SK35794A3 - Microorganism strain of penicillium chrysogenum ccm 8157 - Google Patents

Abstract

This strain of the micro-organism Penicillium chrysogenum CCM 8157 can produce penicillin V and G in higher concentrations than previous strains. The higher concentrations are achieved by a mutant strain prepared by exposure to suitable mutagens, by the passive selection and selection of regulator mutants. Penicillin is produced on a fermenting medium containing a source of carbon, nitrogen, sulphur, phosphorus, trace elements, chalk, the precursor of a side chain and an antifoaming agent. The optimum cultivating temperature is 25 degrees C. Under the above-mentioned optimum conditions the strain produces according to the invention in laboratory conditions about 45,000 units of penicillin V per 1 ml in 168 hours of fermentation.

Description

Technical field

The invention relates to a strain of the microorganism Penicillium chrysogenum CCM 8157 and its selectates which are capable of producing penicillin V and G at higher concentrations than the previous strains.

state of the art

The current production of penicillin is one of the most sophisticated fermentation technologies. The high production of penicillin per unit volume is due to the continuous recruitment of new strains in the process of targeted mutagenesis and the selection achieved by studying the physiology of these new strains, culminating in mastering the fermentation technology of the best strains on an industrial scale.

SUMMARY OF THE INVENTION

The microorganism strain Penicillium. Chrysogenum CCM 8157, internally designated EB XIV BV / 39 (Research Institute of Antibiotics and Biotransformation, Roztoky u Prahy, hereinafter referred to as VUAB), was obtained by a series of mutagenic interventions, passive selections and selections of regulatory mutants from CC -760, with internal designation NMU 2/40 (VÚAB). The immediate parent strain is strain CCM 8121, internally designated EB IX C / 51 (VÚAB), to which a mutagenic intervention of etidium bromide in the growth phase was applied followed by selection of regulatory mutants resistant to high valine concentrations. The breeding strategy results from said genealogical line leading to the strain of the invention.

NMU 2/40 CCM F-760

UV Ji

UV 1/114 NMG \

NMG 1/65

NMU

NMU XXIV / 29 UV

UV LIX B / 48

UV

UV LXIII / 70 (CCM 8012)

AG \

AG II A / 48

UV

UV LXV A / 14 NMG

NMG XVII A / 35 UV

UV LXXXIII B-L / 39 EMS 1 »

EMS XI A / 5 EB V

EB VIII B / 22

NMG

NMG XXIX B / 2 EB

EB IX C / 51 (CCM 8121)

EB \

CC X 8 B-V / 39 (CCM 8157)

Abbreviations' ·

NMU - action of N - nitrosomethyl - ureas

UV - exposure to ultraviolet radiation

NMG - N-methyl-N-nitro-N-nitrosoguanidine treatment

AG - action of azidoglycerol

EMS - effect of ethyl methane sulfonate

EB - action of ethidium bromide

Application of mutagens ^: The source of UV radiation was a germicidal lamp (Philips TUV 40 W). The apparatus was applied to an aqueous spore suspension of the starting strain, placed in a 45 mm diameter Petri dish at a distance of 20 cm from the source of radiation, with continuous stirring in an electromagnetic mixer. At certain time intervals, 0.5 ml of the suspension, diluted in decimal row, was withdrawn and sown on Petri dishes with sporula soil.

N -rososomethyl guanidine (NMG) was applied under both mineral and growth conditions; accordingly, the mutagen concentration in the mutation mixtures varied as well as the duration of action. When applied under mineral conditions to a spore suspension, the concentration of NMG in the mutation mixture was 2 mg / ml and the application time was from 15 to 40 minutes with continuous agitation in closed Erlenmayer flasks. Application under growth conditions was carried out in 500 ml boiling flasks containing 40 ml of soil composed of a source of carbon (glucose), nitrogen (yeast extract, sodium nitrate), sulfur, phosphorus, inorganic salt and mutagen at concentrations of 2.5 to 10 mg / ml. . This mutation mixture was inoculated with 2 ml of inoculum, which was previously prepared by inoculating 40 ml of inoculum medium (composition shown in Example 1) in 500 ml boiling flasks through a spore eye and culturing for 48 hours at 25 ° C on a rotary shaker (25 mm rpm), the mutation mixture was cultured for 24 hours on a rotary shaker at the same flasks.

Nitrosomethylurea (NMU) was applied at a final concentration of 6 mg / ml for 15 to 60 minutes to the spore suspension of the respective strain, sealed with stirring in an Erlenmeyer flask. The mixture was buffered with itophosphate buffer to pH 6.0.

Azidoglycerol (AG) was applied at a final concentration of 0.5 M for 60 to 120 minutes. Mutagenesis was performed in a closed Erlenmayr flask with constant agitation.

Ethyl methane sulfonate (EMS) was applied at a final concentration of 0.05 M for 18 hours at 25 ° C. The mutagenic mixture was buffered with phosphate buffer to pH 7.2 without stirring.

Ethidium bromide (EB) was applied under growth conditions. Mutagenesis was performed on sporulating glycerine n-melasse type sporulation medium containing aatagen content of 5-50 pg / ml for 216 hours. Single colonies were obtained by inoculating colonies onto Petri dishes with sporulation medium without mutagen.

The selection of regulatory mutants resistant to high concentrations of valine was performed by seeding 1 ml of spore suspension obtained from the mutagenic intervention into 10 ml of soil containing 5 mg of valine in 1 ml of soil, and a source of carbon (glucose), nitrogen, phosphorus and inorganic salt. Cultivation was carried out for 24 hours at 25 ° C on a rotary shaker (ezcenter 25 mm, 240 rpm). 1 ml of culture was then harvested, diluted in decimal series and sown on Petri dishes with sporula soil.

