CS236206B1 - Industrial bacterial strain Brevibacterium sp. CCM 3632 - Google Patents

Abstract

The invention relates to the industrial bacterial strain Brevibacterium sp. M27/P28 producing L-Lysine, deposited in the Czechoslovak Collection of Microorganisms of the University J.E. Purkyně tř. Obránců míru 10, Brno, under the designation CCM number 3632. It is characterized by increased production of the essential amino acid compared to the parent strain Brevibacterium sp.

Description

The present invention relates to a novel industrial bacterial strain of Brevibacterium sp, (LCrTMThere is an increased yield of L-lysine product). nitrogen source corn extract »peanut flour hydrolysate and other growth factors including mineral salts.

The present invention provides a new industrial strain of Brevibacterium sp. M27 / P 28 producing L-lysine »deposited in the Czechoslovak Collection of Microorganisms University of J. E. Purkyně, tř. Defenders of Peace 10 »Brno» under the designation CCM number 3632.

Process for preparing a new production strain of Brevibacterium sp. is based on the application of the mutagenic factor N-methyl-N * -nitro-N-nitrosoguanidine, which induces novel mutant strains characterized by another type of pigmentation. Further, the mutant chromogenic mutants are characterized by an increased production of an essential amino acid against the parent parental strain Brevibacterium sp.

In this way, a chromogenic mutant with ocher pigmentation was prepared. As a starting material for mutagenesis the strain Brevibacterium sp. M27 mountain AEC ^r light yellow pigmentation, producing an average of 30.5 g / l L-lysine after 96 hours of cultivation.

The morphological and physiological characteristics of a strain of the invention designated Brevibacterium sp. M27 / P28.

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Morphology of strain: a / colonies on complete agar broth after 72 hours cultivation at 29 ° C:

shape round edges flat surface glossy profile convex with slightly elevated center color yellow eye command bone 2 Φ 3 mm b / microscope:

sticks short thick, sporadically longer size: width 1,0 1,2 yU length 2,0 «3,6 yU Gram coloring

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

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

Substrate Control Test mutant strain tested

M27 M27 / P28 360glucose + lactose sucrose + indole hydrogen sulfide urea + red. NOj + citrate + mannit mobility

The table shows that the basic biochemical characteristics of the mutant strain and the parent strain of Brevibacterium sp. M27 is identical.

Procedure for obtaining mutant ^ZUb strains

In order to obtain mutant strains of Brevibacterium sp. N27 with altered pigmentation and increased production of the essential amino acid was used for the breeding of the parent strain by a mutation process. The active chemical N-methyl-N * -nitrosine-N-nitroguanidine (Kooh-Light, England) was chosen as a mutagen.

Bacterial suspension from the exponential growth phase was prepared by culturing in liquid complete soil for 24 hours at 29 ° C. After washing and resuspending the cells in 0.2 H tris-maleic buffer pH 6.0, the starting cell number was determined by inoculating various dilutions of the culture on a sriri petri dish complete with agarose broth. A mutagen dissolved in buffer was added to a measured portion of the suspension to a final concentration of 1 mg / ml. The reaction mixture was incubated in a water bath at 29 ° C. The mutagen treatment was stopped by adding a 10-fold amount of 4 ° C cold tris-maleic buffer and centrifuging. Several mutagen treatment times were chosen to have cell survival ranging from 10 to 0.1%. After mutagen treatment, the suspension was seeded on complete agar broth and incubated for 72 hours at 29 ° C. The colonies with altered pigmentation were isolated and further evaluated.

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

The following example illustrates the method for fermentation and production of lysine by the mutant strain.

Example

The culture mutant Brevibacterium sp. Inoculate M27 / P28 with 50 ml of inoculum medium in a 100 ml beaker. Ingredients m / dia: sucrose those. 20 g, sodium acetate crystals 40 g, corn extract / 60% wt. drying / 30 g, water dest. ad 1000 ml. The inoculated culture is cultivated on a rotary shaker (240 rpm) at 29 ° C for 18-24 hours. The 10 ml culture is inoculated with 20 ml of fermentation medium (in a 500 ml flask / icebox). Ingredients:

- 4 283 206 sucrose of those. 180 g, peanut flour hydrolysate 200 ml, cornsteep / 60 X dry · 10 g, potassium hydrogen phosphate 1 g, magnesium sulfate crystals. 0.1 g, calcium carbonate ground 30 g, water dest. ad 1000 mt, pH of the fermentation medium after sterilization 6.8 to 7.0. Cultivation is carried out on a rotary shaker at 29 ° C for 96 hours. During fermentation, the pH is adjusted to 7.0 with ammonia solution at pH 7.0. Lysine production in chromogenic mutant Brevibacterium sp. H27 / P28 is 50.1 g Lysine / L fermentation medium / 96 hr fermentation.

Claims (1)

SUBJECT OF THE INVENTION 263 206 Industrial bacterial strain Brevibacterium sp. CCM 3632, producing L-lysine.