CS264856B1 - Brevibacterium flavum strain CCM 3982 - Google Patents

Abstract

The purpose of the solution is the production of the essential amino acid lysine in a medium with sucrose 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 selective media, a mutant resistant to S—(2-aminoethyl)-L-cysteine, dependent on homoserine and leucine and sensitive to sodium 2-fluoropyruvate was isolated. When cultivated for 4 days in a medium with sucrose as a carbon source, peanut flour hydrolysate and corn leachate as sources of organic and ammonium sulfate as an inorganic nitrogen source and with mineral salts, it accumulated 58 g/L L-lysine at high conversion under aerobic conditions at 28 °C and neutral pH in an environment. Lysine, used primarily as a feed supplement, can be produced in this way using microorganisms using conventional technological procedures.

Description

BACKGROUND OF THE INVENTION The present invention relates to a novel 2-fluoropyruvate-sensitive lysine analog-resistant lysine-resistant strain of Brevibacterium flavum which accumulated high amounts of carbon source under aerobic conditions and neutral pH at high carbon conversion rates at a suitable carbon source. lysine.

In the fermentation preparation of lysine, mutants of coryneform bacteria with multiple genetic traits, which are auxotrophic-regulating mutants exhibiting susceptibility to inhibitors of key lysine biosynthesis enzymes, are used as production organisms. In order to isolate such mutants, mutants of Brevibacterium flavum have been isolated which, in addition to resistance to the lysine analog, i.e. S- (2-aminoethyl) -L-cysteine and homoserine and leucine dependence, are susceptible to sodium 2-flourpyruvate. With the S- (2-aminoethyl) -L-cysteine-resistant and fluorpyruvate-sensitive mutant Brevibacterium flavum, 51 g / L L-lysine was produced in glucose medium as a carbon source (Ozaki, H.-Shiio, I .: Agric Biol Chem 47: 1569 (1983).

The amount of lysine production is dependent on the optimal ratio of pyruvic acid dehydrogenase and carboxylase, which, as glucose metabolism enzymes, also represent key enzymes of lysine biosynthesis. These enzymes decide whether pyruvate will be used in energy metabolism ₍ net) O-will be preferentially in the presence of excess biotin and in the inhibition of fluoropyruvate-induced dehydrogenase metabolized by biotin-activated carboxylase to the oxalacetate from which aspartic acid is formed - the starting lysine biosynthesis compound in bacteria (Tosaka, O.-Takinami, K .: Agric. Biol. Chem. 42:95, 1978; Tosaka, CA-Yoshijiara, Y.-Ikeda, S.-Takinami, K,: Agric. Biol. Chem., 49: 1305 (1985).

. The production of lysine exceeds the known strains of Brevibacterium flavum CCM 3982, which is the subject of the invention. The main advantage of the novel strain according to the invention is its ability to produce high amounts of lysine at a high degree of conversion of the carbon source. When culturing a new strain in production medium with sucrose as a carbon source, peanut flour acid hydrolyzate and corn leach as complex and ammonium sulfate as an inorganic nitrogen source and with mineral salts, after 4 days cultivation at 28 ° C and pH maintained by aqueous ammonia solution were recorded 7.0 Lysine production 58 g / L at 40% conversion.

The new strain of Brevibacterium flavum CCM 3982 is characterized by the following morphological-culture and physiological properties:

Gram-positive, immovable, non-sporulating cocci to short sticks, occurring individually and in irregular clusters.

On masopepton agar they form round, yellow colonies with straight edges; produces intense yellow growth over the agar surface on masopepton slant agar; turbidity and sediment can be observed in the broth, blank is not formed.

Physiological properties: optimum temperature - 28-30 ° C, optimum pH - 6,8-7,2, aerobic organism does not liquefy gelatin, does not decompose cellulose, fermentes glucose, fructose, sucrose, starch does not hydrolyze, nitrates reduce, hydrogen sulphide does not, indole does not .

Catalase test: positive.

Voges-Proskauer's text: negative.

