CS265699B1 - Industrial bacterial strain Brevibacterium sp. CCM 4013 - Google Patents

Abstract

The invention relates to an industrial bacterial strain Brevlbacterlum sp. M27 / ZA46 producing L-valine deposited in the Czechoslovak Collection of Microorganisms of the University of J.E. Purkyně, tř. Obránců míru 10, Bmo, under the designation CCM 4013. M27 production of essential amino acid.

Description

The invention relates to a novel industrial bacterial strain Brevibacterium sp. M27 producing L-valine. The cultivation takes place in liquid fermentation media which contain carbohydrates (sucrose or other sugars) as the main source of carbon for growth and production and as a nitrogen source corn extract, peanut flour hydrololyxate and other growth factors including mineral salts.

The present invention provides a new industrial strain of Brevibacterium sp. M27 / A46 producing L-valine, deposited in the Czechoslovak Collection of Microorganisms, J.E.Purkyně University, tř. Defenders of Peace 10,

Brno, under the designation CCM number 4013.

Process for preparing a new production strain of Brevibacterium sp. M27 is based on the application of the mutagenic factor N-methyl-N'-nitro-N-nitrosoguanidine, which induces novel mutant strains characterized by auxotrophy. Some of the obtained auxotrophic mutants are characterized by the production of an essential amino acid.

The Brevibacterium sp. M27 auxotrophic to homoserine and resistant to S-amino-ethyl-L-cysteine (AEC ^r ).

The morphological and physiological characteristics of a strain of the invention designated Brevibacterium sp. M27 / A46

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Strain morphology;

(a) colonies on complete agar medium after 72 hours of cultivation at 29 ° C:

shape round edges flat surface glossy profile convex with slightly elevated center color deep yellow size 2 3 mm

(b) microscopically:

sticks thick short, sporadically longer, occasional pairing size: width 1,0 <2 1,2 µm length 2,0 3,6 µm Gram positive

Genetic characteristics: the strain is dependent on homoserine and cystine and resistant to AEC.

In the parent strain of Brevibacterium sp. M27 and the mutant strain were tested for utilization of some C and N sources and their biochemical properties were determined.

Substrate Control Test mutant strain tested

M27 M27 / A46 hydrogen sulphide mannit lysine indole ornithine

Simmons citrate urea ++

ONPG

Voges-Proskauer inosit

Tween 80 phenylalanine arginine sodium malonate ’

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Substrate Control Test mutant strain tested

M27 / A46

adonit - + celobiosa + + rhamnosa + + sucrose + + • sorbit - - eskulin - - trehalosa + + dulcit - - glucose + + without substrate - -

The table shows that the basic biochemical characteristics of the mutant strain and the parent strain of Brevibacterium sp. M27 is identical except for the ability to ferment adonite in the mutant strain, while the parent strain has a negative reaction.

Procedure for obtaining a mutant strain

Breeding of the parent strain of Brevibacterium sp. M27 to obtain essential amino acid producing strains was performed by chemical mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (Koch-Light, England).

A suspension of cells from the exponential growth phase was prepared by culturing in liquid complete broth for 24 hours at 29 ° C. The Poian culture was washed and resuspended in Tris-maleic buffer pK6

So that the cell concentration is approximately 10 cells / mL and a mutagen solution in the same buffer is added to a final concentration of 0.5 mg / mL or 1 mg / mL. The reaction mixture was incubated in a water bath at 29 ° C for 30 or 20 min so that cell survival ranged from 5 to 0.1%. After mutagen treatment, the culture was washed, diluted in saline, and seeded on complete solid broth and incubated at 29 ° C for 96 hrs.

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Fermentation evaluation of auxotrophic mutants of Brevibacterium sp. M27 was performed on liquid broths in laboratory scale flasks on a rotary shaker at 29 ° C for 96 hours.

The method of fermentation and production of valine by the mutant strain is given in the following example.

Example

The inoculum was prepared by culturing a production mutant strain of Brevibacterium sp. M27 / A46 in 50 mL inoculation medium in a 500 mL beaker. Inoculum medium composition: sucrose of those. 20 g, sodium acetate crystals. 40 g, maize extract (60% w / w) 30 g, distilled water ad 1000 mL. Cultivation was carried out on a rotary shaker (240 rpm ^-1 ) at 29 ° C for 18 45 24 hours. Growing inoculum at 10% volume was seeded with 20 mL of fermentation medium (in a 500 mL brewing flask) of the following composition : sucrose of those. 120 g, peanut flour hydrolyzate 120 mL, cornsteep 5 g, potassium hydrogen phosphate 1 g, magnesium sulfate crystals. 0.1 g, calcium carbonate micronized 20 g, distilled water ad 1000 mL, pH of fermentation medium after sterilization 6.8 £ 7.0. The cultivation was carried out on a rotary shaker at 29 ° C for 96 hours. During the fermentation, the pH was adjusted to 7.0 with 10% ammonia solution. Production of valine and mutant strain of Brevibacterium sp. M27 / A46 is 6.5 g of L-valine / L fermentation medium / 96 hr fermentation.

Claims (1)

Industrial bacterial strain Brevibacterium ap. CCM 4013, characterized in that it produces L-valine.