DD278357A5 - Method of increasing enzyme activity and synthesis efficiency of organisms - Google Patents
Method of increasing enzyme activity and synthesis efficiency of organisms Download PDFInfo
- Publication number
- DD278357A5 DD278357A5 DD32499389A DD32499389A DD278357A5 DD 278357 A5 DD278357 A5 DD 278357A5 DD 32499389 A DD32499389 A DD 32499389A DD 32499389 A DD32499389 A DD 32499389A DD 278357 A5 DD278357 A5 DD 278357A5
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- DD
- German Democratic Republic
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- microorganisms
- atcc
- synthesis
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- excretions
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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Abstract
Die Erfindung betrifft ein Verfahren zur Steigerung von Enzym-Aktivitaeten und der Syntheseleistung von Organismen, welches dadurch gekennzeichnet ist, dass man dieselben mit inaktivierten Elicitoren-haltigen Mikroorganismen, Fragmenten derselben oder Exkretionen Elicitoren-haltiger Mikroorganismen in Kontakt bringt, mit der Massgabe, dass man zur Steigerung von Enzym-Aktivitaeten und der Syntheseleistung nicht mikrobieller Organismen inaktivierte Elicitoren-haltige Bakterien, Fragmente derselben oder Exkretionen Elicitoren-haltiger Bakterien verwendet. Das erfindungsgemaesse Verfahren ermoeglicht es, beispielsweise die Syntheseleistung von Farbstoffe, Antibiotika, Alkaloide oder Phytoalexine bildenden Mikroorganismen oder Pflanzen zu steigern oder die Enzymaktivitaeten von zur Steroidtransformation befaehigten Mikroorganismen zu erhoehen.The invention relates to a method for increasing enzyme activities and the synthesis performance of organisms, which is characterized in that it brings the same with inactivated elicitor-containing microorganisms, fragments thereof or excretions of elicitorium-containing microorganisms in contact, with the proviso that used to increase enzyme activities and synthesis performance of non-microbial organisms inactivated elicitorial-containing bacteria, fragments thereof or excretions of elicitor-containing bacteria. The process according to the invention makes it possible, for example, to increase the synthesis efficiency of dyes, antibiotics, alkaloids or phytoalexins-forming microorganisms or plants or to increase the enzyme activities of microorganisms capable of steroid transformation.
Description
Die Erfindung betrifft ein Verfahren zur Steigerung von Enzym-Aktivitäten und der Syntheseleistung von Organismen, welches dadurch gekennzeichnet ist, daß man dieselben mit inaktivierten Elicitoren-haltigen Mikroorganismen, Fragmenten derselben oder Exkretionen Elicitoren-haltiger Mikroorganismen in Kontakt bringt, mit der Maßnahme, daß man zur Steigerung von Enzym-Aktivitäten und der Syntheseleistung nichtmikrobieller Organismen inaktivierte Elicitoren-haltige Bakterien, Fragmente derselben oder Exkretionen Elicitoren-haltiger Bakterien verwendetThe invention relates to a method for increasing enzyme activities and the synthesis performance of organisms, which is characterized in that it brings the same with inactivated elicitorium-containing microorganisms, fragments thereof or excretions of elicitorium-containing microorganisms in contact, with the measure that to increase enzyme activity and synthesis efficiency of non-microbial organisms inactivated elicitorial-containing bacteria, fragments thereof or excretions of elicitorial-containing bacteria used
Anwendungsgebiete sind die Biotechnologie, Landwirtschaft, Pharmazin und Medizin. Fields of application are biotechnology, agriculture, pharmacy and medicine.
EMcitoren sind bekanntlich mikrobielle oder pflanzliche Wirkstoffe, die, mit Geweben höherer Pflanzen in Kontakt gebracht, deren Enzym-Aktivitäten und Syn'heseleistung steigern. Die so in diesen Pflanzen akkumulierten Inhai sstoffe werden, wenn sie antimikrobiell sind, als Phytoalexine bezeichnet.Emcitors are known to be microbial or herbal agents that, when contacted with tissues of higher plants, increase their enzyme activities and synthesis performance. The Inhai substances thus accumulated in these plants, if they are antimicrobial, are called phytoalexins.
Gegenwärtig sind mehr als 100 Verbindungen, Jie der Phytoalexindefinition genügen, aus verschiedenen Pflanzenarten isoliert worden. Sie gehören verschiedenen Naturstoffgruppen an wie Terpenoiden, Linolensäurederivatcn, Acetylenen und Polyacetylenen, Bihenzylen, Stilbenen, Phenanthrenen und Dihydrophenanthrenen, Benzofuranen und Phenolbenzofuranen, Furocumarinen, Avenaluminen, Flavanen, Phenylbenzofuranen, Benzoxazinonen, Alkaloiden, Isoflavonoiden (Brooks and Watson, Nat. Prod. Reports 2,1985,427).At present, more than 100 compounds satisfying the definition of phytoalexin have been isolated from various plant species. They belong to various natural product groups such as terpenoids, linolenic acid derivatives, acetylenes and polyacetylenes, bihenzyls, stilbenes, phenanthrenes and dihydrophenanthrenes, benzofurans and phenolbenzofurans, furocoumarins, avalanumines, flavanins, phenylbenzofurans, benzoxazinones, alkaloids, isoflavonoids (Brooks and Watson, Nat. Prod , 1985.427).
Eine technische Anwendung hat diese Elicitoren-Wirkung bislang nicht gefunden. Dies hat mehrere Gründe: Es ist, von wenigen Ausnahmen abgesehen, bislanp nicht möglich, Zellen höherer Pflanzen unter wirtschaftlich vertretbaren Bedingungen in Subinerskulturen zu vermehren. Elicitoren in der Fermentation mittels; Mikroorganismen anzuwenden, schien a priori zwecklos zu sein, da man nach der vorherrschenden Lehrmeinung über die Wirkungsweise von Elicitoren (Albersheim: Spectrum der Wissenschaften, 11,1985,85) davor, ausgehen mußte, daß diese die Enzymaktivität und die Stoffwechselvorgänge in Mikroorganismen nicht beeinflussen.A technical application has not yet found this Elicitoren effect. There are several reasons for this: With a few exceptions, it is not possible for bislanp to increase the cells of higher plants in sub-niger cultures under economically justifiable conditions. Elicitors in fermentation by means of; To apply microorganisms seemed to be a priori futile, because one had to assume that they do not affect the enzyme activity and metabolic processes in microorganisms according to the prevailing view on the way in which elicitors work (Albersheim: Spectrum der Wissenschaften, 11, 1985, 85) ,
Bemerkenswert ist auch, daß nach geltender Lehrmeinung Bakterien eine Phytoalaxinbildung bei Pflanzen nur auslösen können, indem sie mittels bestimmter Enzyme aus der pflanzlichen Zellwand Elicitoren freisetzen, die als endogene Elicitoren diü Phytoalexinbildung stimulieren.It is also noteworthy that, according to prevailing theory, bacteria can only induce phytoalaxin formation in plants by releasing elicitors from the plant cell wall by means of certain enzymes, which stimulate phytoalexin formation as endogenous elicitors.
Durch die Erfindung wird ein Verfahren zur Steigerung von Enzym-Aktivitäten und der Syntheseleistung von Organismen bereitgestellt. Das erfindungsgemäße Verfahren ermöglicht es, beispielsweise die Syntheseleistung von Farbstoffe, Antibiotika, Alkaloide oder Phytoalexine bildenden Mikroorganismen oder Pflanzen zu steigern oder die Enzym-Aktivitäten von zur Steroidtransformation befähigten Mikroorganismen zu erhöhen.The invention provides a method of increasing enzyme activities and synthesis efficiency of organisms. The process according to the invention makes it possible, for example, to increase the synthesis efficiency of dyes, antibiotics, alkaloids or phytoalexins-forming microorganisms or plants or to increase the enzyme activities of microorganisms capable of steroid transformation.
Der vorliegenden Erfindung liegt die Aufgabe zugrunde, ein Verfahren zur Steigerung von Enzym-Aktivitäten und der Syntheseleistung von Organismen zu entwickeln.The present invention has for its object to develop a method for increasing enzyme activities and the synthesis performance of organisms.
Es wurde nun gefunden, daß Verbindungen und Zellpräparationen, die im folgenden als Elicitoren bezeichnet werden, überraschenderweise doch in der Lage sind, in Mikroorganismen En*ym-Aktivitäten und deren Syntheseleistung zu steigern.It has now been found that compounds and cell preparations, which are referred to below as elicitors, are surprisingly able to increase enzymatic activities and their synthesis efficiency in microorganisms.
Darüber hinaus wurde gefunden, daß es auch Bakterien gibt, die Elicitoren enthalten, welche keine Enzyme oder nutritive Faktoren sind.In addition, it has been found that there are also bacteria containing elicitors which are not enzymes or nutritional factors.
Erfind mgsgemäß wird die Aufgabe dadurch gelöst, daß man zur Durchführung des Verfahrens inaktivierte Elicitoren-haltige Mikroorganismen verwendet oder Fragmente dieser Mikroorganismen, wie zum Beispiel Zellwandfraktionen, Zellfragmente mechanisch aufgeschlossener oder chemisch bzw. enzymatisch lysierter Zellen oder durch Hilfsstoffe. wie zum Beispiel Ethanol oder Aceton präzipitierte Zeilinhaltsstoffe, da es für den Fachmann offenkundig ist, daß es in der Regel viel zu aufwendig ist, Elicitoren selbst zu isolieren oder zu synthetisieren, um dieso dann zur Beeinflussung des Stoffwechsels anderer Organismen zu verwenden.According mgsgemäß the object is achieved in that one uses for carrying out the process inactivated elicitorium-containing microorganisms or fragments of these microorganisms, such as cell wall fractions, cell fragments mechanically disrupted or chemically or enzymatically lysed cells or by auxiliaries. such as, for example, ethanol or acetone precipitated cell ingredients, as it will be apparent to those skilled in the art that it is generally far too expensive to self-isolate or synthesize elicitors to then use them to affect the metabolism of other organisms.
Wenn der Mikroorganismus Elicitoren in die Kulturbrühe abgibt oder nach Lysis oder Sterilisation wasserlösliche Eliciiorenhaltige Zeilinhaltsstoffe bildet, so kann man auch diese Elicitoren-haltigen Exkretionen der Mikroorganismen zur Durchführung des erfindungsgemäßen Verfahrens verwenden.If the microorganism releases elicitors into the culture broth or forms water-soluble elicitorial cell constituents after lysis or sterilization, it is also possible to use these elicitor-containing excretions of the microorganisms for carrying out the method according to the invention.
