CS253097B1 - Industrial strain of Claviceps fusiformis Loveless F 27 CC-4/85 - Google Patents

Abstract

The subject of the solution is a new industrial strain of the fungus Claviceps fusiformis Loveless F 27 producing the alkaloids elymoclavine and agroclavine by submerged fermentation, stored in the collections of the Institute of Microbiology of the Czechoslovak Academy of Sciences in Prague under No. CC-4/85. Under conditions of deep cultivation, the strain produces high amounts of clavine alkaloids with a favorable representation of elymoclavine, a low level of the original chanoclavine and negligible formation of exopolysaccharides. Another advantage of the new strain is the rich germination of conidia on agar nutrient media. The use of the strain in the fermentation production of elymoclavine and agroclavine alkaloids will make it possible to streamline the fermentation process itself, the extraction and isolation of alkaloids and the subsequent production of polysynthetic medicinal preparations.

Description

The invention relates to a novel industrial strain of the fungus Claviceps fusiformis Lovelles, producing alkaloids elymoclavin and agroclavin by submersal fermetration.

Agroklavin is characterized by extraordinary biological effects, e.g. inhibits lacquering (PG Mantle, Proc. Royal Soc. B. 170, 423, 1968), prevents implantation of a fertilized egg (G. Mantle, J. Reprod. Fertility 18, 81, 1969) , shows antibacterial effects (S. Schwarz and E. Eich, Planta Med. 47, 212, 1983), and cytostatic (E. Eich et al., Biochem, Pharm. 33, 4, 1984).

Elymoclavin is characterized by antibacterial activity (G. Schwarz and E. Eich, Planta Med. 47, 212, 1983) and is a preferred substrate for the preparation of pharmacologically important semisynthetic derivatives of lysergic acid, e.g. nicergoline (Tung-Chung Choong, HR Shough, Tetrahedrom Letters) 19, 1627, 1977, M. Semonsky, N. Kucharzsyk, Coll., Czech. Chem. Commun., 33, 577, 1968; M. Beran et al., Coll., Czech. Commun. 39, 1768, 1974; al., Coll., Czech, Chem. Commun. 42, 1412, 1977; Belg. Pat. 633 438).

Fermentation production of clavin alkaloids is not yet satisfactorily solved. Although described in a number of publications (M. Abe and S. Yamatodani, Progr. Ind. Microbiol. 5, 205, 1964; L. Vinning and PM Nair, Can. J. Microbiol. 12, 915, 1966; A. Tonolo and E. Udvardy-Nagy,

Aota Microbiol. Aoad. Sci. Hung. 15, 29 (1968); Cs. AO 199 986), all of which remains burdened by low yield and process length.

These deficiencies are largely eliminated by a new industrial strain of Claviceps fusiformis Lovelles F 27, producing alkaloids of elymoclavin and agroclavin by submersive fermentation, deposited in the collections of the Institute of Microbiology of the Czechoslovak Academy of Sciences in Prague, No. CC-4/85.

Characteristics of the new strain F27

The new C. fusiformis F27 strain (hereinafter F27 strain) grows on T2 agar nutrient medium slowly to form a continuous, heavily corrugated layer of gray-green airy mycelium that secretes an olive green pigment into the medium. The aerial mycelium sporulates abundantly. It forms 3-4x10? / Cm spores, predominantly (80%) microconidia (2x3-5 µm) and to a lesser extent macroconidia (2x10 µm).

Some mycelium cells form chlamydospores and arthrospores. Submerged mutants of F27 are characterized by long strands of branched multicellular mycelium composed initially of spacellular cells and, in older mycelium, from sclerocyte cells. Submerged culture development is accompanied by the formation of macro- and microconidia.

As shown in Tab. 1, the new strain F27 varies in the parent strain W1 in the intensity and nature of growth on the garage nutrient medium T2, the color of the air mycelium, the formation of a different soluble pigment and the significantly higher germination of conidia. Submerged F27 strains are characterized by higher production of clavin alkaloids, higher elymoclavin levels and lower concomitant chanoclavin levels compared to the parent W1 strain (Table 2).

Tab. 1 - Comparison of the new strain C. fusiformis F27 with the parent strain C. fusiformis W1 on T2 agar medium

Character F2 W1 Grow gradual formation of a heavily corrugated layer of mycelium intense formation slightly wrinkled layers of mycelium Coloring of the air mycelia gray-green mauve Soluble pigment olive green red violet Germination conidia (%) 5.5 1.4

Tab. 2 - Production of clavin alkaloids by the new strain C. fusiformis F27 and the parent strain C. fusidormis W1 under conditions of submersal fermentation ⁺

Strain Mutagen type dose (erg) overall g / i elymoclavin % agroklavin % chanoklavin % F27 UV 1 000 4.4 71 24 3 Wl - - 4.0 57 28 12

⁺ On a rotary shaker in Erlenmeyer flasks (300 ml) with 60 ml medium (Řeháček et. Al. AO 199 986).

