spektralphotometer UV 210 A (SHIMADZTJ, Japan) ermittelt.

Ergebnisse und Diskussion Bei dem verwendeten Zellstamm von NigeUa damascena L. handelte es sich um eine seit 1984 existierende Langzeit-Dunkelkultur. Sie setzte sich aus zwei verschiedenen Gewebetypen zusammen. Der erste Typ wurde von kleinen, plasmareichen meristematischen Zellen gebildet, die in dichten Nestern (Meristemoiden) auftraten (Abb. 1). Der zweite Typ bestand aus wesentlich größeren Zellen von parenchymatischem Charakter, die sowohl einzeln als auch in lockeren Aggregaten vorlagen (Abb. 2). Der Zellstamm bildete unter Schüttelbedingungen im Dunkeln einen gelben, wasserlöslichen Farbstoff, der aber nur intrazellulär auftritt. Eine entsprechende Farbstoffbildung wurde auch bei einer anderen Nigella rfamascena-Zellkultur festgestellt [7], Im wesentlichen verhielt sich der verwendete Stamm bei Anzucht in unbelichteten Airlift-Fermentoren wie unter Schüttelkulturbedingungen. Die beiden beschriebenen Gewebetypen sowie die Farbstoffbildung waren eindeutig nachweisbar. In den belichteten Fermentoren zeigte der Stamm jedoch deutliche Veränderungen: In erster Linie trat eine Lichtschädigung der parenchymatischen Gewebefraktion zutage. Viele Zellen dieses Gewebes zeigten bereits nach zweitägiger Kultivierung im be-

SCHMAUDER,

H.-P.,

DÖBEL,

P., Lichteinfluß auf Fermentorkulturen

Abb. 1. Kleine, plasmareiche meristematische Zellen aus der Nigella (Dunkelkultur, Vergrößerung: 2 0 0 x )

Abb. 2. Große, plasmareiche Zellen aus Nigella (Dunkelkultur, Vergrößerung: 100X) 4

Acta Biotechnol. 8 (1988) 2

161

damascena

domaicerao-Zellkulturen

— Zellkultur

162

Acta Biotechnol. 8 (1988) 2

lichteten Fermentor Degenerationserscheinungen. Nach 5—6 Tagen war unter diesen Bedingungen ein erheblicher Teil der parenchymatischen Zellen abgestorben. Zellteilungen konnten in diesem Gewebe nur während der ersten beiden Tage festgestellt werden. Im Gegensatz zum parenchymatischen Gewebeanteil zeigten die Meristemoide unter Lichteinfluß keine nennenswerten Beeinträchtigungen ihrer Vitalität. Zum überwiegenden Teil vergrößerten sie sich im Laufe der Fermentation durch Zunahme der Zellzahl. Dabei waren bis zum 4. Kulturtag mitotische Zellteilungen beobachtbar. Im Ergebnis der Zellvermehrung wurden Tochter-Meristemoide abgegliedert, die mit den primären Meristemoiden in engem räumlichen Zusammenhang blieben, so daß bis zu 6 mm große, kompakte Aggregate entstanden, die der Fermentor-Lichtkultur ein zunehmend grobdisperses Aussehen verliehen (Abb. 3).

t i

Abb. 3. Meristemoide und mit ihnen verbundene Tochtermeristemoide aus Nigella damascenaFermentorkulturen (Dunkelkultur, Vergrößerung: 200 X)

Bei der Kultivierung im unbelichteten Fermentor wurden weder im Parenchymgewebe noch in den Meristemoiden Schädigungssymptome festgestellt. Beide Typen zeigten unter Lichtausschluß eine deutliche Vermehrung und Massenzunahme. Dabei konnten bis zum 5. Kulturtag mitotische Zellteilungen nachgewiesen werden. Viele Meristemoide wiesen gegen Ende der Fermentation eine Vergrößerung und Parenchymatisierung peripherer Zellen auf. Tochter-Meristemoide wurden ebenso wie in der Lichtkultur gebildet, trennten sich hier aber häufig von den Primär-Meristemoiden ab, so daß die Aggregate nur 1—2 mm groß wurden. Bei der Produktion des gelben Farbstoffes bestanden ebenfalls deutliche Unterschiede zwischen belichteter und verdunkelter Fermentorkultur. Dabei zeigten sich Parallelen zum Verhalten des parenchymatischen Gewebetyps. Der Degeneration und zahlen-

S c h m a t t d e r , H . - P . , D ö b e l , P . , Lichteinfluß auf

Fermentorkulturen

163

mäßigen Verringerung von Parenchymzellen entsprach eine deutliche Abnahme der Gelbfärbung im belichteten Fermentor. Bei der mikroskopischen Überprüfung erwies sich allerdings die Gelbfärbung nicht als spezifisch für die Parenchymzellen. Sie wurde nicht selten auch in Meristemoiden festgestellt. Entsprechend starke Unterschiede zwischen belichteter und unbelichteter Kultur konnten bei der physiologischen Charakterisierung beobachtet werden. Für diese Analysen wurden neben dem Trockengewicht und dem pH-Wert des Kulturmediums auch der Verbrauch wichtiger Nährstoffe, wie Saccharose, Glucose, anorganisches Phosphat, Ammonium-, Nitrationen, sowie der Gehalt der Zellen an gelbem Farbstoff verfolgt (Abb. 4, 5).

Tage

Abb. 4. Wachstum, pH-Wert und spezifische Farbstoffgehalte (OD365/mg TG) von Nigella damaseena Fermentorkulturen O O

O"

Lichtkulturen, „

„

,

x X

, x-.-

Dunkelkulturen, (OD365/mg TG) „ , (TG) „ , (pH)

Der Verlauf der Trockengewichtskurve zeigt, daß unter Lichteinfluß das Wachstum deutlich stagnierte und ab 6. Kulturtag keine neue Biomasse mehr gebildet wurde. Nach 8 bis maximal 10 Kulturtagen war im belichteten Airlift-Fermentor die Kultur weitgehend abgestorben. Ein Indiz für das Absterben der Zellen ist auch der Verlauf des pH-Wertes, der vom 4. Kulturtag an stark in das saure Milieu bis zu Werten von pH 3,4 bis 3,5 absank (Abb. 4). Unterschiede zwischen belichteten und verdunkelten Fermentoren traten auch bei der Analyse des Substratverbrauches hervor (Abb. 5). Der Verbrauch an anorganischem Phosphat ist bei belichteter und bei Dunkelkultur bis zum Absterben der Lichtkultur nahezu gleich, während die Nitrat-Assimilation leicht unterschiedlich abläuft. Die Lichtkulturen verbrauchen geringfügig mehr Nitrat bevor sie absterben. Interessanterweise wird sowohl die verfügbare Menge an anorganischem Phosphat als auch an Ammoniumionen nicht vollständig von den Kulturen ausgenutzt. Der Endphosphatgehalt entspricht etwa 1/3 der Ausgangskonzentration. Der Ammoniumionenanteil im Medium steigt gegen Fermentationsende sowohl bei den Licht- als auch bei den Dunkelkulturen wieder an, vermutlich durch Zellyse und Abbaureaktionen im Bereich der Proteine und Aminosäuren bedingt. Ebenso wie die anderen Nährstoffanalysen zeigten Saccharose4*

164

Acta Biotechnol. 8 (1988) 2

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Abb. 5. Nährstoffverbrauch in Fermentorkulturen von Nigella damascena L. Obere Darstellung: Dunkelkulturen Untere Darstellung: Lichtkulturen

bzw. Glucoseverbrauch nach 6—8 Tagen eine deutlich geringere Aktivität der Lichtkulturen an. Die Geschwindigkeit der Saccharosespaltung wie auch des Glucoseverbrauchs war in den Licht-Kulturen stark reduziert. Der Anteil gelber Farbstoffe in den Kulturen sank bei Belichtung ständig, wogegen die dunkel angezogenen Zellen in den ersten Tagen eine Zunahme des Farbstoffgehaltes, bezogen auf das Trockengewicht, zeigten. Darauf folgte zwischen dem 2. und 6. Kulturtag eine Phase nur geringer Abnahme des Farbstoffanteiles ehe schließlich der stärkere Abbau einsetzte (Abb. 4). Die aus den Zellen mit Ethanol bzw. Ethanol/ 10%ige HCl (95:5) isolierbare Farbstofffraktion zeigte im UV-Bereich die in Abb. 6 wiedergegebenen Absorptionskurven. Diese sind nahezu identisch mit anderen Gemischen gelber Farbstoffe, die aus verschiedenen Organen von Nigella damascena L. auf analoge Weise isoliert werden konnten. Bei Versuchen zur chromatographischen Charakterisierung zeigte das Gemisch in L M A eine einheitliche Zone, so daß für die Gesamtgehaltsbestimmung eine Abtrennung von anderen Zellinhaltsstoffen mit diesem Laufmittelsystem möglich war. Da unter 320 nm zu viele aromatische Verbindungen absorbieren, wurde der Farbstoffgehalt der Zellen bei dem Maximum 365 nm gemessen.

SCHMAUDER, H . - P . , DÖBEL, P . , L i c h t e i n f l u ß auf F e r m e n t o r k u l t u r e n

165

Abb. 6. Absorptionskurven der Farbstoffgemische, isoliert aus gelben Fermentorkulturen ( ), Samenschalen ( ) bzw. Wurzelspitzen ( ) von Nigella damascena L. (Zur Isolation vergleiche unter Material und Methoden)

Bei weiteren chromatographischen Experimenten konnte mit Hilfe des LM B eine Auftrennung der gelben Fraktion in mindestens drei Banden beobachtet werden. Aus den entsprechenden Absorptionen im UV-Bereich (Tab. 1) kann man entnehmen, daß zwischen den Farbstoffgemischen aus der Zellkultur, den Samenschalen und den Wurzelspitzen von Nigella-Vi\&nzen eine große Ähnlichkeit besteht. Es fällt auf, daß sowohl farbstofführende Organteile der Ganzpflanze als auch die farbstoffbildenden Zellkulturen unter Lichtausschluß heranwachsen. Der verhältnismäßig schnelle Farbstoffverlust bei Lichtzutritt zu den Kulturen deutet darauf hin, daß entweder die Farbstoffsynthese im biologischen System lichtempfindlich ist oder der gebildete Farbstoff unter Lichteinfluß schneller angebaut wird. Isolierte Fraktionen mit dem Farbstoff sind unter Lichteinfluß stabiler. Eine gelbe Verbindung aus den Samen einer anderen Nigella-Art (Nigella sativa L.) konnte von E L - D A K H A K H N Y als Thymochinon identifiziert werden. Diese Substanz wurde gleichzeitig als der pharmazeutisch interessante Inhaltsstoff des Samenöls dieser Nigella-Art erkannt, der in einigen Ländern des Orients als wertvoller Wirkstoff der Volks- und traditionellen Medizin angewendet wird. Für das Thymochinon aus dem Samenöl von Nigella sativa L. wurden folgende Absorptionen im UV-Bereich mitgeteilt: 252, 259 und 306 nm [14, 15]. Die von uns erhaltenen UV-Absorptionen stimmen damit teilweise überein (Tab. 1, Stoff 1), so daß durchaus thymochinonähnliche Verbindungen in dem gelben Farbstoffgemisch vorhanden sein könnten. Für die Aufklärung der Strukturen dieser Komponenten sind weitere Untersuchungen erforderlich. Die beschriebenen Ergebnisse zeigen, daß dem Licht als Uniweltfaktor bei der Fermentorkultivierung pflanzlicher Zellen eine wesentliche Bedeutung zukommen kann. Im vorliegenden Fall ist dieser Einfluß negativ. Dies steht in Übereinstimmung mit der früher beschriebenen hemmenden Wirkung des Lichtes auf eine histologisch weniger komplexe Kultur von Nigella damascena L. Bei ihr konnte im Vergleich zu den im Dunkeln angezogenen Kontrollen eine signifikante Reduktion der Zellvermehrung und

166

Acta Biotechnol. 8 (1988) 2 Tab. 1. UV-Absorptionen (in nm) der bei der DC-Trennung in LM B auftretenden drei gelben Zonen, isoliert aus Zellkulturen, Samenschalen bzw. Wurzelspitzen von Nigella damascena L. (Sch = Schulter; Stoff 1 = Rf 0,79, Stoff 2 = Rf 0,69, Stoff 3 = R, 0,60) H e r k u n f t aus

Stoff 1

Stoff 2

Zellkultur

Samenschale

Wurzelspitze

208 222 228 252 258 275 282 308

208 222 228 252 Sch

208 222 228 252 Sch

275 282 Sch 307 Sch

275

Sch Sch

365 Sch

365 Sch

307 Sch 338 Sch 365 Sch

210 230 Sch 238 Sch

210 230 238 265 Sch

210 230 Sch 238 Sch 268 Sch

308 315 365

308 315 365

210 245

210 245 258 Sch

Sch Sch Sch

278 Sch 308 Sch 322 Sch 365 Stoff 3

210 245 258 Sch

265 Sch 285 308 340 365

308 315

308 315

365

365

einiger ausgewählter physiologischer Parameter beobachtet werden [6]. In beiden Fällen erfolgte die Belichtung im 12 Stunden Licht-Dunkel-Rhythmus. Im Falle von Dauerbelichtung, wie sie von verschiedenen Autoren für die Fermentorkultivierung von Pflanzenzellen eingesetzt wird, ist bei Verwendung von Nigella-KuUuren mit verstärkten Hemm- und Schädigungseffekten zu rechnen. Abkürzungen TG DC LM OD Suc

Trockengewicht Dünnschichtchromatographie Laufmittelsystem optische Dichte Saccharose

Eingegangen: 21. 5. 1987

SCHMAUDER, H . - P . , DÖBEL, P . ,

Lichteinfluß auf Fermentorkulturen

167

Literatur [1]

[2]

F., V O G E L M A N N , H.: In: Plant tissue culture and its biotechnological application, Eds.: B A R Z , W . , R E I N H A R D , E., Z E N K , M . H., Springer-Verlag, Berlin, Heidelberg, New York, 1977, 245. S C H M A U D E R , H.-P., R Ö B L I T Z , D.: Abhandlungen der AdW der DDR, N 3 / 1 9 7 9 (publ. 1 9 8 0 ) ,

WAGNER,

205.

M. W . : In: Plant Biotechnology, Eds.: M A N T E L L , S . H . , S M I T H , H . , Cambridge University Press, Cambridge, 1983, 3. S C H M A U D E R , H.-P.: In: Wissenschaftliche Geräte für die Biotechnologie, Hrsg.: LAUCIKNER, G., B E C K M A N N , D . , ZWG-mytron (AdW), Heiligenstadt, 1 9 8 7 , 2 4 5 . R Ö B L I T Z , H., R Ö B L I T Z , D., S C H M A U D E R , H.-P., G R Ö G E R , D.: Plant Cell Reports 2 ( 1 9 8 3 ) , 1 2 2 . S C H M A U D E R , H.-P., D Ö B E L , P., G R Ö G E R , D . : Biochem. Physiol. Pflanzen 1 7 9 ( 1 9 8 4 ) , 6 1 1 . D Ö B E L , P.: Protoplasma (1987), im Druck S C H M A U D E R , H . - P . , G R Ö G E R , D . , B A D E , W . : Acta Biotechnol. 3 ( 1 9 8 3 ) , 9 3 . S C H M A U D E R , H . - P . : In: Wissenschaftliche Geräte für die Biotechnologie, Hrsg.: L A U C K N E R , G., B E C K M A N N , D., ZWG mytron, Heiligenstadt 1 9 8 3 , 4 3 . B A G I N S K I , E. S . , F O A , P. P., Z A K , B . : In: Methoden der enzymatischen Analyse, 3. Auflage, Hrsg.: B E R G M E Y E R , H. U., Verlag Chemie, Weinheim, 1974, 909. F A W C E T , J . R., SCOTT, J. E.: J . Clin. Pathol. 1 3 (1960), 156. H O A T H E R , R . C., R A C K H A M , R . F.: Analyst 84 (1959), 548. Arzneibuch der DDR, Diagnostische Labormethoden. E L - D A K H A K H N Y , M.: Planta Med. 1 1 ( 1 9 6 3 ) , 4 6 5 . E L - D A K H A K H N Y , M.: Arzneimittelforschung 15 (1965), 1227.