The EB XIV B-V / 39 (VUAB) strain was obtained using the above sequence of mutagenic interventions and the method for selecting regulatory mutants for valine.

Isolates were evaluated over time for penicillin production according to the scheme:

isolated colony on Petri dish i

oblique agar

J vegetative inoculum

f ermentation a

After isolation, passive selection or selection for regulatory mutants, the isolates were evaluated for production in two experiments two-well before preservation and in three experiments three-well after preservation. Preservation of the stem material was performed by lyophilization. During the evaluation, isolates were selected whose properties (penicillin production, growth on solid liquid media, sporulation) did not change with preservation.

Penicillin V was determined by high performance liquid chromatography (HPLC).

The CCM Si57 strain object of the invention was obtained by this evaluation system.

The strain forms a colony size of 8-10 mm in 9 days of culture at 25 ° C on monosporic sowing on sporulation medium of glycerine n-melasse type. The center of the colony is collapsed, the outer edge towards the dike is wrinkled. The spores are beige. Culture sporulates well and quickly on different types of sporulation media and on natural substrates (hail). The number of spores in 1 ml of the suspension prepared by washing 30 g of spore barley with 50 ml of water is 8.10 ^s .

The vegetative inoculum is prepared on soils containing sucrose or glucose as the carbon source, and corn extract as the nitrogen source. The optimal vegetative inoculum time is 36-48 hours at an optimum temperature of 25 ° C.

This strain achieves the highest production on fermentation broth containing sucrose or lactose as a carbon source, cotton flour (Pharmamédi a) and ammonium nitrate as a nitrogen source and suitable sources of sulfur, phosphorus and magnesium in the form of inorganic salts, calcium carbonate, penicillin side chain precursor and antifoaming agents. . The optimum temperature for the fermentation of penicillin is 25 ° C.

the microorganism strain Penicillum chrysogenum was 11th 1992 in Cs. collection of microorganisms, flasarsA listed from 13.

University of Technology, Joštova 10, 662 43 Brno, under the number CCM 8157.

EXAMPLES OF THE INVENTION

Example 1

in 500 ml boiling water banks so 40 containing 3% - sucrose than leachate (2,5%), ammonium sulfate (5 inorganic salts nitrate sodium

The vegetative inoculum was prepared by submerged culture (ml of inoculum medium, carbon source, corn%) as the nitrogen source,. d trace potassium hydrogen phosphate, magnesium sulphate, zinc and manganese sulphate in trace amounts and calcium carbonate (0.2%). Said soil was inoculated with slant agar spores or 2.5 ml of a distilled water spore suspension and incubated on a rotary shaker m 1 (eccentric 25 mm, 240 rpm) for 48 h at 25 ° C,

The vegetative inoculum was seeded with 40 ml of fermentation medium in a 500 ml boiling flask. The carbon source was sucrose (1.5%) and lactose (16 Z), the nitrogen source was cotton ammonium nitrate (1.7 Z), the medium further contained a side chain (phenoxyacetic acid at 4%) and a biosynthetic precursor 1), V phenylacetic acid in case of penicillin biosynthesis G), inorganic salts - magnesium sulfate (1%), potassium dihydrophosphate (0.8%), sodium sulfate (1)

%), calcium carbonate (1%) and antifoam (1% soybean oil). The cultivation was carried out under conditions identical to the preparation of the inoculum.

Fermentation lasted 8 days, samples for production determination were taken on 6th and 8th days of fermentation. Production peaked at day 7 of fermentation.

Production comparison of the initial parent strain with the CCM 8157 strain of the invention ^: Penicillin strain (internal designation) (j / ml)

NMU 2/40 25,000

EB XIV B-V / 39 45 000

Example 2

Fermentation of penicillin V was performed on a 1-scale scale in fermenters with a working volume of 180 L. Inoculum with EB XIV BV / 39 isolate was prepared from canned barley on standard seed broth containing 3% corn steep liquor, 2% sucrose, 0.3% sodium nitrate, 0,2% calcium carbonate and 0,05% potassium dihydrogen phosphate, 0,01% magnesium sulphate, 0,002% zinc sulphate, 0,007% manganese sulphate and 0,5% soybean oil, in a fermenter with a working volume of 120 l at 36 hours of cultivation. The fermentation medium used contained 3% cotton flour, 1.5% corn extract, 0.4% calcium carbonate, 0.4% phenoxyacetic acid, 0.2% ammonium sulfate, 0.5% sucrose, 0.1% potassium dihydrogen phosphate and 0.5% soybean oil. During the fermentation, 156 kg of sucrose 50% solution were continually dosed here, the concentration of phenoxyacetic acid was maintained at 10-2% by volume of sodium salt, and the concentration of ammoniacal nitrogen was maintained at 10% - % of ammonium sulfate solution at a value of 0.2 to 0.8 g / l. A 3% potassium dihydrogen phosphate solution was metered in simultaneously with the ammonium sulfate. The fermentation was carried out at a constant temperature of 25 ° C, an overpressure of 0.5 bar, with an air flow of 140 rpm, stirring at 300 rpm. During the fermentation, the pH was maintained in the range of 6.5 to 7 by the addition of ammonia. 4.8 kg of PNC-V were obtained from the stated working volume in 194 hours of the fermentation process.

Industrial usability

The strain of Penicillium chrysogenum CCM 8157 can be used industrially for the biotechnological production of the antibiotics penicillin V and penicillin G.

Claims (1)

PATENT CLAIMS Penicillin chrysogenum strain CCM 8157 producing penicillin V and G.