SU1206306A1 - Pseudomonas species m 35 strain - producer of l-tryptophan on ethanol - Google Patents

Abstract

Shta, mm Pseudomonas species M35 - (collection of the Central Museum of Industrial Microorganisms of the Institute VNIIGenetika, collection number TsMPM B-3038) - producing L-tryptophan on ethanol.

Description

This invention relates to the microbiological industry and relates to the preparation of a new strain producing L-tryptophan.

The purpose of the invention is to obtain the producer strain L-tryptophan, which provides an increase in the yield of the target product using a simpler mineral medium containing ethanol as a source of carbon and energy.

The proposed strain Pseudomonas species M35 - producing L-tryptophan is stored in the Central; the Museum of Industrial Microorganisms of the Institute VNIIGenetika Lod registration number B-3038.

The strain Pseudomonas species M35 is obtained as a mutant as a result of a 4-step selection for the productivity of L-tryptophan using N-methyl-N-nitro-N-nitrosoguanidine as a mutagen - a structural analog of L-tryptophan - 5 -methyl-DL-tryptophan. The starting point for obtaining the mutant is the wild-type strain Pseudomonas species, grown from the wastewater of the Minsk plant of medical equipment.

At each stage of selection, the strain cells in the logarithmic growth phase are treated with L-methyl-K-nitro-K-nitrosoguanidine (100 μg / ml for 30 minutes at 28 ° C in citrate buffer, pH 5.5) on agar medium of the following composition,%; lNa2HP04 (anhydrous) - 0.6 - KHjjP040, NaCl U, U5; 0.1; CaCl 0.0015; MgS04 7H20 0.2; fructose 0,2, agar 1,5; pH 7.0. 5-Methyl-DL-triptophan is dissolved in alcohol and added to the prepared medium before use, to a final concentration of 100, 500, 1000, and 10,000 µg / ml, respectively, at each subsequent selection stage. Bacteria are cultured at 28 ° C for 5-7 days. The grown colonies are passaged repeatedly on the medium of the indicated composition and then checked for the production of L-tryptophan, each time selecting the most productive variants. L-Tryptophan is determined by a known method.

As a result, a mutant distinguished from the wild type by resistance to the inhibitory effect of high concentrations, 5-methyl-1) L-tryptophan, was isolated. In contrast to the well-known properties of the new strain, they will receive a greater yield of L-tryptophan when cultivated on a simpler mineral medium (without vitamins and trace elements) containing ethanol as a carbon source and energy.

The strain Pseudomonas species M35 is characterized by the following features. .

Morphological signs. Cells are straight, rod-shaped with rounded ends, 0.6-2 - 3jn, movable with 2-4 monopolar flagella, gram-negative, do not form spores.

Cultural features. Colonies on agar nutrient media (co-peptone agar, King In agar) are smooth, flat, round, the edge is flat, grayish-white. After 24 hours, growing at 28 ° C forms: coloNII 1-2 mm in size. The growth of the injection in MPA is moderate, mainly on the surface of the medium. In liquid nutrient media forms intense turbidity. It grows well on mineral media of simple composition, does not require growth factors.

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

Pigments. Form a yellow-green water-soluble fluorescent pigment on the environment King. Intracellular pigment does not form.

The reaction to oxidase and catalase is positive. The reaction of Voges-Prosk-ura is negative. Indole does not form. Ammonia forms. Hydrogen sulfide does not emit. On nutrient media containing 5% sucrose solution, Levan does not form. The test for anaerobic denitrification is negative. The reaction to anaerobic arginine dehydrolase is positive, lecithin-like activity is absent.

Does not hydrolyze starch, gelatin, tween-80.

The following carbon sources are used: glucose, glycerin, sucrose, galactose, maltose, fructose, ethanol, methanol, tyrosine, phenylalanine, ornithine, glycine, leucine, glutamic acid, asparagi3. new acid. Does not utilize: arabinose, mannose, xylose, lactose, mannitol, raffinose, dulcitol, sorbitol, cellobiose, inositol, series, anthranilic acid, acetamide, lysine, tryptophan.

According to Bergey, bacteria identification bacteria, Pseudomonas species M35.

Example 1. With a braid loop, sowing the culture of Pseudomonas species M35 in test tubes with aminopeptide broth (pH 7.0, volume 2-3 ml) is done. The seed is grown at 18 hours. The finished seed is introduced in an amount of 3-5% (by volume) into tubes (20 ml in diameter, height 10 cm) containing 5 ml of fermentation medium. The fermentation medium is sterilized at 0.5 atm for 30 minutes. Before use, add the ethanol and then pour it into test tubes. The composition of the fermentation medium is as follows,%:

NajHP04

(anhydrous) 0.6

KHjP04

(anhydrous) 0.3

NaCl0.05

Compiled by N.Afanaseva Editor N.Yatsola Tehred 3, PaliKorrektor S.Shekmar

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VNIIPI USSR State Committee

for inventions and discoveries 113035, Moscow, Zh-35, Raushsk Ha6i, 4/5

Branch PPP Patent, Uzhgorod, Proektna St., 4

06306;

NH4C10.1

CaCla0.0015

MgS04 7H20 0.2 Ethanol 1.5 5 After 72 hours of pulling (pH 7.0) on a rocking chair (240 rotations / min) at 28 ° C, 0.3 g / l of L-tryptophan is accumulated in the culture fluid.

Example 2. Growing a seed material as well as the composition of the fermentation medium as in Example 1. Sowing the seed in an amount of 3-5% (by volume) is made into Erlenmeyer flasks (capacity of 15,250 ml) containing 50 ml of enzyme-- environment.

After 72 hours pulling out on the rocking chair (240 rotations / min.) At 28 ° C, 20 0.63 g / l of L-tryptophan accumulates in the culture fluid.

The use of the Pseudbmonas species M35 strain makes it possible to increase the yield of L-tryptophan by a factor of 20 compared with the known Pseudomonas species strain 5MT when cultured on a simple mineral medium (without vitamins and microelements) containing ethanol as an carbon source and energy.

Claims (1)

STRAIN ΡδΕϋϋΟΜΟΝΑδ 8РЕС1Е8 М 35 PRODUCER OF L-TRIPTOPHANE ON ETHANOL Strain Rzeszkipopaa vresgev M35 (collection of the Central Museum of Industrial Microorganisms of the Institute "VNIIGenetika", collection number CMPM B-3038) - producer of tryptophan. 1 1206306 ²