[3] FOWLER, [4] [5] [6]

[7] [8] [9]

[10] [11] [12] [13] [14]

[15]

A c t a Biotechnol. 8 (1988) 2, 168

Book Review C. W E B B , G . M . BLACK, B . ATKINSON

Process Engineering Aspects of Immobilized Cell Systems R u g b y — W a r w i c k s h i r e : T h e I n s t i t u t i o n of Chemical E n g i n e e r s , 1986. 240 S., 107 A b b . , 48 Tab., 2 5 L I n diesem B u c h w u r d e n v o m Herausgebertrio die V o r t r ä g e z u s a m m e n g e f a ß t , die auf der 1984 in Manchester u n t e r der im B u c h t i t e l g e n a n n t e n T h e m a t i k a b g e h a l t e n e n K o n f e r e n z d a r g e b o t e n w u r d e n . E s gliedert sich f o r m a l in 5 Teile m i t insgesamt 25 Beiträgen (Cell Immobilization / 3 ; Immobilized Cell R e a c t o r s / 3 ; Immobilized Cell Particles / 4 ; I n d u s t r i a l Applications / 4 ; Supplem e n t a r y Contributions / 11). Diese t h e m a t i s c h e Gliederung ist jedoch weder als Wegweiser d u r c h d a s B u c h noch als streng gültig zu b e t r a c h t e n , denn in m e h r e r e n Teilen des Buches sind Hinweise zu den ersten drei „ t o p i c s " zu f i n d e n u n d der letzte Beitrag im 4. Teil b e i n h a l t e t (noch) keine „ i n d u s t r i e l l e " A n w e n d u n g . F a s t alle A u t o r e n sind E u r o p ä e r , h a u p t s ä c h l i c h aus Großbritannien. I m 1. bis 3. Teil, aber a u c h im 5. Teil des Buches werden b e h a n d e l t : Wichtige Merkmale u n d Eigens c h a f t e n v o n Zellimmobilisaten; verschiedene Methoden ihrer Herstellung (für weitergehende Inf o r m a t i o n e n sind z. T. zahlreiche Literaturhinweise a n g e f ü h r t ) ; Möglichkeiten u n d Grenzen der A n w e n d u n g immobilisierter E n z y m e u n d Coenzyme einerseits u n d immobilisierter Zellen andererseits f ü r S u b s t r a t w a n d l u n g e n , die über c o e m z y m e r f o r d e r n d e e n z y m a t i s c h e M e h r s c h r i t t r e a k t i o n e n v e r l a u f e n ; R e a k t o r ( g r u n d ) t y p e n f ü r den E i n s a t z immobilisierter B i o k a t a l y s a t o r e n u n d interessante Vergleiche der Leistung solcher R e a k t o r e n bei Verwendung freier oder immobilisierter vitaler Zellen oder bei Koexistenz beider (bei wuchsassoziierter P r o d u k t b i l d u n g bzw. bei Milieu/Reaktionsbedingungen, die f o r t w ä h r e n d e Zellvermehrung v e r u r s a c h e n ) ; R e a k t o r k i n e t i k bzw. Problem des b e h i n d e r t e n S t o f f a u s t a u s c h e s (einschließlich der Schwierigkeit einer ausreichenden Sauerstoffz u f u h r zu den Zellen); das Problem der A u f r e c h t e r h a l t u n g einer L a n g z e i t s t a b i l i t ä t sowohl der p r o d u k t b i l d e n d e n A k t i v i t ä t immobilisierter vitaler Zellen als auch der Immobili3ierungs(Einschluß)matrix selbst (Abnutzung oder Zerstörung der Matrix d u r c h mechanische B e a n s p r u c h u n g , s t a r k e n Zellzuwachs bzw. reaktionsbedingte Gasbildung) sowie im engen Z u s a m m e n h a n g d a m i t die A n f o r d e r u n g e n an die biologischen/physiologischen E i g e n s c h a f t e n der zu immobilisierenden Zellen, deren V e r ä n d e r u n g e n d u r c h Immobilisierung bzw. E i n s a t z der I m m o b i l i s a t e im R e a k t o r u n d Möglichkeiten zur A u f r e c h t e r h a l t u n g der gewünschten E i g e n s c h a f t e n ; B e t r a c h t u n g e n zur Biomassebeladung verschiedener Immobilisierungsmatrizes (d. h. zu Immobilisierungsmethoden) u n d zur immobilisierten Biomassemenge pro R e a k t o r v o l u m e n e i n h e i t bei verschiedenen R e a k t o r t y p e n u n d Immobilisierungsmatrizes. I m 4. Teil ( „ I n d u s t r i a l Applications") werden beschrieben: Die Abwasserbehandlung in einer zweistufigen (anaerob/aerob) Pilotanlage (Wirbelbett- bzw. F l i e ß b e t t r e a k t o r e n , B a k t e r i e n a n S a n d adsorbiert). Der „ C A P T O R " - A b w a s s e r p r o z e ß (aerob, Mikroorganismen an/in P U - S c h a u m partikel gebunden). Der industrielle E i n s a t z immobilisierter Amyloglucosidase zur Ausbeutesteigerung (Kostensenkung) bei der Gewinnung kristalliner Glucose sowie beim H F C S - P r o z e ß (Diu n d Oligosaccharidspaltung im „ H F C S - R a f f i n a t " ) . Die Immobilisierung von Pflanzen- u n d Tierzellen (Methoden, Besonderheiten, Anwendungsmöglichkeiten). Die T h e m a t i k der im letzten Teil des Buches z u s a m m e n g e f a ß t e n Vorträge variiert vom „biologischen F i l m " (rasche B e s t i m m u n g der Zellbindungsfähigkeit u n d -festigkeit, E i n f l u ß der Beschaffenheit der Zelloberfläche, kontinuierliche E t h a n o l p r o d u k t i o n ) über das physiologische Verh a l t e n verschiedener gelimmobilisierter Zellen bei behinderter Z u f ü h r u n g von 0 2 , N ä h r s t o f f e n oder L i c h t ; die H 2 - bzw. X H 3 - B i l d u n g m i t eingeschlossenen (Alginat, P U ) Zellen bis zur Methode der „ p a s s i v e n " Immobilisierung mikrobieller oder pflanzlicher Zellen in/an hochporöse Medien [,,Biomass S u p p o r t Particles ( B S P ) " , einschließlich Gewebe oder F o r m k ö r p e r aus S t a h l d r a h t , Casaicinu n d kontinuierliche Cellulaseproduktion m i t B S P , weitere Hinweise z u m „ C A P T O R " - A b w a s s e r prozeß]. Obgleich keine E x p e r t e n aus J a p a n bzw. den USA (auch n i c h t aus der U d S S R , B R D , Italien, Frankreich) zu W o r t k o m m e n , ist dieses B u c h zumindest all denjenigen zu empfehlen, die sich m i t „immobilisierten Zellen" befassen, wenn auch n u r a m R a n d e . Sie werden wertvolle Hinweise finden. R . BERGER

Acta Biotechnol. 8 (1988) 2, 1 6 9 - 1 7 5

Microbial Lipases in Biotechnology SzTAJER, H . , ZBOINSKA, E .

Institute of Organic and Physical Chemistry, Technical University, Wybrzeze Wyspianskiego 27, 50—370 Wroclaw, Poland

Summary The lipolytic activity of microorganisms and substrate specificity of lipases are described. The applications of these enzymes in various industrial processes are discussed.

Lipolytic Properties of Microorganisms

Lipases are produced by plants, animals and microorganisms. Earlier investigations were concerned mainly with enzymes which participate in lipid metabolisms in animals. The most thoroughly studied was lipase from the pancreas. Recently, increasingly more attention is being paid to lipases produced by bacteria and fungi (Tab. 1). Lipolytic activity was found in microorganisms which take part in the production of cheeses (i.e. Streptococcus lactis, Lactobacillus casei, Penicillium roqueforti) [14, 15, 31, 32], Certain lipolytic strains were isolated from raw milk. Moreover, many lipolytic strains occur in soil. Pathogenic strains of Staphylococcus aureus also exhibit lipolytic activity. There is little information on the lipolytic activity of Streptomyces.

Substrate Specificity of Microbial Lipases

Lipolytic enzymes of microbial origin hydrolyze various animal fats and vegetable oils as well as synthetic mono-, di- and triglycerides. Synthetic triglycerides are preferred substrates. Microbial lipases may be divided into two groups: non-specific and specific lipases. Enzymes from the first group do not distinguish between the three positions of glycerol esters. Such lipases were isolated from Geotrichum candidum, Staphylococcus aureus and Humicola lanuginosa [41], These non-specific lipases bring about a total hydrolysis of triglycerides to fatty acids and glycerol. The second group of lipases, i.e. the specific enzymes, hydrolyze ester in the 1 and 2 positions of glycerides giving free fatty acids and a mixture of mono- and diglycerides. The 2-monoglycerides and, to a lesser degree, the 1,2- or 2,3-diglycerides are unstable and therefore the enzymatic hydrolysis is followed by acyl group migrations. This leads

170

Acta Biotechnol. 8 (1988) 2 Tab. 1. Lipase producing microorganisms Microorganisms

Ref.

Pseudomonas fluorescens Pséudomonas aeruginosa Propionibacterium acnes Propionibacterium granulosum Acinetobacter calcoaceticus Achromobacter lipolyticum Corynebacterium acnes Proteus sp. Staphylococcus aureus Streptococcus lactis Myxococcus xantus Lactobacillus sp. Bacillus cereus Candida lipolytics, Candida rugosa Candida deformans Candida curvata Saccharomyces lipolytica Rhodotorula pilimonae Rhizopus delemar Rhizopus japonicus Rhizopus microsporus Rhizopus nodosus Rhizopus arrhizus Pénicillium roqueforti Pénicillium cyclopium Fusarium solari Geotrichum candidum Humicola lanuginosa Aspergillus niger

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

t o 1-monoglycerides and 1,3-diglycerides. Therefore t h e e x t e n t i o n of the incubation t i m e m a y result i n a total splitting of triglycerides. E n z y m e s from Aspergillus niger, Rhizopus arrhizus, Rhizopus delemar or Pseudomonas sp. belong to this group of lipases. T h e a c t i v i t y of lipases d e p e n d s o n the chain length a n d saturation ratio of the f a t t y acid in f a t s which serve as substrates [42, 43, 44], J E N S E N [45] described the e n z y m e f r o m Oeotrichum candidum which exhibits a high specificity to oleic acid and linoleic Tab. 2. Releasing of fatty acids from fats occurring in milk by various microbial lipases [46] Fatty acid

Amounts of fatty acid released by lipases from various microorganisms [%] A.

Caprylic acid Laurie acid Palmitic acid Stearic acid Oleic acid Linoleic acid

1.9 1.0 12.9 8.0 47.4 6.5

lipolyticum

0. 1.3 1.9 15.7 1.1 61.0 5.3

candidum

A. niger

P. roqueforti

1.0 3.2 33.7 14.0 28.7 1.6

3.1 3.2 17.8 9.0 47.9 2.1

171

SZTAJEK, H . , ZROINSKA, E . , M i c r o b i a l L i p a s e s

acid independently of their positions in the triglyceride. A lipase from Achromobacter lipolyticum acts also very selectively releasing preferentially linoleic acid during hydrolysis of the fats. A lipase from Aspergillus niger hydrolyzes preferentially fats containing stearic acid. Fatty acids released from fats occurring in milk by various microbial lipases are shown in Tab. 2 [46]. The specificity of lipases to fatty acids was utilized to produce dietetically important polyunsaturated fatty acids. Application of Lipolytic Enzymes Lipases are being used in oil and fat industry to modify fats. Certain fatty acids released from milk are important in cheese ripening giving a characteristic flavour and promoting the process [47, 48, 49] (Tab. 3). Specific flavours of numerous cheeses are due to various microbial lipases. Lipases can also be used to modify fats in the bakery goods (bread, crackers) (Tab. 4). Tab. 3. Amounts of fatty acids released by various lipases from fats occurring in milk Enzymes

Amounts of released fatty acids [ % ]

Lipase from bovine panreas Lipases from Pénicillium, roqueforti Lipases from Achromobacter sp.

17 38 22

Tab. 4. Lipolytic enzymes, useful in modifying of fats suitable for incorporation into baked goods Lipase source Mould lipases Aspergillus niger Oeotrichum candidum Penicillium roqueforti Bacterial lipases Achromobacter lipolyticum Pseudomonas fluorescens

The use of modified fats improves the flavour, colour, softness and structure of bread. Microbial lipases are also utilized for modification of milk products. Partial hydrolvzis of beef fat improves the properties of pet-food. In a process lipases are added to the pet-food to modify beef fat without influencing others components. In leather industry microbial lipases are used for leather defatting. Efforts are under way at present to develop technologies using lipolytic enzymes in laundry detergents [51, 52, 53]. The importance of enzyme in laundry detergents has been enhanced due to a decrease in detergent performance caused by lower levels of phosphate builder required now because of environmental consideration. Another factor decreasing the performance of detergent and thus stimulating the application of enzyme is the tendency towards lower temperature washing to save energy. The prospects for using lipases for laundry purposes appear to be good because the activity of lipases is not adversely affected by most commercial detergents.

172

Acta Biotechnol. 8 (1988) 2

An exciting development is the new focus on enzymes t h a t can be used in the mainstream of oleochemical processing. Three key areas with potential for improvement by enzymology are f a t splitting for f a t t y acid production, lipid synthesis via reversal of hydrolysis and lipid modification by ester interchange or interesterification. The standard technology for f a t t y acid production is high temperature and pressure countercurrent, steam splitting. Recent patents and publications have demonstrated t h a t this reaction can be accomplished by means of lipolytic enzymes [19, 54, 55, 56], I n this case, the reaction proceeds well at ambient pressure and a relatively low temperature on the order of 40°: 60° resulting in much lower energy costs. Because of mild conditions and the high specificity of the enzyme it may be possible to produce polyunsaturated acids from some fats. The lipase from Candida sp. is able to accomplish 98% hydrolysis within 4 hrs. I n this case enzyme hydrolysis achieved results approaching those seen in chemical hydrolysis system, but under much milder reaction conditions. Another area of potential application of lipases is the reversal of hydrolysis reaction in order to synthesize esters. By decreasing the water concentration it is possible to pull the reaction in the direction of esterification. The esterification reaction with lipolytic enzymes goes under much milder conditions than those with chemical catalysts. By choice of enzyme and substrates it is possible to produce very specific products [57, 58, 59, 60]. The lipase from Rhizopus arrhizus was used to esterify acids of various chain lenghts with various alcohols. The Rhizopus sp. enzyme is specific for long chain f a t t y acids (8 to 16 carbon atoms) [61]. Synthesis of esters by Rhizopus enzyme is shown in Tab. 5. Tab. 5. The synthesis of esters catalysed by lipase from Rhizopus arrhizus [61] Procent of various fatty and esters Alcohols

2:0

3:0

4:0

8:0

16:0

n-Propanol n-Butanol Izopentanol Izooctanol

0 0 0 0

0 0 15.0 26.5

0 4.9 29.6 57.4

29.3 35.6 81.8 52.6

40.0 52.6 85.0 78.9

Another area of potential enzyme application is the interesterification. I n this process the f a t t y acid composition of f a t can be varied to fats with desired properties. For example it is possible to exchange only one of the f a t t y acids in the triglyceride molecule for another acid. Microbial lipases are particularly useful in interesterification because of their specificity for particular positions in the triglyceride molecule for f a t t y acids chain lenghts [41]. Interesterification can be performed in the presence of f a t t y acid triglyceride in the reaction mixture but the result will vary with different microbial lipases (Fig. 1). I t was shown that non specific lipases can catalyse the process of synthesis of triglycerides modified at all three positions while the 1,3 specific lipases will produce interesterified triglycerides modified at positions 1 and 3. Interesterfication can be performed in the mixtures containing various glycerides (Fig. 2). The possibility of producing new triglycerides by interesterification is very important for fat industry because it can lead to valuable products that have specific properties. M A C R A E , COLEMAN and M C R A E [ 5 5 , 6 2 , 6 3 ] suggest using microbial lipases to produce cocoa butter substitute which is used in food and cosmetiscs formulations.

