SU1440923A1 - Strain of pseudomanas species as producer of thriptophane synase - Google Patents

Abstract

This invention relates to biotechnology and relates to the production of a new strain-producer of tryptophan synthase, which can be used for the enzymatic synthesis of L-tryptophan from indole and serine. The goal of the invention is to develop a NIN — production of a tryptophan synthase producer strain based on bacteria of the genus Pseudomonas, which has a higher activity. The strain Pseudomonas VKPM B-3835 (Ml5) is trpA. mutant bacteria Pseudomonas sp. M discharged from the sewage of the Minsk plant of medical preparations. Bacteria Pseu- g domonas sp. B5, infused in a mineral medium with a limited amount of L-tryptophan, has a TS-B tryptophan synthase activity that catalyzes the conversion of indole and serine to L-tryptophan equal to 200 nmol / min / mg of protein. (L 4 4i O; o N5 00

Description

The invention relates to biotechnology and relates to the acquisition of a new one. a producer strain of tryptophan synthase (KF 4o2,1o20) j which can be used for the enzymatic sieve of L-tryptophan from indole and serine

The purpose of the invention is to obtain a tryptophan synthase producer strain based on bacteria of the genus PseudomonaSf with a higher activity of UO

The strain of bacteria Pseudomonas sp. BKTIM B-3835 (Ml5) is a trpA mutant of the bacteria Pseudotnonas sp. M discharged from the sewage of the Minsk plant medical preparation. Bacteria Pseudomonas sp. MIS grown in an environment with a limited amount of L-trypto-fun, have a TC-B activity of three tophan synthase, which is much better than that of well-known Brevi-bacterium producers. The medium used to cultivate Pseudoinona s M15 consists of cheap and readily available substances. The time spent on cultivating the strain is only 12 hours, instead of 24 hours from the known pseudomonas sp. Ml5 does not need to create a high degree of aeration of the culture.

Strain Pseudomonas sp. Ml5 has the following characteristics.

Morphological signs. Other cells are rod-shaped with round ends 0,6x2-3 c, motile with 2-4 monopolies with flagella, gram-negative, no spore is formed

Cultural features. Colonies on agar nutrient media (moc-peptone agar, King In agar) smooth flat round edges of smooth grayish-white After 24 hours, 28 ° C, colonies 2–3 mm in size grow. The puncture growth in MPA is moderate, mainly on the surface of the medium, In the liquid nutrient medium forms an intensive cloudiness.

Physiological and biochemical characteristics In relation to oxygen, oylgate aerob. The optimum growth temperature is 28-30 ° C, the pH of the medium is in the range of 6.8-7.3. Nitrogen is utilized in the form of ammonium, urea, and nitrate salts.

Pigments Forms a yellow-green water-soluble fluorescent pigment on the medium King B. Does not form an intracellular pigment.

The reaction to oxidase and catalase is positive. The reaction of Voges-Proskauer is negative. Indole does not form. The test for anaerobic arginyldehydrolase is positive. Reaction to anaerobic denitrification is negative., Lecithin is absent

Does not hydrate starch, gelatin, tween-80.

Uses glucose, glycerol, sucrose, galactose, maltose, fructose, ethanol, methanol, tyrosine, phenylaline, ornithine, glycine, leucine, glutamic acid, aspartic acid as a carbon source. Does not utilize arabinose, mannose, xylose, lactose, mannitol, raffinose, dulcite, sorbitol, cellobiose, inositol, serine, lysine, tryptophan, anthranilic acid, acetamide.

Example 1. Bacteria Pseudomonas sp. M15 is inoculated into 1 min medium, g / l: Na., HP04 - 6; 3; NaCl 0.5; one; CaCl 0.015; 2; 2 l glucose containing L-three tofac in a concentration of 0.005 g / l and cultured at 28 C with aeration for 12 hours. The bacteria Pseudomonas sp. so grown. Ml5 have a tryptophan synthase activity of 200 nmol / min / mg protein

II p and me p 2. Bacteria Pseudomonas sp. Ml5 is inoculated into a known medium g / l: 1; (NH4) aS04 25; MgS04. one; MnS04- 0.008 | glucose 130; fumaric acid 12, acetic acid 3; Biotin. 0,00005; thiamine hydrochloride 0.002 g / l; L-tyrosine 0,; 65; DL Methionine 0.4; CaCOj 50; hydrolyzed soybeans 50, рЫ 6.5 and cultivated at 28 ° С with aeration for 12 hours. The bacteria Pseudomonas sp., thus purified. M15 have tryptophan synthase activity equal to 170 nmol / min / mg protein

And p. and mea 3. Bacteria Pseudomonas sp. M15 is inoculated into the medium used to cultivate members of the genus Pseudomonas, g / l 10; NaNH4HP04-4H O 3.5 | 0.2; citric acid 2 g / l; glucose 2; pH 7.0, containing L-tryptophan at a concentration of 0.005 g / l, and cultured at 28 ° C with aeration for 12 hours. Grown

- 1440923

way bacteria pseudoraonas sp. The tryptophan synthase m15 potency compared with TC-B tryptophan activity with the known one. The time spent in synthesis of 190 nmol / min / mg mine on growing Pseudomonas sp. protein. with M15, 2 times less than for Vvevibacterium flavnm.

Claims (1)

The determination of the TC-B activity of tryptophan synthase was carried out according to the general formula of the invention by the adopted method. The same method was used for the prototype strain. 10 Bacterial Strain Pseudomonas speSo, the proposed strain cies BKIIM B-3835, a trypto producer, has a higher TC-B active-fan synthase.