DD278357A5 - Method of increasing enzyme activity and synthesis efficiency of organisms - Google Patents

Abstract

In the process disclosed, the organisms are brought into contact witn inactivated elicitor-containing microorganisms, fragments of the latter, or excretions of elicitor-containing microorganisms. Only inactivated elicitor-containing bacteria, fragments of the latter, or excretions of elicitor-containing bacteria may be used to activate enzymes and promote synthesis in non-microbial organisms. The process can be used, for example, to promote synthesis in microorganisms or plants which produce pigments, antibiotics, alkaloids or phytoalexins or to activate enzymes in microorganisms capable of transforming steroids.

Description

Field of application of the invention

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 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

 Fields of application are biotechnology, agriculture, pharmacy and medicine.

Characteristic of the known state of the art

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.

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).

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) ,

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.

Object of the invention

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.

Explanation of the essence of the invention

The present invention has for its object to develop a method for increasing enzyme activities and the synthesis performance of organisms.

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.

In addition, it has been found that there are also bacteria containing elicitors which are not enzymes or nutritional factors.

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.

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.

Microorganisms known to possess elicitors include the fungal strains and yeasts listed in Table I below:

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.

Table 1

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 Phomaexigua

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, 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

Table 2

Bacillus iicheniformis ATCC 9945 Brevibacterium butanicum ATCC 21196 Brevibacterium flavum ATCC 13826, ATCC 14067

Brevibacterium lactofermentum ATCC 13655

Brevibacterium glutamigenic ATCC 13747

Brevibacterium ammoniagenes ATCC 6872

Brevibacverium pumilus ATCC 7061

Corynebacterium hydrocarboclastum ATCC 15592

Corynebacterium nephridii ATCC 11425

Corynebacterium paurometabolum ATCC 8368

Corynebacterium lilium ATCC 15990

Corynebacterium striatum ATCC 6940

Corynebacterium xerosis ATCC 373

Corynebacterium diphtheriae (strain Mass. 8 / Behring Worke)

Corynebacterium molassecola ATCC 17965

Corynebacterium glutamicium ATCC 13032

Corynebacterium uratoxidans ATCC 21749

Lactobacillus casei subsp. rhamnosus ATCC 7469

Lactobacillus plantarum DSM 20174

PirnelobactertumescensAJ 1460

Rhodococcus fascians ATCC 12975

Rhodococcus fascians 1 - isolate of Prof. Dr. med. Stolp, Univ. Bayreuth Rhodococcus fascians 2 - sol. Stolp, Univ. Bayreuth

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.

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.

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 ,

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.

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.

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).

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).

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.

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.

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.

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.

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.

embodiments

The following embodiments serve to illustrate the invention.

Example 1

Stimulation of the synthesis of colored ingredients (actinorhodin, piodigiosin) in Streptomycos lividans (ATCC 19844) by cell wall preparations.

80ml of a nutrient medium consisting of

103g sucrose 10g glucose

10.12 g magnesium chloride hexahydrate 0.25 g potassium sulfate

0.1 g Casaminoacids / Difco Labs, Detroit / USA) 800 ml of distilled water

are sterilized (20 minutes, 12O ⁰ C) and sterile supplemented with the following freshly prepared solutions.

1 ml 8rnl 1.5ml 10ml 0.2m!

0.5% igerKaliumdihydrogen phosphate solution

20% 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

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.

Table 3

Tested bacterial cell walls of

Dry Cell Yield (mg)

dye content

rel. absorbance units

without (schedule) B. ammoniagenes (ATCC 6872) B. glutamigenes (ATCC 13747) B. pumilus (ATCC 7061)

Linens (ATCC 19391)

C. diphtheriao (measure 8)

C. 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 44

38 24

34 34 50 41

2 31

G = Brevibacterium C = Corynebacterium Ce = Cellulomonas L = Lactobacillus S = Staphylococcus

Example 2

Stimulation of the synthesis of colored ingredients (actinorhodin, prodigiosin) in Sxreptomyces lividans (ATCC 19844) by cell wall extracts.

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.

Example 3

Stimulation of the synthesis of colored ingredients (actinorhodin, prodigiosin) in Streptomyces lividans (ATCC 19844) by cell extracts.

