CS220217B1 - Microorganism strain Penicillium chrysogenum CCM F-713 - Google Patents

Abstract

The present invention relates to a novel penicillin G and V producing strain of Penicillium chrysogenum CCM F-713. The novel strain was obtained from the production strain used by combining mutagens (p-fluorophenylalanine and UF-light) with passive selection.

Description

(54) Strain of Penicillium chrysogenum CCM F-713

2

The present invention relates to a new Penicillium chrysogenum strain CCM F-713 producing penicillin G and V. The new strain was obtained from the production strain used by combining the action of mutagens (p-fluorophenylalanine and UF-light) with passive selection.

The present invention relates to a strain of Penicillium chrysogenum CGM F-713 producing penicillin G and V.

This strain was obtained from Penicillium chrysogenum strain number 65/41, obtained from Biotika np, by mutagenic treatment and repeated passive selection. PFP use of mutagens (DL-p-fluorophenylalanine. Calbiochem) was applied as follows: A suspension of spores was prepared in the usual way undiluted plated on Petri dishes with sporulation broth (molasses, glycerol, yeast extract and salt) with added PFP concentrations 1.10 ^-4 to 7.5.10 ^-4 M. Petri dishes were incubated at 25 ° C. After sporulation, the spores were washed off with sterile distilled water with a drop of Tween 80 and sown on Petri dishes with the same sporulated soil without mutagen as passive selection was performed.

UV mutant used. The UV light source used was a 4'0W germicidal lamp with a 250W upstream voltage stabilizer and 1% sensitivity. 5 ml of spore suspension in water was irradiated in a 45 mm diameter petri dish. During irradiation, the suspension was stirred on an electromagnetic stirrer. The distance of the dish to the radiation source was 20 cm, irradiation was interrupted at appropriate time intervals by taking 0.5 ml samples which were diluted and sown on petri dishes with the same sporula soil as described in the passive selection procedure.

Spores for passive selection were suspended in distilled water or saline, diluted with a factor of 10 dilution, and inoculated into petri dishes with the same sporula broth. Several dishes were inoculated in parallel from each dilution. After incubation for 7-10 days at 25 ° C, the isolates were sporulated and resuspended on sloping agar with identical sporula broth. The material was also incubated at 25 ° C for 6-8 days.

The above method of alternating mutagens with passive selection yielded isolate UV XLIV / 347-36,

65/41 i PFP + PFP selection XI / 264 i UV UV XLIV / 347 i UV selection XLIV / 347-36

The isolates were evaluated according to the following scheme:

isolated colony on Petri ml as well as sloping agar and vegetative inoculum I of the fermentation itself

The isolates were evaluated twice before lyophilization, i.e. before preservation, after lyophilization two to three times. Lyophilized cultures serve for long-term preservation.

Penicillin potency was determined by the hydroxamate method.

With this evaluation system, a new strain according to the invention was obtained.

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 coffered soil. The culture sporulates well and quickly on various types of co-soils and on natural substrates such as millet, barley, corn, rice, etc. The color of the cultured culture is deep beige. The trunk on natural substrates is stable for several months. His growth abilities are extraordinary. As early as 24 hours in submersible vegetative inoculum preparation, the amount of mycelial mass reaches as much as the other strains in 48 hours, as well as the desired biomass concentration for biosynthesis initiation 24 to 48 hours earlier than in other strains.

Another advantage of the strain is its tolerance to high soil penicillin concentrations. Model addition of 30,000 U / ml penicillin to the soil at the start of fermentation does not affect penicillin biosynthesis, 50,000 U / ml penicillin is acceptable as an addition at 48 hours or later. With this content of penicillin in the soil, the biosynthetic ability of the strain is not yet reduced.

The strain has another important property of minimal sensitivity to iron. For production strains, a 25% reduction in yield with an iron content in the soil of 50 g / ml, which occurs in iron production tanks, is typically a reduction in penicillin recovery of only 8%.

An important advantage of the strain is its tolerance to high mechanical stress during fermentation. Even in a very drastic manner of mixing, there is minimal release of nucleotides from the cells, so that mycelial fibers are not damaged.

Furthermore, it should be noted that the efficacy of penicillin achieved in the filtrate is increased by elution of the remaining sediment by 30% and by disintegration of mycella by another 10%.

Example 1

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

The vegetative inoculum thus prepared was seeded with 500 ml fermentation flasks, 40 ml fermentation broth volume.

The carbon source was lactose and glucose, the nitrogen source was corn extract, soy flour and ammonium salt, the soil also contained a side chain precursor, salt, calcium carbonate to buffer the soil, and a sulfur source.

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

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

Production comparison of strains:

penicillin V strain penicillin G j / ml j / ml

UV XLIV / 347 19,000 20,000

UV XLIV / 347-36 23,000 24,000 (CCM F-713)

Example 2

Fermentation in pilot tanks, 250 liters capacity, was carried out in three stages, the pre-seed medium containing sucrose as the carbon source, the corn source and the inorganic nitrogen source as nitrogen, and the other soil and chalk components. The inoculation broth contained sucrose as the carbon source, the nitrogen source was the peanut flour alkaline eluate, chalk and salts. The carbon source in the fermentation broth was sucrose, the nitrogen source was the peanut flour alkaline eluate, the other soil component was the respective side chain precursor, sulfur source, chalk and other salts.

During fermentation, the soil is supplemented with a carbon source of sucrose, a nitrogen source of ammonium sulfate and urea, a precursor and a phosphate.

The inoculation tank with a volume of 200 liters is filled with 100 liters of inoculation soil, inoculation is carried out with a culture grown to about 88 g of groats, cultivation temperature is 25 ° C, stirring 350 rpm, aeration of 100 liters / min. hours.

Inoculation tank of 250 liters, filling of 150 liters of inoculum broth was inoculated with 10 ° / o inoculum from a pre-seed tank, cultivation temperature 25 ° C, stirring 30 rpm, aeration 150 lt / min, cultivation time 36 hours.

The 250-liter fermentation tank was filled with 150-liter fermentation broth. Inoculation was performed with 10% vegetative inoculum, fermentation temperature 25 ° C, stirring 320 rpm, aeration 150 to 190 liters / min, fermentation time 150 to 222 hours.

During the fermentation, sucrose solutions, nitrogen mixtures with an additional amount of precursor and phosphate, increasing fermentation broth volume and mycelium growth were added continuously or semicontinuously to compensate for soil collection and dilution. Thus controlled fermentation was achieved at the end of the fermentation, ie at about 222 hours up to 40,000 U / ml, while in individual extractions of the fermentation broth the efficiency of penicillin was from 14,000 U / ml in the first to 34,000 U / ml. in the last exhaust. The exhaust is approximately 10 to 10 liters during fermentation. This strain, due to its excellent growth properties, allows the application of the above dilution technology and thus achieves a substantial increase in yields in both U / ml and mia per volume of fermentation broth.

The strain Penicillium chrysogenum, labeled UV XLIV / 347-36, was deposited in Cs. collection of microorganisms (Czechoslovak Colection of Microorganisms), J. E. Purkyně University, Brno, Tř. Defenders of Peace 10, under the number CCM F-713.

Claims (1)

Subject VYNALEZU Penicillium chrysogenum strain CCM F-713, producing penicillin V and G.;