CS262188B1 - Strain of microorganism penicillium chrysogenum ccm f 8012 - Google Patents

Abstract

The purpose of the solution is to obtain a strain producing penicillin in higher concentrations than is achieved by previous tribes, which is more resistant to mechanical stress in compared to previous strains. Penicillin synthesizes a mutant of the Penicillium chrysogenum strain obtained after treatment with suitable mutagens and passive selection in medium containing a suitable source of carbon, nitrogen, sulfur, phosphorus, precursor, antifoam, trace elements and chalk at high conversion carbon sources under aerobic conditions at Deň: 25 ° C. During the 168 hour cultivation strain generates 33,400 j / under laboratory conditions penicillin V or 30,900 µ / ml penicillin G, on a laboratory fermentation scale of tanks produces 35 980 µ / ml penicillin V.

Description

The present invention relates to a strain of Penicillium chrysogenum CCM F-8012 and its selectates (internal designation UV LXIII / 70) producing penicillin G and V.

Penicillin production is three times higher than it was 40 years ago when its production began. This increase is due to the development of breeding methods based on the use of artificially induced variability of biological material.

The strain production program is based on random selection of artificially induced mutants using appropriate chemical or physical mutagens and testing of mutagenized cultures. The range of mutagens to be used is governed by their effects, since it is necessary to eliminate the accumulation of undesirable aberrations that may progressively accumulate, such as deterioration of growth properties, sporulation, loss of resistance to mechanical stress, etc.

The strain Penicillium chrysogenum CCM F8012, internal designation UV LXIII / 70 was obtained by mutagenesis and passive selection from the parent strain CCM F-760, internal designation NMU 2/40. The breeding procedure follows from the following scheme, which shows the genealogical line leading to the new strain.

Penicillium chrysogenum NMU 2/40 »PS

NMU 2 / 40-4 1 UV

UV L / 114 NM NMG

NMG 1/65 1 NMU

NMU XXIV / 29 'UV' UV LIX B / 40

UV

UV LXIII / 70 1 PS

UV LXIII / 70-selectates Explanation of abbreviations:

UV - exposure to UV light

NMG - action of nitrosomethylguanidine

NMU - action of nitrosomethylurea

PS - passive selection

The UV-light source was a Philips TVV 40 W germicidal lamp with a 250 V upstream ST 250 voltage stabilizer and 1 ° / o sensitivity. The spore suspension in water was irradiated in a 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 samples, which were further diluted and sown on petri dishes with sporula soil.

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

Nitrosometylurea was applied at a final concentration of 6 mg / ml. The spore suspension in citric phosphate buffer pH 6.0 was mixed in sealed Erlenmeyer flasks, after the addition of the mutagen, samples were taken at appropriate time intervals, diluted and sown in Petri dishes with sporula broth.

The spores for passive selection were suspended in distilled water or saline, diluted and sown on petri dishes with sporula broth.

In this way, the application of mutagens and passive selection yielded the UV LXIII / 70 isolate and its selectates.

The isolates were evaluated according to the following scheme:

isolated colonies on Petri dish sloped agar t vegetative inoculum and fermentation itself

Isolates were productively evaluated in 2 experiments, preferably lyophilized or stored in liquid nitrogen, after seeding, the isolates were re-evaluated in three experiments. Canned can serve for long-term preservation of strains and allow preservation of acquired properties.

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

By this evaluation system the strain and its selectates have been claimed and claimed.

The colonies of the trunk grow up to approx. 12 mm in size during monosporic sowing within 9 days, the center of the colony is bowl-shaped, from the war to the edge the colonies are wrinkled, the color of the spores is beige. The culture sporulates well and quickly on different types of sporulatory soils and on various natural substrates such as rice, hail, millet, etc. The number of spores in 1 ml of suspension resulting from washing the end spores from the 10 ml end tube is ko262188

Lem 3.10 ⁷ and the number of spores in 1 ml of suspension resulting from washing spores from 30 g of groats of 50 ml of water is 9. 10 ⁷ spore in ml.