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.

Example 4

Stimulation of the synthesis of colored ingredients in Slreptomyces coelicolor (ATCC 13405).

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).

Example 5

Stimulation of the synthesis of colored ingredients in Streptomyces griseoruber (DSM 40275).

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).

Example 6

Stimulation of the synthesis of faked ingredients in Streptomyces purpurascens (DSM 40310).

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).

Example 7

Stimulation of the synthesis of colored ingredients in Streptomyces latericius (DSM 40163).

Under the conditions of Example 1, but using Streptomyces latericius (DSM 40163). This bacterium also achieves a significant increase in dye formation.

Example 8

Stimulation of the synthesis of colored ingredients in Streptomyces violascens (DSM 40082).

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.

Example 9

Stimulation of the formation of β-lactam antibiotics (cephalosporins, penicillin N) by Streptomyces clavuligerus (ATCC 27064).

5g 3- (N-morpholino) -propanesulfonic acid (MOPS)

3.5 g of dipotassium hydrogen phosphate

0.6 g of magnesium sulfate heptahydrate

2g L-asparagine

10g glycerin

1 g of yeast extract (Oxide, Wesel, DE)

1 ml of trace element salt solution - containing per liter

1 g of iron (II) sulfate heptahydrate

1 g of manganese (II) chloride tetrahydrate

1 g of zinc chloride heptahydrate

Iq potassium chloride

are made up to 1 liter with distilled water and sterilized (20 minutes, 120 ^° C.).

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).

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.

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.

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.

Example 10

Stimulation of the formation of alkaloids (sanguinarine, chelirubin, marcarpin and chelerythrine) by cultures of Eschscholtzia californica.

In 241 ml chambers of a polystyrene multi-shell (Nunc, 6200 Wiesbaden 12) are under the J. Berlin et. al.

(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) 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%.

Table 4 below shows the results obtained in this series of experiments.

Table 1

Tested bacterial cell walls% elicitor activity

B'evi> acteriumbutanicum ATCC21196 19

Biev 'lcterium flavum ATCC 13826 16

Brevibücteriumflavum ATCC 14067 30

Brevibacterium glutamigenes ATCC137 113

Brevibacterium lactofermentum ATCC13655 43

Brevibacterium ammoniagenes ATCC 6872 40

, Corynebaoterium hydrocarboclastum ATCC15592 8

Corynebacterium nephridii ATCC11425 123

Corynebacterium paurometabolum ATCC 8368 16

Corynebacteriunlilium ATCC15990 108

Corvnebacteriurr. stnatum ATCC 6940 17

Corynebacterium petrophilum ATCC19030 0

Corynebacterium xerosis ATCC 373 102

Corynebacterium diphtheriae strain Mass. 8 137

Rhodococcusfasciens ATCC12975 27

Rhodococcusfasciansi 11 Isolate of Prof. Dr. med. Stolp, Univ. Bayreuth

Rhodococcusfascians2 7 Isolate of Prof. Dr. med. Stolp, Univ. Bayreuth

Example 11

Stimulation of the formation of indole alkaloids (valiesiacotamine) in cultures of Rauvolfia serpentina.

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.

After incubation for 5 days, the cell mass doubled in both the elicited cultures and controls. The cells are harvested and extracted with methanol.

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.

Example 12

Stimulation of 17-ketosteroid reductase activity of Rhodotorula glutinis IFO 0389.

a) A 2-liter Erlenmeyer flask containing 500 ml of sterile nutrient medium containing 5% glucose monohydrate

2% Cornsteep liquor

- adjusted to pH 6.5 -,

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) A 500 ml Erlenmeyer flask containing 100 ml of sterile nutrient medium containing 1% Cornsteep liquor

5% Nurupan "" (manufacturer Nurupan GmbH, 4000 Dusseldorf 1, DE) 1% Metarin " ¹¹ (manufacturer Lucas Meyer; 2000 Hamburg 28; DE)

- adjusted to pH 6.2 -,

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.

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) 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.

