SK55596A3 - Highly productive strain of penicillium chrysogenum microorganism - Google Patents

Abstract

A high-production strain of the microorganism Penicillium chrysogenum CCM 8196 (NMG FOK 15). The strain was obtained by selecting mutants produced by exposure of the spores to chemical mutagens and an increased concentration of phenyl acetic acid during submerged cultivation. The subsequent passive selection resulted in the strain of Penicillium chrysogenum CCM 8196 (NMG FOK 15) producing more than 35,000 IU PEN G/ml in168 hours of cultivation in aerated flasks at 25 degrees C.

Description

Technical field

The invention relates to a high production strain of Penicillium chrysogenum CCM 8197 (NMG-FOOK 31) producing penicillin V (PEN V) at higher concentrations than the strains used hitherto.

BACKGROUND OF THE INVENTION

Worldwide production of penicillin is steadily increasing, in addition to its direct use in therapy, much of the production is used as a raw material for the production of 6 aminopenicillanic acid and semi-synthetic β-lactam antibiotics. Increasing the productivity of the production strains used is the most economically advantageous way of increasing the production volume. The development of breeding methods that rely on the selection of random mutants induced by suitable mutagens has contributed significantly to increasing the production ability of the strains. Circuit used m. The agens are selected according to their effect so as to increase production without compromising the viability of the microorganisms.

Prior to the present invention, other strains producing PEN V and PEN G, mostly based on Penicillium chrysogenum, were known. An important feature of a production strain is its productivity and maintenance of production activity, as long as possible in the cultivation time. The production strains currently used produce up to about 60,000 IU / ml penicillin in the fermentation medium at a culture time of about 120 to 200 hours.

There is known Penicillium chrysogenum CCM F-558 described in US-A-187216 producing PEN G and PEN V in 120 hours with a yield of 19,000 IU / ml penicline on sucrose and soy flour soil.

Further, a strain of Penicillium chrysogenum CCM F.-569 described in U.S. Pat. No. 199980 producing 24,000 IU / ml PEN G and PEN V in 175 hours of cultivation on pharmamedia and sucrose media is known. A characteristic of this strain is its ability to grow and produce even at high concentrations of ammoniacal ions, represented in nutrient media by nitrogenous substances such as ammonium sulfate, urea and the like. It is known for its ability to achieve high penicillin production even on a depleted carbon source in a culture medium such as lactose. Thereby, a simplified control of the fermentation process over a technology based on continuous dosing of sucrose as a carbon source has been achieved.

Another known strain is the Penicillium chrysogenum CCM F 647 strain described in U.S.A. 200944, which produces 24,000 IU / ml penicillin in 164 hours. cultivation. A characteristic of this strain is its high stability, it does not lose its production ability by passage on solid soils to IV. generations. Its advantage is that it does not have specific requirements for the carbon source.

The Penicillium chrysogenum Thom strain was obtained by long-term treatment with pfluorophenylalanine and irradiation. It produces about 22,000 IU / ml penicillin in 164 hours.

SUMMARY OF THE INVENTION

A new high-production strain of Penicillium chrysogenum CGM 8197 (internal designation NMG-FOOK 31) produces an increased concentration of PEN V as well as PEN G in selected clones of this strain. It is stored in Cs. Collection of Microorganisms at Masaryk University in Brno, Tvrdého 14, under number CCM 8197.

The strain Penicillium chrysogenum CCM 8197 (internal designation NMG-FOOK 31) was obtained by the action of chemical mutagens and the action of phenoxyacetic acid on spores in submerged culture of the starting strain CCM F-760. The following passive selection resulted in a strain of Penicillium chrysogenum CCM 8197 (NMG-FOOK 31) producing 35,000 IU PEN V / ml in 168 hours. Cultivation takes place in flasks under aeration at 25 ° C.

The breeding process is described in the following scheme, which shows the genealogical line leading to a new production strain:

Penicillium chrysogenum

CCM F-760

PS ▼

CCM F-760-19

NMU ▼

NMU XXII / 39

NMG ▼

NMG XXXI / 42

FOOK ▼

NMG FOOK-31

Abbreviations explained:

PS - passive selection

NMU - action of nitrosomethylurea NMG - action of nitrosomethylguanidine FOOK action of phenoxyacetic acid

Nitrosomethylurea was applied at a final concentration of 6 mg / ml. The spore suspension in citric phosphate buffer pH 6.0 was mixed in closed Erlenmeyer flasks. After addition of the mutagens, samples were taken at suitable time intervals for 30-60 min. and after appropriate dilution were inoculated into Petri dishes with sporula soil.