173

S T Z A J E R , H . , ZBOINSKA, E . , M i c r o b i a l L i p a s e s -RI

-RI -R

T

+

-R —

-RI

R2

-R

2

-R

2

non specific R2 +

R3C00H

P LIPA SES

+

—K 0

R2

- R

3

- R

3

-R2 +

R3COOH

R3

R2

R2

—R2

+

2

-R

T

+

+

2

-R

3

R3

R3

-R3

+ -Rt

+ —R2

—Rj

-RI

+

-R

3

-R

-RI

Rx

-RI

+

-RI

-RI +

R«

3

R

I

- R

R2

- R

3

+

+

- R - R

3

-R

-R3

+ -R

—R2

-R

-R

+

R3

—R2 + R3COOH +

+

2

R^OOH

—R,

R3

Fig. 1. Interesterification of triglycerides and f a t t y acids

-RI -RI -RI

R3

-RI

—

R2

—

R2

non specific -R

2

+

RI

—R2 -R

lipases

+

-R

R3

-R2 LRX

+

i , 3 specific R2 -R

3

lipases

3

+

+

—

R

2

—RG

-RI

—R2

-R

—RJ

+

-RI R2

—R3

—R3

-R

3

+

-RI

•R 3

-RX

LR

-Rx

-Rx

-R

-R2

+ -R2

+ -R

Rx

-R3

-R

Fig. 2. Interesterification of triglycerides

-RI

+

3

-R

—

2

I

r

—RJ

-Ri

-RI

R2

3

-RI

-RI

+

R]

-R

-R

+

—

R3

-RI

•"RJ

lipases

2

R2

-R

-RI

1,3 specific

-R

-RI

'-RI

—R 3 -Ri

+

X

-R

+

-RI

+

+

X

-RI + -R

3

-RX +

""RJ

R3

-RX

L

R2

~"R2

—

—R2

+

-R

3

—RJ

—

-R

-R

3

-R

3

3

"RJ R3

+

+

R2

R2

R

3

R2

— R J

HRI

-R +

3

-RX —RG

Acta Biotechnol. 8 (1988) 2

174

Due to the wide application of enzymes in the oil and fat industry is an increase of the number of companies which produce commercial lipolytic enzymes (Tab. 6). The development of immobilization technics particularly using artificial membranes open new possibilities of lipids transformation in continuous processes. Tab. 6. Some companies producing commercial lipolytic enzymes Company

Country

Enzyme

Gist- Brocades NV Hughes and Hughes Novo Industri A/S

Holland U. K. Denmark

J O H N a n d E . STURGE L t d .

U. K.

lipase lipase lipase lipase

from from from from

Mueor miehei Ehizopus arrhizus Aspergillus niger Aspergillus niger

Received May 11, 1987

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Acknowledgements This work was supported by the Polish Scientific Project CPBP 04.11.2.4.

Acta Biotechnol. 8 (1988) 2, 176

Book Review D . L . MTTLCAHY, G . B . M U L C A H Y , E .

OTTAVIANC

Biotechnology and Ecology of Pollen Proceedings of the International Conference on t h e Biotechnology and Ecology of Pollen, 9—11 J u l y 1985 University of Massachusetts, Amherst, MA, USA. New York, Berlin, Heidelberg, Tokyo: Springer-Verlag, 1986. 528 S., 129,— DM Die Forschung a m Pollen der Angiospermen h a t im letzten J a h r z e h n t Portschritte erzielt, die f ü r die Pflanzenzüchtung, -genetik und -physiologie, ebenso aber auch f ü r die Ökologie von erheblicher Bedeutung sind. Dies ist zum großen Teil auf den Einsatz moderner biochemischer und genetischer Untersuchungstechniken zurückzuführen, wurde aber außerdem durch die grundlegende Entdeckung initiiert, d a ß ca. 60% der im Sporophyten zur Ausprägung kommenden Strukturgene auch im Pollen exprimiert werden und dort einer intensiven Selektion unterliegen. I m vorliegenden Symposiumsband werden die neuen Aspekte der Pollenforschung in 6 Kapiteln umfassend und detailliert dargestellt. Das 1. Kapitel („Gene Expression In Pollen"; 7 Beiträge) befaßt sich mit der Genwirkung, Genregulation und Selektion auf der Ebene des Gametophyten und des Sporophyten. Für verschiedene Pflanzenarten wird nachgewiesen, daß die Mehrzahl der Gene, vor allem solche, die essentielle Prozesse des Zellstoffwechsels kontrollieren, sowohl im Pollen als auch im somatischen Pflanzengewebe aktiv ist. — Das folgende Kapitel („Pollen in Biotechnology"; 20 Beiträge) behandelt im wesentlichen den Einsatz von Gen- und Zellmanipulationstechniken am Pollen, außerdem Art- und Gattungskreuzungen und Versuche zur Selektion von Pollenresistenzen gegenüber verschiedenen Streßfaktoren und phytopathogenen Mikroorganismen. Einige der vorgelegten Ergebnisse bieten Ansatzpunkte f ü r die Nutzung in neuartigen Züchtungsstrategien. Weiterhin wird hier über die Eignung des Pollens als Indikator f ü r Luftund Umweltbelastung berichtet. — Das 3. Kapitel („Self-Incompatibility And Pollen-Style Interactions"; 17 Beiträge) bringt eine Fülle von Versuchsergebnissen zur Inkompatibilität und Wechselwirkung zwischen Pollen und Griffelgewebe. Bei der Interpretation der Ergebnisse zeigt sich die ganze Komplexität dieser seit langem diskutierten Problematik, zu der auch in den vorliegenden Beiträgen wieder modifizierte Modellvorstellungen entwickelt werden. — I m 4. Kapitel („Pollen Ultrastructure and Development"; 7 Beiträge) wird vor allem die Zusammenlagerung von Spermazellen und vegetativem Zellkern zur „male germ u n i t " demonstriert und die Möglichkeit der experimentellen Isolierung dieses Komplexes diskutiert. Weiterhin werden strukturelle und biochemische Aspekte der Pollenentwicklung und Pollensterilität behandelt. — Das folgende Kapitel („Pollen Physiology And Metabolism"; 12 Beiträge) befaßt sich mit recht unterschiedlichen Problemen, vom Zusammenhang von Energiebilanz und Wassergehalt mit der Pollenfertilit ä t und dem Pollenstoffwechsel, der Hemmstoffwirkung und dem Einfluß des Narbengewebes auf das Wachstum der Pollenschläuche sowie der Ionenverteilung in Pollenschlauch und Narbe bis zu der Frage, ob die Bienen Substanzen mit keimungshemmender Wirkung auf den gesammelten Pollen ausscheiden. — Schwerpunkt des letzten Kapitels („Gametophytic Ecology"; 15 Beiträge) ist die Pollenkonkurrenz. Bei einer Reihe von Arten wird mit unterschiedlichem Ergebnis gep r ü f t , ob die unter Normalbedingungen auf die Narbe übertragene Pollenmenge tatsächlich so groß ist, daß es zu einer Konkurrenz der Pollenschläuche k o m m t . Die aus dem „genetic overlap" abgeleitete These, daß raschwüchsige Pollenschläuche ihr überdurchschnittliches Durchsetzungsvermögen auf den resultierenden Samen übertragen, wird f ü r verschiedene Objekte bestätigt und kann auch f ü r die Früchte zutreffen, die bei mehreren Arten nach Bestäubung mit geringen Pollenmengen und fehlender Pollenkonkurrenz relativ häufig abgestoßen werden. — Es folgen noch 27 Abstracts von Postern, die sehr unterschiedliche Teilbereiche der Pollenproblematik betreffen und z. T. bereits vorher Beschriebenes wiederholen. Hier, wie auch bei manchen der vorangegangenen Beiträge, wäre die Beschränkung auf Wesentliches sicherlich der Lesbarkeit zugute gekommen. Der Band wird von einem Autorenverzeichnis (172 Namen!) und einem 5 1 /2 s e 'tigen Sachregister beschlossen. P . DÖBEL

Acta Biotechnol. 8 (1988) 2, 1 7 7 - 1 8 7

Enzyme Overproduction: Principle and Application Tripathi,

G.

Department of Zoology Bañaras Hindu University Varanasi-221005, India

Summary The rapprochement between gene physiology and protein chemistry proffered a wishful manipulation or programming of biological blueprint which we now know as recombinant DNA technology. I t s premises are very many and ends are manifold. Until rather recently, the recombinant DNA technique could conceive the idea of engineering enzyme molecules by cloning and selection of the gene in question for enzyme production. At least, in principle, the process is too simple but its underlying mechanism is rather much stringent. Various experimental paradigms have been brought to work for production of enzyme at will by introducing a given gene into a high yielding system of microorganisms. I t facilitates overproduction of enzymes of interest which can be implicated in several important industrial, biomedical, and environmental processes a t a large scale. Such approaches of enzymes made-to-application have already started asserting tremendously in doing their appropriate jobs a t the level of molecular interactions. A rapid progress in this important and interesting area of biocatalytic manipulation will certainly achieve the goal of biocatalysis-made-to-order by altering kinetic and thermodynamic components of enzyme molecules.

Introduction Consistently immense efforts of molecular biologists deciphered a concentric knowledge about the functional integration between DNA, RNA, and protein. Precisely, DNA is the fundamental molecule of life and serves as the repository of genetic information. The stored message in DNA is transcribed as messanger RNA which is subsequently translated into a specific protein. The final protein molecules impart in structural and/or functional design of biological machinery. Essentially some proteins have to speed up the chemical reactions by lowering the activation energy required for molecular interactions. They are well known as enzymes. These catalytic proteins constitute the metabolism of a living cell. Currently, certain antibodies have been reported to be catalytic [1, 2], In addition to protein enzymes, it has been well documented that RNA also acts as an enzyme [3]. Day-to-day valuable investments in biocatalysis and rapid progress in gene cloning technique is substantially trying to couple the genetic engineering and enzyme technology for the advancements of each other. Specially it is a remarkable stride in enzyme technology for overproduction as well as redesigning the enzyme molecules. It reflects 5

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one of the masterplans of modern biology to approach biocatalysis a t the level of the genes. As a result the biocatalysis can be made to order [4]. These added significances approve the importance of a compendious view on the molecular physiology of gene cloning and enzyme overproduction. The information on this aspect is very limited rather much scarce and disjointed. Hence a trickle of reports have been discussed on the basis of past ideas and present approaches for future developments. Concept of Molecular Breeding Gene cloning follows the principle of insertion of a desired gene into the genome of an appropriate host through transport vehicles. The gene may either contain the complete genetic information or external control elements along with the original coding sequences. The host cell is transfected by cloning vehicle followed by transformation, growth, and division of the cell. Concomitantly the multiplication and amplification of the vector occur, as a result, the foreign gene product is synthesized in the host cell in abundance. The vectors may be plasmids, cosmids, or bacteriophages. Plasmid is the most commonly used vector and may be defined as any genetic element which is supplemental to the normal genome of the cell [5]. The expression of cloned gene is mainly dependent on both the type of vector as well as the host cell. The vector should be stable and the host cell should have capacity to reproduce up to several generations. The transcriptional, translational, and post-translational events further influence the expression of the foreign gene in prokaryotic and eukaryotic cells. The accurate gene expression requires transcriptional start signals and transcriptional stop signals. They are promoters and terminators respectively. I n an eukaryotic gene the promoter acts as the recognition site for RNA polymerase and the terminator appears to stabilize the transcribed messenger RNA [6]. The promoter seems to consists of the TATA box and an enhancer sequence which are needed for maximal transcription initiation [7]. The translational signals are characterized by ribosomal binding sites. I t is closely linked to the promoter region in eukaryotes while lies on the mRNA (messenger RNA) representing the Shine-Dalgarno region in prokaryotes as translational initiating site. The differences in nucleotide sequence among different translation initiation regions account for variation in the efficiency of mRNA [8]. I n numerous cases the translational product must be processed to produce an active form of the protein. This posttranslational processing occurs through the cleavage of the leader sequence which has been exemplified in the yeast system (Saccharomyces cerevisiae) comprising appropriate gene encoding the protein of bacterial, plant, or animal systems [8].

Physiology of Enzyme Overproduction Since, in 1977, it was discovered that the genes of higher organisms are split into segments. I t is unlike the case in Escherichia coli where genes are continuous not the split one. Certainly it is not the essential occurrence. Even some eukaryotic organisms do not possess split genes and certain microorganisms have the split genes. Generally the protein coding regions of eukaryotic genes and sometimes prokaryotic genes comprise exons (coding regions) interrupted b y introns (noncoding gaps). Introns allow the recombination of exons [9] and coding as well as noncoding sequences are transcribed completely which results the formation of a precursor m R N A molecule. The latter is stripped off its introns and permits end to end reassembling of exons to give an mRNA. The entire event is known as RNA processing which does not occur in baterial system. The RNA after splicing contains only those nucleotide sequence which can encode the

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final protein molecule. This excision of intronic sequences seems to be organism-specific [8]. The whole process from gene to its product can be influenced by gene cloning technique. Subsequently a desired amount of protein product can be obtained within biological economy and precise molecular accuracy from the host cells. Such gene cloning technique has been employed to produce various types of structural and functional protein molecules. But the cloning of genes encoding enzymes is of paramount importance to biocatalytic processes. The last ten years of appreciable investments in gene cloning of enzymes revealed the involvements of several donor and host systems comprising organisms from bacteria to mammals (Tab. 1). But the reports on overproduction of enzymes (Tab. 2) are much scarce and fragmentary. Most of the relevant and technical advancements in this concern could be made during the last six years. As a result it has been shown that very high level of enzyme production can be achieved when the related genetic information is introduced and maintained in bacterial, plant, and mammalian systems [8, 33]. Only few recently reported examples of enzyme overproduction are discussed below for detail accounts. Tab. 1. Cloning of genes for enzymes in some prokaryotic as well as eukaryotic systems Gene for the enzyme

Donor

Host

Reference

Adenine phosphoribosyl transferase Alkaline phosphatase a-Amylase

Cricetulus larabensis Homo sapiens Escherichia cóli M. musculus

[10] [10] [11] [12]

Catabolic dehydroquinase Chloramphenicol acetyltransferase Citrate synthase Dihydrofolate reductase DNA polymerase Endonuclease Eco R I /S-Galactosidase Glucoamylase ^-Glucosidase Glutamate dehydrogenase Kanamycin nucleotidyltransferase Thymidine kinase

Neurospora crassa E. coli

Mus musculus Mus musculus E. coli Saccharomyces cerevisiae N. crassa Nicotiana tàbacum E. coli E. cali S. cerevisiae

>> 33 33 33 33

8. diastaticus Kluyveromyces fragilis N. crassa Staphylococcus aureus Gallus domesticus

33 33 33 39

N. crassa Bacillus stearothermophilus M. musculus

[13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24]

Citrate Synthase: It is a rate-controlling enzyme which catalyzes a crucial step at the branchpoint of oxidative, lipogenic, and anaplerotic pathways in Escherichia coli. The recombinant DNA technology can be used to alter such rate-controlling steps in biochemical pathways. The bacterial strain DEK15 (a derivative of 23559) and the plasmids pWK401 and pWK452 are constructed to study the expression of citrate synthase gene. DEK 15 is formed by transduction of mutant citrate synthase allele (from E. coli W620) into strain 23559 following contransduction with TnlO. Insertion of a 3.1kb Hind IIIEcoRI git A fragment from pLC26—17 into the same site in pBR322 results the formation of the plasmid pWK401. However, the plasmid pWK452 is constructed by insertion of 250-bp Bam HI fragment containing tac promoter [34] in pWK401. The plasmid pWK401 has structural gene for citrate synthase under control of its own promoter, while pWK452 contains the incorporated tac promoter. RecA - derivative of wild-type strain containing pWK452 plasmid shows 2.0—2.5 fold overproduction of the enzyme 5*

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Acta Biotechnol. 8 (1988) 2 Tab. 2. Level of enzyme production by molecular cloning of the related genes Enzyme

Productive level (% cell protein or fold)

Reference

Invertase Chloramphenicol acetyltransferase Citrate synthase Exonuclease III jS-Lactamase DNA ligase Thymidine kinase Dihydrofolate reductase Glucoamylase Tyrosyl tRNA synthetase

2-fold 2-10-fold

[25] [26]

2-50-fold 125-fold 400-fold 500-fold 1%

[15] [27] [28] [29] [30]

25-30% 50%

[31] [32]

