CS196053B1 - Process for improving microorganism corynebacterium glutamicum and microorganism strain corynebacterium glutamicum ccm 3287 - Google Patents

Description

The present invention relates to a method of breeding a Cooynebacterium glutamicum microorganism and a Corynebacterium glutaoicum CCM 3 microorganism strain. 287, producing L-lysio, resistant to the lysine analog and dependent upon it.

Until 1970, the most commonly used mutants in the fermentative production of lysine were the mutants of auKoorooni, particularly homoseein-dependent mucans. Sano and Shiio / J. Gen, Appl. Míccooio1. 16, 373 (1970) described mutants of the species Brevibacterium flavum which, in the onotonatogenic analogue of lysine S- (2-ammonium) -L-cysteine (AEC) and threonium, grew while the starting finger strain was inhibited. These resistant mucins (AECr-mucins) accumulated in the lysine medium in an amount of about 32 g / l.

The ability of resistant muuant to accumulate high levels of lysine is not only specific to the above species, but has been. other species known as potential lysine producers. In the case of this species, S- (2-amino-ethyl) -L-cystel resistance is usually combined with auxotrophy. Corynebacterium glutaoicuo ATCC 21,526 / AEC ^r , Homoosr ', Leu ' g / liter, Cooynebacterium glutamicum ATCC 21 543 (AEC ^r , Homossr, Leu), production of 32.0 g / 17 and strains listed in German Pat. No. 2 321 461, characterized in addition to AEC resistance, also by dependence on amino acids lines (serine, pito, l, cio, alaio), vitamins (viaaoin B 2) and nucleic acid components (guanini), which accumulate in medium 30 to 30. 40 g / l of lysine.

However, for economical industrial ferro-production of lysine, said levels are low. It has therefore been sought to obtain a strain or ouuant with maximum lysine production within a reasonable time, without the extraordinary demands and essential components of oodium and their onos.

The new strain Cooynebocterium glutomi.cum, deposited in the Czechoslovak collection of microorganisms by J. E. Purkyoe in Brno, Class of the Defenders of mru 10, under the designation CCM 3,287, which is the subject of the invention, surpasses the production of lysine.

The present invention also relates to a method of breeding Corynebacterium glutamicum obtained by a novel strain which comprises treating a starting protophia mhoorgaoim with a solution of 0.05M ethyloethane sulfooate for 18 hours. The resulting muutins, which are grown after the suspension has been treated with a mucilagin, are isolated, and in a minimum medium of 10 .mu.l / l S-.alpha.-thiyl / -L-.

-cysteine. and 0.001 g / L L-hoDoosriou. Preferably, a 0.05 M solution of ethyl ethanesulfonate in a phosphate buffer having a pH of 7.2 is used.

The main advantage of the novel strain according to the invention is the substantially increased production of lysine, which is due both to the resistance to the α and lysine analogs and to the dependence of the lysine. Increased production is evident when two strains of this strain are produced in a production medium with sucrose, peanut acid hydrolyzate and corn liquor, and salts in a fermeotation tank, with suitable mixing and airing, at constant temperature.

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96053 pH of 6.8-7.2 with ammonia at 29 ° C. Under these conditions, after 96 hours of cultivation, lysine production of about 60 g / l medium is achieved.

The new strain of Cooynebacterium glutamicum CCM 3,287 is characterized by the following moophological-curing and physiological properties: Grampositive, non-agglutinous with non-speckling cocci up to short sticks, sizes 1.6 x 1.9 to 3.5 µm, with metachromatic granules, jumping unevenly , in pairs and in irregular clusters.

On masopepton agar it forms round, smooth, shiny, creamy lily colonies with straight edges.

On Masopepton slant agar, it produces an intense lily growth over the entire surface of the agar, leaving no pigmentation.

Turbulence and sedimentation can be observed when this organism grows in the lujon.

On blood agar, they form round, smooth, gray colonies, with a straight lk (nnhnm) lysing.

Physiological properties:

Optimal temperature: 27 to 32 ° C. Optimm ^! pH: 7.0 to 7.2

Temperature: 56 ° C for 1 hour, ° C for 10 minutes.

Aerobic organism.

Gelatin is not ^^. Litmus milk decolourizes, but does not precipitate. Glucose, fats and sucrose are fermented. Scroll hydrolyses only salt.

Cellulose does not decompose. Duucates do not reduce. Hydrogen sulfide does not form.

Indole does not form.

Catalase test: positive.

Methylene blue test: negative. Vooin-Prosrauer test: negative.

