CS246668B1 - Brevibactarium flavum strain CCM 3868 - Google Patents

Abstract

The purpose of the solution is the production of the essential amino acid lysine in a medium with molasses as a carbon source. Lysine is synthesized and accumulated in the medium by a mutant of the bacterial species Brevibacterium flavum. By the action of a mutagen and the application of media with increasing molasses concentrations, a mutant resistant to S—(2-aminoethyl)-L-cysteine, dependent on homoserine and leucine and growing in a medium with a high molasses content was isolated. When cultivated for 96 h in a medium with molasses as a carbon source, peanut flour hydrolysate as a nitrogen source and mineral salts under aerobic conditions at 28 °C and neutral pH, it accumulated 46 g/1 L-lysine in the medium. Lysine, used primarily as a feed supplement, can be produced in this way using microorganisms using conventional technological procedures.

Description

(54) Brevibactarium flavum CCM 3868 strain

The purpose of the solution is to produce the essential amino acid lysine in a medium with molasses as a carbon source. Lysine synthesizes and accumulates mutants of the bacterial species Brevibacterium flavum in the medium. Mutoser and application of media with increasing molasses concentration isolated a mutant resistant to S- (2-aminoethyl) -L-cysteine, dependent on homoserine and leucine, and growing in a high molasses content that was cultured in a molasses medium as a carbon source , a peanut flour hydrolyzate as a nitrogen source, and with mineral salts under aerobic conditions at 28 ° C and neutral pH, accumulated L-lysine in an environment of 46 g / L. Lysine, used primarily as a feed supplement, can be produced using microorganisms by conventional technological processes.

The present invention relates to a novel strain of Brevibacterium flavum species resistant to lysine analogues and dependent on homoserine and leuoin, growing in a medium with high molasses content and accumulating in a medium with molasses as carbon source and acid hydrolyzate of peanut flour and ammonium sulphate as aerobic nitrogen conditions and at neutral pH high levels of lysine.

Suitable lysine producers used in the fermentation process are coryneform bacterial mutants, selected as mutants resistant to amino acid analogs and amino acid dependent. Such mutants are, for example, mutants of Brevibacterium lactofermentum resistant to S- (2-aminoethyl) -L-cysteine and dependent on alanine or leucine and accumulating in glucose medium 30-40 g / l of lysine (Tosaka, O.-Takinami, K.-). Hirose & Y .: Agr Biol Chem 42: 1181, 1978; Tosaka, O.-Enei, H.-Hirose, Y .: Trends in Biotechnology 1:70, 1983).

Brevibacterium flavum and Corynebacterium glutamicum mutants that have been resistant to antibiotics, such as penicillin or bacitracin, in addition to resistance to amino acid analogs and amino acid dependence, can be used in the fermentation preparation of lysine using molasses as a carbon source. 1972; Swiss Pat. 535,831 (1973). Lysine production achieved by Brevibacterium flavum mutants varied between 30-35 g / l.

The new strain of Brevibacterium flavum, deposited in the Czechoslovak Collection of Microorganisms of the University of J. E. Purkyně in Brno, class Defender of Peace 10, under the designation CCM 3,868, which is the subject of the invention, exceeds the production of lysine.

The main advantage of the novel strain according to the invention is its ability to grow in a medium with a relatively high molasses content and in a medium with a molasses as a carbon source to produce lysine in relatively high amounts. Cultivation of a new strain in production medium with molasses, peanut flour acid hydrolyzate and ammonium sulphate as a nitrogen source and mineral salts in flasks incubated on a rotary shaker at 28 ° C and pH maintained with aqueous ammonia solution to pH 7.0 can be achieved after 4 days cultivation lysine 46 g / l.

The new strain of Brevibacterium flavum CCM 3,868 is characterized by the following morphological-culture and physiological properties:

Gram-positive, immovable, non-sporulating coca to short sticks.

On masopepton agar they form round, smooth, yellow colonies with straight edges.

On blood agar they form round, yellow colonies with straight edges that do not haemolyz. when growing in the broth, turbidity and sediment are formed, but not blank.

Physiological properties:

Optimal temperature: 28-30 ° C.

Optimal pH: 6.8-7.2.

Aerobic organism.

Gelatin does not liquefy.

It does not break down cellulose.

Glucose, fructose, sucrose fermentes.

Starch hydrolyses poorly.

Reduces nitrates.

Hydrogen sulfide does not form.

Indole does not form.

Catalase test: positive.

Voges-Proskauer test: negative.

It requires biotin and thiamine from vitamins, homoserine and leucine from amino acids.

It is able to grow in medium with 60 molasses.

