CS199775B1 - Strain of microorganism brevibacterium flavum producing l-lysine - Google Patents

Description

Invention ss. relates to a new strain of Brevibacterium flavum, dependent on houosarla and resistant to a lysine analog, accumulating in culture in a β medium by a suitable source of 0, a source of N and mineral salts under aerobic conditions and at an unopposed pH of L-lysine.

In the fermentative preparation of L-lysine, auxotrophic mutants of bacteria, in particular those requiring homology to grow, were most commonly used on an industrial scale. Sano and Sbilo (Seno, K.-Shiio, I.t. Gen.Appl.Microbiol.16: 373.1970) published a paper describing mutants of the species Brevibaoterium-flavum resistant to the sulfur analog of lysine ie S- (2-aminoethyl) - L-oyeteine (AEC), which accumulates in the medium of about 52 g / l L-lysine. In r. 1973 and Japanese labor com Nakamori and Shiio (Nakamorl, S. - Shiio,

I.:Agr. Biol-Chem. 37: 653 (1973)) to isolate lysine (20 g / l) -breaking mutants of Brevibacterium flavum that were resistant not only to ABC but also to the threonine analogue, i.e. kyaeline-alpha-amino-beta-hydroxivalere mutants. Brevibaoterium flavum has recently proved resistant and auxotrophic ea as suitable lysine producers (Sapadone Patent 2,321,461, 1975). Bromading in 65-70 g / l lysine medium.

The known strains of the above-mentioned strains exceed the strain of the present invention, namely the new strain of the microorganism Brevibaoterium flavum, deposited in the Czech Republic999975

199 77S of the Collection of Microorganisms of the University of J.B. in Brno, Tr. Defenders of Mar 10 under CCM 251 ·

The main advantage of the new strain is the increased production of L-lysine, which is caused both by depenedenoia to homoserine and by resistance to lysine anology, found in two-stage cultivation of this strain in production medium with aaoharosis, acid hydrolyzate of peanut flour, corn and thiamine in a fermentation tank suitably aerated and agitated, in which the pH of the ammonia water is maintained at 7.0-7.3 during cultivation and the temperature at 29 ° C - 96 hours. 1-L-lysine.

The new strain is characterized by the following morphological, cultivation and physiological properties:

morphological-cultivation properties:

Gram-positive, immovable, non-spherical to short rods, occurring individually and in irregular angles, on masopeptone agar form round, smooth, lined colonies with straight edges , blanka does not have high biological properties:

optimally pH 6.8 - 7.2 optimal temperature 28 - 30 ° 0 aer & bny organism gelatin does not drain cellulose does not decompose starch does not hydrolyze sjcvaeaje: glucose, fructose, mannose, maltose, aacharose due to reduce hydrogen sulphide do not indole do not test for urine - test on urine - positive Voges-ProskaUerov test - negative for growth required with vitamins - biotin and thiamine from amino acids - homoserine

New. strain induced for 18 h by impregnating with a 0.05 ^M solution of ethylmethanesulfonite on a cell suspension of the Brevibacterium flavum strain in phosphate buffer (pH 7.2). which was supplemented in addition to biotin and thiamluu also by Ir-honoserine (0.01 mg / ml) and S- (2-aminoethyl) -L-systeine (10 mg / ml) and validated were incubated at 26 ° C for 120 h. Colonies that appeared on the plates were inoculated on sloping mazopeptone agar. Cultures that have grown to agaroob have been tested o and dependent on homossrin for rszistsnoie n <* _α and the ability to produce L-lysine in the medium ·

199 779

The method of fermentative preparation of lysine using this strain is described for illustrative purposes in the following case:

Example 1 by culture of strain Brevibaoterium flavum CCM 251 which grew on carnopeptene slurry agar and inoculate a 500 ml boiling flask with 60 ml of inoculum medium of the following composition »saoharose 2 * 5 96, corn extract (65% solids) 5%, pH 7 * The inoculated flask is incubated on a rotary shaker (220 rpm at 28 ° C for 84 h. 50 ml inoculum is seeded with 800 ml production medium in a 2 liter fermentation tank, Composition of production medium »sucrose 18 96, araeid flour acid hydrolyzate 25 96, corn extract (65 96 dry matter) 1 96, EHg DO ^ 0 * 2 96, UgSO ₄ · 7H ₂ 0 0 * 005 96, CaCO 2% and a trace amount of BeSO 4 · 7 ^ 0 (20 mg / kg) 1000 ml) * MnSO4 · AHgO (20 mg / 1000 µl), biotin (50 mog / 1000 ml) * thlamine hydrochloride (200 mog / 1000 ml) soybean oil (5 ml / 1000 ml) * pH 7 * 0 · Inoculated medium was mixed 800 ipm and air 0 * 8 L air / ml · at 29 ° C · used for soybean and during cultivation soybean oil · pH a and adjusts pavkem of 6 hours' lntervaloch to 7.0 to 7 * 2 · While the culture is added rostok sucrose and 50 ml of 70 $> a solution of legs 50, 42 * 52, 62, 72 hours · · kultiváele

Culture time 96 h · - Lysine aa is determined manometrically using lysine decarboxylase · Produkoia at 96 h. 96 g / l of L-lysine.

Claims (1)

Brevibaoterium flavum CCM 251-homoserine-dependent enzyme resistant to S- (2-aminoethyl) -L-cyateine, producing L-lysine