CS253986B1 - Microorganism strain Penioillium chrysogenum CCM P-786 - Google Patents

Abstract

The purpose of the solution is to obtain a strain producing penicillin in higher concentrations than achieved by previous strains. Penicillin is synthesized by a mutant of the Penioillium chrysogenum strain obtained after the action of suitable mutagens and passive selection in a medium containing a suitable source of carbon, nitrogen, sulfur, phosphorus, precursors, chalk, antifoam and trace elements at a high conversion of the carbon source under aerobic conditions at 25 °C. During 168 hours of cultivation, the strain for which patent protection is applied produces 31,000 U/ml of penicillin V or 25,800 U/ml of penicillin under laboratory conditions.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a penicillin V and G strain of Penicillium chrysogenum CCM F-786 (internal designation UV LV / 20-I / 12).

Today's industrial penicillin production technology is one of the best-managed fermentation technology technologies. .

Three factors contribute to the increase in penicillin yield, namely the breeding of the strains, the knowledge of their physiology and the engineering of the process.

The breeding of strains has a 4-year tradition. · Since the initial empiric, genetic work is currently being transferred on a scientific basis, replaced by a rational selection technique. The main indicator of the breeding effect other aspects, eg sporulation ability, morphology, higher density growth, shear yield, soil filterability after fermentation, carbon conversion capacity, inorganic nitrogen sources instead of organic sources, in-product resistance, ie · penicillin, according to the incorporation of the side chain into the penicillin molecule, according to the resistance to the toxic action of the precursors, according to the appropriate technological properties, such as foaming, sticking to the walls of the tanks, etc. selected selection.

The strain of Chickweed chrysogenum CCM F-786, internal designation UV LV / 20-I / 12, was obtained by treatment with mutagens and passive selection from the starting strain CO F-760, internal designation OU 2/40. is listed

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a genealogical line leading to the claimed strain.

Penicillium chrysogenum NMU 2/40

UV

Ψ

UV LII / 55

NMG

NMG II / 116

UV> *

UV LV / 20

PS

UV.

.V / 20-I / 12

Explanation of abbreviations: UV - exposure to UV light

NMG - action of nitrosomethylguanidine PS - passive selection

Application of mutagens: sources of light were a germicidal lamp

Philips TW 40 W with 250V ST 250 line voltage stabilizer and 1% sensitivity. The suspension spore in water, 5 ml, was irradiated in a 45 mm diameter Petri dish. During irradiation, the suspension was stirred on an electromagnetic stirrer. The distance of the dish from the source of irradiation was 20 cm, the radiation was interrupted at appropriate time intervals by taking 0.5 ml of the sample, which were further diluted, and sown on Petri dishes with sporula soil.

A concentration of 2 mg / ml in the final mutation mixture was used for nitrosomethylguanidine mutagenization. The application time was from 15 to 40 minutes with stirring in closed Erlenmeyer flasks. The pH of the mutation mixture was buffered with a pH 6.0 citric phosphate buffer. Samples were taken at time intervals and diluted to an ineffective NMG concentration and sown on sporulated soil petri dishes.

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The spores for passive selection were suspended in distilled water or saline, diluted with a number and factor of 10. Several dishes were spiked with sporula broth in parallel from each dilution.

The above-described application method, mutagens and passive selection resulted in UV LV / 20-I / 12 isolate.

The isolates were evaluated according to the following scheme:

isolated colony on Petri dish 1 slant agar

Φ vegetative inoculum of yeast ^ fermentation

The isolates were evaluated in 2 pre-conservation experiments and 3 per post-conservation experiments. Canned food is used for long-term preservation of spore material and allows the preservation of suitable growth and production properties.

Penicillin potency was determined using an automated system using the hydroxamate colorimetric method.

This evaluation system resulted in the strain to which the claim is claimed.

The trunk forms a colony of about 12 mm in 9 days of cultivation at 25 ° C during monosporic sowing on sporulation medium of glycerin-molasses type, during the 9 days of cultivation, the center of the colony is sunken. The culture sporulates well and quickly on different types of sporulating soils and on natural substrates (millet, hail, etc.) · The number of spores in 1 ml suspension resulting from washing the End's 10 ml water tube is * 7 around 2.10 '/ ml and spores in 1 ml suspension resulting from washing spores 30 g hail 50 ml water is 8.10? spore / ml.

Soils containing glucose or sucrose as the carbon source can be used for the preparation of the vegetative inoculum, with the same growth and production values being achieved. A suitable nitrogen source for the inoculum preparation is corn extract. Optimal age

- 4 253 986 vegetative inoculum to reach maximum production is 48 hours at an incubation temperature of 25 ° C, fermentation broth is inoculated with 10 $ vegetative inoculum7 The strain has considerable demands on sufficient oxygen supply even in the preparation of vegetative inoculum.

Maximum production is achieved by this strain in the lactose or sucrose -Pharmamedia fermentation broth. For this strain, the form of sulfur and inorganic nitrogen source is of key importance; Soy or peanut flour can be used in place of Pharmamedia without a significant decrease in productivity. Utilization of carbon source, resp. lactose is most economical at a concentration of 4 - 8 $, for maximum production is optimum 13 $, from 1 mg of lactose is produced on a laboratory scale of 250 U / ml penicillin.

The strain's sensitivity to mechanical stress is at the level of good strains, the strain has high oxygen transfer requirements and is sensitive to dissolved oxygen dropping or to stop stirring. Penicillin biosynthesis in this strain is not stopped in the presence of 50,000 U / ml of model-added penicillin, during a standard fermentation a maximum of $ 25 penicillin is decomposed, producing about $ 2-hydroxy hydroxyl foams.

Example 1

The vegetative inoculum was prepared by submersive cultivation in 500 ml boiling flasks with 40 ml inoculum broth (carbon source sucrose, nitrogen corn extract, ammonium sulphate and inorganic salts). The vegetative inoculum broth was inoculated with a sloping agar spore loop or 2.5 ml spore suspension resulting from the washing of groats (30 g) with 50 ml water and incubated on a rotary shaker for 48 hours.

The vegetative inoculum thus prepared was inoculated with a 10 ml fermentation flask containing 500 ml, a fermentation broth volume of 40 ml. The carbon source was sucrose and lactose, the nitrogen source Pharmamedia or soy or peanut flour, ammonium salt, the soil further contained a side chain precursor, salts, calcium carbonate to buffer the soil, and a sulfur source.

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Preparation of vegetative inoculum and fermentation were carried out on rotary shakers at 230 rpm, eccentric of the machine at 25 mm, at 25 ° C.

Fermentation under these conditions lasted 8 days, samples for efficiency determination were taken at 6, 7 and 8 days of fermentation. · Maximum production was achieved on 7 days of fermentation.

Production comparison of the starting strain with the claimed strain;

strain Penicillin V penicillin G (internal label) j / ml j / ml NMU 2 / 4o 25 000 23 000 UV LV / 20-I / 12 31 000 25 800

SUBJECT OF THE INVENTION

Claims (1)

SUBJECT OF THE INVENTION Penicillium chrysogenum strain CCM-F-786 producing penicillin V and G.