When nitrosomethylguanidine was applied, a concentration of 1 mg / ml mutagens in the final mutation mixture was used. The application time was from 15-60 min. with stirring in sealed Erlenmeyer flasks, the pH of the mutation mixture was buffered with citric phosphate buffer pH 6.0. At certain time intervals, samples were taken, diluted to an ineffective concentration of NMG, and seeded in Petri dishes with sporula soil.

40 ml of broth containing sucrose, comsteep, ammonium sulfate, sodium nitrate, calcium carbonate, inorganic nitrogen salt and 0.25% phenoxyacetic acid are poured into a 500 ml boiling flask. After sterilization, the soil is inoculated with 2 ml of a suspension of spores of the Penicillium chrysogenum strain. Allow to cultivate on a shaker at 230 rpm at 25 ° C. After 48 hours of culture, the culture is seeded on sporulated agar medium in Petri dishes and allowed to cultivate at 25 ° C for 8-10 days.

Spore suspensions in sterile distilled water were prepared for passive selection.

By the described application of mutagens, phenoxyacetic acid treatment and passive selection, the NMG FOOK 31 isolate was obtained.

The isolates were evaluated according to the following scheme:

isolated colony on Petri dish 1 slant agar vegetative inoculum ▼

submerged cultivation

The production activity of the isolates was determined in two pre-preservation experiments and three post-preservation experiments. Canned can serve for long-term preservation of spore material and allow preservation of growth and production abilities.

Penicillin potency was determined by the spectrophotometric imidazole method.

Morphological characteristics

The strain forms a molar colony of approximately 12 mm in size over 9 days of cultivation at 25 ° C on monosporic sowing on glycerin sporulation medium. A circular crater forms in the center of the colony, from the central circular crater to the edge the colony is wrinkled. The color of the spores is beige. Culture sporulates well and quickly on various types of sporulatory soils and on natural substrates eg. barley, rice and the like. The number of spores in 1 ml of suspension obtained by washing the spore coating on slanted agar in an End-tube of 10 ml of water is about 1. 10 ⁷ . The strain stored at 5 ° C on agar soils is stable for 5 to 6 weeks, on natural substrates for several months, minima. however, 3 months, in the lyophilized state for about 5 years and in liquid nitrogen for more than 5 years. Soil containing sucrose, glucose as a carbon source, and corn extract as a source of organic nitrogen may be used to prepare the inoculum. Inoculum growth is about 48 hours. at 25 ° C. Maximum production in this strain is achieved on fermentation broth containing lactose or sucrose, soy flour, corn extract or pharmamedia, a source of sulfur, inorganic nitrogen and a precursor. The strain exhibits good resistance to mechanical stress, has relatively high oxygen transfer requirements, and is sensitive to a drop in soluble oxygen in the medium below 10% saturation.

Physiological characteristics

Optimum temperature 25 ° C, optimum pH 6.3 to 7.0. It is an aerobic microorganism that fermentes glucose, fructose, sucrose, lactose. Hydrolyzes starch and proteins well, reduces nitrates. It requires biotin for growth, phenylalanine among amino acids.

The advantages of the high-production strain Penicillium chrysogenum CCM 8197 are that it achieves higher production than the parent strain. It is less sensitive to changes in phenoxyacetic acid concentration in the nutrient medium and is therefore advantageous for PEN V biosynthesis under industrial production conditions, with the main advantage being a higher production capacity when a yield of 35,000 IU / ml penicillin can be achieved in 168 hours. The strain is not enormously sensitive to mechanical stress, which is advantageous in industrial applications, since the mycelium is not damaged. For these reasons, it is suitable for use in industrial fermenters.

Examples

Example # 1.

Sucrose inoculum broth in 500 ml 60 ml flasks is inoculated with a spore suspension and cultured at 25 ° C on a rotary shaker for 40 hours. Growth vegetative inoculum was inoculated and cultured on a rotary shaker production flasks containing nutrient media containing lactose, pharmamedium, ammonium sulfate, calcium carbonate, and precursor.

The fermentation lasted 8 days. The maximum production was achieved on the 7th day of fermentation The production capacity of the claimed strain is as follows:

tribe

Internal strain identification

PENV PENG

IU / ml IU / ml

CCM 8197

NMG-FOOK 31

100

100

Industrial usability

The new high-production strain of Penicillium chrysogenum CCM 8197 (NMG FOOK 31) can be used in the industrial production of penicillin V and penicillin G.

Claims (1)

Penicillium chrysogenum high-strain strain CCM 8197 (NMG FOOK 31) producing penicillin V and penicillin G.