CS234909B1 - Industrial bacterial strain Brevibacterium sp. CCM 3633 - Google Patents

Abstract

The invention relates to the industrial bacterial strain Brevibacterium sp. CCM 3633, stored in the Czechoslovak Collection of Microorganisms of the J. E. Purkyně University, tř. Obránců míru 10, Brno, under the designation CCM number 3633. It is characterized by increased production of essential amino acids compared to the initial parent strain Brevibacterium sp.

Description

The invention relates to a novel industrial bacterial strain Brevibacterium sp. CCM 3633, producing L-lysine in increased yield. The cultivation takes place in liquid fermentation media containing, as the main source of carbon for growth and production of the essential amino acid, carbohydrates (sucrose or other sugars) and as the source of nitrogen, corn extract, peanut flour hydrolyzate and other growth factors including mineral salts.

The present invention provides a new industrial strain of Brevibacterium sp. M27 / P34 producing L-lysine, deposited in the Czechoslovak Collection of Microorganisms University J. E. Purkyně, tř. Obránců míru 10, Brno, CCM number 3633.

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 new mutant strains characterized by another type of pigmentation. The obtained chromogenic mutants are further characterized by increased production of an essential amino acid against the parent parental strain Brevibacterium sp.

In this way, a chromogenic mutant with pink pigmentation was prepared. The Brevibacterium sp. M27 hom ^_ AEC ^r light yellow pigmentation, producing an average of 30.5 g / 1 of L-lysine after 96hod. cultivation.

The morphological and physiological characteristics of the strain of the invention designated Brevibacterium sp. M 27 / / P34.

Substrate glucose lactose sucrose indole hydrogen sulfide urea red. NOs citrate mannit mobility

Tribal morphology:

a) colonies on complete agar medium after 72 hours cultivation at 29 ° C shape rounded edge slightly serrated surface matt profile convex with slightly distinctive »center color pink size 2 to 3 mm« bj microscopic sticks thick short and longer, frequent arrangement in pairs size:

width 1.0 to 1.2 μ length 2.0 to 3.6 μ Gram positive staining

Genetic characteristics:

the strain is homoserine dependent and AEC resistant.

In the parent strain of Brevibacterium. properties 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.

Control

M27 +

+ +

+

Test, production, mutant strain M27 / P34 CCM 3633

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 strains

In order to obtain mutant strains of Brevibacterium sp. M27 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‘-nitroso-N-nitroguanidine (Kooh-Light, England) was chosen as a mutagen.

A suspension of bacteria 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 M tris-maleic buffer pH 6.0, the starting cell number was determined by inoculating various dilutions of the culture on a series of complete agar broth Petri dishes. 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 maintain cell survival in the range of 10 to 0.1 ·%. After mutagen treatment, the suspension was seeded on complete agar broth and incubated for 72 hours at 29 ° C. Then, 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 of fermentation and production of lysine by the mutant strain. Example

The culture mutant Brevibacterium sp. Inoculate M27 / P34 with 50 ml of inoculation medium in a 500 ml beaker. Media composition: sucrose ones. 20 grams, sodium acetate crystal. 40 g corn steep liquor (60 wt.% Dry matter. J 30 g Water dist. Ad 1000 ml. The inoculated culture was cultivated on a rotary shaker (240 rev. Min ^-1) at 29 ° C for 18-24 hours. Grew In a culture of 10% by volume, 20 ml of fermentation medium (in a 500 ml flask) are inoculated as follows: sucrose 180 g, peanut flour hydrolysate 200 ml, cornsteep (60% dry) 10 g, potassium hydrogen phosphate 1 g , magnesium sulfate crystals 0.1 g, calcium carbonate ground 30 g, water dist 1000 ml, pH of 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 was adjusted to 7.0 with 10% ammonia solution, and the lysine production of the chromogenic mutant Brevibacterium sp. M27 / P34 was 52.3 g lysine / l fermentation medium / 96 hr fermentation.

Claims (1)

pRedmE Industrial bacterial strain Brevihacte INVENTION sp. CCM 3633 producing L-lysine.