6%

[16]

citrate synthase. Whereas, E. coli harbored with the plasmid pWK401 represents 50-fold overproduction of the enzyme. This overproduction can be varied from 10% to 5000% in wild-type E. coli [15]. Thymidine kinase: Foreign gene products expressed in prokaryotes are generally not expected to have post-transcriptional and post-translational modifications as occurs in eukaryotes. Sometimes these modifications are essential for activation of the protein products. If a cloned gene can be expressed at high levels in an eukaryotic system the functional product may be produced in abundance. Hence, the experiments have been conducted to introduce the thymidine kinase gene of herpes simplex virus (HSV) type I into an adenovirus vector system and thus the resulting hybrid viruses carrying thymidine kinase (TK) gene when infected to human or monkey cells produce a very high level of the enzyme product [30]. Construction of the hybrid adenovirus (Fig. 1) is performed by digestion of DNAs of the plasmids (pmY591, or pmY599) and the adenovirus 2/5 recombinant (1 X51) with Bam H I following ligation with T4 DNA ligase. Then it is mixed with the adenoviral 1 x 5 1 DNA to produce hybrid viruses containing SV40 DNA. At first the TK gene is sandwiched between adenovirus and SV40 DNAs to form pmY591, and the TK fragment in this plasmid represents the entire continuous 1128-nucleotide sequence [35]. The TK segment also possesses 57 nucleotides of 5' untranslated leader sequence but does not have TK promoter and the proximal 50nucleotides of the 5' untranslated leader [36], This TK segment also comprises the polyadenylation signal which seems to be 60—90 nucleotides downstream from the termination codon. Infection of human or monkey cells with these hybrid viruses carrying TK gene produce very high levels of TK protein showing 10% of the newly synthesized protein in late infected cells, and this protein is accumulated to represent 1% of the total cell protein under optimal conditions [30]. Consequently, it has also been suggested that this vector system may be used in a procedure by which a variety of gene products (biologically active and properly modified) could be produced at high levels in mammalian cells [30]. Nitrogenase: The nitrogenase enzyme complex is involved in biological nitrogen fixation. In addition, the nitrogen fixation process also requires ATP and a strong reductant [37]. The bacterium, Klebsiella -pneumoniae has 15 linked nitrogen fixation (nif) genes. Three nif genes (nif H, nif D, nif K) are conserved in the journey of evolutionary history that cloned K. pneumoniae nif HDK DNA [38], The nif HDK DNA hybridize to the DNA sequences of every nitrogen fixing bacterium including Rhodopseudomonas capsulata [39]. R. capsulata is a purple, non-sulphur bacterium which is reported to contain multiple copies of the genes for nitrogenase components, and some of the extra nif gene

181

TRIPATHI, G., Enzyme Overproduction Aderto

TK

ATO

SV

TGA

ATO

iO-T

TA A pmYS91

Bom HI IBollì Bglïï Eco RI(HpoH) (M-SI IADENOVIRUS-TK-SV 40 FRAGMENT)

Bom HI

J

Adenovirus Bom HI

(29}

R R L J T

1

Bom HI

I Precursor 1

NS\\
n

IN VIVO DNA

r

vector

(59.5)

} P

frogment

AdTK

SVR591

Number inside indicotes

mop units

Promoter Bom HI site of pBR 322

1

> Direction of transcription

n mu

Segments

of tripartite leader

Fig. 1. Construction of a hybrid adenovirus containing thymidine kinase (TK) gene of herpes simplex virus (HSV) type I [30]

sequences can be functionally activated [40] for high levels of enzyme production. The attempt has been undertaken using cloned K. pneumoniae nif H D K DNA; and the plasmids, p 103 and p 108, have been constructed which contain 11.8-kbp Hind I I I and 4.9-kbp Hind I I I fragments respectively [40]. 11.8-kbp Hind I I I fragment from R. capsulata comprises the functional nif H D K operon and known as copy I. These two Hind I I I fragments are subcloned into mobilizable vector pRK292 [41]. At this step, the site-specific mutagenesis of R. capsulata gene is performed to study the functional properties of the cloned nif genes [40]. Briefly, a 2.4-kbp xho I fragment (the cartridge) bearing the gene for kanamycin resistance Km (expresses in R. capsulata) is ligated in vitro in a cloned target fragment and the constructed plasmid transforms E. coli and then it is mobilized into a gene transfer agent (GTA) — overproducing strain of R. capsulata. The Kmr cartridge is inserted into the nif H gene of copy I. Using these techniques, one insertion and two deletions are produced in copy I, and the physical map of copy I nif H D K region in different strains of R. capsulata is shown in Fig. 2. The strains having KmT marker insertion are Nif"; and Nif + pseudorevertants which are obtained from both insertion as well as deletion mutations. Now it is clearly evident that nif HDK genes are present in several copies in the genome of R. capsulata. The two important possibilities have been discussed [40] to evaluate the physiological significance of these extra copies of nif genes — the extra copies are pseudogenes, and the extra copies are actually silent but a controlled gene rearrangement can bring genes or segment of genes to the copy I region encoding nitrogenase which has different properties (i.e., ammonia switch-off, substrate affinity, cofactor requirement and regulation) from the copy I nitrogenase. Protein kinase C: The protein kinase C phosphorylates proteins at serine or threonine residue in the presence of Ca 2+ and membrane phospholipids [42, 43]. Diaeylglycerol (DAG) increases affinity of protein kinase C for Ca2+ and phospholipid which in turn

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Acta Biotechnol. 8 (1988) 2

ScoRISHindm I nifH and most of nifDI fragments in 3.7kbp 7.6 kbp wild - type P.CAPSULATA 11-8 kbp Wild strain

tnifK) 5.0kbp

»

0 * t'-'h T~A T T ^ T h * lEcoRI! iHindHI) (AvalI (SalUlXhol} X A R RH RA S X S H R Kmr cartridge insertion / ' Soli site in the nifH X X H R A S ££ S H ON 51 . f. 750-bp deletion of x ^ x X h o ! n ' fD 306 R H R AR S H R /"\ r 7.0 kbp deletion /Km ^\ between Ava I sites

581003

Kmr - Gene for konomycin resistance from in 5 Fig. 2. Physical map of the copy I nif HDK region in different strains of Rhodopseu-

domonas capsulata [40]

activates the^enzyme at resting intracellular Ca 2+ levels [44], Protein kinase C is the intracellular receptor of phorbol esters [45]. Both DAG and phorbol esters interact with the enzyme at the same site [46] for its activation. The rat brain protein kinase C contains a single polypeptide chain which gives two fragments ( ~ 3 2 and 51 Kd) after proteolysis [42], The fragment of 51 Kd is catalytic domain while the 32 Kd fragment is the regulatory domain of the enzyme. The function of former fragment is independent of phospholipid, Ca 2+ , or DAG whereas the latter binds phorbol esters or DAG in a phospholipid — and Ca 2+ -dependent manner. Subsequently, three different protein kinase C related cDNA clones (PKC-I, PKC-II, PKCI I I ) have been isolated and two (PKC-I and PKC-II) of them have been subcloned into the expression vector PMT-2 to study the expression of protein kinase C activity [47], The cells transfected with either PMT-2PKC-I or PMT-2PKC-II subclones bind at least five times more phorbol ester than cells transfected with PMT-2 alone (control cells). The membrane fraction of cells transfected with PMT-2 PKC-I has 3-fold higher levels of Ca 2+ , phosphatidylserine, and DAG or phorbol ester dependent protein kinase activity than the control cells. Hence, the expression of PKC-I and PKC-II clones in COS cells results several-fold induction of high affinity phorbol ester binding [47]. Other enzymes: Genes for several other enzymes have also been cloned to achieve their high-level production. The E. coli bacterium expresses tyrosyl tRNA synthetase (Tyr TS) gene of Bacillus stearothermophilus as 50% of the total soluble proteins in the cell [32]. Glucoamylase gene from Aspergillus awamori species synthesizes 25—30% of the total supernatant proteins as glucoamylase in the yeast, Saccharomyces cerevisiae [31]. The glycosylated secreated (S) and non-glycosylated cytoplasmic (C) forms of invertase are synthesized by separate transcripts from SUC gene is S. cerevisiae. Certain overproducing mutants have been constructed which can overproduce S as well as C forms of invertase [25]. The resolvase gene (tnp R) of yd transposon, when cloned into pA8, expresses 2—3% of the total cell protein as yd resolvase in bacterial cell [48]. Recombinant plasmids, pCV29 and pCV32, containing E. coli D H F R gene encode 6% of the total soluble

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bacterial protein [16]. I t has been reported that certain polynucleotides are able to form Z-DNA structure (Z-conformer) which can be used to stimulate the expression of a cloned gene. Some potential Z-conformers have been shown to stimulate the expression of bacterial Chloramphenicol acetyltransferase (CAT) gene up to 2—10-fold in mouse cells [26].

Gene Dosage Effect and Temperature-Induced Replication The high copy number of vectors per host cell impact gene dosage effect causing overproduction of protein products. An increased dosage of a given gene and its efficient transcription largely influence a high-level enzyme production. For instance, cloning of the positive regulator gene (pho B) for alkaline phosphatase of E. coli into pBR322 vector represents multiple copies of pho B gene in host cell. As a result a large quantity of alkaline phosphatase is produced [11]. In certain cases, temperature also induces the formation of multiple copies of a gene. 125-fold higher exonuclease I I I is obtained in bacterial cells when the plasmid pKC16 carrying E. coli exonuclease I I I gene is exposed to higher temperature [27], It is due to temperature induced increase in copy number of the gene. Similarly, 400-fold production of /9-lactamase can be obtained when the plasmid pKN410 possessing E. coli -lactamase gene is exposed to higher temperature forming multiple copies of the gene [28]. Such temperature induced enzyme overproductions are due to 'runaway replication'.

Transposon as Vector Now-a-days, plasmids, cosmids, and bacteriophages are used as vectors to increase the production of enzymes in genetic engineering processes. I n addition, transposons also function as a broad host range vectors. Transposons can be inserted into plasmids or bacterial genomes and this insertion process is independent of host cell recombination. I t permits the stable insertion of a desirable gene into the genome of other life form, and certainly does not provide any extra-genomic load on the host organism. As a result, transposons as broad host range vectors may be used in many genetic engineering techniques now applicable in industrial processes due to their extra advantages over other vectors [49]. Further it is believed that transposons, in near future, may become a powerful tool for genetic manipulations [50].

Perspectives and Accomplishments I t is the only mid 1970s when the term biotechnology gained popularity as a contraction of biological technology. I n the last seven years various definitions of biotechnology came into existence, and the most appropriate one seems to be the application of scientific and engineering principles to the processing of materials by biological agents to provide goods and services [51]. Agents include a wide range of biological substances, such as enzymes, as well as whole cells or multicellular organisms and mentioning services as well as goods cover processes such as waste and water treatment. Now enzymes at work have broaden their scope in almost all major sectors of biotechnological avenues comprising gene splicing, hybridoma production, agricultural and biological enterprises, protein and microbial engineerings etc. This is how the classical enzymology invaded the world of slicing and splicing. As a result many of the promises

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Acta Biotechnol. 8 (1988) 2

of today's biotechnology are trying to be fulfilled with the help of day-to-day advancing technical knowledge and know-hows. Meanwhile the increasing and wider use of enzymes invited DNA recombinant technique to come and solve the problem wisely and easily. Only in the recent past this powerful probe for gene manipulation strengthened the skill of our bioengineerings to make newer approaches to produce further biotechnological marvels as well as to rectify the existing processes at a large. Certain useful approaches are concisely presented as follows: Industrial Assignments: A number of biochemical processes in chemical, pharmaceutical, and food industries are assigned to enzymes for production as well as processing of many needful compounds to meet the present demand. A set of multibillion Dollar industry is established in the concern involving nearly 150 companies all over the world. Enzymatic production of L-amino acids and 6-aminopenicillanic acid (6-APA) are the well known examples of chemical and pharmaceutical industries respectively. The former comprises the synthesis of L-tyrosine, L-tryptophan, L-citrulline, L-ornithine, L-aspartic acid, L-glutamic acid and so on. While the latter includes the industrial manufacturing of semisynthetic penicillin as well as some other novel penicillins. One of the greatest commercial success of biocatalysis is concerned with food technology. It prevails the production of high-fructose corn syrup (HFCS) by enzymatic hydrolysis of corn starch. Consequently, the current annual production of organic chemicals is estimated to be around 100 million metric tons, many of these, could be produced by microbial fermentation with or without chemical processing [52], Unlike biological systems, the chemical reactions in enzyme reactors are not reasonably optimum and coordinated. In such cases, generally there occurs a single rate-limiting step which controls the reaction system as a whole. This rate limitation is imposed oftenly in ease of a complex reaction sequence (e.g., amino acids and antibiotics syntheses) and hampers the productivity which may be improved either by genetically allevating the rate-limiting step in the cell or increasing the cell concentration [53], On the otherway the DNA recombinant technique could be applied to increase the production of rate limiting enzymes and alter the metabolic regulatory schemes in order to maximize the flow of raw materials towards the formation of desired products and simultaneously keep away from the formation of unwanted by-products [53]. Analytical Implications: Enzymes have unique substrate specificity thereby have got the property to act on one substrate out of many. This fundamental property of enzyme has given a concept for an easy and quick detection of a particular compound in a fluid comprising several chemical substances. A method is already in practice for grafting enzymes on solid support like glass rods etc. and the designs are commonly referred to as biosensors or enzyme electrodes. I t facilitates the measurement of a desired component in a complex substance as blood samples, waste streams, and so forth. I t involves the determination of a particular chemical just by putting an enzyme electrode in sample medium and reading the concentration of the compound in question. Generally one type of enzyme electrode can be used for determination of only one chemical compound. Few coupled enzyme electrodes have also been constructed for the measurement of more than one compound. There are limited number of enzyme electrodes that to a very narrow range of species i.e. H + , 0 2 , NH 4 + , C0 2 , glucose, L-aminoacids, and so on. Any how its principle has a general applicability, and has already been used to assay a wide variety of diagonistically, environmentally, or otherwise important compounds including urea, sugar, cholesterol, phenol etc. [54], At least, in principle, a given compound can be determined by a biosensor composed of an immobilized enzyme and an electrochemical devise to sense a chemical species. Biomedical Importances: Medical importance of certain enzymes have been reported in detection as well as rectification of diseases. Monitoring of the level of lactate dehydro-

TRIFATHI,

G., Enzyme Overproduction

185

genase (LDH) in blood of patients having cancer or liver diseases are clearly evident in diagnosis and prognosis. The diseases caused by defects in enzyme systems can be substituted to normalize the metabolic set up of body. As a result the disease may be cured. I t can be performed either by administering enzymes or immobilized enzymes into the biological system or by applying enzymes in many extracorporeal devices [55] and constitute the branch enzyme therapy. Directly or indirectly there are three prominent techniques — organ transplantation, substitution of exogenous enzymes, administration of genes to replace missing enzymes — generally employed for enzyme therapy. The treatment of a-galactosidase A deficiency ( F A B R Y ' S disease) and GATJCHER'S disease has been attempted unsuccessful by kidney and spleen transplantation respectively. Although the approach of enzyme replacement therapy by administration of genes is theoretically relevant but may impose several major hurdles in implementation. I t is only the administration of exogenous enzymes [56] which bears some potential applications. I t is largely based on the concept of semipermeable microcapsules [57] which are used for enzyme administration. The idea of nutritional deprivation leading to death of tumour cells is being used for treatment of cancer. Circulatory level of asparagine can be depleted by exogenous administration of asparaginase which in turn inhibit the growth and survival of sensitive tumour cells in lymphocytic leukemia. Mutations in certain genes coding lysosomal enzymes or proteins cause lysosomal storage diseases as genetic diseases which may be treated by administering entrapped enzymes that can degrade the accumulated substances in lysosomes. Some in vitro studies are under progress employing fibroblast cells and by using /^-hexosaminidase A, «-galactosidase, glucocerebrosidase etc. Similarly clotting disorders (hemophilia A and hemophilia B) are the genetic defects, in which, the fibrinolytic system is activated resulting the dissolution of hemostic plug. Certain enzymes are also being experimented to tackle this intricate problem as well as some other problems including the treatment of neonatal jaundice and poisoning. In Europe, uricase enzyme is used to treat the toxicity caused by high level of uric acid in body so also the enzyme carboxypeptidase G is applied to reverse the effect of methotrexate. Further, the enzyme therapy seems to be equally important in surgery to avoid the blood clotting while transplanting the kidney and heart as well as other cases of open surgeries. In condern a blood filter containing immobilized heparinase enzyme is used in pump-oxygenator to remove the heparin from blood which is administered alongwith heparinized blood during transplantation. Environmental Detoxifications: The rapid growth of industrial enterprises are regularly disposing a good number of non-biodegradable compounds in human environment. Its long-term fate and toxicological properties cause risks and hazards to the biosphere in general. In the last eight years appreciable investments have been designed to look forward for environmental protection by genetic engineering of enzymes. Consequently, a rapid evolution of catabolic pathways is eagerly desired to prevent the accumulation of toxic chemical in the environment. I t could be performed in two important ways [58]Enzyme overproduction through a gene dosage effect, inactivation or modification in the stringent control of the regulatory genes involved, or enzyme production having altered specificity by mutational divergence. Derivation of novel enzyme activities from heterologous or pre-existing genes for related enzymes by gene-recruitment or genetic rearrangement etc. The former may be applicable if wastes are to some extent chemically analogous to the natural substrates, while the latter is applicable to the non-analogous wastes as substrates. Ultimately the use of genetically engineered microorganisms in environment seems to be of a great importance in metabolizing toxic compounds. Several degradative

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genes have been cloned in microorganisms in the view of environmental detoxifications. B u t their field applications are much restricted and need permission from the Recombinant D N A Advisory Committee (RAC).