Mikroorganismen, von denen bekannt ist, daß sie Elicitoren besitzen, sind unter anderem die in der nachfolgenden Tabelle I aufgelisteten Pilzstämme und Hefen:Microorganisms known to possess elicitors include the fungal strains and yeasts listed in Table I below:
In eigenen Versuchen wurden proteolytisch mittels Trypsin gereinigte Zellwandpräparate von Gram-positiven Bakterienstämmen der Gattungen Bacillus, Corynebacterium, Brevibacterium, Cellulomonas, Lactobacillus Pimelobacter, Rhodococcus und Staphylococcus und in Wasser hitzesterilisierte Mikroorganismen dieser Gattungen und deren Filtrate daraufhin untersucht, ob sie Elicitoren besitzen. In der nachfolgenden Tabelle 2 sind die Bakterienstämme aufgeführt, in denen Elicitoren nachgewiesen werden konnten.In our own experiments, proteolytically purified by trypsin cell wall preparations of Gram-positive bacterial strains of the genera Bacillus, Corynebacterium, Brevibacterium, Cellulomonas, Lactobacillus Pimelobacter, Rhodococcus and Staphylococcus and in water heat sterilized microorganisms of these genera and their filtrates were examined to see if they have elicitors. Table 2 below lists the bacterial strains in which elicitors could be detected.
Alternaria carthami Botrytis cinerea (ATCC 48345) Ceratocystis f imbriata Ceratocystisulmi Chondrostereum purpureum Cladosporium fulvum (ATCC44961) Colletotrichumlindemuthianum (ATCC 52471) Fusariumsolani Fusariumsolanifspmori (ATCC 44934) Ganoderma applanatum Glomerella cingulata Helmithosporium carbonum (ATCC 24962) Moniliniafructicola Nectria haematococca PhomaexiguaAlternaria Carthami Botrytis cinerea (ATCC 48345) Ceratocystis f imbriata Ceratocystisulmi Chondrostereum purpureum Cladosporium fulvum (ATCC44961) Colletotrichumlindemuthianum (ATCC 52471) Fusariumsolani Fusariumsolanifspmori (ATCC 44934) Ganoderma applanatum Glomerella cingulata Helmithosporium carbonum (ATCC 24962) Moniliniafructicola Nectria haematococca Phomaexigua
Phytophihora cannabivora Phytophthora capsici (ATCC 52771) Phytophthorainfectans (ATCC 44776) Phytophthora megasperma var glycinea Phytophthora infectans Phytophthora megasperma Phytophthora nicotiane Pucciniacoronata Pyricularia oryzae (ATCC 15923) Saccharomyces cerevisiae Verticill.;um albo-atrum Verticillium dahliae (ATCC 26289)Phytophthora capsici (ATCC 52771) Phytophthorainfectans (ATCC 44776) Phytophthora megasperma var glycinea Phytophthora infectans Phytophthora megasperma Phytophthora nicotiane Pucciniacoronata Pyricularia oryzae (ATCC 15923) Saccharomyces cerevisiae Verticill. ; around albo-atrum Verticillium dahliae (ATCC 26289)
Arch. Bioch. Biop. 229,1984,136 Physiol. Plant Pc ~i. 11,1977,287 Phylochemistry23,1984,759 Phytochemistry23,1984,383 J. Chem. Soc. Perkin Trans 1,1984,14341 Physiol. Plart Pathol. 16,1980,391 Eur. J. Biochem. 129,1983,593 Plant Physiol. 76,1984,833 Nat. Prod. Rep. 2,1985,439 Phytochemistry 22,1983,1039 Physiol. Plant. Pathol. 21,1982,171 Z. Naturforsch. Sect. C 38,1983,899 J. Am. Chem. Soc, 84,1962,1919 Phytochemistry 22,1983,2291 Phytochem.21,1982,1818 Z. Naturf. Sect. C39,1984,217 Physiol. Plat. Pathol. 18,1981,379 Phytochem.23,1984,537 Arch. Bioch. Bioph. 229,1984,136 Phytpathol.Z,27,1956,237 J. Biol. Chem. 259,1984,11341 Phytopathology 71,1981,864 Physiol. Plant Pathol. 20,1982,189 Agric. Biol. Chem. 48,1984,253 Plant Physiol. 62,1978,107 Nat. Prod. Rep. 2,1985,429 ATCC-Katalog, 16. Aufl. 1984Arch. Bioch. Biop. 229,1984,136 Physiol. Plant Pc ~ i. 11, 1977, 2887 Phylochemistry 23, 1984, 599 Phytochemistry 23, 1984, 383 J. Chem. Soc. Perkin Trans 1,1984,14,341 Physiol. Plart pathol. 16.1980.391 Eur. J. Biochem. 129,1983,593 Plant Physiol. 76,1984,833 Nat. Prod. Rep. 2,1985,439 Phytochemistry 22,1983,1039 Physiol. Plant. Pathol. 21,1982,171 Z. Naturforsch. Sect. C 38,1983,899 J. Am. Chem. Soc., 84, 1962, 1919 Phytochemistry 22, 1983, 2991 Phytochem. 21, 1982, 1818 Z. Natur. Sect. C39,1984,217 Physiol. Plat. Pathol. 18,1981,379 Phytochem. 23,1984,537 Arch. Bioch. Bioph. 229,1984,136 Phytpathol.Z, 27,1956,237 J. Biol. Chem. 259,1984,11341 Phytopathology 71,1981,864 Physiol. Plant pathol. 20,1982,189 Agric. Biol. Chem. 48, 198, 253 Plant Physiol. 62,1978,107 Nat. Prod. Rep. 2,1985,429 ATCC catalog, 16th ed. 1984
Bacillus iicheniformis ATCC 9945 Brevibacterium butanicum ATCC 21196 Brevibacterium flavum ATCC 13826, ATCC 14067Bacillus iicheniformis ATCC 9945 Brevibacterium butanicum ATCC 21196 Brevibacterium flavum ATCC 13826, ATCC 14067
Brevibacterium lactofermentum ATCC 13655Brevibacterium lactofermentum ATCC 13655
Brevibacterium glutamingenes ATCC 13747Brevibacterium glutamigenic ATCC 13747
Brevibacterium ammoniagenes ATCC 6872Brevibacterium ammoniagenes ATCC 6872
Brevibacverium pumilus ATCC 7061Brevibacverium pumilus ATCC 7061
Corynebacterium hydrocarboclastum ATCC 15592Corynebacterium hydrocarboclastum ATCC 15592
Corynebacterium nephridii ATCC 11425Corynebacterium nephridii ATCC 11425
Corynebacterium paurometabolum ATCC 8368Corynebacterium paurometabolum ATCC 8368
Corynebacterium lilium ATCC 15990Corynebacterium lilium ATCC 15990
Corynebacterium striatum ATCC 6940Corynebacterium striatum ATCC 6940
Corynebacterium xerosis ATCC 373Corynebacterium xerosis ATCC 373
Corynebacterium diphtheriae (Stamm Mass. 8/Behring Worke)Corynebacterium diphtheriae (strain Mass. 8 / Behring Worke)
Corynebacterium melassecola ATCC 17965Corynebacterium molassecola ATCC 17965
Corynebacterium glutamicium ATCC 13032Corynebacterium glutamicium ATCC 13032
Corynebacterium uratoxidans ATCC 21749Corynebacterium uratoxidans ATCC 21749
Lactobacillus casei subsp. rhamnosus ATCC 7469Lactobacillus casei subsp. rhamnosus ATCC 7469
Lactobacillus plantarum DSM 20174Lactobacillus plantarum DSM 20174
PirnelobactertumescensAJ 1460PirnelobactertumescensAJ 1460
Rhodococcus fascians ATCC 12975Rhodococcus fascians ATCC 12975
Rhodococcus fascians 1 - lsolat von Prof. Dr. Stolp, Univ. Bayreuth Rhodococcus fascians 2 - lsolat von Prof. Dr. Stolp, Univ. BayreuthRhodococcus fascians 1 - isolate of Prof. Dr. med. Stolp, Univ. Bayreuth Rhodococcus fascians 2 - sol. Stolp, Univ. Bayreuth
Im Rahmen der vorliegenden Erfindung wurden bislang nur Bakterienstämme weniger Gattungen von Gram-positiven Eubacterien daraufhin untersucht, ob sie Eücitoren besitzen. Auch unter den Pilzstämmen inklusive der Hefen wurden - soweit man den Vorpubükationen entnehmen kann - meist nur solche auf das Vorhandensein von Elicitor-Aktivitäten untersucht, von denentekannt ist, daß sie phytopathogen sind. Es ist deshalb zu erwarten, daß zusätzlich noch zahlreiche Mikroorganismen, wie zum Beispiel Bakterien der Gattungen Mycobacterium oder Pseudonocardia, aufgefunden werden können, die ebenfalls Elicitoren besitzen.In the context of the present invention, only bacterial strains of less genera of Gram-positive eubacteria have been investigated to date for their possession of effervescence. Also among the fungal strains including the yeasts, as far as one can see from the prepubications, only those that have been known to be phytopathogenic are usually examined for the presence of elicitor activities. It is therefore to be expected that in addition numerous microorganisms, such as, for example, bacteria of the genera Mycobacterium or Pseudonocardia, can be found, which also have elicitors.
Die Prüfung von Mikroorganismen auf Elicitor-Aktivität kann problemlos mittels der üblichen Screening-Versuche, wie sie dem Fachmsnn geläufig sind, durchgeführt werden.The testing of microorganisms for elicitor activity can easily be carried out by means of the usual screening experiments, as known to those skilled in the art.