To grow, it requires a vitamin of vitamins and an amino-crystals of clay.

In the inventive breeding process, the starting trichlorobacterium Cooynebacterium glutamicum is initially ruthenium 24 hours in complete copper, followed by washing the slurry with phosphate buffer 8 at pH 7.2 and exposing the mutagen to half-fermentation, in this case 0.05 M of a solution of ethyl mmehansulfonate, preferably again in a buffer having a pH of 7.2. After this operation, the suspension washed with distilled water is cultured on a minimal medium with the addition of S- / 2-apinolnethyl-L-cysteine and L-holpplsein. Growths that are grown are inoculated on sloping masopepton agar and tested for the degree of analogue, for the dependence on hω-olenin, and for the ability to produce lysine.

РоИгоЬпс »^! The invention will be apparent from the following examples. .

Example 1 (salt solution is a solution of 0.5 g iron sulfate heptahydrate in 100 ml water).

Incubate on a reciprocating shaker / swing number 91 / min, swing length 9 cm / at 28 ° C for 24 hours. The cell suspension was then washed with phosphate buffer (pH 7.2) and, after washing, exposed to a half-life of 0.05 M ethyl methanesulfonyl for 18 hours. Thereafter, the cellular suspension is washed twice with sterile distilled water with water, diluted and the diluted suspension is inoculated into plates with the following medium (in g):

glucose. 1.0 sodium sodium tartrate0.4 prim, potassium phosphate7.0) sec. 02 g agar25.0 distilled water 1000 ml

The medium is supplemented with 10 mg / ml S- / 2-aminoethyly-L-cysteine and 0.001 mg / ml L-homooeein. The plates were incubated at 28 ° C for 120 hours and the colonies which appeared after that time on the surface of the plates were inoculated on sloping masopepuono agar. The agar-grown isolates were tested for degree of analog resistance, aluminum deposition, and lysine-producing ability. '

EXAMPLE 2

To the strain of Corynebacterium glutamicum CCM 3,287, 24 hours old. inoculate 60 ml of the circular mode in a 750 ml Erlenmeyer flask of the following composition (in g):

sucrose 25.0 corn steep liquor (65 dry matter) 30.0 water 1000 ml pH 7.0; sterilization at 120 ° C for 30 min. The inoculated soil is inoculated on a reciprocating shaker (91 rockers / min, swing length 9 cm) at 28 ° C for 18-24 hours. Inoculated with a culture of 50 ml, 800 ml of production medium is inoculated in a two-liter fermentation tank of the following composition (in g):

sucrose180.0 peanut flour acid hydrolyzate 300.0ml corn steep liquor / 65% dry matter / 10.0 primary potassium phosphate2.0 magnesium sulphate crystal.0.3 calcium carbonate20.0 ferrous sulphate crystal.0 ^ 2 manganese sulphate crystal.0 li ^ in0.004 soybean oil1 ml water 1000 ml pH7.0

Protltrophile with Cooynnelaterium glutamicum is inoculated with 60 ml of fashion in a 750 ml Erlenmayer flask of the following composition (in g):

glucose 0.5 casein hydrolyzate 10.0 extract from yeast 5.0 prim, potassium phosphate 3.0 sec. potassium phosphate 1, o sHi solution 1 ml seaweed agar 25.0 water 1 000

Sterilization 3x for 30 min at 121.5 kPa. Aeration 0.8 l / mn, stirring at 800 ° C / ° C, temperature 29 ° C. Foaming during soybean oil cultivation The pH is adjusted to 7.0 with ammonia water at pH intervals. ml of 70 Z solution at 30, 42, 54, 66 and 74 hours of culture 96 hours of culture Lysine content was determined by lotto using lysine decarloxylase The production of lysine after 96 hours of culture was 61.5 gramsM.

Claims (3)

P ft E D M Ě Т Method for breeding Corynebacterium glutamicum, characterized in that a culture of the initial prototrophic microorganism is treated with a 0.05 M solution of ethyl methanesulfonate for 18 hours, after which the obtained mutants grown after inoculation of the mutagen-treated suspension on minimal medium with 10 mg / ml S- (2-aminoethyl) -L-cysteine and 0.001 mg / ml L-homoserine. OF THE INVENTION 2. A process according to claim 1, wherein a 0.05 M solution of ethyl methanesulfone tu in a phosphate buffer having a pH of 7 ^ 2 is treated. 3. Lysine producing strain of Corynebacterium glutamicum CCM 3287 resistant to lysine analogue and homoserine dependent, obtained by the method of item 1