CS204301B1 - Industrial mutant of fungi claviceps purpurea cp 7/5 ccmf-630 with producing alcaloides agroclavine and elymoclavine - Google Patents

Description

The present invention relates to a novel industrial mutant of Claviceps purpurea strain CP7 / 5 CCM F-630 with high submersible production of pharmaceutically important agroclavin alkaloids (M. Abe et al., Jap. Pat. 178 336) of structural formula I

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

(II)

Mutant samples are deposited in the Czechoslovak Collection of Microorganisms at the University of J. E. Purkyně in Brno under S. CCM F-630.

The mutant protected by the invention was obtained from 664 isolates prepared by the mutation described below by the action of ethyl methanesulfonate (EMS) and UV radiation on the laboratory strain Claviceps purpurea CP7 (Institute of Microbiology of the Czechoslovak Academy of Sciences in Prague 4).

Preparation of High Production Mutant:

All cultures were incubated in the dark at 24 ° C. CP7 maternal culture spores that were culminated for 3 to 4 weeks on sloping T2 agar broth (C. Spall, in Genetics of Industries! Microorganisms. Eda. Z. Vanek, Z. Hostalek, J. Cudlin. Elsevier, Amsterdam 1973, p. 393) were suspended in 0.066 M phosphate buffer pH 7.2 and in saline. The resulting suspension always contained 1 to 4.10? spor.ml ^- , of which only 0.5% to 1% of germination. Agar broth T2 contained the following components (gl ^- dast.H2O): sucrose 100, L-asparagine 10, Ca (NO3) 2 · 4 H2O 1, fermented extract 0.1, KH2PO4, 0.25 MgSO4 ·. 7 H2O, 0.25, 0.12 KC1, peso ₄ .7 H2O, 0.02, ZnSC> 4.7 H2O, 0.015, agar 20 pH 5.2 after autoclaving at 105 ° C for 15 min.

The spore suspension in 0.066 M phosphate buffer pH 7.2 was exposed for 9 hours to 0.02-0.075 M EMS (J. Nesvera, Fol. Microbiol. 18 352 (1973)), washed twice with said buffer, plated on Petri dishes ( ¢ 1 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 physiological saline, 5 ml was transferred to a Petri dish (gf 10 cm) and irradiated with a UV source set to 10 J, b, ^{3, with} doses of 150 to 1000 Jm @ 2, at 10 second intervals with constant motion. 0.5 ml of spore suspension was collected. After suitable dilution with physiological saline, the resulting joint 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 joints on agar medium in Petri dishes were individually transferred to slanted T2 agar broth and incubated for 3-4 weeks. The grown cultures were P ^and k tested for their ability to form alkaloids under submarsal farming conditions (cf. 199 199).

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

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

T a b u 1 k a 1

Mutant type Mutagen dose J.m-Z conc. (M) Total alkaloids (g agroclavin ml '’) Agroklavin % Elymoklavin% ' CP7 / 5 EMS 0.05 3.9 77 23

The CP7 / 5 mutant is morphologically identical to the parental CP7 strain. Under submerged fermentation conditions it grows abundantly (12 to 20 mg dry weight of myeelia.ml ^- ), intensively spores macro and microconidia and produces extracellular glucans and extracellular brown-red pigment.

On agar medium T2 they form a low layer of gray-white airy myeelium richly branched with side hyphae topped with conidiogenic cells. For the CP7 / 5 mutant, the weight ratio of acroclavin / elymoclavin in the produced alkaloid mixture of Sini 3.3.

The production mutant is stored in Endo tubes on T2 agar broth in the dark at 4 ° C. They are re-vaccinated every 3-4 months and used after 30 days incubation at 24 ± 1 ° C for inoculum preparation or stored as a stock culture at 4 ° C.

The mutant Claviceps purpurea, designated CP7 / 5, provides a key section of the large-scale production of agroclavin and elymoclavin alkaloids by submerged fermentation.

Claims (1)

SUBJECT OF THE INVENTION Industrial mutant of Claviceps purpurea CP7 / 5 CCM F-630 with production of agroclavin and elymoclavin alkaloids. Severography. n. p- Introduction 7. Μοβ »