So kann man beispielsweise in Reihenversuchen die Mikroorganismen, deren Enzymaktivität oder Syntheseleistung gesteigert werden soll, in Submerskulturen anzüchten, den einzelnen Kulturen inaktivierte Mikroorganismen unterschiedlicher Spezies oder Subspezies zusetzen und nach erfolgter Fermentation analytisch ermitteln, in welchen Kulturen eine Steigerung der Enzymaktivität oder der Syntheseleistung erzielt wird.Thus, for example, the microorganisms whose enzymatic activity or synthesis efficiency is to be increased can be cultivated in submerged cultures, microorganisms of different species or subspecies inactivated, and, after fermentation, analytically determined in which cultures an increase in enzyme activity or synthesis efficiency is achieved ,
Die Durchführung des erfindungsgemäßen Verfahrens ist, soweit es die Fermentation mittels Mikroorganismen betrifft, für den Fachmann unproblematisch. Der Mikroorganismus, dessen Enzymaktivität oder dessen Syntheseleistung gesteigert werden soll, wird unter den bekannten Bedingungen angezüchtet; dann setzt man der Kultur die inaktivierten Elicitoren-haltigen Mikroorganismen, Fragmente derselben, Zellextrakte derselben oder Exkretionen derselben zu und setzt die Fermentation wie üblich fort. Die Zugabe der inaktivierten Mikroorganismen der Fragmente oder Extrakte dieser Organismen oder der Exkretionen von Elicitoren-haltigen Mikroorganismen kann bereits zu Fermentationsbeginn erfolgen. Die optimale Zugabezeit ist naturgemäß von der Art des angezüchteten Mikroorganismus abhängig, insbesondere vom Verlauf seiner exponentiellen Wachstumsphase, und muß im Einzelfall ermittelt werden. So erweist es sich bei Bakterien beispielsweise oft als zweckmäßig, diese Zugabe 4 bis 30 Stunden nach Fermentationsbeginn durchzuführen. Bei der Zugabe inaktivierter Mikroorganismen oder Fragmente derselben verwendet man pro Kubikmeter Fermentationsbrühe üblicherweise 1 bis 1000g (vorzugsweise 10 bis 200g) inaktivierten Mikroorganismus oder 0,1 bis 100g (vorzugsweise 1 bis 30g) des Fragments dieses Organismus. Verwendet man Exkretionen von Elicitoren-haltigen Mikroorganismen, so wird es in der Regel ausreichend sein, pro Kubikmeter Fermentationsvolumen 1 bis 5Ol der Ev!;retions-Lösung anzuwenden. Wenn das erfindungsgemäße Vorfahren dazu dient, die Enzymaktivität eines Mikroorganismus, der zur enzymatischen Umwandlung von Substraten angewendet wird, zu steigern, wird man die Zugabe des Substrates in der Regel 0 bis 10 Stunden nach erfolgter Zugabe des Elicitoren-haltigen inaktivierten Mikroorganismus oder dessen Fragmenten oder Exkretionen beginnen.The implementation of the method according to the invention, as far as the fermentation by means of microorganisms, for the expert unproblematic. The microorganism whose enzyme activity or synthesis efficiency is to be increased is grown under the known conditions; then the culture is added to the inactivated elicitor-containing microorganisms, fragments thereof, cell extracts thereof or excretions thereof and the fermentation is continued as usual. The addition of the inactivated microorganisms of the fragments or extracts of these organisms or the excretions of elicitor-containing microorganisms can be carried out already at the beginning of fermentation. The optimal addition time is naturally dependent on the nature of the microorganism grown, in particular on the course of its exponential growth phase, and must be determined on a case-by-case basis. For example, it often proves useful in bacteria to carry out this addition 4 to 30 hours after the start of fermentation. When adding inactivated microorganisms or fragments thereof, from 1 to 1000 g (preferably 10 to 200 g) of inactivated microorganism or 0.1 to 100 g (preferably 1 to 30 g) of the fragment of this organism are usually used per cubic meter of fermentation broth. If excretions of elicitor-containing microorganisms are used, it will generally be sufficient to use from 1 to 50 μl of the evaporation solution per cubic meter of fermentation volume. When the ancestor of the present invention serves to increase the enzyme activity of a microorganism used for the enzymatic conversion of substrates, the addition of the substrate will usually be from 0 to 10 hours after the addition of the elicitor-containing inactivated microorganism or its fragments or Excretions begin.
Die optimalen Fermentationsbedingungen sind von der Art des verwendeten Mikroorganismus, den Fermentationsbedingungen, der Art und Menge des Elicitoren-haltigen Materials usw. abhängig; sie müssen im Einzelfall durch Vorversuche, wie sie dem Fachmann geläufig sind, ermittelt werden.The optimum fermentation conditions depend on the type of microorganism used, the fermentation conditions, the type and amount of elicitor-containing material, etc .; In individual cases, they must be determined by preliminary tests, as are familiar to the person skilled in the art.
Zur Herstellung der inaktivierten Elicitoren-haltigen Mikroorganismen werden diese unter den üblichen Bedingungen angezüchtet, sodann durch Zentrifugieren oder Filtration von der Kulturbühne getrennt, gewünschtenfalls gewaschen und nochmals isoliert. Zur Inaktivierung der Mikroorganismen können unterschiedliche Verfahren angewendet werden. Mögliche Inaktivierungsmethoden bestehen darin, auf diese Mikroorganismen typische Zellgifte wie Ethylenoxide, Formaldehyd, Ozon, Quecksilberverbindungen, organische Lösungsmittel, wie Methanol, Ethanol oder Aceton, einwirken zu lassen oder die Mikroorganismen durch Erhitzen auf 90° bis 140° durch Einwirkung extremer Druckdifferenzen (Desintigration), Einwirken hochfrequenter elektrischer Feldsr oder durch UV-Bestrahlung, Bestrahlen miί γ-Strahlen oder Ultraschalleinwirkung abzutöten. Die Bedingungen, unter denen die Inaktivierung durchgeführt werden kann, sind dem Fachmann wohl bekannt. (K.H.Wallhäuser, H.Schmidt: Sterilisation, Desinfektion, Konservierung, Chemotherapie, Georg Thieme Verlag, Stuttgart [DEJ, 1967).To prepare the inactivated elicitorium-containing microorganisms they are grown under the usual conditions, then separated by centrifugation or filtration from the culture stage, washed if desired and again isolated. Different methods can be used to inactivate the microorganisms. Possible inactivation methods include acting on these microorganisms typical cell toxins such as ethylene oxides, formaldehyde, ozone, mercury compounds, organic solvents such as methanol, ethanol or acetone, or the microorganisms by heating at 90 ° to 140 ° by the action of extreme pressure differences (desintigration) , Exposure to high-frequency electrical field or by UV irradiation, irradiation miί γ-rays or ultrasound exposure kill. The conditions under which inactivation can be carried out are well known to those skilled in the art. (K.H.Wallhäuser, H. Schmidt: Sterilization, Disinfection, Conservation, Chemotherapy, Georg Thieme Verlag, Stuttgart [DEJ, 1967).
Fragmente Elicitoren-haltiger Mikroorganismen kann man beispielsweise durch Lysieren der Mikroorganismen durch Einwirken von osmotischem Schock oder Temperaturschock, durch Autolyse der Mikroorganismen, durch Behandeln der ZeIiBn mit Ultraschall oder durch Zerreiben der Mikroorganismen mit Glasperlen, Glasstaub oder Quarzsand und anschließendos differenziertes Zentrifugieren erhalten (Hughes, D. E., Wimpenny, J. W. T., and Lloyd, D.: The disintegration of micro-organisms. In: Methods in Microbiology, Vo113, (Norn's, J. R., and Ribbons, D.W., eds.) pp. 1-54, Academic Press, New York, London, 1971). Aus diesen Zellfragmenten können beispielsweise durch Trypsin-Behandlung er reinigte Zellwandfraktionen dargestellt werden. Die bereits erwähnten und in den nachfolgenden Ausführungsbeispiolen verw ndeten Zellwandfraktionen wurden nach dem von Schleifer und Kandier (Arch. Mikrobiol. 57,1967,335-365) beschriebenen Verfahren hergestellt.Fragments of microorganisms containing elicitors can be obtained, for example, by lysing the microorganisms by the action of osmotic shock or temperature shock, by autolysis of the microorganisms, by sonication of the cells, or by trituration of the microorganisms with glass beads, glass dust or quartz sand, followed by differential centrifugation (Hughes, et al. DE, Wimpenny, JWT, and Lloyd, D .: The disintegration of micro-organisms., In: Methods in Microbiology, Vo113, (Norn's, JR, and Ribbons, DW, eds.) Pp. 1-54, Academic Press, New York, London, 1971). From these cell fragments, for example, by trypsin treatment he purified cell wall fractions can be displayed. The cell wall fractions already mentioned and disclosed in the following examples were prepared according to the method described by Schleifer and Kandier (Arch. Mikrobiol 57, 1967, 335-365).
Andererseits ist es aber auch möglich, aus wasserlöslichen Zellbestandteilen durch Ausfällen beispielsweise mit Ethanol oder Aceton Elicitorenhaltige Präzipitate herzustellen (Kocourek, J., and Ballou, C.E., J.Bacteriol. 100,1969,1175-1181). Exkretionen Elicitoren-haltiger Mikroorganismen sind aktiv abgegebene, durch Lyslalen Bleaky machen Extraktion mit überkritischen verflüssigten Gasen (z. B. Kohlendioxid) oder Sterilisation von Zollen erhaltene Zollbestandteilc ,vasserlösliche oder durch Abfiltrieren oder Abzentrifugieren der Organismen erhaltene Kulturbrühen. Diese können bei Bedarf weiter aufgereinigt werden, beispielsweise durch Extraktion lipophiler Substanzen, Absorption stark färbender Substanzen usw. Wie die bislang durchgeführten Versuche, die in den Ausführungsbeispielon näher beschrieben werden, zeigen, scheint das erfindungsgemäße Verfahren zur Steigerung von Enzym-Aktivitäten oder der Syntheseleistung von Mikroorganismen sehr vielseitig anwendbar zu sein. So konnte durch Zugabe von Zellwandpräparationen von in der Tabelle 2 aufgeführten Mikroorganismen die Farbstcffbildung des Streptomyces lividans (Actinorhodin, Prodigiosin), sowia die Farbstoffbildung Streptomyces ccelicolor, des Streptom/ces griceorubar, des Streptomyces latericius, des Streptomyces purpurascens und dos Streptomyces violascens stimuliert werden. Ferner war es möglich, die Bildung von ß-Lactam-Antibiotika des Streptomyces calvuligerus und oie Alkaloidsynthese von Claviceps paspali zu stimulieren. Eine derartige Steigerung der Syntheseleistung von Mikroorganismen kann man nicht nur mittels der Zellwandpräparationen von den in der Tabelle 2 aufgeführten Bakterien erzielen, sondern es ist auch zu erwarten, daß auch mittels Elicitoren-haltigen Pilzen und Hefen, wie sie in der Tabelle 1 aufgeführt sind, eine Steigerung von Enzym-Aktivitäte.i und der Syntheseleistung von Mikroorganismen erzielt wer 1en kann. Es gelang ferner mit Hilfe von Zellwandpräparationen von in der Tabelle 2 aufgeführten Mikroorganismen in Zellkulturen höherer Pflanzen, wie Eschscholtzia californica oder Rauvolfia serpentina, eine signifikante Steigerung der Alkaloidbildung zu erzielen.On the other hand, it is also possible to produce elicitor-containing precipitates from water-soluble cell constituents by precipitation, for example with ethanol or acetone (Kocourek, J., and Ballou, C.E., J. Bacteriol., 100, 196, 175, 81). Excretions of elicitor-containing microorganisms are actively released, by Lyslale Bleaky, extraction with supercritical liquefied gases (eg., Carbon dioxide) or sterilization of tar obtained Zollbestandteilc, water-soluble or obtained by filtering off or centrifuging the organisms culture broths. These can be further purified if necessary, for example, by extraction of lipophilic substances, absorption of strong coloring substances, etc. As the experiments carried out so far, which are described in more detail in the exemplary embodiment, show the method of the invention for increasing enzyme activities or the synthesis of Microorganisms to be very versatile. Thus, by adding cell wall preparations of microorganisms listed in Table 2, it was possible to stimulate the color formation of Streptomyces lividans (actinorhodin, prodigiosin), as well as the dye formation Streptomyces ccelicolor, the streptome / ces griceorubar, Streptomyces latericius, Streptomyces purpurascens and Streptomyces violascens. Furthermore, it was possible to stimulate the production of β-lactam antibiotics of Streptomyces calvuligerus and alkaloid synthesis of Claviceps paspali. Such an increase in the synthesis efficiency of microorganisms can be achieved not only by means of the cell wall preparations of the bacteria listed in Table 2, but it is also to be expected that also by means of elicitorium-containing fungi and yeasts, as shown in Table 1, an increase of enzyme activities and the synthesis performance of microorganisms can be achieved. It was also possible with the help of cell wall preparations of microorganisms listed in Table 2 in cell cultures of higher plants, such as Eschscholtzia californica or Rauvolfia serpentina, to achieve a significant increase in alkaloid formation.