Acknowledgements Financial supports from Council of Scientific and Industrial Research, New Delhi, India is gratefully acknowledged. The author thanks Dr. T. GRODZICKER (Cold Spring Harbor Laboratory, New York) and MACMILLAN Journals Ltd., London, for granting permission to reproduce Fig. 1 and 2 (given in the present article) from Proc. Natl. Acad. Sei. USA 8 2 : 3 5 6 7 - 3 5 7 1 ( 1 9 8 5 ) and Nature 3 0 7 : 2 8 9 - 2 9 2 ( 1 9 8 4 ) respectively. Received April 20, 1987

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G.: Curr. Genet. 8 ( 1 9 8 4 ) , 333. BARNES, G., RIÑE, J . : Proc. Nat. Acad. Sei. USA 82 (1985), 1354. P A N T H I E R , J . J . , F O U R N I E R , P . , H E S L O T , H . , RAMBACH, A . : Curr. Genet. YAMASHITA, I., FUKUI, S.: Agrie. Biol. Chem. 47 (1983), 2689. SPANOS, A . , S E D G W I C K S , S .

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RAYNAL, A . , GUERINEAU, M . : M o l . G e n . G e n . 1 9 5 ( 1 9 8 4 ) , 1 0 8 .

Gen. Mol. Cell. Biol. 4 ( 1 9 8 4 ) , 1 1 7 . Proc. Nat. Acad. Sei. USA 8 3 ( 1 9 8 6 ) , 5 7 6 . W I G L E R , M . , P E L L I C E R , A . , S I L V E R S T E I N , S . , A X E L , R . : Cell 14 ( 1 9 7 8 ) , 7 2 5 . P E R L M A N , D . , R A N E Y , P . , HALVORSON, H . 0 . : Proc. Nat. Acad. Sei. USA 8 3 ( 1 9 8 6 ) , 5033. B A N E R J E E , R., G R U N B E R G E R , D . : Proc. Nat. Acad. Sei. USA 8 3 ( 1 9 8 6 ) , 4 9 8 8 . R A O , R . N., R O G E R S , S. G.: Gene 3 ( 1 9 7 8 ) , 2 4 7 . U H L I N , B . E., MOLIN, S., GUSTAFSSON, P., NORDSTRÖM, K . : Gene 6 (1979), 91. K I N S E Y , J . A., RAMBOSEK, J . A . :

L I A O , H . , MC K E N Z I E , T . , HAGEMAN, R . :

PANASENKO, S . M . , CAMERON, J . R . , D A V I S , R . W . , LEHMAN, I . R . : S c i e n c e 1 9 6 ( 1 9 7 7 ) , YAMADA, M . , L E W I S ,

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K . W . K . , GELFAND, D . H . , HOLLAND, J . P . , MEADE, J . H . : S c i e n c e 2 2 8 ( 1 9 8 5 ) , 2 1 .

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[ 3 2 ] WINTER, G . , FERSHT, A. R . , WILKINSON, A . J . , ZOLLER, M . , SMITH, M . : N a t u r e 2 9 9 (1982), 156.

[33] HARRIS, T. J . R . : Expression of eukaryotic genes in E.coli. (Ed. R. WILLIAMSON). Academic Press, London 1983, 127.

I n : „Genetic Engineering 4 "

[ 3 4 ] D E BOER, H . A . , COMSTOCK, L . J . , VASSER, M . : P r o c . N a t . A c a d . Sei. U S A 8 0 ( 1 9 8 3 ) , 2 1 .

[35] WAGNER, M. J . , SHARP, J . A., SUMMERS, W . C.: Proc. N a t . Acad. Sei. U S A 78 (1981), 1441. [36] [37] [38] [39] [40] [41]

MC KNIGHT, S. L . : Cell 3 1 (1982), 3 5 5 . MORTENSON, L . E . , THORNELEY, N . F . : A n n . R e v . B i o c h e m . 4 8 (1979), 3 8 7 . CANNON, F . C., RIEDEL, G . E . , AUSUBEL, F . M . : M o l . G e n . G e n . 1 7 4 (1979), 59. RUVKUN, G . B . , AUSUBEL, F . M . : P r o c . N a t . A c a d . Sei. U S A 7 7 (1980), 191. SCOLNIK, P . A . , HASELKORN, R . : N a t u r e 3 0 7 (1984), 2 8 9 . DITT, G . , STANFIELD, S., CORBIN, D . , HELINSKI, D . R . : P r o c . N a t . A c a d . Sei. U S A 77 ( 1 9 8 0 ) ,

7347. [42] INOUE, M., KISHIMOTO, A., TAKAI, Y., NISHIZUKA, Y . : J . Biol. Chem. 252 (1977), 7610.

[43] NISHIZUKA, Y.: Nature 308 (1984), 693. [ 4 4 ] KISHIMOTO, A . , TAKAI, Y . , MORI, T . , KIKKAWA, U . , NISHIZUKA, Y . : J . B i o l . C h e m . 2 5 5 (1980),

2273. [ 4 5 ] NISHIZUKA, Y . : S c i e n c e 2 2 5 (1984), 1365. [ 4 6 ] SHARKEY, N . A . , LEACH, K . L . , BLUMBERG, P . M . : P r o c . N a t . A c a d . Sei. U S A 8 1 ( 1 9 8 4 ) , 6 0 7 . [ 4 7 ] KNOPF, J . L . , L E E , M . , SULTZMAN, L . A . , KRIZ, R . W . , LOOMIS, C. R . , HEWICK, R . M . , BELL,

R . M.: Cell 46 (1986), 491.

[48] REED, R . : The resolvase protein of the transposon. I n : „Methods in Enzymology" (Ed. R. W u , L. GROSSMAN, K . MOLDAVE). Vol. 100 (Part B). Academic Press, New York 1983,191. [49] OLD, R. W., PRIMROSE, S. B.: Principles of Gene Manipulation: An Introduction to Genetic Engineering. Blackwell Scientific Publications, Oxford 1985, 155. [ 5 0 ] TRIPATHI, G . , SHUKLA, S. P . : J . Sei. I n d . R e s . 4 5 (1986), 2 1 .

[51] BULL, A. T., HOLT, G., LILLY, M. D.: Biotechnology. International Trends and Perspectives. Organization for economic Co-operation and Development. (1982). [ 5 2 ] ABELSON, P . H . : S c i e n c e 2 1 9 (1983), 6 1 1 .

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[57] CHANG, T. M. S.: Science 146 (1964), 524. [58] GHOSAL, D., Y o u , I.-S., CHATTERJEE, D. K . , CHAKRABARTY, A. M.: Science 228 (1985), 135.

Acta Biotechnol. 8 (1988) 2, 188

Book Review L I M , H . C. ; K .

VENKATASUMBRAMANIAN

Biochemical Engineering IV Annals of the New York Academy oï Sciences; Vol. 469 New York: The New York Academy of Sciences, 1986. 448 pages, 112 S

Dieser Sammelband enthält 38 Vorträge, die auf der 4. Internationalen Konferenz über biochemisches Engineering im Oktober 1984 in Galway, Irland, gehalten wurden. Der Band beinhaltet Arbeiten zu weitgefächerten Themenkreisen auf dem sich ständig ausbreitenden Gebiet der Biotechnologie. Dazu gehören Beiträge zur Synthese, zum Cloning und zur Expression von Genen biologisch wichtiger Moleküle. Das sind: erstens extracelluläre Enzyme von und durch Bacillii, zweitens Conalbumin- und Ovalbuminexpression in E. coli und abschließend die chemische Herstellung von Genen f ü r insulinähnliche Wachstumsfaktoren (menschliches Somatomedin C und 59 Val-Somatomedin) und deren Expression in E. coli. Weiter folgen Beiträge zur derzeitigen Gestaltung und Durchführung von Bioprozeß-Systemen und zwar zu Analysemethoden zur Bioreaktorsicherheit, zu neuen Seperationsmöglichkeiten f ü r tierische Zellen, zu Scale-up-Fermentation mit rekombinierten Mikroorganismen, zur Dynamik von Colicin-Plasmiden in kontinuierlichen Kulturen, zur Automatisierung von Bioprozessen und zur Stabilität von kontinuierlichen Kulturen mit rekombinanten Organismen. Der 3. Abschnitt stellt ausgesuchte Arbeiten zum Massentransfer und Transport in Bioreaktoren vor. Hier geht es unter anderem um die Nutzung von mikrobiellen Kulturen als Sauerstoffsensoren, um Gelöstsauerstoffmessung unter 100 ppb, lim gekoppelte Membran-elektrophoretische Trennung und um Membranreaktoren. I m Teil Biokinetik und Bioreaktorentwicklung geht es hauptsächlich um Plasmid-Wirt-Wechselwirkungen, Plasmidstabilität, um Modelle der Kinetik von immobilisierten Enzymsystemen und die Anwendung der Elektronen-Paramagnetischen-Resonanz-Spektroskopie zum Studium von immobilisierten Enzym-Systemen. Die folgenden Kapitel gehen auf verschiedene pflanzliche und tierische Zellkulturtechniken, auf die biologische Produktion von Energie zur Optimierung und automatischen Kontrolle von Bioreaktoren, vorrangig bei Fermentationen zur Produktsynthese und zu interessanten Selektionsmöglichkeiten mittels Stress in Chemostaten, ein. Jeder Beitrag ist mit meistens sehr übersichtlichen Abbildungen und einem weiterführenden Literaturverzeichnis versehen worden. Dieser Band kann, wie schon seine Vorgänger, als ein nützlicher Führer für biochemische Ingenieure. Mikrobiologen, Biochemiker, Genetiker und andere auf diesen Gebieten Tätige dienen. E . SPANIER

Acta Biotechnol. 8 (1988) 2, 1 8 9 - 1 9 6

The Influence of Control Parameters of Extraction and Concentration over Oxidation and Degradation of Amino Acids POBEDIMSKI, D . G . , PEVZNER, I . L . , SADUIKOV, R . A .

Kazan Chemikal Industry Institute, 420015 Kazan, Karl-Marx-Street 68 USSR

Summary The detailed thermal decomposition characteristics of several amino acids in different fase state — dry and moist crystals (Trp, Arg HC1, Lys HC1), water solutions (Trp) are determined. It is found that thermodecomposition of Trp in water solutions is inhibited by Na 2 S 2 0 3 and oxyethylidendiphosphonic acid. The method of finding optimal control parameters (temperature of moist crystals, heating surfaces, drying agent) during amino acid drying on the bases of their thermodecomposition kinetics is given. The recieved results can be used for technolodical optimization of different stages of amino acid extraction and concentration processes.

On different stages of production, amino acid solutions and crystals are subjected to the influence of different physical and chemical factors, as the result of such treatment some amount of product is lost. The dyed products appearing during that process, for example in Trp production [1], give the necessity to bleach the solution, that draw the additional losses. For practical aims (the elaboration of amino acid concentration technology, the optimization of drying and so on) it is necessary to know the kinetic parameters of amino acid decomposition reactions. The questions concerning thermal stability of dry amino acid crystals in the atmosphere of air in literature are practically not elaborated. The information about melting points of natural amino acids was published in reference books, e.g. [2], P. G. O L A F S S O N and A. M. B B Y A N published the results of thermal analysis of 19 L — amino acids in atmosphere of N 2 (differential scanning calorimeter DSC — I B „Perkin Elmer" USA) [3—7]. The kinetic parameters recieved from DSC and TG curves were reported in works [5—8]. These results showed that melting point characterizes not the beginning of thermodecomposition, but the temperature of maximal reaction rate and that the difference between the beginning and maximum of reaction may give 30—40 °C and is dependent on the conditions of experiment. But it is difficult to use quoted works for technological calculations because there is no information concerning the dynamics of thermooxidation and sublimation processes. I n the previous works it was shown, that several amino acids (Thr, Trp, Tyr and others) [9, 10] in dry state are thermooxidized during heating on air, in these works the attempt to use obtained results for evaluation of guarantied thermostability time, which is important for drying, was undertaken. I t was difficult to treat DTA and DTG curves using the method [6—8] because of the several processes superposition during exothermal oxidation reaction.

190

Acta Biotechnol. 8 (1988) 2

As for moist crystals, there is information only about the influence of temperature on drying of cultural media containing Trp [11]. I t is reported in the work [12] about the increase of reaction rate between amino acids and sugars in condition of increasing moisture of crystals from 0 to 1 kg/kg. I t shows the necessity of investigation the influence of moisture content and composition of soluvent over amino acid decomposition reaction. At last, the thermal stability of amino acid water solutions is well investigated in alkaline and acidic p H values [13—15]. The authors of work [16] having analysed the decomposition of Ala and Gly in water solution showed the maximum of reaction rate in isoelectric point, but for more complex amino acids (Val, He, Asn) [17, 18] it was not confirmed. Our previous experiments showed that Lys HC1 and Arg HC1 were sufficiently stable during heating over 10 hours at 95 °C in the area of p H 1—13, but the losses of Trp reached 10—25 mass % [19]. The aim of present work is the systematic investigation of thermochemical behaviour of amino acids in solid state (dry and moist crystals) and in water solutions, the identification of main chemical processes and their kinetics, the elaboration of possible ways of amino acid stabilization from chemical destruction. This task was realized on the following objects: Arg HC1, Lys HC1, Trp. The experiments with dry crystals were carried out according to the method which had been used for investigation of thermal polymerization of free Lys [20]. Experimental Methods Purified amino acids were used in the experiments: Trp and Lys HC1 — produced in USSR, Arg HC1 — in Hungary ("Reanal"). An automatic amino acids analyzer AAA-881 (CSSR) was used for the determination of amino acid concentration on the shot (7X60 mm) Ostion LG — KS08—03 column with buffer at 5.28 (reproducibility of amino acid determination ± 2 % ) . Dry Crystals: The 10 mg portion of dry amino acid (moisture content — 0.005 kg/kg) were situated in glass vessels (15 X 50 mm) and endured in air thermostat ( ¿ 1 °C) for 0.5, 2, 4, 8 hours. Then samples were dissolved in 500 ml H 2 0 and introduced in analyser. Moist Crystals: The 50 mg portion of dry amino acid were mixed in hermetic vessels with water or ethanol — water mixture and endured for 48 hours at 10 °C to receive the homogeneous moisture content. Then vessels with crystals were endured in water thermostat (±0.5°C). After 5 and 10 hours the samples were taken out, dryed under vacuum, dessolved in 2 500 ml water and introduced in analyzer. Water Solutions of Trp: Water solution of Trp (concentration 5 • 10~3 M) were poured in glass vessels (volume —15 ml), closed hermeticaly and endured in water thermostat ( i 0 . 5 o C ) . The samples were taken out every 60 min. when the determination of the reaction order was carried out and after 20 hours in others experiments. After endurance the content of the vessels was dryed under vacuum dissolved in 10 ml of water and after dilution introduced in analyzer. p H measurements were carried out at 20 ± 2°C by means of ionomer EV — 74 (USSR) (reproducibility ¿ 0 . 0 5 pH). The essential p H value was prepared with solutions of NaOH and HC1 with precision 0.05 pH.

