CS273134B1 - Strain of "penicillium chrysogenum ccm 8013" microorganism - Google Patents

Abstract

The aim of the invention is to acquire the strain producing penicillin in higher concentrates that with previous strains. Penicillin is synthesised by the mutant Penicillium chrysogenum, acquired after the action of the selected mutagens and after passive selections, in a medium containing a suitable source of carbon, nitrogen, sulphur, phosphorus and trace elements, chalk, a precursor of the side-chain of penicillin and the defoamer, at high conversion of the source of carbon, under aerobic conditions at 25 degrees C. In the course of 168 hours cultivation in laboratory conditions this strain forms 35,300 j of penicillin V per 1 ml.

Description

(57) The purpose of the solution is to obtain a penicillin-producing strain at higher concentrations than that achieved by the previous strains. Penicillin synthesizes a Penicillium chrysogenum mutant, obtained after treatment with selected mutagens and passive selections, in a medium containing a suitable source of carbon, nitrogen, sulfur, phosphorus and trace elements, chalk, penicillin side chain precursor and antifoam, at high carbon source conversion, under aerobic conditions at 25 ° C. This strain generates 35,300 [mu] l of penicillin V per ml over 168 hours of cultivation under laboratory conditions.

CS 273134 Bl

The invention relates to a strain of the microorganism Penicillium chrysogenum CCM 8013 (internal designation UV LXV A / 24) producing penicillin V and G and selectates thereof.

Penicillin production is currently three orders of magnitude higher than it was forty years ago when penicillin production began. This development is largely due to the development of breeding methods based on the use of artificially induced variability of biological material.

The strain production program relies on random selection of mutants induced artificially by appropriate chemical or physical mutagens, and □ testing of mutagenized cultures obtained. The range of mutagens used is selected according to its effects in such a way as to increase production without compromising the viability of the microorganism.

The strain of Penicillium chrysogenum CCM 8013, internally labeled UV LXV A / 24, was obtained by mutagenic treatment and passive selection from the starting CCM F-760, internally labeled NMU 2/40. The breeding procedure is shown in the following diagram, which shows the genealogical line leading to the claimed strain.

Penicillium chrysogenum

NMU 2/40 J. PS

NMU 2 / 40-4 UV

UV L / 114]. NMG

NMG 1/65 NMU

NMU XXIV / 29 UV

UV LIX B / 783

Juv

UV LXV A / 24

Jps

UV LXV A / 24-selectates

Explanation of abbreviations: UV .....

NMG ....

PS .....

action of UV light action of nitrosomethylguanidine action of nitrosomethylurea passive selection

Application of mutagens:

The UV light sources were a Phillips TUV 40W germicidal lamp with a 1% ST 250 line voltage stabilizer. The spore-in-water suspension in the Petri dish, stirred with an electromagnetic stirrer, was irradiated with UV light. The distance of the dish from the radiation source was 20 cm. Radiation was interrupted at selected intervals, samples were taken and sown on sporulated soil petri dishes after appropriate dilution.

For nitrosomethylguanidine mutagenization a mutagen concentration of 2 mg / ml in the resulting mutation mixture was used. The mutagen administration time was between 15 and 45 minutes. The mixture was stirred on an electromagnetic stirrer in light-protected Erlenmeyer flasks, the pH of the mutation mixture was stabilized with a pH 6.0 citric phosphate buffer.

Samples were taken at time intervals, diluted to an ineffective mutagen concentration, and seeded on sporulated soil petri dishes.

CS 273134 B1 i

Nitrosometylurea was applied at a final concentration of 6 mg / ml. The mutagenized spore suspension in citric phosphate buffer pH 6.0 was stirred with an electromagnetic stirrer in light-protected Erlenmeyer flasks, sampled at appropriate time intervals, and sampled onto sporulated soil petri dishes after dilution.

For passive selection, spores were resuspended in distilled water or saline, diluted and sown in sporulated soil petri dishes.

In this way, the application of mutagens and passive selection yielded a UV LXV A / 24 isolate and its selectates.

The isolates were evaluated according to the following scheme:

isolated colonies on petri dish i

oblique agar

I vegetative inoculum

proper fermentation

Isolates after mutagenic or passive selection were evaluated in two experiments, preferably lyophilized or stored in liquid nitrogen. After seeding the cans, the isolates were re-evaluated in three experiments. Preservation serves for long-term preservation of strains and allows preservation of acquired properties.

Penicillin determination was performed by an automated system based on the use of the hydroxamate colorimetric method.

This evaluation system yielded the claimed strain and its selectates.

The colonies of monosporic sowing grow in size over a 9 day period of cqa 11 mm in diameter, the center of the colony is formed by a cup-shaped depression, between the top of the barrel and the edge are colonies creased. The color of the spores is beige. Culture sporulates well and quickly on various types of sporula soils and on natural substrates such as hail, rice, millet, etc.

The number of spores in a 1 ml suspension resulting from washing the spores from the End tube 10 ml of water is about 10? and the number of spores in 1 ml of the suspension resulting from washing

The vegetative inoculum can be prepared on soils containing sucrose or glucose as a carbon source. A suitable nitrogen source for inoculum preparation is corn extract. The optimum age of the vegetative inoculum is 36 to 48 hours, the optimum temperature is 25 ° C.

The highest production with this strain is achieved in fermentation broth containing lactose or sacharpse as a carbon source. The most suitable nitrogen source is cotton flour in combination with an inorganic nitrogen source. Furthermore, the fermentation broth must contain a source of sulfur, phosphorus and magnesium, calcium carbonate, a penicillin side chain precursor, and an antifoam. The temperature optimal for penicillin biosynthesis is 25 ° C.

Example

The vegetative inoculum was prepared by submerged strain cultivation in 500 ml beakers with 40 ml inoculum broth containing sucrose as a carbon source, corn extract as a nitrogen source, ammonium sulfate and other inorganic salts. The soil for preparing the vegetative inoculum was inoculated with a loop of sloping sporulation agar spores or 2.5 ml of spore suspension resulting from the washing of 30 g of groats in 50 ml of distilled water, and incubated on a rotary shaker for 48 hours.

CS 273134 Bl

The vegetative inoculum thus prepared was seeded at a 1:10 ratio in a 500 ml fermentation flask containing 40 ml of fermentation broth. Lactose in combination with sucrose served as carbon source in the fermentation broth, cotton flour in combination with ammonium nitrate served as nitrogen source. Furthermore, the soil contained a source of magnesium, sulfur, phosphorus, a side chain precursor, calcium carbonate, and an antifoam.

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

The fermentation time was 8 days, penicillin samples were taken on days 6, 7 and 8. fermentation. Maximum production was achieved on the 7th day of fermentation.

The production comparison of the starting strain with the claimed strain is shown in the following table.

internal strain identification penicillin V (j / ml) NMU 2/40 32 300 UV LXV A / 24 35 300

Claims (1)

SUBJECT OF THE INVENTION A microorganism strain of Penicillium chrysogenum CCM 8013, characterized in that it produces penicillin V and G and selectates thereof.