Weiterhin gelang es mit Hilfe dieser Zellwandpräparationen, die Fähigkeit von Bacillus lentus, Steroide in der 1,2-Position zu dehydrieren und die Fähigkeit von Rhodoturulaglutinis 17-Ketosteroide selektiv zu reduzieren und die Fähigkeit von Penicillium raistrickii Steroide in iler 15-a-Position zu hydroxylieren, deutlich zu steigern. Erfolglos blieb bislang lediglich der Versuch, mit Hilfe dieeer Zeilwandpräparation die Fähigkeit von Curvularia lunata zur 11-ß-Hydroxylierung von Steroiden zu steigern. Diese Versuche lassen die Hoffnung berechtigt erscheinen, daß es mit Hilfe des erfindungsgernäßen Verfahrens möglich sein wird, auch die Bildung zahlreicher anderer gewerbsmäßig nutzbarer mikrobieller Inhaltsstoff') zu stimulieren und v/eitere Enzymaktivitäten von Mikroorganismen, die technisch nutzbar sind, zu steigern.Furthermore, with the help of these cell wall preparations, Bacillus lentus' ability to dehydrate steroids at the 1,2-position and the ability of rhodoturulaglutinis to selectively reduce 17-ketosteroids and the ability of Penicillium raistrickii steroids in the iler 15-a position hydroxylate, significantly increase. To date, there has been no attempt to increase the ability of Curvularia lunata to 11-ß-hydroxylate steroids with the help of this cell wall preparation. These attempts give hope for the hope that it will be possible with the aid of the process according to the invention to also stimulate the formation of numerous other industrially useful microbial constituents and to increase further enzyme activities of microorganisms which are technically utilizable.
Derartige mikrobielle Inhaltsstoffe sind beispielsweise Antibiotika, wie die Penicilline, Cephalosporine, Cyclosporine, Actinomycine, Gramicidine, Neomycine, Gentamycine, Nystatine, Tetracycline, Nikomycine oder das Lincomycin, das Erythromycin, das Chloramphenicol, das Griseofulvin oder die Fusidinsäure u.a.. Mutterkornalkaloide, wie das Ergocryptin, das Ergotamin, das Ergosin, das Ergocristin, das Ergocomin, das Argroclavin, das Chanoclavin, das Festuclavin, die Paspalinsäure oder die Lysergsäure-Derivate, Vitamine, wie das Vitamin B12, das Riboflavin oder las ß-Carotin, Enzyme wie die Amylasen, Glycoseisomeraocn, Proteasen, Pectinasen, Lipasen, Penicillinacylusen oder die Lgctase, Nucleoside, wie die Guanyisäure oder Inosylsäure oder beispielsweise auch Aminosäuren, wie das Cystein, die Glutaminsäure, das Tryptophan, oder das Lysin. Mikroorganismen, die wegen ihrer Enzymaktivitäten technisch angewendet werden, sind oeispielswei'ie solche, die Steroidtransformationen wie die 11-a-, 11-ß- oder 15-a-Hydroxylierung, die Δ'-Dehydrierung, 17-a, 17 ß-Ketoreduktionen oder den Seitenkettenabbau von Sterinen oder Antibiotikatransformationen, wie Penicillinspaltung, bew'rken. Es ist nicht unwahrscheinlich, daß pc ι nit Hilfe des erfindungsgemäßen Verfahrens gelingen könn'd, neue technisch verwertbare mikrobielle Inhaltsstoffe, wie zum Beispiel neuo Antibiotika aufzufinden, indem man den zu tecienden Mikroorganismen inaktive Elicitoren-haltige Mikroorganismen zusetzt. Diese Hoffnung isi deshalb nicht unbegründet, weil bekannt ist, daß zahlreiche höhere Pflanzen Phytoaloxine nur dann in nennenswerten Mengen bilden, wenn sie mit Elicitoren-haltigen Mikroorganismen infiziert sind.Such microbial ingredients are, for example, antibiotics such as penicillins, cephalosporins, cyclosporins, actinomycins, gramicidines, neomycins, gentamycins, nystatins, tetracyclines, nicomycins or lincomycin, erythromycin, chloramphenicol, griseofulvin or fusidic acid, etc. Ergot alkaloids, such as ergocryptine , Ergotamine, Ergosin, Ergocristin, Ergocomin, Argroclavin, Chanoclavin, Festuclavin, Paspalic Acid or Lysergic Acid Derivatives, Vitamins like Vitamin B12, Riboflavin or las ß-Carotene, Enzymes like Amylases, Glycose isomeraocn, proteases, pectinases, lipases, penicillin acyls or the Lgctase, nucleosides, such as guanyic or inosilicic or, for example, amino acids, such as cysteine, glutamic acid, tryptophan, or lysine. Microorganisms that are used industrially for their enzymatic activities are, for example, those which include steroid transformations such as 11-a, 11-β or 15-a hydroxylation, Δ'-dehydrogenation, 17-α, 17β-ketoreductions or promote side chain degradation of sterols or antibiotic transformations such as penicillin cleavage. It is not unlikely that pc ι nit by means of the method according to the invention könn'd to find new technically useful microbial ingredients such as neuo antibiotics by adding to the tecienden microorganisms inactive elicitors-containing microorganisms. This hope is not unfounded because it is known that many higher plants only form phytoaloxins in significant amounts when they are infected with elicitor-containing microorganisms.
Erfindungsgemäß können inaktivierte Elicitoren-haltige Bakterien, Fragmente derselben oder Exkretionen Elicitoren-haltiger Bakterien auch zur Steigerung von Enzym-Aktivitäten und der Synthesoleistung in Geweben, Gewebekulturen, Zellkulturen höherer Organismen wie Pflanzen, Tieren oder des Menschen angewendet werden.In the present invention, inactivated elicitor-containing bacteria, fragments thereof, or excretions of elicitor-containing bacteria can also be used to enhance enzyme activities and synthesis performance in tissues, tissue cultures, cell cultures of higher organisms such as plants, animals or humans.
Es wurde be1, eits erwähnt, daß die Anwendung von enzymfreien Elicitoren-haltigem Material der Bakterien zur Steigerung der Leistung zur Synthese von Inhaltsstoffen höherer Pflanzen exp irimentell belegt werden konnte; dies könnte eventuell für die Anwendung pflanzlicher Zellkulturen zur Herstellung von Arzneimittelwirkstoffen von Bedeutung sein (M.H.Zenk in: Pharmazie heute, 103,1982, Band 3, 131-138). Grundsätzlich scheint es auch denkbar, daß Elicitoren-haltiges Material von Bakterien auch zur Steigerung der Leistung zur Syruhese von Inhaltsstoffen in Kulturen tierischer oder menschlicher Gewebe oder Zellen dienen könnte oder in der Therapie - beispielsweise bei Wundbehandlungen - Anwendung finden könnte.It has be 1, EITS noted that the use of enzyme-free elicitor-containing material of the bacteria to increase the performance for the synthesis of substances of higher plants could be irimentell is exp; this could possibly be of importance for the use of plant cell cultures for the preparation of active pharmaceutical ingredients (MHZenk in: Pharmazie heute, 103, 1982, Volume 3, 131-138). Basically, it also seems conceivable that elicitorial-containing material from bacteria could also serve to increase the performance of syruhese of ingredients in cultures of animal or human tissue or cells or in the therapy - for example, in wound treatments - application.
Ausführungsbeispieleembodiments
Die nachfolgenden Ausführungsbeispiele dienen zur näheren Erläuterung der Erfindung.The following embodiments serve to illustrate the invention.
Baispiel 1Example 1
Stimulation der Synthese von gefärbten Inhaltsstoffen (Actinorhodin, Piodigiosin) bei Streptomycos lividans (ATCC 19844) durch Zellwandpräparate.Stimulation of the synthesis of colored ingredients (actinorhodin, piodigiosin) in Streptomycos lividans (ATCC 19844) by cell wall preparations.
80ml eines Nährmediums, bestehend aus80ml of a nutrient medium consisting of
103g Saccharose 10g Glucose103g sucrose 10g glucose
10,12 g Magnesiumchlorid-Hexahydrat 0,25 g Kaliumsulfat10.12 g magnesium chloride hexahydrate 0.25 g potassium sulfate
0,1 g CasaminoacidslDifco Labs, Detroit/USA) 800 ml destilliertes Wasser0.1 g Casaminoacids / Difco Labs, Detroit / USA) 800 ml of distilled water
werden sterilisiert (20 Minuten, 12O0C) und mit folgenden frisch bereiteten Lösungen steril supplemented.are sterilized (20 minutes, 12O 0 C) and sterile supplemented with the following freshly prepared solutions.
1 ml 8rnl 1,5ml 10 ml 0,2m!1 ml 8rnl 1.5ml 10ml 0.2m!