POBEDIMSKI,

D. G.,

PEVZNBR,

I. L. et al., Influence of Control Parameters

191

Results and Discussion 1. The kinetics of amino acids thermal decomposition. Dry crystals: The results of our investigations have shown, that all examined amino acids in dry state are thermooxidized during heating in atmosphere of air, though Arg HC1, according to the results of the work 10 should melt with decomposition in the interval of temperature 205—230 °C. The curves of amino acid losses are satisfactory described by kinetic equation for zero — order reactions. The curves of Lys HC1 mass lossing at 210—220°C have unclear nature: they are characterized by stepped changes of mass loss rate, for example, at 220 °C after 2 hours of endurance the samples contain 90.5 i 6 mass % Lys HC1, but after 4 hours only 3 ± 1 mass %. I t was described the thermal polymerization of free Lys at 195 °C (atmosphere of N 2 ), with convertion 100% in 1 hour [20]. The extraordinary behaviour of Lys HC1 during heating is explained, perhaps, by stepped course of thermal oxidation and polymerization processes. The rate constants of thermal oxidation (K) were calculated as: K = —

t

—,

where

Ci, C — initial and final mass concentration of amino acid [mass %]. r — time of endurance [s] The calculated rate constants, activation energies (Ea) and logarithm A [21] are placed in Tab. 1. Tab. 1. The

ARRHENITJS

parameters of dry amino acid crystals thermooxidation

Amino acid

Temperature [°C]

Rate constant [mass %/s]

Ea [kJ/mole]

In A

Arg HC1

180 200 210

(7.6 ± 2.1) 10-4 (3,4 ± 0 . 1 ) 10"3 (6.9 ± 0.6) IO"3

136 ± 22

29 ± 5.5

Lys HC1

200 210 220

(7.6 ± 0.8) 10-4 (2.3 ± 0.2) IO"3 (6.7 ± 0 . 1 ) 10"3

207 ± 15

45.5 ± 3.7

Trp

180 200 220

(9.9 ± 3.3) 10-5 (1.3 ± 0.2) 10-3 (1.4 ± 0.04) 10- 2

229 ± 18

51.6 ± 4.4

Moist Crystals: The thermostability of moist crystals investigations were carried out in conditions, modelling conductive drying. The results of experiments showed, that after 10 hours of endurance at 95 °C the moist crystals (moisture content U = 0.3—1.0 kg/kg) reaches 15—25 mass %. The thermodecomposition reaction has the first order. The rate constant was calculated as: [S-LL

The values of K, Ea and InA are placed in Tab. 2. I t was found t h a t K values are strongly dependent on moisture content and composition of ethanol-water mixture (Fig. 1). I n the case of Lys HC1 and Trp K values are correlated with moisture content, but the

192

Acta Biotechnol. 8 (1988) 2 Tab. 2. The ARRHENIUS parameters of moist amino acid thermodecomposition Amino acid

Arg HC1

Temperature [°C]

R a t e constant, [s-i]

70

(2.8 ± 0.6) 10-« (4.5 ± 0.6) 10" 6 (1.1 ± 0.08) 10- 5

57.9 ± 12.2

7.5 ± 4

(7.6 ± 4.2) 10- 7 (2.05 ± 0.7) 10- 6 (8.3 ± 0.75) lO"6

107.8 ± 22.9

23.6 ± 7.6

(1,0 ± 0,3) 10- 6 (2.0 ± 0.5) 10-« (4.7 ± 0.33) 10- 6

65,0 ± 15

8.9 ± 4.9

80 95 Lys HC1

70

80 95 Trp

70

80 95

Ea [kj/mole]

In A

decomposition rate of Arg HC1 at U = 0.6 kg/kg and U = 1.0 kg/kg has the similar value. That fact can be explained by full dissolving of Arg HC1 during heating at such conditions. When Lys HC1 and Arg HC1 were treated with ethanol — water mixture the reaction rate was greatly decreased in comparison to pure water, but in the case of Trp the reaction rate increased. The dependence of K on U is apparently connected with solulibility of amino acids, for example, the character of curves dependence K on ethanol volume % is similar to the character of amino acid solulibility curves in ethanol — water mixtures: the solubility of Lys HC1 and Arg HC1 dicreases with the increase of ethanol volume in mixture, at the same time it is known, that solubility of Trp in hot ethanol — water mixtures is higher then in pure water at the same temperature. Water Solutions of Trp: The result of experiments showed, that the rate constants were dependent on pH value, i.e. — are effective (seeming) (Fig. 2). The minimum of reaction rate is found to be in the range of pH 1—3 and 12—13, maximum rate is at 10 (the decomposition of Trp during 10 hours at 95 °C makes up — 4 0 % ) . At pH 4—6 rate constant is independent on pH. 12 r

0

40 Ethanol content U 102

80

[kg/kg]

Fig. 1. The dependence of decomposition rate constant on moisture content and composition of ethanol-water mixture ({7 = 1 kg/kg) 1. The dependence K on U: a. Arg HC1, b. Lys HC1, c. Trp 2. The dependence K on ethanol volume % in mixture: a. Arg HC1, b. Lys HC1, c. Trp

POBEDIMSKI, D . G . , PKVZNER, I . L . e t a l . , I n f l u e n c e of C o n t r o l P a r a m e t e r s

«r

193

I

Fig. 2. The rate constants of Trp decomposition reaction in water solution at 95 °C and different p H values (Trp concentration- 5 • 10~3 M)

It was found that oxygen influences thermal decomposition of Trp: the blowing through solution for 10 min by 0 2 increases the reaction rate 1.5—2 times, the analogous operation with helium dicreases reaction rate 1.4—1.5 times as compared with control. The kinetic curve of decomposition show that reaction is of first order. The influence of some synthetic antioxidants, oxyethylidendiphosphonic acid (OEDF) and S — containing salts of Na + on Trp thermodecomposition was examined with the aim to stabilize Trp during heating. From the Tab. 3 it is seen that a little antioxidative abbility is shown by antioxidants 3 and 2 the addition of other antioxidants into the solution catalyses the decomposition reaction. Good antioxidante properties are shown by Na 2 S 2 0 3 in concentration 6.3 • 10 3 — 1 • 10" 3 M, but Na 2 S and Na 2 S0 4 (1 • 10" 2 to 1 • 10~4 M) catalyse the decomposition of Trp. C(CH3)3 I It is clear, that the main antioxidative fragment of 1—4 H O — C H 2 — does not I C(CH3)3 "work" and the effect of stabilization is due to acetic and benzoic acids, tlie appearence in complex propionic acid and dimethylphosphoric acids activates the decomposition process. Therefore, the decomposition of Trp is an oxidative non — radical process. An other phosphorous containing acid- OEDF in concentration 1 • 10" 2 has inhibiting qualities. The addition of typical catalyzer of liquide-fase oxidant — F e S 0 4 (1 • 10" 3 — 1 • 10" 4 M) to the solution increases 1.7—1.8 times K value. The simultaneous presence of OEDF and FeS0 4 in the solution inhibites decomposition on 87.2% in comparison with Trp solution with FeS0 4 only. So the addition in Trp solution Na 2 S 2 0 3 and OEDF in equimolar proportion dicreases decomposition rate 5—6 times in comparison with pure Trp. The Ea and In A of Trp decomposition reaction for pH 1 — 13 is placed in Tab. 4. 2. The selection of concentration and extraction control parameters on the basis of decomposition kinetics. The obtained kinetic parameters of Trp decomposition in water solutions can be used for selection of control parameters of amino acid extraction and concentration and for 6

Acta Biotechnol. 8 (1988) 2

194

Acta Biotechnol. 8 (1988) 2

Tab. 3. The effect of Trp decomposition inhibition and activation in water solution in presence of different additions Substance

Molecular mass

Kkx • 10« [s-1]

Concentration [M]

Stabilization effect AS -^cont -^es 100,

[%]

C(CH3)3 1.

HO——CH 2 -CH 2 -C(0)0K

300

1 • 10- 2 1 • 10~3 1 • 10- 4

6.2 ± 0.6 6 ± 0.6 6 ± 0.6

H O — C H 2 - N ( C H 3 ) 2 H O O C — ^ ^ 384

1 • IO"2 1 • 10"3 1 • 10- 4

3.5 ± 0.3 3.2 ± 0.3 5.7 ± 0.6

27.7 32.5 -10

323

1 • io-2 1 • 10- 3 1 • io- 4

3.3 ± 0.3 3.2 ± 0.3 5.3 ± 0.2

32 32,5 -10

428

1 • io- 2 1 • IO"3 1 • io- 4

20 ± 2 18 ± 1.5 17.7 ± 1-5

C(CH3)3

-29 -25 -25

C(CH3)3 2.

C(CH3)3 C(CH3)3 3.

HO—CH2-N(CH3)2HOOCCH3 C(CH3)3 C(CH3)3

4.

HO—C3 - 'CH2"N(CH 3 ) 2 -.(CH 3 0) 2 P0H C(CH3)3

5.

0 OH 0 H0\|| | ll/OH /P-C-Pv XOH HO I CH3

206

-316 -275 -268

1 • io-2 1 • 10- 3 1 • 10- 4

1.7 ± 0.15 65 5.1 ± 0.5 -6 5.5 ± 0 . 6 -15

78

1 • IO-3 1 • IO-4

10 ± 1 7.4 ± 0.7

158

6.3 • 10- 3 1 •io-3 1 • 10- 4

8. Na 2 S0 4

142

1 • IO"3 1 • 10- 4

5.2 ± 0.5 8.7 ± 0.7

-8 -81

9. F e S 0 4

278

1 • IO-3

8.3 ± 0.7

-73

6. Na,S

1 •

10. F e S 0 4 + OEDF 11. Trp (pH 6, pure)

IO-3

+ 1 • 10-2

206

1 • io- 3

-108 -54

0.6 ± 0.15 88 3.7 ± 0.3 23 7.6 ± 0.7 -56

1 ± 0.15 87.2 (•^cont = 8.3) 4 ± 0.3 (-^cont)

Pobedimski, D. G., Pbvznbr, I. L. et al., Influence of Control Parameters

195

Tab. 4. The Abbhenitts parameters of Trp decomposition in water solution at different pH values

pH 4 5

6 7 9

10

Ea [kJ/mole] 110 103 84.4 62.4 89.5 103

± ± ± ± ± ±

15 16 21 19 17.5 13.8

In A 23 21.4 15.1 7.8 18.3 22.4

± ± ± ± ± ±

4.9 5 7 6.2 4.8 4.2

estimation of losses on these stages. For example, as is seen from Fig. 2, on the stage of ion exchange, which occures at alkali values of pH, the elution of Trp must be carried out a t p H higher than 11. More over, the Ea and In A values are possible to use for determination of Trp losses in injection solutions during durative conservation. The results of kinetic studies of thermodecomposition of crystals can be used for selection of the method and temperature parameters of drying process. The amino acid losses during drying may be as follows: 1. Thermodecomposition of moist crystals 2. Thermooxidation of dry crystals 3. Mechanic losses (carrying away, glueing on the dryer walls and so on). Mechanic losses — is the characteristic of dryer and may be found from their technical characteristics. Thermooxidation of dry crystals may be carried out during the contact of already dry material with high temperature surface or drying agent. The degree of decomposition of moist amino acids is dependent on U, temperature of product and duration of drying. Next formula may be given for determination of amino acid losses (AC) during drying, taking into consideration that the decomposition reaction of moist crystals of the first order and dry crystals — zero order: AC = 100 -

100 exp

-A1

exp (-Ea^RT)

•

((a0 + aU) (b0 + bx) d r )

o

Aj, EaJ, T j — parameters of moist crystals A2, Ea 2, T2 — parameters of dry crystals a 0 , a - T r p : 1.25, ( - 0 . 1 ) ; ArgHCl: 1.09, ( - 0 . 2 ) ; LysHCl: 0.78, ( - 0 . 4 6 ) b0, b - T r p : 1.06, 0.33; ArgHCl: 0.91, ( - 0 . 9 ) ; LysHCl: 0.19, 0.95 x — ethanol content, volume % • 10"2 From the analysis of formula it is seen that during conductive drying (for example in drying case) which is remarkable for a long period of process (near 10 hours) the use of high temperature ( > 80 °C) causes significant losses of product, for example during 10 hours drying of Trp at 95 °C with initial moisture content 0.5 kg/kg, the losses reach 5%. Therefore, to preserve the intensity of drying and quality of Trp it is necessary to use vacuum and temperature 50—60 °C. 6*

196

Acta Biotechnol. 8 (1988) 2

More effective method of amino acid drying is the vacuum — oscillation regime [22], based on the changes of convective and vacuum drying. In this case the use of high temperature drying agent is possible. Having established the admited losses of amino acid (AC ad) one can select the admited temperatures of material and drying agent (T2). For Trp, if AC ad = 0.15 mass %, U = 0.25 kg/kg r = 3600 s, the maximal admitted temperature of moist crystals over the drying process is 64 °C and the temperature of drying agent and heating surfaces may be 150 °C. The analogous calculation may be made for other amino acids. Received F e b r u a r y 2, 1987

References V . G., S T R U C H A I L I N , T . I . , A Y M U H A M E T O V A , T . B . : Amino acids for agriculture, food industry, medicine and scientific recearches. Conference materials, Frunze, 1981. P. 29. The properties of organic compounds. Refference book. Leningrad, Khimiya, 1984. P. 520. OLAFSSON, P. G., BRYAN, A. M.: Anal. L e t t . 2 (1969), 505. O L A F S S O N , P. G . , B R Y A N , A . M . : Polym. Lett. 9 ( 1 9 7 1 ) , 5 2 1 . O L A F S S O N , P . G., B R Y A N , A. M . : Mikrochim. Acta 5 ( 1 9 7 0 ) , 8 7 1 . OLAFSSON, P. G., BRYAN, A. M.: Anal. L e t t . 4 (1971), 596. O L A F S S O N , P . G., B R Y A N , A. M . : Geoch. cosm. A. 3 5 ( 1 9 7 1 ) , 3 2 7 . H U N G , G . W. C.: Thermochim. Acta 2 3 (1978), 233. POBEDIMSKI, D . G . , SADUIKOV, R . A., P E V Z N E R , I . L . : Journal of AllUnion Chemical

[1] OZEREDENKO,

[2] [3] [4] [5]

[6] [7]

[8] [9]

S o c i e t y (1985) 6, 3 9 5 . [ 1 0 ] P O B E D I M S K I , D . G . , SADUTKOV, R . A . , P E V Z N E R , I . L . , I V A N O V , I . U . :

Biotechnology

6 (1985),

596.

[11] D A M B E R G , B. E., S T O L O V A , S H . V.: Prikl. Biokhim. Mikrobiol. 2 (1970), 178. [12] BEKER, M. E . : Drying of microbial biomass and extracellular products of metabolism. Riga, Zinatne, 1967, P. 202. [ 1 3 ] M U R R A Y , K . , R A S M U S S E N , P . , N E U S T E D T E R , J . , L U C K , J . M . : J . Biol. Chem. 2 ( 1 9 6 5 ) 2 4 0 . [ 1 4 ] S T E W A R T , M . , N I C H O L L S , C. H . : Aust. J . Chem. 25 ( 1 9 7 2 ) , 1 5 9 5 . [ 1 5 ] S T E W A R T , M . , N I C H O L L S , C. H . : Aust. J . Chem. 2 5 ( 1 9 7 2 ) , 2 1 3 9 . [16] HARIN, L. V., SHANGAREV, K. P . : Fermentn. Spirt. Prom-st. 7 (1970), 21. [ 1 7 ] MATVEEV, V . E . , SKVORTCOV, G . E . , KUYAN, N . V . : B i o t e c h n o l o g y 1 (1986), 5 3 .

Biotechnology 3 ( 1 9 8 6 ) , 4 3 . I. L., S A D U I K O V , R. A . : Biotechnology of Recycling, International Symposium, Leipzig, 1986. P. 49. [ 2 0 ] M I L T O N , R . H . , D U A N E L , R . , E S T E R , B.: Arch. Biochem. Biophys. 1 3 0 ( 1 9 6 9 ) , 4 4 1 . [21] EMANUEL, N. M., KNORRE, D. G.: Course of chemical kinetics. Moskva, Higher scool, 1984. P. 463. [ 1 8 ] MATVEEV, V . E . , SKVORTCOV, G . E . , K U Y A N , N . V . :

[19] POBEDIMSKI, D . G., PEVZNER,

[ 2 2 ] SADUIKOV, R . A . , MIGUNOV, V. V . , KARPOV, A . M., e t a l . : A c t a B i o t e c h n o l . 5 (1985) 363.

Acta Biotechnol. 8 (1988) 2, 197-205

Characterization of Biotechnological Processes and Products Using High-Performance Liquid Chromatography (HPLC) I. Separations of Carbohydrates, Organic Acids and Lipids GEY,

M.

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

Summary Suitable and optimized chromatographic separation systems for HPLC analyses of mono-, di- and oligomeric carbohydrates, organic acids (e.g. gluconic acid, a-ketoglutaric acid), phospholipids (PE, LPE, LPC, PC, SPH) and neutral lipids (squalene, cholesterol) are demonstrated. Applications of HPLC technique are separation examples of sugars from hydrolyzed starches which were isolated from potatos, calculations of organic acids in fermentation mediums and determinations of neutral lipids and phospholipids which were isolated from microbial biomass. The liquid chromatographic separations are based on self-packed highly efficient (approximately 80000 theoretical plates per meter, N/m) glass columns.