Example 13

Stimulation of the steroid A'-dehydrase activity of Bacillus lentus (ATCC 13805).

a) A 2-liter Erlenmeyer flask containing 500 ml of sterile nutrient solution containing 0.5% Cornsteep liquor

0.05% glucose monohydrate 0.1% yeast extract

- adjusted to pH 7.0 -,

is inoculated with a spill of Bacillus lentus (ATCC 13805) and shaken for 48 hours at 30 ° C at 190 revolutions per minute.

b) A 500 ml Erlenmeyer flask containing 100 ml of sterile nutrient solution containing 3.0% soya powder

0.5% consteep liquor

0.1% yeast extract

0.05% glucose monohydrate

- set to /? H 7.3 -,

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.

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) 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).

Example 14

Stimulation of the formation of alkaloids (lysergic acid amide and isolysergic acid amide) of Claviceps paspali (ATCC 13895).

a) A 500 ml Erlenmeyer flask containing 50 ml of a sterile nutrient solution containing 4% sorbitol (technically pure)

1% glucose monohydrate

2% succinic acid 0.6% aluminum sulfate

0.5% yeast extract (Difco ^IRI from Difco Labs, Detroit, USA) 0.1% potassium dihydrogen phosphate, 0.03% magnesium sulfate heptahydrate

adjusted to pH 5.2 with sodium hydroxide solution,

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) A 500 ml Erlenmeyer flask containing 50 ml of a sterile nutrient solution containing 8% sorbitol (technically pure)

6% succinic acid

0.9% ammonium sulfate

0.1% calcium nitrate tetrahydrate

0.05% dipotassium hydrogen phosphate

0.03% magnesium sulfate heptahydrate

0.02% yeast extract (Difco ^(R) from Difco Labs., Detroit / USA)

0.0007% ferric sulfate heptahydrate

0.0006% zinc sulfate heptahydrate

adjusted to pH 5.2 with sodium hydroxide solution,

is inoculated with 5 ml preculture of Claviceps paspali and shaken for 250 hours at 24 ⁰ C at 240 revolutions per minute.

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.

This gives 35 mg of a mixture of lysergic acid amide and isolysergic acid amide (yield 700 mg / l of culture).

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).

Example 15

Stimulation of Penicillium raistrickii 15α-hydroxylase activity (ATCC 10490).

a) 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

- adjusted to pH 6.0 -,

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) 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

- adjusted to pH 6.0 -,

is inoculated with 10ml of the prepared according to a) Penicillium preculture.

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.

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) 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.

Claims (6)

1. A method for increasing enzyme activities and the synthesis performance of organisms, characterized in that one brings the same with inactivated elicitorium-containing microorganisms, fragments thereof or excretions of elicitorium-containing microorganisms in contact, with the proviso that to increase the enzyme Activities and synthesis performance of non-microbial organisms inactivated elicitorial-containing bacteria, fragments thereof or excretions of elicitorial-containing bacteria. 2. A method for increasing enzyme activities and the synthesis performance of microorganisms according to claim 1, characterized in that it brings the same with inactivated Elicitorenhaltigen microorganisms, fragments thereof or excretions of elicitorium-containing microorganisms in contact. 3. A method for increasing the synthesis power of microorganisms according to claim 2, characterized in that one is capable of dye formation, alkaloid formation or antibiotic Br rien, fungi and yeasts with inactivated elicitor-containing bacteria, fungi or yeasts, fragments thereof or excretions of this microorganism brings in contact. 4. A method for increasing enzyme activities of microorganisms according to claim 2, characterized in that one brings for steroid transformation capable of bacteria, fungi or yeasts with mactivated elicitor-containing bacteria, fungi or yeasts, fragments thereof or excretions of these microorganisms in contact. 5. A method for increasing the synthesis performance of organisms according to claim 1, characterized in that one brings cell cultures of the dye synthesis, for the alkaloid synthesis or the Phytoalexinsynthese capable of higher plants with inactivated elicitor-containing bacteria, fragments thereof or excretions of these Baktetisn in contact. 6. A method for increasing enzyme activities and Syninselesung of organisms according to claim 1 to 5, characterized in that they are brought into contact with water heat sterilized elicitorium-containing microorganisms or with filtrates thereof.