CS240712B1 - Lysine producing stem of microorganism brevibacterium flavum ccm 3736 - Google Patents

Description

The present invention relates to a novel strain of Brevibacterium flavum, resistant to lysine analog and dependent on homoserine and leucine, accumulating high amounts of lysine in culture with a suitable source of carbon, nitrogen and mineral salts under aerobic conditions and at neutral pH.

In the fermentative preparation of lysine, regulatory mutants resistant to amino acid analogs have been found to be suitable production organisms, replacing the previously used auxotrophic mutants, especially homoserine-dependent mutants.

Among the lysine producers, mutants with multiple genetic traits are mentioned as the most suitable in the literature, i.e., mutants resistant to the lysine analog, which, in addition to this resistance, also showed dependence on certain amino acids.

(Sano K. - I. Shiio: J. Gen. Appl. Microbiol. 16: 373, 1970) found that some of the mutants of the species Brevibacterium flavum resistant to the lysine analog, i.e. S- (2- aminoethyl β-L-cysteine (AEC) accumulates relatively high amounts of lysine (about 30 g / L) in the medium.

Other important lysine producers were also Brevibacterium lactofermentum mutants that were resistant to AEC and required leucine to grow (Tcsaka O. - K. Takinami - Y. Hirose: Agr. Biol. Chem. 42:

(1181, 1978).

By the production of lysine, the presently known strains exceed the new strain of Brevibacterium flavum, deposited in the Czechoslovak Collection of Microorganisms of the University of J. E. Purkýně in

- Brno, Peace Defender Class 10, under the designation CCM 3736, which is the subject of the invention.

The main advantage of the new strain according to you. of the finding is significantly increased production of lysine, caused by resistance to anaV, log lysine, and dependence on homoserine and leucine. Increased lysine production is evident in the two-stage cultivation of this strain in production medium with sucrose, peanut flour acid hydrolyzate, corn steep liquor, and mineral salts in >. flasks incubated on a rotary shaker at 28 ° C and pH maintained by ammonia water at 7.0. Under these conditions, after 96 hours of cultivation, 46 g / l of production is achieved.

The new strain of Brevibacterium flavum CCM 3736 is characterized by the following morphological-culture and physiological properties:

Gram-positive, immovable, non-sporulating (ingot to short sticks, occurring individually, in pairs and in irregular clusters).

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

On Masopepton sloping agar, the yellow growth covers the entire surface of the agar.

When the organism grows in the broth, turbidity and sediment are formed, but not the blank.

On blood agar, this organism forms round, yellow colonies, non-haemolyzing, with straight edges.

Physiological properties:

Optimal temperature - 28 to 30 ° C.

Optimal pH - 6.8 to 7.2.

Aeorobic organism.

Gelatin does not liquefy.

It does not break down cellulose.

Glucose, fructose, sucrose fermentes. Starch hydrolyzes very poorly. Reduces nitrates.

Hydrogen sulfide does not form.

Indole does not form.

Catalase test: positive.

Voges-Proskauer test: negative.

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

The starting strain of Brevibacterium flavum CCM 251 is cultured for 24 hours in complete medium and the cell suspension after washing with phosphate buffer (pH -7.2) is treated with a 0.05 M solution of ethyl methanesulfonate in buffer (pH -7.2) for 18 hours. The mutagen suspension was washed 2X with sterile distilled water 2X and seeded, after dilution into plates with minimal medium supplemented with 10 mg / ml S- (2-aminoethyl) -L-cysteine, 0.01 mg / ml L in addition to biotin and thiamine. -homoserine and-0.01 mg / ml L-leucine.

Colonies grown on homoserine and leucine plates were seeded on sloping masopepton agar. The isolates grown on sloping agar are tested for resistance, homoserine and leucine dependence, and the ability to produce lysine is examined.

The details of the invention follow from the following examples.

Example 1

Brevibacterium flavum CCM 251 is inoculated into 60 ml of complete medium in a 750 ml conical flask of the following composition:

glucose i 0.5% casein hydrolyzate 1.0 yeast extract 0.5 potassium dihydrogen phosphate 0.3 potassium hydrogen phosphate 0.1 salt solution 1.0 ml per 1000 ml medium per 1000 ml medium agar 2.5

240 (Salt solution is (a solution of 0,5 g ferrous sulphate heptahydrate and 0,5 g manganese sulphate pentahydrate in 100 ml water)

Incubate the inoculated flask on a reciprocating shaker (number of rockers 91 / min, swing length = 9 cm) at 28 ° C for 24h. The cell suspension was then washed twice with phosphate buffer (pH -7.2) and the wash was exposed to a 18 hour 0.05 M solution of ethyl methanesulfonate in phosphate buffer (pH -7.2). The cell suspension is then washed 2X with sterile distilled water, suitably diluted and diluted with the diluted suspension.

eats plates with minimal composition: the medium follows- glucose 0.1% sodium citrate hydrogen phosphate 0.04 Potassium 0.2 dihydrogenphosphate 14 g solution Potassium 100 ml water; 50 ml solution per 1000 ml medium ammonium sulfate 0.1 rat magnesium sulphate. 0.01 solution of Fe and Mn salts as above agar 2.5 Minimal media sh with solution biotin and thiamine. To 1000 ml medium with

Add 1 ml of a solution of ¹ .02 g of biotin in 100 ml of water and 1 ml of a solution of 0.04 g thiamine in 100 ml of water. The medium is then supplemented with 10 mg / ml S- (2-aminoethyl) -L-cysteine and 0.01 mg / ml L-homoserine.

Plates are incubated for 96 h at 28 ° C and colonies grown on sterile punch plates are transferred to plates with minimal medium containing only homoserine in addition to biotin, thiamine and to plates with 0.01 mg / ml L-homoserine and 0.01 mg / ml L-leucine. Colonies that grew on plates containing homoserine and leucine but not on plates containing only homoserine were seeded on masopepton agar. Isolates grown on chic12 agar are tested for resistance to the lysine analog, homoserine and leucine dependence, and lysine-producing ability.

Example 2

A culture of Brevibacterium flavum CCM 3736, 24 h old, is inoculated with 60 ml of inoculum medium in 500 ml flasks. Inoculate the control, which is the starting strain of Brevibacterium flavum CCM 251 in the same way. The composition of the inoculation medium is as follows: · sucrose 2.5% corn steep liquor (05% dry matter) 3.0 pH 7.0

The inoculated flasks were incubated on a rotary shaker (220 rpm, eccentric - 2.5 cm) for 24 h at 28 ° C. Inoculate 2% (g) with a 500 ml boiling flask with 20 ml production medium as follows:

sucrose 18.0% acid hydrolyzate ‘ 30.0% peanut flour (obj.) corn extract (65% dry) 1.0% dihydrogenphosphate Potassium 0.2 magnesium sulfate crystals. 0.03 calcium carbonate 2,0 ferrous sulphate ikryst. 0,002 manganese sulfate crystals. 0,002 biotin 0.00005 thiamine (hydrochloride) 0.0002 pH - 7.0

During the cultivation, the pH is adjusted to 7.0 to 7.2 at 24 hour intervals with ammonia water. The culture time is 90 h. Lysine is determined manometrically using lysine decarboxylase.

Lysine production at 96 h culture was 46 g / l (control production 40 g / l).

Claims (1)

PREDMST Lysine-producing strain of Brevibacterium flavum CCM 3736, resistant to homoserine and leucine, S- (2-aminoethyl) -L-cysteine.