The microbial production of single cell proteins (SCP), syntheses of organic acids for the food industry and isolation procedures of enzymes, lipids and other valuable substances including their chromatographic purification are some applications in the field of modern biotechnology. Analytical investigations of these products and their fermentation procedures involve a great variety of tasks. Chromatographic "high-performance techniques" such as HPLC become more and more important for biotechnology because many different substances can be analyzed and preparatively isolated in short times. In most cases derivatization procedures are not necessary and the substances to be analyzed must only be soluble in a liquid phase. This is the reason why 80% of organic compounds known today can be examined by HPLC [1]. I n this article suitable and optimized chromatographic separation systems for analyses of some biochemical, biological or biotechnological substances of interest such as mono-, di- and oligomeric carbohydrates, organic acids, phospholipids and neutral lipids are demonstrated. The chromatographic separations were based on self-packed highly efficient glass columns [2], Glass becomes more and more important especially for "bioanalytical" investigations because of its inertness. Material and Methods Apparatus: For analytical investigations a high-performance liquid chromatograph ( K N A U E R , F. R. G.) equipped with a 750/04 high-pressure pump, 87.00 UV/VIS monitor, TY recorder, refractive index monitor (Aerograph, VARIAN, U. S. A.), Rheodyne 7010 in-

198

Acta Biotechnol. 8 (1988) 2

jector (RHEODYNE, C A , U . S . A . ) and a 3 3 7 0 B integrator (HEWLETT PACKARD, U . S. A.).

For preparing glass columns a filling apparatus (KNAUER) coupled with another highp r e s s u r e p u m p (CHROMPTON PARKINSON, G. B.) w a s u s e d .

Stationary and Mobile Phases: As stationary phases LiChrosorb R P 18,5 fim; LiChrosorb RP8, 5 /¿m; LiChrosorb NH 2 , 5 nm (E. MERCK, F. R. G.); Silasorb NH 2 , 5 and 7.5 ¡im\ Silasorb 600, 5 fim (CHEMAPOL, Czechoslovakia) and an anion-exchange resin for high pressure liquid chromatgraphy (CHEMIEKOMBINAT B I T T E R F E L D , G. D. R.) were used. For elution and preparing the slurries tetrachloroethylene, dioxan, ammonium sulfate, sodium hydroxide, phosphoric acid of analytical grade (LABORCHEMIE APOLDA, G. D. R.) and double distilled water were available. HPLC-grade acetonitrile was purchased from PCK SCHWEDT (G. D. R.). Glass Columns [2]: The glass columns had an internal diameter of 3.8 mm and an external diameter of 12 mm. They could be employed in different lengths, e.g. 50, 100, 150 and 250 mm. I n to the frontages of the columns grooves were milled. These grooves and the resulting smaller areas are the main reasons why good sealing against high pressure (approximately 40 MPa) was achieved. The pressure stability of the glass tubes could be drastically improved (5: 60 MPa) by a 24 hour ion exchange procedure using a melt of potassium nitrate containing 1% potassium carbonate a t a temperature of 430 °C. Column Packing Procedure [3, 4]: A glass column was put in a cartridge, "screwed" and coupled with a packing apparatus by means of an adapter. For fillings with different stationary phases also slurries of different compositions were necessary. The amounts of stationary phase for 50 mm or 250 mm columns were approximately 0.4 and 2.4 g, respectively. slurry 1: reversed-phase material or silica gel in 20ml tetrachloroethylene/dioxan 17:3 v/v, slurry 2: amino phases in 20ml tetrachloroethylene, slurry 3: anion-exchange resins in 20ml double distilled water.

Characterization of Glass Columns: For the characterization of self-packed glass columns the number of theoretical plates (N) was calculated (equation 1). (1)

tR: retention time [mm] w: peak width a t half height [mm] Fig. 1 shows a reversed-phase separation of test substances eluted on LiChrosorb RP18 (particle size: 5 ¡um). As mobile phase acetonitrile/water 70:30 v/v was used.

GEY, M., High-Performance Liquid Chromatography

199

?

w, w2 w3

3 J LA/

>R3

10

fR2

fR1

Time [mini

Fig. 1. Chromatogram of a test mixture 1: phthalic acid, 2: dimethyl phthalate, 3: unknown, 4: diethyl phthalate, 5: diphenyl, 6: dibutyl phthalate, glass column: 150 X 3.8 mm, stat. phase: LiChrosorb RP18,5(j.m; mob. phase: acetonitrile/water 70: 30 v/v, flow rate: 0.7 ml/min, pressure: 6 MPa, detection: UV, 254 nm

For a column packed under optimum conditions about 83000 theoretical plates per meter with reference to biphenyl were calculated. This value was attained at a flow rate of 0.7 ml/min. The theoretical plate height (H) of 12 /¿m again with reference to biphenyl calculated by equation 2 also demonstrates the high quality of this glass column. H

=

^

M

(2)

L : column length [/¿m].

Results and Discussion Carbohydrates: HPLC analyses of carbohydrates in food [5] and also in polysaccharides (e.g. cellulose, regenerated fibre) hydrolyzed under enzymatic or acid conditions have already been reported [2], Ion-exchange resins and silica gels modified chemically with y-aminopropyl groups [6, 7] or impregnated in situ with a so-called amine modifier such as T E P and piperazine [8, 9] are the most commonly used stationary phases for liquid chromatographic separations of sugars. As eluents for stationary "amine phases" acetonitrile/water mixtures are used.

200

Acta Biotechnol. 8 (1988) 2

Mobile phases with a small water content allow the chromatography of the most important monosaccharides, e.g. rhamnose, xylose, arabinose, fructose, mannose, glucose and galactose. Separations of oligomeric carbohydrates up to a degree of polymerization (dp) of 11 are possible when the mobile phase contains 30.. .50% water. A volume ratio of 80% acetonitrile and 20% water is suitable for the determination of monosaccharides as well as disaccharides, as seen in Fig. 2. 12 sugars — 7 monomeric

Fig. 2. Chromatogram of a test mixture ef: eluent front, 1: rhamnose, 2: xylose, 3: arabinose, 4: fructose, 5: mannose, 6: glucose, 7: galactose, 8: sucrose, 9: cellobiose, 10: maltose, 11: lactose, 12: melibiose, glass column: 150 X 3.8 mm, stat. phase: LiChrosorb NH 2 , 5 ¡xm; mob. phase: acetonitrile/water 80: 20 v/v, flow rate: 0.6 ml/min, pressure: 10 MPa, detection: R I

and 5 dimeric compounds — could be separated in nearly 30 minutes on a 150 mm glass column packed with Silasorb NH 2 of 5 fim particle size. However this chromatogram shows a poor resolution of glucose from galactose. A drastic improvement in separation is possible if highly efficient silica gel columns impregnated with piperazine are available [2], Also a reduced water content (approximately 10%) in the eluent is necessary. A very time-consuming group-type separation of a pentose, hexose, di- and trimeric oligosaccharide is shown in Fig. 3. Rhamnose, glucose, cellobiose and raffinose could be eluted on a 100 mm glass column at a flow rate of 1.7 ml/min in about 3 minutes. Such optimized separation systems are employed for quick determinations of oligomeric carbohydrates under routine conditions.

GEY, M., HiGH-Performance Liquid Chromatography

201

g

Fig. 3. Chromatogram of a test mixture ef : eluent front, 1 : rhamnose, 2 : glucose, 3 : eellobiose, 4 : raffinose, glass column: 100 x 3.8 mm, stat. phase: Silasorb NH 2 , 5 fj.m; mob. phase: acetonitrile/water 75: 25 v/v, flow rate: 1.7 ml/min, pressure: 9 MPa, detection: R I i

2 Time

0 [mini

Starch

Degradation

0.1 N HCl120

°C

15 min

20 min

-ef

8

6 Time

4

2

ef

0

[mini

Pig. 4. Chromatograms of hydrolyzed starches from potatos ef: eluent front, 1: glucose, 2 : maltose, 3 . . . 7 : dp3...dp7 (dp: degree of polymerization), glass column: 100 x 3.8 mm, stat. phase: Silasorb NH 2 , 7.5 |un; mob. phase: acetonitrile/water 65: 35 v/v, flow rate: 1.0 ml/min, pressure: 5 MPa, detection: R I

202

Acta Biotechnol. 8 (1988) 2

The chromatogram in Fig. 4 shows separation examples of oligomeric sugars from hydrolyzed starches which were isolated from potatos. Here the HPLC method offers good possibilities for determinations of optimum conditions of starch degradation regarding reaction time, temperature and also type and concentration of different mineral acids

[10].

Organic Acids:| Organic acids are produsecible by microbial syntheses based on sugar or paraffin containing fermentation mediums [11], Depending on the microorganisms and fermentation conditions it is possible to produce these acids with concentrations between 30 and 1000 g/1. -ef

2

=i

5

I

I

50 W

I

30 Time

I

L_ 1

Fig. 5. Chromatogram of a fermentation solution (organic acids) ef: eluent front, 1, 3, 4, 6: unknown, 2: gluconic acid, 5: 2-ketogluconic acid, glass column: 250 x 3.8 mm, stat. phase: anion-exchange resin, 10—20 (j.m; mob. phase: 0.32 M (NH 4 ) 2 S0 4 , pH = 9.5; flow rate: 0.6 ml/min, pressure: 8 MPa, detection: UV, 210 nm

20 10 0 [mini

Organic acid analyses by HPLC have become more and more important since ion pair chromatography [12—15] and ion exchange chromatography (IEC) were developed. A further essential advantage especially of IEC is the possibility of direct on-column injection. Also, in most cases time-consuming derivatization procedures are not necessary. I n what follows some applications of organic acid separations on an anion-exchange resin will be demonstrated. For elution a 0.32 M ammonium sulfate buffer, p H = 9.5, was used. The assignments of the elution curves were carried out at a wavelength of 210 nm. Fig. 5 shows a chromatogram of a separation of gluconic acid from 2-ketogluconic acid. Both substances were produced by a microbial synthesis based on a mannitol containing fermentation solution. Other compounds (peaks 1, 3, 4, 6), which could not be identified, were also present in this sample. I n another case a microbial synthesis was carried out on a paraffin medium. As is seen in Fig. 6 citric acid, isocitric acid and «-ketoglutaric acid could be separated. For qualitative and quantitative analyses appropriate test substances were used. A self-packed glass column of only 50 mm length was sufficient to realize this separation with good resolutions in less than 15 minutes.

GEY, M., HiGH-Performance Liquid Chromatography

ef

\M 15

10

203

Fig. 6. Chromatogram of a fermentation solution (organic acids) ef: eluent front, 1: unknown, 2: citric acid, 3: isocitric acid, 4: a-ketoglutaric acid, glass column: 50 X 3.8 mm, stat. phase: anion-exchange resin, 10—20 [¿m; mob. phase: 0.32 M (NH 4 ) 2 S0 4 , pH = 9.5; pre column: 1 5 0 x 3 . 8 mm packed with anion-exchange resin, flow rate: 0.5 ml/min, pressure: 7 MPa, detection: UV, 210 nm

5

Time [mini

Lipids:

Phospholipids and neutral lipids as, e.g., fatty acids, hydrocarbons, glycerides and sterols are characteristic compounds of single cell proteins and other microbially produced biomasses. The isolation of lipids is based on an extraction of biomass using chloroform/methanol (1:1 v/v) as reported by Folch et al. [16]. Acetone precipitation yields a soluble fraction (neutral lipids) and a fraction not soluble in acetone (phospholipids). The detectability of most lipids is problematic. One reason is that detections in the middle UV-range are not possible because of the absence of chromophoric groups in their moleculs. Another is that assignments of elution curves by a refractive index monitor are not sensitive enough. On the other hand, eluents such as chloroform or acetic acid, which

Fig. 7. Chromatogram of phospholipids from yeast biomass 1: phosphatidyl ethanolamine (PE), 2: lysophosphatidyl ethanolamine (LPE), 3: lysophosphatidyl choline (LPC), 4: phosphatidyl choline (PC), 5: sphingomyeline (SPH), glass column: 150 X 3.8 mm, stat. phase: Silasorb 600, 5 [j.m; mob. phase: methanol/acetonitrile/water (100:100:3 v/v/v), flow rate: 0.3 ml/min, pressure: 4 MPa, detection: UV, 203 nm 20

10

Time [mini

204

Acta Biotechnol. 8 (1988) 2

are the preferred mobile phases especially for phospholipid separations by thin layer chromatography, show too strong absorptions in the near UV-range (200.. .220 nm). In Fig. 7 a chromatogram of phospholipids [17—20] from microbial biomass is shown. A mobile phase consisting of methanol/acetonitrile/phosphoric acid was suitable to separate phospholipids on glass columns packed with silica gel and to detect them at a wavelength in the near UV-range (e.g. 203 nm).

2

•«i

50

40

30 Time

20 [mini

10

0

Fig. 8. Chromatogram of neutral lipids from yeast biomass ef: eluent front, 1: squalene, 2 : cholesterol, glass column: 150 X 3.8 mm, stat. phase: LiChrosorb R P 8 , 5 pim; mob. phase: methanol/phosphoric acid (100: 1.5 v/v), flow rate: 0.6 ml/min, pressure: 6 MPa, detection: UV, 203 nm

Analyses of neutral lipids [21—24] from microbial biomass suggest much more complex compositions. The chromatographic separation of such a fraction is shown in Fig. 8. Only squalene and cholesterol could be identified by a comparison with the retention times of corresponding test substances. Most compounds of this neutral lipid fraction were not identifiable. A semipreparative HPLC technique using glass columns with an internal diameter of 8 mm [25] is one way to isolate unknown substances and to identify their structures by means of other analytical methods (MS, GC-MS, F T I R , NMR etc.). Received March 27, 1987

References [1] EPPERT, G.: Mitt. bl. Chem. Ges. DDR 30 (1983), 146. [2] GEY, M., MÜLLER, W . : Die Nahrung-Food, in press. [3] EPPERT, G.: „Einführung in die schnelle Flüssigchromatographie", Akademie-Verlag, 1979.

GEY, M., High-Performance Liquid Chromatography

205

[4] MEYER, V.: „Hochleistungsflüssigchromatographie", Verlag Moritz Diesterweg, Verlag Sauerländer, 1984. [5] GEY, M.: Lebensmittelindustrie 32 (1985), 184.

[ 6 ] LENDEN, J . C., LAWHEAD, L . : J . C h r o m a t o g r . 1 0 5 ( 1 9 7 5 ) , 1 2 5 .

[7] SCHWARZENBACH, R.: J . Chromatogr. 117 (1976), 206. [8] AITZETMÜLLER, K . : J . Chromatogr. 156 (1978), 354.

[ 9 ] BOUMAHRAZ, M . , DAVYDOV, V . Y A . , KIESELEV, A . V . : C h r o m a t o g r a p h i a 1 5 ( 1 9 8 2 ) , 7 5 1 .

[10] GEY, M., BECKER, U.: Unpublished results.

[ 1 1 ] STOTTMEISTER, U . , BEHRENS, U . , WEISSBRODT, E . , BATH, G . , FRANKE-RINKER, D . , SCHULZE,

E.: Z. Allg. Mikrobiol. 22 (1982), 399.

[12] QUVESHI, A. A., ELSON, C. E . , LEBECK, L . A . : J . Chromatogr. 273 (1982), 333. [ 1 3 ] HAYASHI, T . , TSUCHIYA, H . , NARUSE, H . : J . C h r o m a t o g r . 2 7 3 ( 1 9 8 3 ) , 2 4 5 . [ 1 4 ] K I E B E R , D . J . , MOPPER, K . : J . C h r o m a t o g r . 2 8 1 ( 1 9 8 3 ) , 1 3 5 . [ 1 5 ] HARA, S . , TAKEMORI, Y . , YAMAGUCHI, M . , NAKAMURA, M., OHKTTRA, Y . : J . C h r o m a t o g r . &44 (1985), 33. [ 1 6 ] FOLCH, J . , L E E S , M., SLOANE STANLEY, G . M . : J . B i o l . C h e m . 2 2 6 ( 1 9 5 7 ) , 4 9 7 . [ 1 7 ] HSIEH, J . Y . - K . , WELCH, D . K . , TÜRCOTTI, J . G . : J . C h r o m a t o g r . 2 0 8 ( 1 9 8 1 ) , 3 9 8 . [ 1 8 ] NASNER, A . , KRAUS, L j . : J . C h r o m a t o g r . 2 1 6 ( 1 9 8 1 ) , 3 8 9 . [ 1 9 ] YANDRASITZ, J . R . , B E R R Y , G . , SEGAL, S . : J . C h r o m a t o g r . 2 2 5 ( 1 9 8 1 ) , 3 1 9 . [ 2 0 ] NISSEN, H . P . , KREYSEL, H . W . : J . C h r o m a t o g r . 2 7 6 ( 1 9 8 3 ) , 2 9 . [ 2 1 ] KINCHI, K . , OHTA, T . , EBINE, H . : J . C h r o m a t o g r . 1 3 3 ( 1 9 7 7 ) , 2 2 6 .