0,5%igerKaliumdihydrogen-Phosphat-Lösung0.5% igerKaliumdihydrogen phosphate solution
20%igerL Prolin-Lösung 5,73%igerTES-Puffer-Lösung(pH7,2) Spurenelementen-Lösung-enthaltend pro Liter 40mgZink(ll)-chlorid 200 mg Eisen(lll)-chlorid-Hexahydrat 10mgKupfer(ll)-chlorid-Dihydrat 10mgMangan(ll)-chlorid-Tetrahydrat 10 mg Dinatriumtetraborat-Pihydrat lOmgHexaammonium-heptamolybdat-Tetrahydrat 1 N-Natronlauge20% Proline solution 5.73% TES buffer solution (pH 7.2) Trace element solution containing per liter 40 mg zinc (II) chloride 200 mg iron (III) chloride hexahydrate 10 mg copper (II) chloride Dihydrate 10 mg Manganese (II) chloride tetrahydrate 10 mg Disodium tetraborate pihydrate 10 mg Hexaammonium heptamolybdate tetrahydrate 1 N sodium hydroxide solution
Je 1,8ml dieser Nährlösung werden steril in die 24 jeweils 3ml fassenden Kammern einer Polystyrol-Multischale (Multidish, Fa. Nunc, 6200 Wiesbaden 12) eingebracht. Je 2 mg derauf Eücitor-Gehalt zu testenden Zellwandpräparate werden in bidestilliertem Wasser 20 Minuten bei 1200C sterilisiert und die erhaltenen Suspensionen den Kammern zugesetzt. 2 Kammern erhalten keine Zusätze, sie dienen als Kontrollen. In allen Kammern wird das Volumen einheitlich mit bidestilliertem Wasser steril auf 2 ml eingestellt. Jede Kammer wird identisch mit 5^l einer Sporensuspension von Streptomyces lividans (ATCC 19844) steril beimpft. Die Inkubation des Versuchsansatzes erfolgt aerob (Tablarschüttler; 100 Umdrehungen pro Minute) bei 26°C. Nach 96 Stunden zentrifugiert man die Zellen ab, wäscht sie mit physiologischer Kochsalzlösung, trocknet sie im Vakuum über Kalziumchlorid und erhält so die in der Tabelle aufgeführten Zellausbeuten. Die bei de Zenlrifugation erhaltenen Überstände werden auf einen pH-Wert von 7 eingestellt, auf 4ml mit Wasser verdünnt und ihre Absorptionspektren zwischen 180 und 800nm ermittelt. Die relativen Mengen der synthetisierte gelösten Sekundärstoffe Actinorhodin und Prodigiosin werden näherungsweise durch Wiegen der Absorptionsgipfel der automatisch aufgezeichneten Spektren ermittelt. Die nachfolgende Tabelle 3 zeigt die in dieser Versuchsreihe erzielten Ergebnisse.Each 1.8 ml of this nutrient solution are sterile in the 24 each 3ml chambers of a polystyrene multi-shell (Multidish, Fa. Nunc, 6200 Wiesbaden 12) introduced. Each 2 mg derauf Eücitor content to be tested cell wall preparations are sterilized in double-distilled water for 20 minutes at 120 0 C and the resulting suspensions were added to the chambers. 2 chambers receive no additives, they serve as controls. In all chambers, the volume is adjusted uniformly with bidistilled water sterile to 2 ml. Each well is inoculated sterile with 5 μl of a spore suspension of Streptomyces lividans (ATCC 19844) sterile. The incubation of the experimental batch is carried out aerobically (tablet shaker, 100 revolutions per minute) at 26 ° C. After 96 hours, the cells are centrifuged off, washed with physiological saline, dried in vacuo over calcium chloride to obtain the cell yields listed in the table. The supernatants obtained in de Zenlrifugation be adjusted to a pH of 7, diluted to 4ml with water and their absorption spectra between 180 and 800nm determined. The relative amounts of the synthesized solutes actinorhodin and prodigiosin are approximated by weighing the absorption peaks of the automatically recorded spectra. Table 3 below shows the results obtained in this series of experiments.
Getestete Bakterienzellwände vonTested bacterial cell walls of
Trockenzellausbeute (mg)Dry Cell Yield (mg)
Farbstoffgehaltdye content
rel. Absorptionseinheitenrel. absorbance units
ohne(Kontiolle) B. ammoniagenes (ATCC 6872) B. glutamingenes (ATCC 13747) B. pumilus (ATCC 7061)without (schedule) B. ammoniagenes (ATCC 6872) B. glutamigenes (ATCC 13747) B. pumilus (ATCC 7061)
B. linens (ATCC 19391)Linens (ATCC 19391)
C. diphtheriao (Mass. 8)C. diphtheriao (measure 8)
C. melassecola (ATCC 17965) C. glutamicum (ATCC 13032) C. IiIiUm(ATCC 15990) Ce.cellasea (ATCC 14359) L.plantarum(DSM20174) S.aureusStammHC. melassecola (ATCC 17965) C. glutamicum (ATCC 13032) C.IiIum (ATCC 15990) Ce.cellasea (ATCC 14359) L.plantarum (DSM20174) S.aureus strain H
22 25 32 34 23 24 26 43 33 36 32 4422 25 32 34 23 24 26 43 33 36 32 44
38 2438 24
34 34 50 4134 34 50 41
2 312 31
G = Brevibacterium C = Corynebacterium Ce= Cellulomonas L = Lactobacillus S = StaphylococcusG = Brevibacterium C = Corynebacterium Ce = Cellulomonas L = Lactobacillus S = Staphylococcus
Stimulation der Synthese von gefärbten Inhaltsstoffen (Actinorhodin, Prodigiosin) bei Sxreptomyces lividans (ATCC 19844) durch Zellwandextrakte.Stimulation of the synthesis of colored ingredients (actinorhodin, prodigiosin) in Sxreptomyces lividans (ATCC 19844) by cell wall extracts.
Unter den Bedingungen des Beispieles 1, jedoch unter Verwendung der Sterilfiltrate der in bidestilliertem Wasser 20 Minuten bei 12O0C sterilisierten 2 mg Zellwandpräparate, wird eine fast gleich starke Stimulation der Farbstoffbildung bei Streptomyces lividans (ATCC 19844) erzielt, wie bei der Verwendung von Suspensionen dieser sterilisierten Zellwände.Under the conditions of Example 1, but using the sterile filtrate of the 2 mg cell wall preparations sterilized in double distilled water for 20 minutes at 12O 0 C, an almost equally strong stimulation of dye formation in Streptomyces lividans (ATCC 19844) is achieved, as with the use of suspensions of these sterilized cell walls.
Stimulation der Synthese von gefärbten Inhaltsstoffen (Actinorhodin, Prodigiosin) bei Streptomyces lividans (ATCC 19844) durch Zellextrakte.Stimulation of the synthesis of colored ingredients (actinorhodin, prodigiosin) in Streptomyces lividans (ATCC 19844) by cell extracts.
Unter den Bedingungen des Beispiels 2, jedoch unter Verwendung von je 20 mg Zellmasse anstelle von 2 mg Zellwandpräparat, wird eine etwa gleich starke Stimulation der Farbstoff bildung erzielt wie bei der Verwendung von Suspensionen der sterilisierten Zellwände.Under the conditions of Example 2, but using each 20 mg cell mass instead of 2 mg cell wall preparation, an approximately equal stimulation of dye formation is achieved as in the use of suspensions of the sterilized cell walls.
Stimulation der Synthese von gefärbten Inhaltsstoffen bei Slreptomyces coelicolor (ATCC 13405).Stimulation of the synthesis of colored ingredients in Slreptomyces coelicolor (ATCC 13405).
'Unter den Bedingungen des Beispiels 1, jedoch unter Verwendung von Streptomyces coelicolor (ATCC 13405), erzielt man bei diesem Baktorium ebenfalls eine signifikante Steigerung der Farbstoffbildung (vermutlich ebenfalls Actinorhodin).Under the conditions of Example 1 but using Streptomyces coelicolor (ATCC 13405), this bacterium also achieves a significant increase in dye formation (presumably also actinorhodin).
Stimulation der Synthese von gefärbten Inhaltsstoffen bei Streptomyces griseoruber (DSM 40275).Stimulation of the synthesis of colored ingredients in Streptomyces griseoruber (DSM 40275).
Unter den Bedingungen des Beispiels 1, jedoch unter Verwendung von Streptomyces griseoruber (DSM 40275), erzielt man bei diesem Bakterium ebenfalls eine sehr deutliche Steigerung der Farbstoffbildung (vermutlich Anthracyclin-Antibiotika).Under the conditions of Example 1, but using Streptomyces griseoruber (DSM 40275), this bacterium also achieves a very marked increase in dye formation (presumably anthracycline antibiotics).
Stimulation der Synthese gefäibter Inhaltsstoffe bei Streptomyces purpurascens (DSM 40310).Stimulation of the synthesis of faked ingredients in Streptomyces purpurascens (DSM 40310).
Unter den Bedingungen des Beispiels 1, jedoch unter Verwendung von Streptomyces purpurascens (DSM 40310), erzielt man bei diesem Bakterium ebenfalls eine starke Steigerung der Farbstoffbildung (vermutlich ebenfalls Anthracyclin-Antibiotika).Under the conditions of Example 1, but using Streptomyces purpurascens (DSM 40310), this bacterium also produces a large increase in dye formation (presumably also anthracycline antibiotics).
Stimulation der Synthese gefärbter Inhaltsstoffe bei Streptomyces latericius (DSM 40163).Stimulation of the synthesis of colored ingredients in Streptomyces latericius (DSM 40163).
Unter den Bedingungendes Beispiels 1, jedoch unter Verwendung von Streptomyces latericius (DSM 40163). erzielt man bei diesem Bakterium ebenfalls eine signifikante Steigerung der Farbstoffbildung.Under the conditions of Example 1, but using Streptomyces latericius (DSM 40163). This bacterium also achieves a significant increase in dye formation.
Stimulation der Synthese gefärbter Inhaltsstoffe bei Streptomyces violascens (DSM 40082).Stimulation of the synthesis of colored ingredients in Streptomyces violascens (DSM 40082).
Unter d#en Bedingungen des Beispiels 1, jedoch unter Verwendung von Streptomyces violascens (DSM 40062), erzielt man bei diesem Bakterium ebenfalls eine signifikante Steigerung der Farbstoffbildung.Under d # s conditions of Example 1, but using Streptomyces violascens (DSM 40062), one also achieves a significant increase in dye formation in this bacterium.
Stimulation der Bildung von ß-Lactam-Antibiotika (Cephalosporine, Penicillin N) durch Streptomyces clavuligerus (ATCC 27064).Stimulation of the formation of β-lactam antibiotics (cephalosporins, penicillin N) by Streptomyces clavuligerus (ATCC 27064).
5g 3-(N-Morpholino)-propansulfonsäure (MOPS)5g 3- (N-morpholino) -propanesulfonic acid (MOPS)
3,5g Dikaliumhydrogenphosphat3.5 g of dipotassium hydrogen phosphate
0,6g Magnesiumsulfat-Heptahydrat0.6 g of magnesium sulfate heptahydrate
2g L-Asparagin2g L-asparagine
10g Glycerin10g glycerin
1 g Hefeextrakt (Oxid, Wesel, DE)1 g of yeast extract (Oxide, Wesel, DE)
1 ml Spurenelementsalz-Lösung - enthaltend pro Liter1 ml of trace element salt solution - containing per liter
1g Eisen(ll)-sulfat-Heptahydrat1 g of iron (II) sulfate heptahydrate
1 g Mangan(ll)-chlorid-Tetrahydrat1 g of manganese (II) chloride tetrahydrate
1 g Zinkchlorid-Heptahydrat1 g of zinc chloride heptahydrate
Iq KaliumchloridIq potassium chloride
werden mit destilliertem Wasser auf 1 Liter aufgefüllt und sterilisiert (20 Minuten; 1200C).are made up to 1 liter with distilled water and sterilized (20 minutes, 120 ° C.).
