CS200944B1 - Strain of microorganism penicillium chrysogenum ccm f-647 - Google Patents

Description

The invention relates to a strain of the microorganism Penicillium chrysogenum UV XXVII / 74 increasingly producing penicillin V, G and 6-aminopenicillinic acid (6-APK). The advantage of the strain is that it does not have specific carbon source requirements.

This strain was obtained from Penicillium chrysogenum strain number 2/170 by a series of repeated mutagenic interventions - nitrosomethylurea, N-methyl-N-nitrosquanidine, UV-light, parafluorophenylalanine - and subsequent periodically assigned passive selections.

Xzolates after mutagenic and passive selection were evaluated according to the following scheme:

isolated colony on Petri dish t 'slant agar vegetative inoculum 4 * self fermentation

The vegetative inoculum was prepared submersibly in 500 ml beakers with 60 ml inoculum broth containing basic nutrients, ie carbon source (sucrose), nitrogen (corn extract in combination with inorganic nitrogen source) and salt. Vegetative

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200 944 - 2 - flasks were inoculated with a spore agar loop and incubated on a rotary shaker for 48 hours ·

The vegetative inoculum thus prepared was inoculated with 500 ml fermentation flasks, 40 ml soil volume · Sources of carbon were added in the form of dragees, soya flour was a nitrogen source, the soil also contains a precursor, a chalk and a sulfur source ·

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

Isolates are evaluated several times before and after lyophilizaol for long-term culture preservation *

Such a rating system has resulted in a claimable strain.

Production comparison of strain UV XXVII / 74e with control strain CCM F-558 (UV XII / 46).

Fermentation procedure for penicillin V biosynthase »

The UV XII / 46 control strain and the UV XXVII / 74 strain were fermented in 500 ml flasks with 40 ml of CBS-labeled fermentation broth on a 230 rpm rotary shaker, 25 mm shot, corresponding to 0.81 ml oxygen transfer / ml / hr · Inoculation is performed with 10% vol vegetative inoculum. Composition of ferment. CBS soil (in% by weight ·):

soy flour 6.0 saoharosis 0.75 saoharose in doses 13.75 phenoxyacetic acid 0.67 calcium carbonate 0.9 sodium thiosulphate 0.8 20% sodium hydride solution 0,2% vol. All ingredients are dissolve in boiling water and after two hours of standing pH is trolled, eventually adjusted to 8.0. After sterilization tends to be 7.5 to 7.7 · rilizaoe is 20 minutes at 120 ° o. Saoharose is dosed in the form of dragee sterilized by gamma radiation according to the scheme: added substance % wt. 24 48 Hod lny 72 95 192 Overall amount Sucrose 1.25 2.5 3.75 3.75 2.5 13.75

Composition of fermentation broth LS ^ MQD (in% by weight):

soy flour 6.0 lactose 10.0 saoharose in dosages 3.74 foxyoxoic acid 0.67

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- 3 soybean soya oil thiosulphate

20% sodium hydroxide solution

0.6% ammonia water

0.8

0,1% vol. (Flask

0,2% vol.

0.1% v / v, added at 168 hours

The soil is prepared identically (192 hours) and from a 50% solution (24.

is dosed in dragee form with GNS _t Sucrose and 48 * hr,) as follows:

added substance Hours Total amount in% wt. 24 48 192

sucrose 0.62 0.62 2.5 3.74 Composition of fermentation broth 16 (in% by weight) » pharmamedia 7.9 lactose 14.0 sucrose in doses 3.74 phenoxyacetic acid 0.75 + 0.2 added 10% sodium salt at 96 * hour calcium carbonate 0.9 sodium thiosulphate 0.8 soybean oil 0.1% v / v 20% Hydroxide solution sodium 0,2% vol. 0.6% ammonia water 0.1% vol added at 168, o'clock acid phosphate Potassium 0,067%

The soil is prepared in accordance with the CNS soil, and sucrose is added in accordance with the LS ^ MOD soil.

Permentation procedure for penicillin 6 biosynthesis *

The penicillin G biosynthesis process is identical to that for penicillin V, except that the fermentation broths do not contain phenoxyootic acid as a precursor, but a gray salt of phenylootic acid, which is dosed during the fermentation as a dragee or 5% solution as follows. (% by weight).

Permentation soil 24 48 Clock The total amount of % 72 96 120 CHS 0.19 0.19 0.19 0,095 - 0,665 LS.jMOD 0.19 0.19 0.19 0,095 0,085 0.75 16 0.19 0.19 0.19 0.19 0.19 0.95

Permentation procedures for biosynthesis 6 — APK,

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Fermentation procedures for blosynthesis of 6-APK are identical to those for penicillin blosynthesis, but differ in that the fermentation broths contain no precursor.

Fermentaoes are run at 25 ° C for 11 days, samples for determination of efficacy are taken at 9, 10 and 1f. days of fermentaoe. The determination is carried out by the hydroxamate method with appropriate standards. The content of the resulting products was monitored by hydromatographs, in particular the formation of minor penioillins at 6-APK, detected biologically.

Production comparison.

strain fermentation soil* product* produkoe J / ul Contents % UV ZXX / 46 CNS 0 18000 99 IN 19500 98 6-APK 8000 85 IS ₁ M0D IN 16500 98 6-APK 9500 90 16 0 17000 99 IN 18000 98 UV ZZVII / 74 CNS 0 20000 99 IN 22000 98 6-APK 10000 85 LS ₁ MCE IN 19500 98 6-APK 12500 90 16 G 23000 99 IN 24500 98

The increase in UV strain ZZVII / 74 is about 10% on saoharose soil for 0 1 V penicillin, 30% for 6-APK, on lactose soil the increase in production is significantly more significant, and by $ 30 for this reason. UV ZIX / 46 on lactose soils decreases production.

Strain morphology * Strain UV XX7XI / 74, i.e. Penioillium ohxysogenum CCM P 647, forms a colony size of 11 mm colony size in 10 to 14 days on monosporocular sowing on glycerin molasses.

At the edge of the colonies, they form radial folds not higher than 1 µm with a circular cylinder in the center at a diameter of 7 µm and 2 mm high. On the edge of the low white zone. The colonies have an ellpsold to spherical shape with a size of 4 to 5 µm ^x 3 to 4 µl and are colorless on microscopic observations. The culture sporulates well on various types of sporulatory soils, the color of the sporulated culture is deep green, after a long period of cultivation.

The UV strain ZZVII / 74 is highly stable, it does not lose production capability by passing it on

- 5 200 944 solid soils up to IV. generation.

The strain of Penicillium chrysogenum, designated UV XXVII / 74, was deposited in £ s. collection of microorganisms (Czechoslovak Collection of Microorganism), J.E.Purkyně University, Brno, tř. Defenders of Peace 10, under number

Claims (1)

SUBJECT OF THE INVENTION A strain of Penicillium chrysogenum CCM F-647 producing penicillin V, G and 6-aminopenicillanic acid.