The starting strain, Brevibacterium flavum CCM 3,736, resistant to S- (2-aminoethyl) -L-cysteine and dependent on homoserine and leucine, was submerged for 24 h in complete medium and the cell suspension was washed with phosphate buffer (pH 7.2). exhibits 18hod. treatment with a 0.05 M solution of ethyl methanesulfonate in phosphate buffer (pH 7.2). The cell suspension after mutagen application is washed with sterile distilled water and 5% (v / v) of the washed suspension is seeded with complete medium flasks and incubated for 24 hours. Inoculate flasks with MeM medium containing 50 µl molasses, 1 S (v / v) inoculum.

After 4 days of culture, the culture is inoculated (again using 1% inoculum) into MeM medium flasks with 60% molasses. After a 4-day cultivation, the culture is seeded on complete medium plates. Colonies grown after 2 days on plates are seeded on sloping agar and isolates grown on sloping masopepton agar are tested for both S- (2-aminoethyl) -L-cysteine resistance and homoserine and leucine dependence, and tested for their ability produce lysine in the medium with molasses as the carbon source.

The details of the invention follow from the following examples:

Example 1

Brevlbacterium flavum CCM 3736 is inoculated with 60 ml of complete medium in a 500 ml boiling flask. The complete media is composed as follows:

glucose casein hydrolyzate yeast extract KH ₂ ^p ° 4 ^K 2 ^HPO 4 solution of agar salts (for solid media)

0.5%

1.0

0.5

0.3

0.1 ml / 1000 ml medium of 2.5% (salt solution is a solution of 0.5 g FeSO ^ .7 HJO and 0.5 g of MnSO ^ .4 H ₂ O in 100 ml water).

The inoculated flask is incubated on a rotary shaker (rotational speed - 3.7, eccentric 25 mm) for 24 h at 28 ° C. The cell suspension is then washed twice with phosphate buffer (pH 7.2) and exposed to the wash for 18 hours. treatment with a 0.05 M solution of ethyl methanesulfonate in phosphate buffer (pH 7.2). After application of the mutagen, the suspension is washed twice with sterile distilled water and a 5% v / v suspension is inoculated with a 500 ml boiling flask with 60 ml of complete medium, which is incubated for 24 hours at 28 ° C on a rotary shaker. Inoculate a 1% (v / v) culture fluid with a 500 ml boiling flask with 20 ml MeM medium as follows:

molasses 50.0% Peanut flour hydrolyzate 2,0 urea 0.5 (nh ₄ ) ₂ Sat ₄ 0.5 kh ₂ Mon ₄ 0.04 MgSO ₄ .7 H ₂ O 0.05 CaCO, 3.0 pH 7.5

The inoculated flask was incubated at 28 ° C for 4 days. 1% (v / v) culture fluid is inoculated again with a 500 ml boiling flask with 20 ml MeM medium containing 60% molasses and after 4 days incubation on a rotary shaker at 28 ° C, complete medium plates are inoculated after dilution. The plates were incubated at 28 ° C for 2 days and the colonies grown on the plates were seeded on sloping agar with masopepton medium. Grown isolates are tested for the degree of resistance to S- (2-aminoethyl) -L-cysteine, for homoserine and leucine dependence, and for the ability to produce lysine in media with molasses as a carbon source.

Example

A culture of Brevibacterium flavum CCM 3868, 24 h old, is inoculated with 60 ml of CSL-B-S inoculum medium in 500 ml flasks. In the same way, the control, i.e. the starting strain of Brevibacterium flavum CCM 3,736; The composition of the inoculation medium is as follows:

sucrose corn steep liquor (65% dry matter) pH

2.5% 3.0

7.0

The inoculated flasks are incubated on a rotary shaker (rotational speed - 3.7, eccentric 25 mm) for 24 h at 28 C. 10% (v / v) B-Me medium:

inocula are inoculated with a 500 ml boiling flask with a 20 ml production flask

molasses 30.0% Peanut flour hydrolyzate 15% (nh ₄ ) ₂ Sat ₄ 1.5% kh ₂ Mon ₄ 0.2 MgSO ₄ .7 H ₂ 0 0.03 CaCOj 2,0 FeSO ₄ . 7 H ₂ O 0,002 MnSO ₄ .4 H ₂ O 0,002 thiamine (hydrochloride) 0.0002 biotin (technical) 0.00005 PH 7.0

During the 4-day cultivation, the pH is adjusted at 24h. intervals with an aqueous ammonia solution of 7.0-7.2. At the end of the culture, the lysine and sugar content of the culture fluid is determined. Lysine is determined manometrically using lysine decarboxylase and sugar as glucose using a Glucose Analyzer 2 (Beokman, U.S.A.).

Lysine production after 4 days of culture was 46 g / l (control production 30 g / l).

Claims (1)

SUBJECT OF THE INVENTION A strain of Brevibacterium flavum CCM 3,868 resistant to S- (2-aminoethyl) -L-cysteine, dependent on homoserine and leucine, growing in a medium with high molasses content and producing lysine in a medium with molasses as a carbon source.