Je 1,8ml dieser Ni.nrlösung werden steril in 3ml fassende Kammern einer sterilen Polystyrol-Multischale (Multidish; Fa. Nunc, 62 Wiesbaden" 12) eingebracht. Je 2 mg der auf Elicilor-Aktivität zu testenden Zellwandpräparate oder 10mg der zu testenden Zellen >verjen als in bidesiilli«vtem Wasser sterilisierte (20 bzw. 45 Minuten 12O0C) homogene Suspensionen zugesetzt. Zwei Kammern, die als Kontrolle dienen, erhalten keine Zusätze. In allen Kammern wird sodann das Volumen einheitlich mit bidestilliertem Wasser steril auf 2 ml eingestell:. Jede Kammer wird identisch mit 5 μΙ einer Sporensusoension vo ι Streptomyces clavuligerus (ATCC 27064) beimpft.1.8 ml of this Ni.nr solution are introduced into sterile 3 ml chambers of a sterile polystyrene multiwell (Multidish, Nunc, 62 Wiesbaden, 12), 2 mg each of the cell wall preparations to be tested for Elicilor activity or 10 mg of the cells to be tested > ve r jen than in bidesiilli "VTEM water sterilized (20 and 45 minutes, 12O 0 C) homogeneous suspensions added. Two chambers that serve as controls, received no supplements. then the volume is uniformly sterile double-distilled water in all chambers 2 ml: Each chamber is inoculated identically with 5 μl of a spore suspension of Streptomyces clavuligerus (ATCC 27064).
Die Inkubation der Versuchsreihe wird unter aeroben Bedingungen (Tablarschüttler; 160 Umdrehungen pro Min>ite) bei 260C vorgenommen. Die Inkubationszeit beträgt 24-48 Stunden.The incubation of the test series is under aerobic conditions (Tablarschüttler; 160 revolutions per min> ite) carried out at 26 0 C. The incubation period is 24-48 hours.
Die Antibiotikumbildung wird im Vergleich mit den Kontrollen mit Hilfe des Plattendiffusionstests überprüft. Die in Soft-Nähragar suspendierten Detektororganismen sind Miciococcus luteus bzw. Bacillus subtilis (106 Zellen pro ml). Auf Standardfillerplättchen (0,9cm Durchmesser) werdon jeweils 25μΙ zentrifugierte (48000 χ g) Kulturbrühe aus den Testkammern appliziert. Nach einer Diffusionszeit von 4 Stunden bei 4°C wird der Biotest für 24 Stunden bei 3O0C inkubiert.The antibiotic formation is checked in comparison with the controls using the plate diffusion test. The detector organisms suspended in soft nutrient agar are Miciococcus luteus and Bacillus subtilis, respectively (10 6 cells per ml). On standard filler plates (0.9 cm in diameter), 25 μΙ of centrifuged (48,000 × g) culture broth are applied from the test chambers in each case. After a diffusion time of 4 hours at 4 ° C, the bioassay for 24 hours at 3O 0 C is incubated.
Bei den Kulturen, die unier Zusatz abgetöteter Zellen von Brevibacterium flavum ATCC 13826, oder von Zeilwandpräparationen dieses Bakteriums beziehungsweise von Zeilwandpräparationen von Corynebacterium diphtheriae (Stamm Mass. 8) angezüchtet wurden, zeigten eindeutig vergrößerte Hemmhöfe im Vergleich zu den Kontrollen eine vermehrte Bildung von ß-Lactam-Antibiotika (Penicillin N, Cephalosporine) durch Streptomyces clavuligerus an.In the cultures grown with the addition of killed cells of Brevibacterium flavum ATCC 13826, or of cell wall preparations of this bacterium or of cell wall preparations of Corynebacterium diphtheriae (strain Mass. 8), clearly increased inhibition sites showed an increased formation of β- compared to the controls. Lactam antibiotics (penicillin N, cephalosporins) by Streptomyces clavuligerus.
Stimulation der Bildung von Alkaloiden (Sanguinarin, Chelirubin, Marcarpin und Chelerythrin) durch Kulturen von Eschscholtzia californica.Stimulation of the formation of alkaloids (sanguinarine, chelirubin, marcarpin and chelerythrine) by cultures of Eschscholtzia californica.
In 241-ml-Kammern einer Polystyrol-Multischale (Fa. Nunc, 6200 Wiesbaden 12) werden unter den von J.Berlin et. al.In 241 ml chambers of a polystyrene multi-shell (Nunc, 6200 Wiesbaden 12) are under the J. Berlin et. al.
(Z. Naturforsch., 38c, 1983,346-352) als optimal beschriebenen Bedingungen jeweils Gewebekulturen von Eb'hscholtzia californica angezüchtet. Eine der Kammern bleibt ohne weiteren Zusatz als Kontrolle, eine Kammer erhält 266ipn/| hitzeextrahiorten ur.d ethanolgefällten Hefeelicitor (hergestellt nach Kocourek, J., and Ballou, C.E., J.Bucieriol 100,1969,1175-(Z. Naturforsch., 38c, 1983, 344-352) cultivated tissue cultures of Eb'hscholtzia californica as optimally described conditions. One of the chambers remains without further addition as control, a chamber receives 266ipn / | ethanol-precipitated yeast elicitor (prepared according to Kocourek, J., and Ballou, C.E., J. Bucieriol 100, 969, 175-
1181) die übrigen je 266mg/l Zellwandpräparat der Bakterien, die in Tabelle 3 aufgeführt sind. Dann werden die Kulturen 72 Stunden lang bei 240C inkubiert und anschließend der Alkaloidgehalt der Kulturen photometrisch ermittelt, wobei der durch den Hefeelicitor induzierte Alkaloidgehalt als 100% gewertot wird.1181) the remaining 266 mg / l cell wall preparation of the bacteria listed in Table 3. The cultures are then incubated for 72 hours at 24 0 C and then determined the alkaloid content of the cultures photometrically, wherein the induced by the yeast elicitor alkaloid content is rated as 100%.
Die nachfolgende Tabelle 4 zeigt die in dieser Versuchsreihe erzielten Ergebnisse.Table 4 below shows the results obtained in this series of experiments.
Getestete Bakterienzellwände % ElicitoraktivitätTested bacterial cell walls% elicitor activity
B'evi >acteriumbutanicumATCC21196 19B'evi> acteriumbutanicum ATCC21196 19
Biev' lcteriumflavumATCC 13826 16Biev 'lcterium flavum ATCC 13826 16
BrevibüCteriumflavum ATCC 14067 30Brevibücteriumflavum ATCC 14067 30
Brevibacteriumglutamingenes ATCC137 113Brevibacterium glutamigenes ATCC137 113
Brevibacterium lactofermentum ATCC13655 43Brevibacterium lactofermentum ATCC13655 43
Brevibacterium ammoniagenes ATCC 6872 40Brevibacterium ammoniagenes ATCC 6872 40
. Corynebaoteriumhydrocarboclastum ATCC15592 8, Corynebaoterium hydrocarboclastum ATCC15592 8
Corynebacterium nephridii ATCC11425 123Corynebacterium nephridii ATCC11425 123
Corynebacteriumpaurometabolum ATCC 8368 16Corynebacterium paurometabolum ATCC 8368 16
Corynebacteriunlilium ATCC15990 108Corynebacteriunlilium ATCC15990 108
Corvnebacteriurr. stnatum ATCC 6940 17Corvnebacteriurr. stnatum ATCC 6940 17
Corynebacterium petrophilum ATCC19030 0Corynebacterium petrophilum ATCC19030 0
Corynebacterium xerosis ATCC 373 102Corynebacterium xerosis ATCC 373 102
Corynebacterium diphtheriae Stamm Mass. 8 137Corynebacterium diphtheriae strain Mass. 8 137
Rhodococcusfasciens ATCC12975 27Rhodococcusfasciens ATCC12975 27
Rhodococcusfasciansi 11 Isolat von Prof. Dr. Stolp, Univ. BayreuthRhodococcusfasciansi 11 Isolate of Prof. Dr. med. Stolp, Univ. Bayreuth
Rhodococcusfascians2 7 Isolat von Prof. Dr. Stolp, Univ. BayreuthRhodococcusfascians2 7 Isolate of Prof. Dr. med. Stolp, Univ. Bayreuth
Stimulierung der Bildung von Indolalkaloiden (Valiesiacotamin) in Kulturen von Rauvolfia serpentina.Stimulation of the formation of indole alkaloids (valiesiacotamine) in cultures of Rauvolfia serpentina.
Die Suspensionskultur von Rauvolfia serpentina (Stöckigt, J., A.Pfitznerand J.Firl: Plant Cell Rep. 1,36-39 [19811) wird in Linsmaier und Skoog (LS)-Medium (Physiol. Plantarum 18,100-127 [1965]) auf Rotationsschüttlern (100 Umdrehungen pro Minute) bei 23°C und Daueriicht (600 Lu>:) kultiviert. Zur Elicitierung werden 200g Zellfrischgewicht/I LS-Medium angeimpft. Als Elicitoren-haltige Fragmente von Mikroorganismen werden Zellwandp.äparate der in Tabelle 4 aufgeführten Mikroorganismen in einer Konzentration von 130mg/l Medium verwendet.The suspension culture of Rauvolfia serpentina (Stöckigt, J., A.Pfitznerand J.Firl: Plant Cell Rep. 1,36-39 [19811] is used in Linsmaier and Skoog (LS) medium (Physiol Plantarum 18, 1-127 [1965]). ) on rotary shakers (100 revolutions per minute) at 23 ° C and Daueriicht (600 Lu> :) cultivated. For elicitation 200g fresh cell weight / I LS medium are inoculated. As Elicitoren-containing fragments of microorganisms Zellwandp.äparate the microorganisms listed in Table 4 are used in a concentration of 130mg / l medium.
Nach einer Inkubation von 5 Tagen hat sich sowohl in den elicitierten Kulturen als auch in den Kontrollen die Zellmasse verdoppelt. Die Zellen werden geerntet und mit Methanol extrahiert.After incubation for 5 days, the cell mass doubled in both the elicited cultures and controls. The cells are harvested and extracted with methanol.
Die Menge des Indolalkaloide Valiesiacotamin wird über eine HPLC-Auftrennung der Extrakte bestimmt. Während die unbehandelten Kontrollkulturen nur 1,16mg/l Medium enthalten, beträgt die Ausbeute bei den elicitierten Kulturen maximal 56ml/l. Dies entspricht einer Steigerung um das 50fache durch den Elicitor.The amount of indole alkaloid valiesiacotamine is determined by HPLC separation of the extracts. While the untreated control cultures contain only 1.16 mg / l medium, the yield in the elicited cultures is a maximum of 56 ml / l. This corresponds to an increase of 50 times by the Elicitor.
