CS239242B1 - Microorganism strain Penicillium chrysogenum CCM F-760 - Google Patents

Abstract

A strain of the microorganism Penicillium chrysogenum CCM F-760, producing penicillin G and V. This strain enables increased production of penicillins during submerged fermentation, is resistant to mechanical stress, grows rapidly on solid and liquid media, and is highly stable in subsequent spore generations.

Description

The present invention relates to a strain of Penicillium chrysogenum CCM producing penicillin G and V.

Although the production of penicillin has been carried out in production fermentation tanks for more than 30 years, the volume of penicillin produced is constantly increasing. An overall increase in production can be achieved either by increasing the production capacity or by increasing the yield of penicillin in its production. Currently, the second method is the only possible way to increase yield, where the main benefit is to improve the production properties of the strain. These strains are obtained by a number of established procedures in long-term breeding programs. The resulting strains then exhibit a number of significant distinct properties, of which the most important are those which manifest themselves in increased penicillin production in large-volume fermenters.

Tribe Warbler i llium chrysogenum CCM 3? - Wo.

was obtained by the action of mutagens and repeated.

passive selections to the parent strain N 9541. The method of breeding the strains is illustrated in the following scheme, which shows the genealogical line leading to the claimed strain.

Penicillium chrysogenum N 9541 PS

9541-16 UV 1

UV 1/47 f PFP 1

PFP 1/17

AND

- 2 ψ ps

ΡΡΡ 1 / 17-30 | NMU 1

NMU 1/27 | NMU 2

NMU 2/40 239 242

Abbreviations: PS - passive selection - selection without mutagenic action UV - exposure to UV-radiation

PPP - action of parafluorophenylalanine NMU - action of nitrosomethylurea.

The last mutagenic effect was on the strain NMU 1/27. The NMU mutagen used was applied at a concentration of 6 mg / ml of the buffered buffer solution of c-itr tf buffer. During the application the mixture stirred mutagenic electromagnetic stirrer at 25 ^e C. At time intervals samples were removed and diluted to a concentration ineffective ha.

Spores for passive selection were suspended in distilled water, diluted with a factor of 10 dilution, and seeded in sporulated soil petri dishes. After. After 14 days incubation at 21 ° C, the isolates were sporulated and resuspended on slant agar. Incubation of this basic sporulating material was carried out at the same temperature for 9 days.

The NMU 2/40 isolate was obtained as described above for the application of mutagens and passive selection. The isolates were evaluated according to the following scheme:

oblique agar

Vegetative inoculum inherent fermentation

Isolates were evaluated in 2 experiments before lyophilization and 3 experiments after lyophilization. · Lyophilized cans are used for long-term preservation of spore material and allow preservation of suitable growth and production properties of the strain.

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

239 242

The monosporic strain forms a colony of 20-22 mm with an elevated center diameter of 5 < 6 &gt; mm in 7 to 10 days of cultivation at 25-25 mm cultivation on sporulated glycerin-molasses sporulation medium. . On minimal soil of the Czapek-Dox type, colonies of up to 25 mm are formed, with the central part of the cylinder being 3 to 5 mm in diameter. At the edge of the colony and on this soil, radial folds are formed, which are not as distinct as on the 3-glycerin-molasses-like broth. The color of the spore colony is beige on sporulation soil, older colonies begin to darken, on minimal soil Czapek-Dox the colonies are white and when aging towards the center it shows a shade of beige. The strain belongs to the strains rapidly growing at all temperatures used for Penicillium chrysogenum strains, both on solid and liquid media.

The advantage of the strain is its high stability in subsequent dispute generations. This strain, without any significant production drop, tolerates booster up to the 4th generation.

The strain is tolerant of increased concentrations of penicillin added to the medium at the start of fermentation.

A suitable property of the strain is the improved concen- tration of the main carbon source - sucrose to penicillin.

Another important characteristic of the strain is its tolerance to the mechanical stresses to which the culture is subjected during mixing in a fermentation vessel. Under high mechanical stress, this strain causes minimal damage to mycelium fibers. This advantage is applied especially in large volume fermentations. The main and most important characteristic of the strain Penicillium chrysogenum CCM P- is the increased production of penicillin during submersal fermentation.

Example 1 v

Fermentation in flasks on rotary shakers.

The vegetative inoculum was prepared submersibly in 500 ml boiling flasks with 40 ml of inoculum broth (carbon source of sucrose, nitrogen corn steep liquor, ammonium sulfate and inorganic salts). The soil for preparing the vegetative inoculum was inoculated with a slanted agar spore loop and incubated on a rotary shaker for 30 to 40 hours.

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The vegetative inoculum thus prepared was inoculated with 500 ml fermentation flasks containing 40 ml of fermentation broth (carbon source lactose and glucose, nitrogen source corn steep liquor, soybean flour and ammonium salt, side chain precursor, inorganic salts, calcium carbonate and sulfur source). 9 days, maximum production was generally achieved on day 8 of cultivation. · Penicillin production under the above conditions was 25,000 U / ml.

Example 2

Permentators in pilot plant fermenters ·

The pre-seeded soil contained sucrose as a carbon source, the source of nitrogen was corn steep liquor, cotton flour and an inorganic source, the other soil components were soluble salts and calcium carbonate. The seed medium was of the same composition. Permentation broth contained sucrose, corn steep liquor, cotton seed, side chain precursor and other salts ·

The permentation tank was filled with 150 L of soil and inoculated with 10% by volume of a seed fermenter, fermentation temperature 25 ° C, stirring 300-320 rpm, aeration 150-180 l / min, fermentation time 200 hours. The course of the fermentation was controlled by feeding the sources of carbon, nitrogen, phosphorus and precursor.

Increasing fermentation broth volume and culture growth were compensated and maintained by soil sampling and appropriate dilution.

This controlled fermentation resulted in a 38,000 U / ml penicillin stroke in the fermenter at 174 hours, with an economic coefficient of 0.140 m / m @ 2 / h. and very favorable conversion parameters of sucrose to penicillin (5.8 kg / kg) were achieved.

Claims (1)

SUBJECT OF THE INVENTION 239 242 Microorganism strain. Penicillin V and G producing cil lium chrysogenum CCM P— Ho ·