CS206930B1 - Strain of microorganism penicillinum chrysogenum ccm f-648 - Google Patents

Description

The present invention relates to a strain of the microorganism Penicillium chrysogenum CCM F-648, which increasingly produces penicillin V and G and 6-aminopenicillanic acid (6-APK). Another advantage of this strain is the considerable resistance to mechanical stresses in the fermentation vessels.

The new strain was obtained from the strain Penicillium chrysogenum (registration number 2/170 of the Research Institute of Antibiotics and Biotransformation in Roztoky u Prahy) by a series of repeated mutagenic interventions (nitrosomethylurea, N-methyl-N-nitrosoguanidine, ultraviolet light, p-fluorophenylalanine). classified passive selections.

The last mutagenic intervention was the action of p-fluorophenylalanine. Petri dishes with sporulated glycerine melase medium with p-fluorophenylalanine added at a concentration of 1. 10 ^-4 M were inoculated with an undiluted suspension of spores (10 ⁶ to 10 ⁷ spores / ml) strain and incubated at 25 ° C until spore (17 days). The scored colonies were washed with water with 0.1% surfactant added. The resulting spore suspension was diluted and plated on sporulation agar plates.

Isolates were evaluated according to the following scheme: isolated colony on Petri dish - »sloping agar -» vegetative inoculum - »self fermentation.

The vegetative inoculum was prepared submerged in 500 ml flasks with 60 ml inoculum broth containing basic nutrients, ie a source of carbon (sucrose), nitrogen (corn extract in combination with an inorganic source of nitrogen) and nutrient salts. The vegetative inoculum was inoculated with a slanted agar spore loop and cultivated on a rotary shaker for 48 hours.

The vegetative inoculum thus prepared was seeded with 500 ml fermentation flasks with 40 ml of fermentation broth. The carbon source was lactose and soybean oil in combination with dosed sucrose; the source of nitrogen was soy flour. The soil further contained a precursor, a chalk and a sulfur source.

The preparation of the vegetative inoculum and the fermentation itself were carried out on rotary shakers at 230 revolutions per minute, with an eccentricity of 25 mm, at 25 ° C.

Production evaluation of the isolates was repeated two to three times, after which the best isolates were preserved by lyophilization and re-evaluated after inoculation (2 to 3 times). Lyophilized cans fold as a stock for further work, are stable for several years. The Penicillium chrysogenum CCM F-648 strain was obtained using the scoring system described.

Production comparison of CCM strain F-648 with the control strain Penicillium chrysogenum CCM

206930.

F-558 was carried out in penicillin v7G and 6-APK biosynthesis on several types of soil fermentations.

The Penicillium chrysogenum CCM F-648 strain forms a colony of 8-10 mm in size on 12 days of cultivation at 25 ° C on monosporic sowing on glycerin-molasses sporulation broth. On the edge is formed radial folds, the center of the colony is inverted, the edge of the colony is lined white. The spores have a spherical to ellipsoidal shape of usual dimensions, they are colorless in the wet monitoring. Culture sporulates well on various types of sporulating soils, on natural substrates (millet, corn, oats, etc.), especially when enriched with sources of nitrogen, resp. Of a 1% solution of enzymatic hydrolysis stopper of cotton flour. The color of the cultured culture is gray-green. Strain CCM F-648 is stable, does not lose production ability passages to IV. gene-; race. ,.

The strain Penicillium tnikroorganismu chrysoge- _(num, kteijý object of the present invention was! Deposited in Qs. Collection of Microorganisms JEPurkyně University, Brno, tr. Obrancu 10 degree under the number CCM F-648.

Further details can be seen from the following examples.

Example I

Biosynthesis of Penicillin

Fermentations were carried out in 500 ml flasks with 40 ml of the respective fermentation broth, on a rotary shaker at 230 rpm, eccentricity 25 mm, with an oxygen transfer rate of 0.81 ml O ₂ / ml / h. Inoculation was performed with 10% vol of vegetative inoculum.

(a) Composition of fermentation broth (% by weight) soy flour 6.0 sucrose, 0.75 sucrose in doses Γ i 13.75 phenoxyacetic acid 0.67 calcium carbonate 0.9 sodium thiosulphate 0.8 20% sodium / sodium hydroxide solution ¹ 0,2% vol.