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

A cell suspension of homoserine and leucine-dependent Brevibacterium flavum CCM 3736 resistant to S- (2-aminoethyl) -L-cysteine and grown in complete medium is suspended in phosphate buffer (pH 7.2) for 18 hours with 0.05M of ethyl methanesulfonate and the suspension after application of the mutagen and washing with sterile distilled water, inoculate a flask with complete medium. 24hod. The washed medium and the undiluted suspension were seeded with minimal medium plates supplemented with homoserine and leucine and plates with minimal medium equally enriched but containing 2-fluoropyruvate sodium. Fluoropyruvate-sensitive mutants are subjected - after determining the degree of S- (2-aminoethyl) -L-cysteine resistance, homoserine and leucine dependence, and fluorpyruvate sensitivity - to production evaluation in production medium with sucrose, peanut flour hydrolyzate, corn steep liquor and mineral salts inoculated with inoculum grown in medium with sucrose, sodium acetate and corn steep liquor.

The details of the invention follow from the following examples:

Example 1

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

glucose 0.5% casein hydrolyzate 1.0 yeast extract 0.5 kh ₂ after ₄

K ₂ HP ° 4 agar salt solution (for solid media) (The salt solution is a 0,5 g solution

0.3

0.1 ml / 1000 ml medium

2.5

FeSO ^ .7 H ₂ O and 0.5 g MnSO ^ .4 H ₂ O in 100 ml water).

The inoculated flask is incubated on a rotary shaker (frequency - 3.7 Hz, eccentric - 25 mm) h at 28 ° C. The cell suspension is 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 beaker with 60 ml of complete medium which is incubated for 24 h at 28 ° C on a rotary shaker. The cell suspension is washed 2X with sterile distilled water and the appropriately diluted suspension is seeded with minimal medium plates supplemented with 0.1 mg / ml L-homoserine and 0.05 mg / ml L-leucine. The composition of the minimum medium is as follows:

glucose 0.1% sodium citrate 0.04 kh ₂ Mon ₄ 0.7 to ₂ hp ° ₄ 0.2 (nh ₄ ) ₂ Sat ₄ 0.1 MgSO ₄ .7 H ₂ O 0.01 biotin solution 2 mg / 1000 ml medium salt solution 1 ml / 1000 ml medium agar 2.5%

(Salt solution: 0.5 g FeSO ₄ -7 H ₃ O and 0.5 g MnSO _{4 <} 4 H ₂ O in 100 ml water.)

Colonies grown on the plates after 3 days incubation at 28 ° C with sterile punches are transferred to minimal medium plates supplemented with homoserine and leucine and homoserine, leucine and 0.1 mg / ml sodium 2-fluoropyruvate. Fluorpyruvate-sensitive mutants are seeded on sloping masopepton agar and the agar-grown isolates are tested for both S- (2-aminoethyl) -L-cysteine resistance, homoserine and leucine dependence, and sodium 2-fluoropyruvate sensitivity, respectively. their ability to produce lysine in a production medium with sucrose, peanut flour hydrolyzate, corn steep liquor and mineral salts is investigated.

Example 2

A culture of Brevibacterium flavum CCM ..., 24 h old, is inoculated with 50 ml of inoculation medium in 500 ml flasks.

• Other inoculum medium: sucrose 2.0% sodium acetate 5.0 corn liquor (60% dry) 3.0 pH 7.0

The inoculated flasks are incubated on a rotary shaker (frequency - 3.7 Hz, eccentric - 25 mm) for 24 h at 28 ° C. A 12% (v / v) inoculum is inoculated with a 500 ml boiling flask with 20 ml production medium of the following composition:

sucrose 15.0% peanut flour hydrolyzate 20.0% (v / v) corn steep liquor (60% dry matter) 1.0% (nh ₄ ) ₂ sat ₄ 1.0 k ₂ hpo ₄ 0.1

MgSO ₄ .7H ₂ 0 0.01

CaCO ₃ 3.0

PH, 7.0

During a 4-day culture, the pH is adjusted to 7.0 with 10% ammonia solution. At the end of the culture, the lysine and sugar content of the culture fluid is determined. Lysine is determined oscilopolarographically and sugar as glucose using a Glucose Analyzer 2 (Beckman,

USA).

Lysine production after 4 days of cultivation was 58 g / l (40% conversion).

Claims (1)

SUBJECT OF THE INVENTION S- (2-aminoethyl) -L-cysteine resistant strain of Brevibacterium flavum CCM 3982, dependent on homoserine and leucine, producing lysine at high conversion, with susceptibility to sodium 2-fluoropyruvate.