Stimulierung der 17-Ketosteroid-Reduktase-Aktivität von Rhodotorula glutinis IFO 0389.Stimulation of 17-ketosteroid reductase activity of Rhodotorula glutinis IFO 0389.
a) Ein 2-l-Erlenmeyerkolben mit 500ml sterilem Nährmedium, enthaltend 5% Glucose-Monohydrata) A 2-liter Erlenmeyer flask containing 500 ml of sterile nutrient medium containing 5% glucose monohydrate
2% Cornsteep liquor2% Cornsteep liquor
- eingestellt auf pH 6,5 -,- adjusted to pH 6.5 -,
wird mit einem Abstrich aus einer Schrägagarkultur von Rhodotorula glutinis IFO 0389 beimpft und 40 Stunden lang bei 3O0C mit 190 Umdrehungen pro Minute angezüchtet.is inoculated with a swab from a slant culture of Rhodotorula glutinis IFO 0389 and cultured for 40 hours at 3O 0 C with 190 revolutions per minute.
b) Ein 500-ml-Erlenmeyerkolben mit 100ml sterilem Nährmedium, enthaltend 1 % Cornsteep liquorb) A 500 ml Erlenmeyer flask containing 100 ml of sterile nutrient medium containing 1% Cornsteep liquor
5% Nurupan"" (Hersteller Nurupan GmbH, 4000 Düsseldorf 1; DE) 1 % Metarin"11 (Hersteller Lucas Meyer; 2000 Hamburg 28; DE)5% Nurupan "" (manufacturer Nurupan GmbH, 4000 Dusseldorf 1, DE) 1% Metarin " 11 (manufacturer Lucas Meyer; 2000 Hamburg 28; DE)
- eingestellt auf pH 6,2 -,- adjusted to pH 6.2 -,
wird mit 10ml der gemäß Beispiel 13a hergestellten Rhodotorula-Vorkultur beimpft und 7 Stunden lang bei 300C und 180 Umdrehungen pro Minute angezüchtet.is inoculated with 10ml of the prepared according to Example 13a Rhodotorula preculture and grown for 7 hours at 30 0 C and 180 revolutions per minute.
Danach setzt man der Kultur 10mg 3-Hydroxy-1,3,5(10),7-estratetraen-17-on zu und fermentiert weitere 210 Stunden lang. Dann extrahiert man die Kultur mit Methylisobutylketon, engt den Extrakt ein und reinigt das erhaltene Rohprodukt durch Chromatographie über eine Kieselgelsäule. Man erhält so 5,9mg 1,3,5(10),7-Estratetraen-3,17a-diol = 59% der Theorie.Thereafter, the culture is added with 10 mg of 3-hydroxy-1,3,5 (10), 7-estratetraene-17-one and fermented for a further 210 hours. Then, the culture is extracted with methyl isobutyl ketone, the extract is concentrated and the crude product obtained is purified by chromatography on a silica gel column. This gives 5.9 mg 1,3,5 (10), 7-estratetraene-3,17a-diol = 59% of theory.
c) Unter den Bedingungendes Beispiels 13 b werden 10 mg 3-Hydroxy-1,3,5(10),7-estratetraen-17-on mit einer Kultur von Rhodotorula glutinis fermentiert, jedoch mit dem Unterschied, daß man dieser Kultur unmittelbar vor der Substratzugabe 5 ml einer sterilen Suspension von 50 mg einer Zellwandpräparation von Bacillus licheniformis (ATCC 9945) in Wasser zusetzt. Nach Aufarbeitung der Kultur erhält man 6,8mg 1,3,5(10),7-Estratetraen-3,17-diol = 68% der Theorie.c) Under the conditions of Example 13b, 10 mg of 3-hydroxy-1,3,5 (10), 7-estratetraene-17-one are fermented with a culture of Rhodotorula glutinis, but with the difference that immediately before this culture the substrate addition 5 ml of a sterile suspension of 50 mg of a cell wall preparation of Bacillus licheniformis (ATCC 9945) in water. After work-up of the culture, 6.8 mg of 1,3,5 (10), 7-estratetraene-3,17-diol = 68% of theory are obtained.
Stimulierung der Steroid-A'-Dehydrase-Aktivität von Bacillus lentus (ATCC 13805).Stimulation of the steroid A'-dehydrase activity of Bacillus lentus (ATCC 13805).
a) Ein 2-l-Erlenmeyerkolben mit 500ml steriler Nährlösung, enthaltend 0,5% Cornsteep liquora) A 2-liter Erlenmeyer flask containing 500 ml of sterile nutrient solution containing 0.5% Cornsteep liquor
0,05 % Glucose-Monohydrat 0,1% Hefeextrakt0.05% glucose monohydrate 0.1% yeast extract
- eingestellt auf pH 7,0 -,- adjusted to pH 7.0 -,
wird mit einer Abschwemmung von Bacillus lentus (ATCC 13805) beimpft und 48 Stunden lang bei30°C mit 190 Umdrehungen pro Minute geschüttelt.is inoculated with a spill of Bacillus lentus (ATCC 13805) and shaken for 48 hours at 30 ° C at 190 revolutions per minute.
b) Ein 500-ml-Erlenmeyerkolben mit 100ml steriler Nährlösung, enthaltend 3,0% Sojapuderb) A 500 ml Erlenmeyer flask containing 100 ml of sterile nutrient solution containing 3.0% soya powder
0,5% Consteep liquor0.5% consteep liquor
0,1 % Hefeextrakt0.1% yeast extract
0,05 % Glucose-Monohydrat0.05% glucose monohydrate
- eingestellt auf /?H 7,3 -,- set to /? H 7.3 -,
wird mit 10ml der Bacillus lentus-Vorkultur beimpft und 7 Stunden lang bei 300C mit 180 Umdrehungen pro Minute geschüttelt. Dann .istzt man der Kultur eine sterilfiltrierte Lösung von 40mg 6a,9a-Difluor-11ß,17a-dihydroxy-16o.-methyl-4-pregnen-3,20-dion in 4ml Dimethylformamid zu und inkubiert weitere 41 Stunden lang.is inoculated with 10 ml of Bacillus lentus preculture and shaken for 7 hours at 30 0 C at 180 revolutions per minute. Then .istzt the culture a sterile-filtered solution of 40mg 6a, 9a-difluoro-11ß, 17a-dihydroxy-16o.-methyl-4-pregnene-3,20-dione in 4ml dimethylformamide and incubated for a further 41 hours.
Danp extrahiert man die Kultur mit Methylisobutylketon, engt den Extrakt im Vakuum ein und rainigt den Rückstand durch Chromatographie über eine Kieselgelsäule. Man erhält so lemgea.ga-Difluor-iiß.na-dihydroxy-iea-methyl-M-pregnadien-3,20-dion (= 40% der Theorie).The mixture is then extracted with methyl isobutyl ketone, concentrated in vacuo and the residue is purified by chromatography on a silica gel column. This gives lemgea.ga-difluoro-iis.na-dihydroxy-iea-methyl-M-pregnadiene-3,20-dione (= 40% of theory).
c) Unter den Bedingungen des Beispiels 14b werden 40mg 6a,9a-Difluor-11ß,17a-dihydroxy-16a-methyl-4-pregnen-3,20-dion mit einer Kultur von Bacillus lentus fermentiert, jedoch mit dem Unterschied, daß man dieser Kultur unmittelbar vor der Subsiratzugabe 5ml einer sterilen Suspension von 50mg Zellwandpräparation von Corynebacterium diphtheriae (Stamm Mass. 8) in Wasser zusetzt. Nach Aufarbeitung der Kultur erhält man 21mg 6a,9a-Difluor-11ß,17a-dihydroxy-16a-methyl-1,4-pregnadien-3,20-dion (= 52,5% der Theorie).c) Under the conditions of Example 14b, 40 mg of 6a, 9a-difluoro-11ß, 17a-dihydroxy-16a-methyl-4-pregnene-3,20-dione are fermented with a culture of Bacillus lentus, with the difference that 5 ml of a sterile suspension of 50 mg cell wall preparation of Corynebacterium diphtheriae (strain Mass. 8) in water is added to this culture immediately before the addition of the subsirium. After workup of the culture to obtain 21mg 6a, 9a-difluoro-11ß, 17a-dihydroxy-16a-methyl-1,4-pregnadiene-3,20-dione (= 52.5% of theory).
Stimulierung der Bildung von A'kaloiden (Lysergsäureamid und Isolysergsäureamid) von Claviceps paspali (ATCC 13895).Stimulation of the formation of alkaloids (lysergic acid amide and isolysergic acid amide) of Claviceps paspali (ATCC 13895).
a) Ein 500-ml-Erlenmeyerkolben mit 50ml einer sterilen Nährlösung, enthaltend 4 % Sorbit (technisch rein)a) A 500 ml Erlenmeyer flask containing 50 ml of a sterile nutrient solution containing 4% sorbitol (technically pure)
1 % Glucose-Monohydrat1% glucose monohydrate
2 % Bernsteinsäure 0,6% Aluminiumsulfat2% succinic acid 0.6% aluminum sulfate
0,5 % Hefeextrakt (DifcoIRI der Difco Labs. Detroit/USA) 0,1 % Kaliumdihydrogenphosphat, 0,03 % Magnesiumsulfat-Heptahydrat0.5% yeast extract (Difco IRI from Difco Labs, Detroit, USA) 0.1% potassium dihydrogen phosphate, 0.03% magnesium sulfate heptahydrate
- eingestellt mit Natronlauge auf pH 5,2 -,adjusted to pH 5.2 with sodium hydroxide solution,
wild mit einer auf - 700C tiefgefrorenen Kultur von Claviceps paspali (ATCC 13895) beimpft und 5 Tagelang bei 24°Cund 240 Umdrehungen pro Minute geschüttelt.with a wild at - 70 0 C frozen culture of Claviceps paspali (ATCC 13895) and shaken for 5 days at 24 ° C and 240 revolutions per minute.
b) Ein 500-ml-Erlenmeyerkolben mit 50ml einer sterilen Nährlösung, enthaltend 8 % Sorbit (technisch rein)b) A 500 ml Erlenmeyer flask containing 50 ml of a sterile nutrient solution containing 8% sorbitol (technically pure)
6% Bernsteinsäure6% succinic acid
0,9% Ammoniurnsulfat0.9% ammonium sulfate
0,1 % Calziumnitrat-Tetrahyd.-at0.1% calcium nitrate tetrahydrate
0,05% Dikaliumhydrogenphosphat0.05% dipotassium hydrogen phosphate
0,03 % Magnesiumsulfat-Heptahydrat0.03% magnesium sulfate heptahydrate
0,02 % Hefeextrakt (Difco(R) der Difco Labs., Detroit/USA)0.02% yeast extract (Difco (R) from Difco Labs., Detroit / USA)
0,0007 % EisendO-sulfat-Heptahydrat0.0007% ferric sulfate heptahydrate
0,0006 % Zinksulfat-Heptahydrat0.0006% zinc sulfate heptahydrate
- eingestellt mit Natronlauge auf pH 5,2 -,adjusted to pH 5.2 with sodium hydroxide solution,
wird mit 5 ml Vorkultur von Claviceps paspali beimpft und 250 Stunden lang bei 240C mit 240 Umdrehungen pro Minute geschüttelt.is inoculated with 5 ml preculture of Claviceps paspali and shaken for 250 hours at 24 0 C at 240 revolutions per minute.
