CS248612B1 - Industrial mutant of sponge claviceps fusiformis w1 cc-1-85 - Google Patents

Abstract

The ripping involves a new industrial mutant fungi Claviceps fusiformis W1 producing alkaloid elymoclavin, deposited in collection of microbiological microorganisms Institute of the Czechoslovak Academy of Sciences in Prague 4 under the 6th CC 1/85. Submerged fermentation of this strain is possible increase the production by keyboard alkaloids, particularly pharmacologically significant alkaloid elyaoclavine.

Description

The invention relates to a novel industrial mutant of the strain Claviceps fusiformis Wi producing a pharmaceutically important alkaloid elyaoclavin (M.Abe et al., US Pat. 2835675) of the structural formula

Mutant samples are deposited in the collection of microorganisms of the Institute of Microbiology of the Czechoslovak Academy of Sciences in Prague 4 under the designation Claviceps fusiformis under No. CC 1/85.

The mutant was obtained from 70 isolates prepared by the UV radiation mutation described below on laboratory strain Claviceps fusiformis MI525 (Institute of Microbiology of the Czechoslovak Academy of Sciences in Prague 4).

Preparation of high-production mutant. All static cultures of the strain were performed in the dark at 24 + 1 ° C on T2 agar broth (C.Spalla, in Genetics of Industrial Microorganisms. Eds.

Z.Vanek, Z.HoStalek, U.Cudlin. Elsevier, Amsterdam 1973, p.393). Soil T2 contained the following components (gL ^-1 5: sucrose 00, L-asparagine 10, Ca (NO 2) _{2 <} 4H ₂ 0, yeast extract 0.1,

KH ₂ PO ₄ 0.25, MgSO ₄ .7H ₂ 0 0.25, KCl 0.12, FeSO ₄ .7H ₂ O 0.02, ZnSO ₄ .7H ₂ O 0.015, agar 20, dest.

H 2 O, pH 5.2 after autoclaving 15 · min. at 105 ° C.

The spores of the 21 day parental culture of Claviceps fusiformis HI525, grown on sloping T2 agar broth in Endo tubes, were washed off with the surface of the culture with distilled water. 5 ml of the resulting slurry containing about 1.5.-10 'spores in 1 ml was transferred to a petri dish (0 10 cm) and irradiated with UV source set at 10 uJ.m ^"2 .s'' dose of 0.1 mUvm" ² (1000 erg).

The irradiated spore suspension was suitably diluted with dist. water and spores contained in 5 ml of the resulting suspension were sown on the surface of T2 agar broth in a Petri dish (0 10 cm) and incubated for 21 days.

Colonies grown from autogenized spores were then individually transferred to slanted T2 agar broth and incubated for 21-28 days.

Growth cultures were tested for their ability to form elymoclavin and other clavin alkaloids under submersive fermentation conditions (Seháček.et, al., Cz. AO 199986).

The alkaloids were determined in fermentation broths of 21-day submersible cultures by calorimetry (G. J. Banks et al., J.gen. Microbiol.

Characteristics of high-production mutant. A comparison of the basic traits of the high-production strain W 1 with the traits of the parental strain MI 525 is given in Tab. 1.

(a) Cultivation on T 2 agar medium.

Mutant W 1 grows on T 2 agar medium at 24 + Ί ° C in a continuous richly creased layer consisting of gray mycelium and oval conidia. It exudes a violet pigment.

b) Submerged cultivation

Submerged cultivation of both the new strain W 1 and the parent strain MI 525 was carried out at 24 + 1 ° C on a rotary shaker and in a laboratory fermenter on a conditioned fermentation medium CS2 thous. A0 199986). Submerged cultures of strain W 1 are characterized by filamentous, branched, multicellular mycelia which produce numerous micro- and macrocorridia and at the end of fermentation

240612 disintegrates into blastoarthrospores. Submerged development of strain W 1 is accompanied by the formation of a violet exocellular pigaent.

Compared to the parental strain Claviceps fusiformis WI 525, the mutant W t is characterized by almost three times higher olymoclavin production and half the content of unwanted exogenous glucans. Under submersive fermentation conditions, 4.2 g of clavin alkaloids, this 2.5 g of elymoclavin, are produced in 1 L of medium. The accompanying alkaloids are agroclavin and chanoclavin. A comparison of the basic traits of the high-production mutant W 1 with those of the parental strain C.fusiformis W1525 is shown in Tab. 1. The mutant according to the invention is stored in the dark at 4 ° C, protected against drying out of the medium in the form of 21 day cultures grown on seedlings of medium T2 in Endo tubes at 24 + 1 ° C. Preserved cultures are regularly revaccinated at three-month intervals.

The high-production mutant Claviceps fusiformis W 1 protected by the invention provides a key section of the large-scale preparation of elymoclavin by submerged fermentation.

Table 1

Culture on agar medium T2 Submerged culture ^

Character

strain W1 strain MT525 strain W1 strain MI525 Increase rich in the coherent layer scarce in individual colonies good rich Coloring air mycelia gray brown-white - - Konidie oval oval Macroconidia microconidia protáhle oval Soluble pigment violet brown-red dark violet brown-pink Exogenous glucans - -1, 20 May 40

tg.L-1

Overall alkaloids (g.L ”’) 4.2 1,2 Elymokla- vin (g.L- ’) 2.5 0.9 § In conditioned fermentation broth CS2 (Czech author's certificate 199986)

SUBJECT V Y H i L SZU

Claims (1)

Industrial mutant Claviceps fusiformis W1 CC - 1/85 producing elymoclavin alkaloid.