Sucrose is dosed in the form of dragee sterilized by gamma irradiation according to the scheme:

hours total quantity

24. 48. 72. 96. 192. % wt. 1,25 2.5 3.75 3.75 2,5 13,75

Composition of fermentation broth II (% by weight): soy flour 6.0 lactose 10.0 sucrose in doses 3.74 phenoxyacetic acid 0.67 calcium carbonate 0.9 sodium thiosulphate 0.8

soybean oil

20% sodium hydroxide solution

2.0% v / v

0,2% vol 0,1% vol

added at 168. idu

0.6% ammonia water

Sucrose is added to the fermentation flasks at 24 and 48 hours as 50% mites of 0.62% by weight. and at 192. 2.5 wt. in the form of a sterile dragee.

(c) Composition of fermentation broth III (in% by weight):

soy flour 2.25 dried sweet whey 17.00 sucrose in doses 3.74 phenoxyacetic acid 0.67 calcium carbonate 0.9 sodium thiosulphate 0.8 soybean oil 0.1% 20% hydroxide solution obj./baňka sodium i / 0,2% vol 0.6% ammonia water 0,1% vol

added at 168, hour

Sucrose is added as in case b).

d) Composition of fermentation broth IV (% by weight) an enzymatic hydrolyzate cotton flour 7.0 lactose 14.0 sucrose in doses 3.74 phenoxyacetic acid 0.75 calcium carbonate 0.9 sodium thiosulfate · 0.8 soybean oil 0.1% - · --------- obj./baňka % \ 2 (|% hydroxide hydroxide solution, 0,2% vol.  sodium i  04]% vol., 0.6% ammonia water Γ "Added in _______— 7- 168. hodin acidic potassium phosphate 0,067 Sucrose is added as in case b).

e) Composition of the fermentation broth V (in% by weight): enzymatic hydrolyzate of cotton flour 5.0 ammonium carbonate 0.75 lactose 14.0 sucrose in doses of 3.74 phenoxyacetic acid 0.75 phenoxyacetic acid in the form of a sodium salt solution 10 , 2 added ^- -------------- at 96 hours calcium carbonate, 0.9 sodium thiosulphate 0.8 soybean oil 0.1% v / v sodium hydroxide solution 0,2% vol.

potassium potassium phosphate 0.067 Sucrose is added as in b).

f) Composition of fermentation broth VI enzymatic hydrolyzate of cotton flour lactose • phenoxyacetic acid calcium carbonate sodium thiosulphate calcium sulphate soybean oil and 20% hydroxide / sodium solution potassium phosphate (in% by weight):

7.0

14.0

0,8 1,2 0,9 ’

0.25 2.2% v / v

0,2% v / v 0,067

Example 2 ϊ

Penicillin G Biosynthesis

The fermentation of the control strain CCM-558 and CCM F-648 for penicillin G biosynthesis was carried out analogously to penicillin V biosynthesis procedures using fermentation broth II, except that the soil did not contain phenoxyacetic acid as a precursor but phenylacetic acid sodium salt which was fed during the fermentation: dragees or in the form of a 5% solution of the _((in wt.%):

% by weight

clock

- 24. 48. 72. 96. : o, i9 0.19 0.19 0,095 0,665

the soil contains no precursor, i.e. phenoxyacetic acid.

All fermentation broths are prepared by dissolving their components in boiling water and checking the pH after 2 hours of standing and optionally adjusting to 8.0. After sterilization it is 7.5 to 7.7. The sterilization time is 20 minutes at 120 ° C.

Fermentations are carried out at 25 ° C for 11 days, samples for efficiency determination are taken on days 9, 10 and 11 of fermentation. The determination is carried out by the hydroxamate method with appropriate standards. The content of formed by-products, especially during biosynthesis of 6-APK, was monitored by chromatography, detection was performed biologically, degradation products of 6-APK, were monitored by paper and thin layer chromatography, detection was performed by densitometry.

The following table summarizes the results achieved:

Strain Ferm. soil Product Production j / ml CCM F-558 AND IN 18 000 II in 16 500 II G 1.6 000 II 6-APK 8 000 IV IN 18 000 CCM F-648 AND IN 19,000 ’ II in 22 000 II G 17 000 II 6-APK 13 000 III IN 23 000 IV IN 24 600 in in 2'2 000 VI in 25 200

Example 3

Biosynthesis of 6-aminopenicillanic acid The fermentation procedure of 6-APK biosynthesis is analogous to that of penicillin biosynthesis

V using fermentation broth II except that

The increase in penicillin V production during fermentation with CCM F-648 is 5% on sucrose soil, at least 20-35% on lactose soils, 5% on penicillin G and 60% on lactose soils.

Claims (1)

SUBJECT OF THE INVENTION Penicillium chrysogenum strain CCM F-648 producing penicillin V, G and 6 aminopenicillanic acid. .