Dann setzt man der Kultur soviel Natronlauge zu, daß ein pH-Wert von mindestens 10 erreicht ist, extrahiert mit Methylisobutylketon, engt die Extrakte im Vakuum ein und reinigt sie durch Chromatographie über eine Kieselgelsäule.Then the culture is added to enough sodium hydroxide solution to reach a pH of at least 10, extracted with methyl isobutyl ketone, the extracts concentrated in vacuo and purified by chromatography on a silica gel column.
Man erhält so 35mg eines Gemisches aus Lysergsäureamid und Isolysergsäureamid (Ausbeute 700mg/l Kultur).This gives 35 mg of a mixture of lysergic acid amide and isolysergic acid amide (yield 700 mg / l of culture).
c) Unter den Bedingungen der Beispiele 5b wird eine Kultur von Claviceps paspali angezüchtet, jedoch mit dem Unterschied, daß man der Kultur nach 72 Stunden 5ml einer sterilen Suspension von 25mg Zellwandpräparation von Lactobacillus casei subsp. rhamnosus (ATCC 7469) in Wasser zusetzt. Nach Aufarbeitung der Kultur erhält man 45mg eines Gemisches aus Lysergsäureamid und Isolysergsäureamid (Ausbeute 900my/I Kultur).c) Under the conditions of Example 5b, a culture of Claviceps paspali is grown, but with the difference that after 72 hours 5 ml of a sterile suspension of 25 mg cell wall preparation of Lactobacillus casei subsp. rhamnosus (ATCC 7469) in water. After working up the culture, 45 mg of a mixture of lysergic acid amide and isolysergic acid amide (yield 900my / l culture).
Stimulierung der 15-a-Hydroxylase-Aktivität von Penicillium raistrickii (ATCC 10490).Stimulation of Penicillium raistrickii 15α-hydroxylase activity (ATCC 10490).
a) Ein 2-l-Erlenmeysrkolben mit 500ml sterilem Nährmedium, enthaltend 3% Glucose-Monohydrat 1 % Cornsteep liquor 0,2% Natriumnitrat 0,05 % Magnesiumsulfat-Hcptahviirat 0,05% Kaliumchlorid 0,002 % EisendD-suifat-Hexahydrat 0,1 % Kaliumhydrogenphosphat 0,2% Oikaliumhydrogenphcsphata) A 2-L Erlenmeyer's flask containing 500 ml of sterile nutrient medium containing 3% glucose monohydrate 1% Cornsteep liquor 0.2% sodium nitrate 0.05% magnesium sulfate Hcptahviirat 0.05% potassium chloride 0.002% Iron end D-suifate hexahydrate 0.1 % Potassium hydrogen phosphate 0.2% ocalium hydrogenphosphate
- eingestellt auf pH 6.0 -,- adjusted to pH 6.0 -,
wird mit einem Abstrich aus einer Schrägagarkultur von Penicillium raistrickii (ATCC 10490) beimpft und 48 Stunden lang bei 300C mit 180 Umdrehungen pro Minute angezüchtet.is inoculated with a smear from a slants culture of Penicillium raistrickii (ATCC 10490) and grown for 48 hours at 30 0 C at 180 revolutions per minute.
b) Ein 500-ml-Eflenmeyerkolben mit 100ml sterilem Nährmedium, enthaltend 1 % Cornsteep liquor 3% Glucose-Monohydrat 0,1 % KaliumdihydrogenphOophat 0,2 % Dikaliumhydrogenphosphat 0,05 % Magnesiumsulfat-Heptahydratb) A 500 ml Eflenmeyer flask containing 100 ml of sterile nutrient medium containing 1% Cornsteep liquor 3% glucose monohydrate 0.1% potassium dihydrogen phosphate 0.2% dipotassium hydrogen phosphate 0.05% magnesium sulfate heptahydrate
- eingestellt auf pH 6,0 -,- adjusted to pH 6.0 -,
wird mit 10ml der gemäß a) hergestellten Penicillium-Vorkultur beimpft.is inoculated with 10ml of the prepared according to a) Penicillium preculture.
Danach setzt man der Kultur 300mg 13-Ethyl-4-gonen-3,17-dionzu und fermentiert 120 Stunden bei 30°C mit 180, Umdrehungen pro Minute.Thereafter, 300 mg of 13-ethyl-4-gene-3,17-dione are added to the culture and fermented at 30 ° C. for 120 hours at 180 rpm.
Dann extrahiert man die Kultur mit Methylisobutylketon, engt den Extrakt ein und reinigt das erhaltene Rohprodukt durch Chromatographie über eine Kieselgelsäule. Man erhält so 180mg IS-Ethyl-iöa-hydroxy^-gonen-S.n-dion.Then, the culture is extracted with methyl isobutyl ketone, the extract is concentrated and the crude product obtained is purified by chromatography on a silica gel column. This gives 180 mg of IS-ethyl-hydroxy-α-n-dione.
c) Unter den Bedingungen von b) werden 300mg 18-Methyl-norandrostendion mit einer Kultur von Penicillium raistrickii fermentiert, jedoch mit dem Unterschied, daß man dieser Kultur unmittelbar vor der Substratzugabe 5ml einer sterilen Suspension zugibt. Diese 5ml enthalten 50mg einer Zellwandpräparation von Corynebacterium diphteriae (Stamm Mass. 8) in Wasser. Nach Aufarbeitung der Kultur erhält man 210mg 13-Ethyl-15a-hydroxy-4-gonen-3,17-dion.c) under the conditions of b), fermenting 300 mg of 18-methyl-norandrostenedione with a culture of Penicillium raistrickii, but with the difference that 5 ml of a sterile suspension are added to this culture immediately before addition of the substrate. These 5ml contain 50mg of a cell wall preparation of Corynebacterium diphteriae (strain Mass. 8) in water. After working up the culture, 210 mg of 13-ethyl-15a-hydroxy-4-gonen-3,17-dione are obtained.
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Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA1259938A (en) * | 1985-12-05 | 1989-09-26 | Friedrich Constabel | Repeated elicitor treatment, a method for semicontinuous metabolite production by plant cells cultured in vitro |
-
1988
- 1988-01-13 DE DE3801023A patent/DE3801023A1/en not_active Withdrawn
-
1989
- 1989-01-11 EP EP89100337A patent/EP0325933B1/en not_active Expired - Lifetime
- 1989-01-11 DE DE89901715T patent/DE58907274D1/en not_active Expired - Lifetime
- 1989-01-11 AT AT89901715T patent/ATE103325T1/en not_active IP Right Cessation
- 1989-01-11 HU HU89975A patent/HU208341B/en not_active IP Right Cessation
- 1989-01-11 WO PCT/EP1989/000015 patent/WO1989006687A1/en active IP Right Grant
- 1989-01-11 JP JP1501513A patent/JPH02502881A/en active Pending
- 1989-01-11 DD DD32499389A patent/DD278357A5/en not_active IP Right Cessation
- 1989-01-11 AU AU32159/89A patent/AU3215989A/en not_active Abandoned
- 1989-01-11 EP EP89901715A patent/EP0354945B1/en not_active Expired - Lifetime
- 1989-01-11 ES ES89100337T patent/ES2051894T3/en not_active Expired - Lifetime
- 1989-01-12 CA CA000588110A patent/CA1317247C/en not_active Expired - Fee Related
- 1989-01-12 PT PT89427A patent/PT89427B/en not_active IP Right Cessation
- 1989-01-13 CN CN89100184A patent/CN1034756A/en active Pending
- 1989-01-13 IL IL8895389A patent/IL88953A/en not_active IP Right Cessation
- 1989-01-13 IE IE7989A patent/IE66500B1/en not_active IP Right Cessation
- 1989-01-13 ZA ZA89304A patent/ZA89304B/en unknown
- 1989-09-07 DK DK442289A patent/DK442289D0/en not_active Application Discontinuation
- 1989-09-12 FI FI894304A patent/FI97302C/en not_active IP Right Cessation
- 1989-09-12 NO NO89893643A patent/NO893643L/en unknown
- 1989-09-13 BG BG89739A patent/BG60596B1/en unknown
-
1992
- 1992-11-05 AU AU28183/92A patent/AU2818392A/en not_active Abandoned
Also Published As
Publication number | Publication date |
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NO893643D0 (en) | 1989-09-12 |
CA1317247C (en) | 1993-05-04 |
ES2051894T3 (en) | 1994-07-01 |
EP0325933A1 (en) | 1989-08-02 |
BG60596B1 (en) | 1995-09-29 |
JPH02502881A (en) | 1990-09-13 |
IL88953A0 (en) | 1989-08-15 |
NO893643L (en) | 1989-09-12 |
DK442289A (en) | 1989-09-07 |
CN1034756A (en) | 1989-08-16 |
HUT57270A (en) | 1991-11-28 |
EP0354945B1 (en) | 1994-03-23 |
EP0325933B1 (en) | 1994-03-09 |
ATE103325T1 (en) | 1994-04-15 |
IE890079L (en) | 1989-07-13 |
IE66500B1 (en) | 1996-01-10 |
FI894304A0 (en) | 1989-09-12 |
DE58907274D1 (en) | 1994-04-28 |
WO1989006687A1 (en) | 1989-07-27 |
AU2818392A (en) | 1993-01-14 |
ZA89304B (en) | 1989-10-25 |
FI97302C (en) | 1996-11-25 |
IL88953A (en) | 1995-03-30 |
HU208341B (en) | 1993-09-28 |
FI97302B (en) | 1996-08-15 |
DK442289D0 (en) | 1989-09-07 |
PT89427A (en) | 1990-02-08 |
AU3215989A (en) | 1989-08-11 |
BG89739A (en) | 1993-12-24 |
EP0354945A1 (en) | 1990-02-21 |
PT89427B (en) | 1993-09-30 |
DE3801023A1 (en) | 1989-07-27 |
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