The strain P27 was bred from an industrial strain of C. fusiformis W1 (Czechoslovak AO 000 000). All strains were cultured in the dark at 24 ° C. C. fusiformis W1 maternal culture spores that were cultured for 3 to 4 weeks in Endo tubes on sloping T2 agar broth (C. Spalla, in Genetics of Industrial Microorganisms. Eds. Z. Vanek, Z. Hostalek, J. Cudlin, Elsevier (Amsterdam 1973, p. 393), were suspended in saline. The resulting slurry contained about 1 to about 10.4 ^{2 2} .ml spores. Medium pozůstávalo T2 the following components (g / 1 dist. H ₂ O): 100 sucrose, L-asparagine 10, Ca _{(NO3> 2} .4 ,, Η Ο 1, yeast extract 0.1, KH ₂ PO ₄ 0 25, MgSO ₄ .7 H ₂ O 0.25, KCl 0.12, FeSC> ₄ .7 H ₂ O 0.02, ZnSO ₄ -7 H ₂ O 0.015, agar 20, pH 5.2 after 15 min sterilization in an autoclave at 105 ° C.

From the spore suspension in saline, 5 ml containing approximately 1.5x10 spore 2 was transferred to a Petri dish (0 10 cm) and irradiated with a dose of 0.1 mJ / m (1000 erg) 2 from a UV source set to 10 µJ / m / sec. 0.5 ml of spore suspension was withdrawn at 10 second intervals. After appropriate dilution with saline, the resulting spore suspension was transferred to Petri dishes (0 10 cm) with agar broth T2, enriched with 0.02% casein hydrolyzate (Bacto-Casamino Acids Difco). The inoculated broth was incubated for two weeks.

Colonies grown from mutagenized spores on garage medium in Petri dishes were individually transferred to sloped T2 agar broth in Endo tubes and incubated for 3-4 weeks. The grown cultures were then tested for their ability to form alkaloids under submersive fermentation conditions (Řeháček et al. AO 199 986).

Prior to use for the industrial production of alkaloids, the novel strain F27 of the invention is stored in the dark at 4 ° C, protected against drying out of the medium in the form of 25 day cultures grown on inclined T2 agar medium in Endo tubes at 24 ± 1 ° C. Preserved cultures are regularly revaccinated at three-month intervals. For a long time, strain F27 is stored either in liquid nitrogen at -176 ° C in inactivated horse serum, enriched with 20% glycerin, or by a specific lyophilization procedure.

The starting inoculum for submersive fermentation of clavin alkaloids is prepared by washing spores from a 3 to 4 week culture of strain F27 grown on T2 agar medium (C. Spalla, Genetics of Industrial Microorganisms. Eds. Z. Vanek, Z. Hostalek and J. Cudlin, Elsevier, Amsterdam) 1973, p. 393). The resulting spore suspension is adjusted to an approximate concentration of 1 to 4-10 ² spores / ml.

1.5 to 2 ml of this suspension is transferred to 60 ml of liquid broth in Erlenmeyer flasks (300 ml containing the following components (g / 1 dest. Η ₂ <3): sucrose 150, Ca (NO ₃ ) ₂ .4 H ₂ O, cornsteep (60%) 10, KH ₂ PO ₄ 0.25, MgSO ₄ .7 H ₂ <3 0.3, ZnSO ₄ .7 H ₂ O 0.2, succinic acid 10, pH 5.2 The inoculated medium is incubated for 7 to 9 days in the dark at 24 ± 1 ° C on a rotary shaker (240 rpm, eccentric 5.5 cm) and 3-5 ml is transferred from the submersible inoculum thus obtained. to Erlenmeyer flasks (300 ml) containing 60 ml of fermentation medium with the following components (g / l ² dest. H ₂ O): sahcarose 100, Ca (NO ₃ > ₂ .4 H ₂ 0 1, CaCl ₂ 1,1 ( NH ₄ ) ₂ SO ₄ 10, KH ₂ PO ₄ 0.25, MgSO ₄ .7 H ₂ O 0.25, ZnSO ₄ -7 H ₂ 0 0.015, FeSC> ₄ .7 H ₂ O 0.02, succinic acid 10, phenobarbital 0.5, pH 5.2 (adjusted with NaOH) The inoculated fermentation medium is incubated for 21 days in the dark at 24 ° C on a rotary shaker (240 rpm). min, eccentric 5.5 cm).

The alkaloids secreted into the fermentation broth are determined by colorimetry (GJ Banks et al., J. Gen. Microbiol. 82, 345, 1974) and by high performance liquid chromatography (M. Wurst et al., J. Chromate. 150, 477, 1978). . The 21 day culture of the new strain F27 contains 4 g / l of clavin alkaloids, of which 70% elymoclavin, 23% agroclavin and 3% chanoclavin.

The fermentation broth obtained is extracted and the alkaloids elymoclavins and agroclavins are isolated from the extract. Elymoclavin is used for further production of semi-synthetic medicinal products.

The main advantages of the new strain F27 are: increased production of clavin alkaloids (4.4 g / l on average), advantageous elymoclavin content in the alkaloid mixture (71%), low concomitant chanoclavin level (3%), reduced incidence of exopolysaccharides (0.3 g) / 1), intensive sporulation, rich germination of conidia cultures on agar nutrient medium (5.5%) and possibility of strain application in treatment of chanoclavin producers.

The use of strain F27 in the fermentation production of elymoclavin and agroclavin makes it possible to streamline the fermentation process itself, the extraction and isolation of alkaloids and the possible subsequent production of semi-synthetic medicinal preparations.

Claims (1)

Industrial strain of Claviceps fusiformis Loveless F27 CC-4/85 producing alkaloids elymoclavin and agroclavin by submersal fermentation.