The vegetative inoculum can be prepared on soils containing glucose no sucrose as the source of carbon, achieving consistent growth values. A suitable nitrogen source for inoculum preparation is corn extract. Optimal age of vegetative wetland; it is 3 to 43 hours, the optimum temperature is 25 degrees Celsius.

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

The selection of strains was also made with regard to resistance to mechanical stress.

Example 1

The vegetative inoculum was prepared by submerged cultivation in 500 ml beakers with 4.0 ml inoculum broth (carbon source sucrose, nitrogen corn steep liquor, ammonium sulfate and inorganic salts). Soil for Table strain (internal designation)

NMU 2/40 UVLXIII / 70 Example 2

The fermentation process was the same as in Example 1 except that either a 6 mm long, 4 mm wide ceramic cylinder or a 4 mm diameter glass ball was added to a portion of the fermentation flasks. Ceramic rollers were ferpenicillin in J / ml on day 4

300

400 vegetative inoculum preparation was inoculated with a spore loop on sloping agar with sporulated soil or 2.5 ml spore suspension resulting from the washing of groats (30 g) of 50 ml distilled water and incubated on a rotary shaker for 48 hours.

The vegetative inoculum thus prepared was seeded with a 10% 500 ml fermentation flask with 40 ml of fermentation broth. The carbon source in the fermentation broth was lactose in combination with sucrose, the nitrogen source Pharmamedia in combination with ammonium nitrate, the soil also contained a source of magnesium, sulfur, phosphorus, side chain precursor, calcium carbonate and antifoam. In penicillin G biosynthesis, the precursor was added during fermentation.

The preparation of the vegetative inoculum and the fermentation itself were carried out on rotary shaking machines (230 rpm, eccentric 25 milliliters) at 25 ^r C.

The fermentation time was 8 days, penicillin potency samples were taken on days 6, 7 and 8 of the fermentation. The maximum production was reached on day 7 of fermentation.

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

penicillin G j / ml

600 30 900 mentions sterile removed from flasks. Samples for penicillin determination were taken from day 3 at 24-hour intervals until day 8 of fermentation. The added bodies were used to detect the mycelium resistance to mechanical stress, their effect on penicillin production expressed in j / ml is shown in the following table.

T & B conditions in the flask days standard 1st shrub, roller - * 1 glass ball

On day 4 the strain was removed

NMU 2/40 UV LXIII / / 70 NMU 2/40 UV LXIII / / 70 NMU 2/40 UV LXIII // 70 3. 5 500 1400 3 800 4 000 4 800 5 300 4. 11 300 15 600 2 200 3 500 4 800 8 900 5. 20 500 23 700 1 800 3 200 4 500 12 300 6. 28 000 33 400 1 800 12 300 8 800 19 300 7. 29 500 33 400 2 200 17 800 10 200 23 000 8. 32 200 30 600 2 600 25 300 13 100 26 600

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The strain to which the patent is claimed is subject to the minimum negative impact of mechanical stress and, moreover, has a considerable regenerative capacity as shown in the results in the previous table.

Example 3

Penicillin V fermentation was performed on a quarter-scale scale in a volume of 201-liter laboratory fermentation tanks (LFT).

The inoculum with the UV LXIII / 70 strain was prepared from the operating barley spore can also in LFT on standard inoculation broth after 32 hours of culture.

Penicillin V fermentation on soil containing 4% soy flour, 1% CSL with 60% dry matter, precursor, limestone and other salts was carried out under usual cultivation and technological conditions with semicontinuous addition of other nutrients (sucrose, ammonium sulphate, soybean oil, phenoxyacetic acid) for 211 hours. Penicillin V production determined by iodometry of 35 980 IU / ml was achieved, which was 118.7% compared to the control NME 2/40 strain.

Claims (1)

I will invent the object Pcnicillium chrysogenum strain CCM F- 8012, producing penicillin V and G. Saveroxrafla, N. Plant 7, Moat