CS235447B1 - Microorganism strain Peniciliium chrysogenum CCM F-747 - Google Patents

Abstract

The invention relates to a strain of the microorganism Peniciliium chrysogenum CCM F-747 producing penicillin G and V. The new strain was obtained from the strain Peniciliium chrysogenum F 713 by the action of mutagens and repeated passive selections.

Description

The invention relates to a penicillium chrysogepura strain CCM F-747 (internal designation NMU XIX / 40) producing penicillin G and V.

Penicillin production, which has been carried out in production fermentation tanks for more than 30 years, is steadily increasing. The increase in production is related to the increased production requirements of semisynthetic penicillins derived from 6-aminopenicillanic acid obtained biologically or chemically from penicillin V or G. The increase in production is either realized by extending the production fermentation plants or by increasing the fermentation yield.

The first alternative requires significant investment costs, the second is made possible either by improving the fermentation conditions based on improved control of the fermentation process or by increasing the production capability of the strains used for biosynthesis. The breeding line of penicillin biosynthesis strains has long been followed by a number of established procedures.

The Penicillium chrysogenum CCM F-747 strain was obtained from the Penicillium chrysogenum CCM F-713 strain by treatment with mutagens (each nitrosomethylurea) and repeated passive selections. The last mutagenic effect was on the NMU strain XVIII / 81. The mutagen-nitrosome.-urea (NMU) used was applied at a concentration of 6 mg / ml citrate phosphate buffer (pH 6.0). The NMU charge was dissolved in the buffer while stirring, then spores suspended in the same buffer were added. During the treatment, the mutation mixture was stirred on an electromagnetic stirrer at 25 ° C. Samples of 1 ml were taken at time intervals and diluted to an ineffective NMU concentration.

Spores for passive selection were suspended in distilled water or physiological saline, diluted with a factor 10e dilution series and inoculated onto sporulated soil petri dishes. Several dishes were inoculated in parallel from each dilution. After incubation for 7 to 14 days at 26 ° C, the isolates were sporulated and resuspended on sloping agar with identical sporula broth. The sporulation material is incubated at 26 ° C for 6 to 10 days.

By the above method of application of mutegens and passive selection, isolate CCM-747 was obtained.

The isolates were evaluated according to the following scheme:

isolated colony on petri dish

Also inclined * agar vegetative inoculum of own fermentation

Isolates were evaluated in two pre-lyophilization (preservation) experiments and in 3 post-lyophilization experiments. lyophilized cans serve for long-term preservation of spore material and allow the preservation of suitable growth and production properties.

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

The strain according to the invention was obtained by this evaluation system. The most important characteristic of the Penicillium chrysogenum CCM-F-747 strain is an increased penicillin production of at least 5% to a total production value of 24,500 j / ml penicillin V compared to the GCM-F-713 strain, which produces about 23,000 j / ml.

The strain forms a colony of 10-15 mm colony size on 7 to 10 days of cultivation at 25 ° C on monosporic sowing on glycerin-molasses sporulation medium. Upon observation of growth, up to day 6 of cultivation, the rate is virtually identical to the most fast growing strains, both at 25 ° C and 28 ° C. Significant differences occur only on day 13 of cultivation, when for example the strain CCM-F-713 appears to be a faster growing strain. Culture sporulates well and quickly on various types of sporulating soils and on natural substrates. The color of the cultured culture is beige. The strain on natural substrates is stable for several months. The growth ability of this strain in submerged fermentation is suitable. After 24 hours in the preparation of the submerged vegetative inoculum, it achieves as much mycelial mass as other strains in twice the time. In the actual fermentation, the desired biomass concentration is achieved to initiate penicillin biosynthesis in 24 to 48 hours than in other strains.

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The advantage of the strain is its tolerance to the high concentrations of penicillin present in the fermentation of the soil. Model addition of 30,000 U / ml penicillin at the start of fermentation to soil will not affect penicillin biosynthesis. The addition of 50,000 U / ml penicillin is tolerable from 48 hours of fermentation. At this penicillin content of the soil added during the above hours, the strain biosynthetic capacity is not reduced.

The strain is tolerant of high iron concentrations in the fermentation medium. At 50 gamma / ml Fe, biosynthesis is reduced by less than 10%, while most industrial strains are reduced by up to 25% at the same iron concentration.

An important advantage of the strain is its tolerance to significant mechanical stress during fermentation. Even with a drastic mixing process, mycelial fibers are minimally damaged. This damage is monitored by the release of nucleotides from the cells into the fermentation medium during fermentation.

It should be noted that the amount of penicillin recovered in the filtrate can be increased by eluting the remaining sediment by 30% and by disintegrating the mycelium by another 10%.

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The vegetative inoculum was prepared submersibly in 500 ml faithful flasks with 60 ml inoculum broth containing basic nutrients, i.e. a source of carbon sucrose, nitrogen corn extract and ammonium sulfate and salts. The soil for preparing the vegetative inoculum was inoculated with a loop of sloping agar spores and incubated on a rotary shaker for 32-36 hours.

The vegetative inoculum thus prepared was seeded with 500 ml fermentation flasks, with a fermentation broth volume of 40 ml. The carbon source was lactose and glucose, the nitrogen source was corn extract, soy flour and ammonium salt, the soil further contained a side chain precursor, salt, calcium carbonate to buffer the soil, and a sulfur source.

The vegetative inoculum as well as the fermentation were prepared on rotary shakers at 230 rpm, eccentricity 25 mm, at 25 ° C.

Fermentations were carried out for 7 days, efficacy samples were taken on the 5th, 6th and 7th days of fermentation, maximum production was usually reached on the 6th day of fermentation.

The production of 24 500 J / ml penicillin V and 25 000 J / ml penicillin G is achieved as described above.

Example 2

Fermentation in pilot plant fermenters, volume 250 l, was three-stage. The pre-seed medium contained sucrose as the carbon source, the corn source, the cotton flour and the inorganic source were nitrogen sources, the other soil components were soluble salts and calcium carbonate. The vaccine soil had the same composition. The fermentation broth contained sucrose, corn extract, alkaline cotton flour eluate, side chain precursor, chalk and longer salts.

The course of the fermentation was controlled by dosing sucrose, ammonium sulfate, urea, precursor and potassium phosphate.

The inoculation fermenter was filled with 100 liters of soil, inoculated with a culture grown to about 80 g of groats, cultivation temperature 25 ° C, stirring 300 rpm, aeration, 00 l / min, cultivation time 36 hours.

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The seed fermentor was filled with 150 L of soil and seeded with 10% vol of the pre-seed fermenter, 25 ° C culture temperature, 300 rpm stirring, 150 L / min aeration, 18 hours culture time.

The fermentation tank was filled with 150 L of soil and inoculated with 10% by volume of a seed fermenter, fermentation temperature 25 ° C, stirring 300 to 320 rpm aeration 150 to 180 l / min, fermentation time 150 to 200 hours.

During fermentation, solutions of sucrose, precursor, nitrogen and phosphorus sources were added continuously or semi-continuously, increasing fermentation broth volume and culture growth were compensated and maintained by soil sampling and dilution. The fermentation controlled in this way reached a level of 40,000 j / ml in the fermenter before 200 hours, with a penicillin level ranging from 27,000 to 36,000 j / ml in 4 samples of fermentation broth.

The strain, due to its excellent growth and regeneration properties, enables to achieve acceleration of the fermentation process (the economic parameters were optimal between 150. · and 170. hours).

Claims (1)

Penicillium chrysogenum strain CCM F-747, producing penicillin V and G. Severography, n. P., MOST