CS218514B1 - Microorganism strain Penicillium chrysogenum CCM F-720 - Google Patents

Abstract

The invention relates to a new strain of Penicillium chrysogenum CCM F-720, which produces penicillin G and V in significantly higher yields under conventional cultivation conditions.

Description

BACKGROUND OF THE INVENTION The present invention relates to a penicillin G and V strain of Penilecillium ch-rysogenum CCM F-720.

This strain was obtained from the Penicillium chryso strain, genes of registration number 65/41 by mutagen treatment and repeated passive selection.

Mutagens used: UV-light · and nitromethylurea (NMU).

Application of UV-light: The light source was a 40 W germicidal lamp with a pre-supply voltage stabilizer in 5 ml of distilled water and irradiated in a 45 ml petri dish. During irradiation, the suspension was stirred on an electromagnetic stirrer. The distance of the dish from the radiation source was 20 cm, during irradiation, 0.5 ml samples were taken at appropriate time intervals, which were further diluted and sown on petri mills with sporulated soil of molasses, glycerin, yeast extract and salts as described for passive selection procedure. , 1 '

Application of NMU: NMU (&. & Λ K. Laboratories, Inc.) was applied at a concentration of 5. · g / ml. The mutagen was dissolved in citrate-phosphate buffer pH 6.0 with stirring, then slurry suspended in the same buffer was added. The mutation mixture was stirred on an electromagnetic stirrer at 25 ° C. At appropriate time intervals, 1 ml samples were taken and diluted to dilute the MNU to an ineffective concentration. Samples were sown on Petri dishes as described in the passive selection procedure.

Passive selection: Passive selection pores were suspended in distilled water or saline, diluted with dilution series and factor 10 and inoculated into petri dishes with the same sporula broth. Several dishes were inoculated in parallel from each dilution. After 6 - 9 days of incubation at 25 ^Q C was completed in isolates sporulation and it was possible přeočkoivat is sustained on slants of sporulation with the same soil. Incubation of this material also occurred at 25 ° C for 6 to 8 days.

By appropriate alternation of mutagens and passive selection, Isolate 43 / 28-80 D was obtained.

The isolates were evaluated according to the following:

isolated colony on Petri dish i

oblique agar l

vegetative inoculum l

proper fermentation

The high-production isolates obtained are kept in the form of lyophilized cans.

Penicillin potency was determined by the hydroxamalt method.

This evaluation system resulted in the strain being protected.

The strain forms a colony size of 7 - 10 mm at a cultivation temperature of 25 ° C during monosporic sowing of the glycerol-molasses sporulation medium over 6 days. The culture intensively sporulates on different types of sporulating soils and on natural substrates such as millet, barley, corn, rice, etc., the sporulation is completed within 7 days of cultivation; The color of the cultured culture is deep green. Colonies on solid soils are milled with breast grooves, which are also visible on the underside of the colony. The center of the colony is embossed, the edge of the colony is white edged at a width of about 2 mm. The conidiophore is about 300 _[ mu] m long and has smooth walls. The brushes are asymmetric, with bunches of whiskers growing on the branches. The conidionic neck is narrowed. The conidia are oval, about 2.5 to 3 .mu.m in size. several weeks, on natural substrates for several months.

Other characteristics of the strain: Mycelium of the strain is moderately sensitive to intense strain during fermentation, iron at 50 χ / ml inhibits biosymthesis by 30-40%, penicillin biosynthesis is inhibited by the product itself at penicillin concentrations above 30,000 U / ml The strain will neutralize various carbon sources, preferably lactose, chess, glucose, and various oils. Suitable nitrogen sources in addition to organic sources are the ahorganic nitrogen compounds, preferably ammonium sulfate. During fermentation about 3% of secondary penicillins are formed. '<>

Example 1 ’i

The vegetative inoculum was prepared submersibly in 500 ml beakers with 60 ml inoculum broth containing basic nutrients, i. carbon source of sucrose, nitrogen, corn extract and ammonium sulfate and salts. The soil for preparing the vegetative inoculum was a looped agar spore loop and incubated on a rotary shaker for about 40 hours.

The vegetative inoculum thus prepared was inoculated with 500 ml fermentation flasks, 40 ml of fermentation broth. The source of carbon was lactose, sucrose and soybean oil, the source of nitrogen was soy flour and ammonium salt, the soil also contained a precursor of the side chain of penicillin, salt, calcium carbonate and a source of sulfur.

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 11 days. Samples for potency determination were taken at 8, 9,

10th and 11th days of fermentation. Maximum production was generally achieved on day 10 of fermentation. Under these fermentation conditions, 16,500 U / ml was achieved on a laboratory scale.

Penicillium chrysogenum strain, labeled 43 / 28-80 D, was deposited in MS. collection of microorganisms (Czechoslovak Collečtion of Microorganisms), J. E. Purkyně University, Brno, Tř. Defenders of Peace 10, under no. CCM F-720.

Claims (1)

SUBJECT OF THE INVENTION Penicillium chrysogenum strain CCM F-720 producing penicillin V and G.