CS204302B1 - Industrial mutant of fungi claviceps purpurea cp 7/63 ccm f-631 - Google Patents

Description

The invention relates to a novel industrial mutant of the strain Clavlceps purpurea CP7 / 63 with high submerged production of pharmaceutically important agroclavin alkaloids (M. Abe et al., Japanese Patent 178 336) of structural formula I

(I) and elymoclavin (M. Abe et al., US Pat. 2,835,675) of structural formula IX

Mutant samples are stored in the Czechoslovak collection of microorganisms at the University of J. E. Purkyns in Brno under δ. CCM F-631

The mutant was obtained from 664 isolates prepared by the mutation treatment of ethyl methanesulfonate (EMS) and UV radiation on the laboratory strain Clavioeps purpurea CP7 (Microbio204302) as described below.

204302 2 logical institute of the Czechoslovak Academy of Sciences in Prague 4). ' In the preparation of the high production mutant, all incubations of the cultures in the dark were performed at 24 ± 1 ° C. CP7 maternal culture spores that have been cultured for 3 to 4 weeks on sloping T2 agar broth (C. Spalla, in the Genetics of Industrial Microorganisms. Eds. 393) were. suspended in 0.066 M phosphate buffer pH 7.2 and in saline. The resulting suspension always contained 1 to 4.10? spore ml -1, of which only 0.5% to 1% germinable. Agar broth T2 contained the following components (g. 1 * * dest.H ₂ O): sucrose 100, L-asparagine 10, Ca (NO 3) ₂ .4 H ₂ O 1, yeast extract 0.1, KH ₂ PO4 0.25 MgSC> 4.7 H ₂ 0 0.25, KCl 0.12, FeSO 4.7 H ₂ O 0.02, ZnSO 4 7 H ₂ 0 0.015, agar 20, pH 5.2 after autoclaving at 105 ° C 15 min.

The spore suspension in 0.066 M phosphate buffer pH 7.2 was exposed for 19 hours, effect 0.02-0.075 M EMS (J. Nesvera, Fol. Microbiol. 18 (1973)), washed twice with said buffer, plated on petri dishes (tf). 10 cm) with T2 agar broth enriched with 0.02% casein hydrolyzate (Bacto-Casamino acids Difco) and incubated for 2 weeks.

From the spore suspension in saline, 5 ml was transferred to a Petri dish (d 10 cm) and irradiated with a UV source set to 10 µm "2.s" f dose while moving. 150 to 1,000 µm 2. 0.5 ml of the spore suspension was withdrawn at 10 second intervals. After suitable dilution with saline, the resulting spore suspension was transferred to Petri dishes (/ 10 cm) with T2 agar broth enriched with 0.02% casein hydrolyzate (Bacto-Casamino acids Difco). The inoculated broth was incubated for 2 weeks.

Colonies grown from mutagenized spores on agar medium in Petri dishes were individually transferred to sown T2 agar broth and incubated for 3-4 weeks. The grown cultures were then tested for their ability to form alkaloids under submerged fermentation conditions (cf. No. 199 986).

Alkaloids were determined colometrically in fermentation fluid of 14-day cultures (GT Banks et al., J. Gen. Microbiol. 82, 345 (1974)) and by high performance liquid chromatography (M. Wurst et al., J. Chromate. 150. 477). (1978).

Characteristics of high-production mutant:

The ability of the mutant to form an extracellular mixture of agroclavin and elymoclavin under submerged fermentation conditions is shown in Table 1.

Tabulkal

Mutant type Mutagen dose J.m-2 conc. (M) Total alkaloids (g agroclavin ml ^- 1) Agroklavin Elymoklavin% CP7 / 63 UV 720 3.2 94 6

The CP7 / 63 mutant is morphologically identical to the parental CP7 strain. Under conditions of submerged fermentation it grows abundantly (12 to 20 mg dry weight of mycelia.ml “)), intensively eporulates macro and microconidia and produces extracellular glucans and extracellular brown-red pigment.

On the T2 agar medium they form a low layer of off-white airy mycelium richly branched with conspicuous cell-ending hyphae. For the CP7 / 63 mutant, the weight ratio of acroclavin / elymoclavin in the produced alkaloid mixture is 15.6.

The production mutant is stored in Endo tubes on T2 agar broth and in the dark at 4 ° C. Every 3-4 months they are re-vaccinated and after 30 days incubation at 24 l 1 ° C they are used to prepare the inoculum or stored as a stock culture at 4 ° C. The mutant Claviceps purpurea, designated CP7 / 63, provides a key portion of the large-scale production of alca-, agroclavin, and elymoclavin by submerged fermentation.

Claims (1)

SUBJECT MATTER Industrial mutant of the fungus Glaviceps purpurea CP7 / 63 deposited in the Czechoslovak Collection of Microorganisms of the University of J. E. Purkyně in Brno under the number CCM P-631 with the production of agroclavin and elymoclavin alkaloids.