[22] AITZETMÜLLER, K . , KOCH, J . : J . Chromatogr. 145 (1978), 195. [23] PRIVETT, 0 . S., ERDAHL, W . L . : Methods Enzymol. 72 (1981), 56.

[ 2 4 ] FRICKE, H . S . G . , OEHLENSCHLÄGER, J . : J . C h r o m a t o g r . 2 5 2 ( 1 9 8 2 ) , 3 3 1 .

[25] GEY, M.: Z. Chem. 27 (1987), 377.

Acta Biotechnol. 8 (1988) 2, 2 0 6 - 2 0 8

Book Reviews H . C . D U B O U R G U I E R , G . ALBAGNAC, J . M O N T R E U I L , C . R O M O N D , P . S A U T I È R E , J . GUILLAUME

Progress in Biotechnology, Volume 2 : Biology of Anaerobic Bacteria Amsterdam, Oxford, New York, Tokyo: Elsevier Science Publisher, 1986. 270 S., 71 $, 160 DfL

Der zweite Band der Publikationsreihe Progress in Biotechnology erithält die ungekürzten Originalbeiträge zu einem internationalen Seminar zur Biologie anaerober Bakterien, veranstaltet 1986 von Conseil Régional Nord/Pas de Calais, Délégation Régionale à la Recherche et à la Technologie und Pôle des Anaerobes in Lille (Frankreich). Die 33 Beiträge behandeln spezielle, aktuelle Probleme der Physiologie, Biochemie, Taxonomie, Genetik (genetische Grundlagen der Antibiotikaresistenz und Möglichkeiten der Einführung von Transposons aus aeroben Mikroorganismen in Bacteroidesstämme sowie Analysen der Organisation des genetischen Materials methanogener Archaebakterien) und Ökologie (speziell Fragen der Entwicklung der Biozönosen im tierischen Verdauungstrakt) ausgewählter Gruppen anaerober Mikroorganismen (methanogene Archaebakterien, sulfatreduzierende und acetogene Eubakterien sowie einige Arten der Gattungen Clostridium und Bacteroides mit industrieller und medizinischer Bedeutung). Größeren Raum nehmen anwendungsorientierte Beiträge ein : Diskutiert werden verschiedene Aspekte der Methanbildung, besonders aus Abprodukten, Möglichkeiten der biotechnologischen Produktion kurzkettiger Fettsäuren (z. B. mit anaeroben methylotrophen Bakterien aus Methanol), die weitere Nutzbarmachung des Abbaupotentials anaerober Bakterien zur Abproduktbeseitigung sowie neue Ansatzpunkte zur Produktion von Säuren und Lösungsmitteln mit Clostridien. L. WÜNSCHE

CHUM, H . , B A I Z E R , M . M .

The Electrochemistry of Biomass and Derived Materials ACS Monograph 183 Washington: American Chemical Society, 1985, 314 S., 107.95 $

The ACS Monograph Series, in which the discussed book is published, intends to serve two principal purposes: the thorough treatment of selected research areas by competent scientists and the stimulation of further research in the field treated. C H U M and B A Z E B ' S book meets both expectations. Two basic technologies usually not covered together in one book are biomass conversion and electrochemistry. The successful coupling of them by the authors opens an unusual insight in making feedstocks for the chemical and fuel industries. Starting with a comprehensive survey in Chapter 1 the term biomass is defined. Data on composition and availability of various types of biomass, including primary and secondary potential, are given. There are also representations of the main processes for technical chemo- and bioconversion of renewable raw materials. Chapter 2

Book Reviews

207

introduces basic electrochemical principles necessary for the non-specialist to gain an understanding of their practical applications. I t explains particularly the basis of classical liquid phase electrolysis using direct current. Wherever possible, illustrative reactions are given as examples. In Chapters 3 — 7 the electrochemical behaviors of plant biomass substances and derived materials are described in the order of increasing complexity of the substrates. The contents include carbon dioxide, biomass derived alcohols, carboxylic acids, aldehydes and ketones, alkanes, alkenes, aromatic compounds, and moreover some aspects of the electrochemistry of monosaccarides, polysaccharides, and lignin. Written in clear and explicit language, the information is easily understood and can be applied by non-specialists as well as experienced researchers. In a short form Chapter 8 deals with synthetic photoelectrochemistry and other techniques gaining increasing importance. In the final chapter suggestions are presented concerning future research and development to show possibilities for further work. Literaturs references for each of the chapters and an exhaustive subject index are given. In summary, the book can be recommended, as of great value for all scientists engaged in or preparing for work in this economically and ecologically important field. C. HARTIG

MIZRAHI, A . , A . L . VAN W E Z E L

Advances in Biotechnological Processes; Vol. 5 New York: Alan R. Liss, Inc., 1985. 336 S., 60.00 t

Hier liegt uns nun der 5. Band einer inzwischen recht erfolgreichen Reihe zu Erfolgen der Biotechnologie vor. Die Autoren haben sich an das bewährte Konzept gehalten und verfahren nach dem Motto: „Wer vieles bringt, wird manchem etwas bringen!" So weit gefächert wie die Anwendungsmöglichkeiten biotechnologischer Verfahren sind auch hier wieder die Beiträge des Bandes. Jedes Kapitel ist dabei ein abgeschlossener, selbständiger Artikel. Sie besitzen alle eine ausführliche und fundierte Einführung in die Thematik, so daß es auch einem „Nichtspezialisten" möglich ist, den Werdegang des jeweiligen Produktes oder Verfahrens zu verfolgen. Nach einer kurzen Einleitung durch die Herausgeber beginnt das Buch mit einem Kapitel, das sich mit der Bedeutung der Kulturmedien für die Verbreitung von Säugerzell- und Bakterien-(Virus-)kulturen beschäftigt. Neben einem Kapitel, das sich mit Pilzfermentation und ihrer industriellen Anwendung, speziell mit exotischen Arten auseinandersetzt, befassen sich 3 Kapitel mit Aspekten des biologischen Abbaus von Schadstoffen und Abprodukten. Dabei werden in einem neue Aspekte des biologischen Ligninabbaus und des Aufschlusses cellulosehaltiger Materialien vorgestellt, während zwei andere die Rolle der Biodegradation von Abwasser analysieren, und zwar einmal vom Gesichtspunkt abzubauender Schad- bzw. Inhaltsstoffe, und zum zweiten werden rotierende biologische Sammler für die Abwasserbehandlung aus technologischer Sicht vorgestellt. In anderen Kapiteln versuchen die Autoren alte Probleme mit neuen Methoden anzugehen und stellen neue Trends vor; z. B. in den Beiträgen zur Produktion von Geflügelvaccinen in Hühnerembryonen und zur biologischen Produktion des Plasminogen-Aktivators. Neue Anwendungsmöglichkeiten auf zwei anderen Gebieten sind ebenfalls Teil dieses Buches. In einem werden die vorhandenen und denkbaren Möglichkeiten pflanzlicher Zellkulturen zur Ertragssteigerung und Stammzüchtung vorgestellt und im anderen die vielfältigsten Varianten der Proteinkonstruktion mittels Computergraphik. Es ist zu hoffen, daß dieses Buch recht vielen Lesern neue Tinregungen geben kann und eventuell vorhandene Wissenslücken schließen hilft.] E . SPANIER

208

Acta Biotechnol. 8 (1988) 2

H . WALTER, D . E . BROOKS, D .

FISHER

Partitioning in Aqueous Two-Phase Systems Orlando, San Diego, New York, Austin, London, Montreal, Sydney, Tokyo, Toronto: Academic Press, inc., 1985. 704 pp 39.95 $ Mit diesem Buch, das etwa zur gleichen Zeit wie die aktualisierte dritte Auflage von P . - A . A L B E R T S „Partition of Cell Particles and Macromolecules" (Erscheinungsjahr 1986) erschienen ist, liegt nunmehr eine noch umfassendere Beschreibung der Grundlagen und Anwendungen der Verteilung von Makromolekülen und Partikeln in wäßrigen Mehrphasensystemen vor. Ohne an vorausgegangene Auflagen gebunden zu sein, folgten die Herausgeber offensichtlich nur der Maxime einer möglichst adäquaten Darstellung des bislang bekannten Wissensstandes. Daß sie sich in diesem Bestreben der Mitarbeit der wohl bekanntesten Vertreter der einzelnen Teildisziplinen versicherten, spricht für ihre seriösen Absichten und verleiht dem Werk eine kaum zu übertreffende Authentizität. So sind dem mit der Literatur vertrauten Leser die Namen G. J O H A N S S O N (Partitioning of Proteins), P. T. S H A R P E , H. W A L T E R , D. E. B R O O K S , J . A. S U T H E R L A N D (Countercurrent Distribution and Continuous Flow Chromatography Techniques), D. E. B R O O K S , K. A. S H A R P , D. F I S H E R (Theoretical Aspects of Partitioning), H. W A L T E R , D. F I S H E R (Partitioning of Red Blood Cells and Mammalian Cell Populations), K. E. M A G N U S S O N , O. S T E N D A H L (Partioning of Bacteria, Virus, and Phage) und H . H U S T E D T , K . H. K R O N E R , M. R. K U L A (Applications of Phase Partitioning in Biotechnology) — um nur einige zu nennen — wohl bekannt. Nicht zuletzt blieb es P.-A. A L B E R T S S O N vorbehalten, eine Darstellung der Geschichte der Polymerphasenverteilung zu geben. So wird der nach Vollständigkeit trachtende Leser kaum Defizite bemerken können. Es fehlt zum Beispiel ein besonderes Kapitel über die Phasensysteme, mit denen sich wiederum P.-A. A L B E R T S S O N eingehend beschäftigt hat. Eine wesentliche Bereicherung jedoch erfährt das Buch durch den umfassenden bibliographischen Teil, der auch in eine Datenbank aufgenommen wurde und nach dem Willen der Herausgeber jedes Jahr ergänzt werden soll. Ohne Einschränkung kann somit dieses Buch allen an der Polymerphasenverteilung von gelösten Stoffen oder Partikeln in wäßrigen Systemen Interessierten empfohlen werden. SONS

H . WAND

H.

BÜHLER

Messen in der Biotechnologie Heidelberg: Dr. Alfred Hüthig Verlag, 1985. 115 S.; 42 Abb.; 45 DM Parallel mit dem Aufschwung der Biotechnologie fand auch eine stürmische Entwicklung der Analytik statt. Während die Produktanalyse in der Vergangenheit meist an einzelnen Proben vorgenommen wurde, sind in den letzten Jahren eine Reihe von kontinuierlich arbeitenden Meßfühlern entwickelt worden, die innerhalb oder außerhalb des Bioreaktors eingesetzt werden. Das vorliegende Buch beschreibt aus der Sicht des Meßtechnikers die Meßfühler, aber auch Verfahren, die als Grundlage für das Verständnis der einzelnen Meßfühler dienen. Die biologische Bedeutung der einzelnen Meßgrößen sowie die daraus abgeleiteten Parameter sind bewußt nur am Rande erwähnt, denn es sollen vorwiegend die Prinzipien und die speziellen Probleme der Meßfühler vermittelt werden. So sind neben Standardverfahren auch viele neue, zum Teil noch wenig bekannte Techniken beschrieben. Die Darstellung der physikalischen und chemischen Grundlagen der beschriebenen Meßverfahren ist knapp und präzise, aber gut verständlich. Deshalb sollte dieses Buch für breite Interessengruppen, wie Laboranten, Ingenieure, Biologen oder Chemiker, die auf dem Gebiet der Biotechnologie arbeiten und mit Meßproblemen konfrontiert werden, als Arbeitsmaterial dienen und in keiner Fachbibliothek fehlen. Positiv ist zu vermerken, daß zu jedem Stichwort zahlreiche weiterführende Literaturstellen, leider vielfach ohne Originaltitel, angegeben werden. E . SCHIEMENZ

BIOMETRICAL JOURNAL Journal of Mathematical Methods in Biosciences Edited at Karl-WeierstraB-Institut of Mathematics, Academy of Sciences of the GDR, by Erna Weber (Berlin), Editor-in-Chief Editors: Heinz Ahrens (Berlin), Klaus Bellmann (Berlin) Assisted by an international Editorial Board The Biometrical Journal was founded for the purpose of publishing contributions on mathematical control of biosciences. The scope includes papers on new theoretical aspects of mathematics and its application to biological sciences in the widest sense of the definition (including biology, medicine, agricultural science, forestry) or on the application of known mathematical and statistical methods to new areas. These may be methods of mathematical statistics an approaches to mathematical cybernetics model building of biological systems with due consideration of electronic data processing. Accepted for publication: original papers, summary reports on the latest developments in the above areas, proceedings and book reviews.

Of interest to: Biologists, anthropologists, psychologists, physicians, agricultural and forestry theorists, and mathematicians dealing with statistics. Biometrical Journal is indexed in Current Contents/Physical, Chemical & Earth Sciences, Science Citation Index Automatic Subject Citation Alert, CompuMath Citation Index and Journal Contents in Quantitative Methods. Published yearly, 1 volume (8 numbers). Volume 30, 1988. 17 cm by 24 cm. Annual list of contents. Annual subscription DM 336,—; single number DM 42,—. Order number: 1060. ISSN 0323-3847.

Orders can be sent — in the GDR: to Postzeitungsvertrieb or to the Akademie-Verlag Berlin, Leipziger Str. 3—4, PF 1233, DDR-1086 Berlin; — in the other socialist countries: to a bookshop for foreign language literature or to the competent news-distributing agency; — in the FRO and Berlin (West): to a bookshop or to the wholesale distributing agency Kunst und Wissen, Erich Bieber OHG, Wilhelmstraße 4, D-7000 Stuttgart 1; — in the other Western European countries: to Kunst und Wissen, Erich Bieber GmbH, General Wille-Straße 4, CH-8002 Zürich; — in USA and Canada: to VCH Publishers, Inc., 303 NW 12th Avenue, Deerfield Beach, FL 33442-1788, USA; — in other countries: to the international book- and journalselling trade; to Buchexport, Volkseigener Außenhandelsbetrieb der DDR, Postfach 160, DDR-7010 Leipzig; or to AkademieVerlag Berlin, Leipziger Str. 3 - 4 , PF 1233, DDR-1086 Berlin.

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Ada Biotedinologica Volume 8

1988

Number 2

Contents Growth Kinetics of Top and Bottom Cultures of Saccharomyces spp. in a Chemostat Using Sugarbeet Molasses 115 ALEXANDER, D . B . ; ZAJIC, J . E . ; JONES, L . P . :

GETTSZCZ, P.; STABZAK, M . ; MICHALSKI, H . : A kLa Identification Technique for a Dynamic Model of the Couplet System: Air-Lift Fermenter — Oxygen Probes 125

M. S . ; J U S U P O W , T. A . ; Biochemical Reactor (in Russian) NUBBTJSIN,

YEMELJANOW, V .

M.: The Multicriteric Optimization of . 139

B.; MUND, K . : Identification of Process Specific lag Times and Consideration in Calculation of Phenomenological Growth Parameters (in German) 147 REIMANN,

EMELYANOV, V.M.; B I L A L O V A , Z . M.; Y U . N . Z U : Utility of Oxygen Carrier in the Processes of Microorganisms Cultivation (in Russian) 151

The Influence of Light on Nigella demascena L. Cells Cultivated in Fermentersystems (in German) 159 SCHMAUDEB, H . - P . ; D Ö B E L , P . :

SZTAJEB, H . ; ZBOI£SKA, E . : TBIPATHI, G . :

Microbial Lipases in Biotechnology

Enzyme Overproduction: Principle and Application

169 177

D. G . ; P E V Z N E B , I. L.; SADUIKOV, R . A.: The Influence of Control Parameters of Extraction and Concentration over Oxidation and Degradation of Amino Acids 189 POBEDIMSKI,

GEY, M.: Characterization of Biotechnological Processes and Products Using Hight-Performance Liquid Chromatography (HPLC) I. Separations of Carbohydrates, Organic Acids and Lipids 197 Book Reviews

124, 138, 168, 176, 188, 206, 207, 208

Acta Biotechnologica is indexed or abstracted in Current Contents/ET & AT; Chemical Abstracts; Biological Abstracts; Biotechnology Abstracts; Excerpta Medica