SU1242524A1 - Streptomuces bikiniensis 17-5 strain used for producing inhibitor of trypsin - Google Patents

Abstract

STEPMM Streptomyces bikiniensis 17-5 CMPM S-644 (Central Museum of Industrial Microorganisms of the Institute VNIIGenetics) used to obtain the trypsin inhibitor .. s 4 1 hE to 14 ;:

Description

The invention relates to biotechnology and can be used to obtain protease inhibitors.

The purpose of the invention is to increase the yield of trypsin inhibitor.

Strain Streptomyces bikiniensis isolated from a sample of brown mountain soil from the region of Maikop.

The strain Streptomyces bikiniensis 17-5 is stored in the central museum of industrial microorganisms of the Institute of All-Russia Research Institute of Genetics under the number of CMPM S-644 and is characterized by the following features.

Cultural and morphological images of coal, elution acidic

naki.

The spore chains are straight, the spore shell is smooth.

On Wednesday No. 1 Gauze, the substrate mycelium is uncolored, aerial white-gray, gray. Soluble pigment is missing.

On the environment of čapek, the substrate mycelium is colorless, in the center of the colony is blackened, aerial mycelium is whitish, to light gray, there is no soluble pigment.

On a glucose-aspartic medium, substrate mycelium is thin, gr zno-creamy, aerial mycelium is whitish, there is no soluble pigment. On Lindenbain medium, substrate mycelium is thin, colorless, aerial mycelium is weak, white, and there is no soluble pigment.

On medium No. 2, the substrate mycelium is thin, dark brown, copious aerial mycelium, light gray soluble, bistrovium pigment.

On oats, the substrate mycelium is thin, colorless on the edge is brownish brown, aerial mycelium is abundant, powdery, medium gray, soluble pigment is weak, light nut. Melaninogenic.

Physiological and biochemical properties. Gelatin does not thin, milk peptoneizes, hydrolyzes starch, and grows on fiber. Absorbs glucose raffinose, mannitol; does not assimilate sucrose, rhamnose, arabinose, xylose, inositol.

Antagonistic properties: inhibits the growth of gram-positive and gram-negative bacteria.

Example 1-. Three-day. culture of strain 17-5 grown on the medium of the following composition,%:

peptone 0.5; Hottinger digest 3 vol.%, glucose 1; NaCl 0.5; tap water pH 6.8-7.2, sterilization 0.5 atm 30.min., seeded in the medium of the same composition in the amount of

ten%. After 28 hours of growth at 28 ° C

mixing the culture fluid. centrifuged.

The inhibitor activity at this time is 130 units / ml.

Characteristics of the target product; trypsin inhibitor preparation, partially purified from the filtrate of the culture liquid by adsorption on active

alcohol, desalting, and partial bleaching on Dowex 1 V resin (Serva) and KM cellulose chromatography, is a mixture of

three peptides with mol.mae. 400-500, alkaline and neutral nature. Active drug 25 units / mg.

Example 2, As a seed, use a three-day culture of the strain on the medium of the following composition,%: peptone 0.5; Kotar Hottinger 3 (by volume); NaCl 0.5, glucose 1; tap water, pH 6.8-7.2, sterilization at 0.5 atm 0.5 h. The seed material is seeded in an amount of 10% in the described medium without glucose. Cultivated for 28 hours at 28 ° C with stirring. The culture fluid is separated by filtration.

The inhibitor activity is determined by suppressing the hydrolysis of 1% casein with 5 µg trypsin in 2 ml of 0.1 M borate buffer pH 7.4 in 30 minutes at 37 s. The reaction is stopped by adding 2 ml of 10% trichloroacetic acid. The amount of hydrolyzed casein is determined spectrophotometrically to the absorption of the trichloroacetic acid-soluble components at 280 nm. Per unit of activity take this amount of inhibitor, which causes a 50% inhibition of casein hydrolysis.

The activity in the culture fluid reaches 130 units / ml as compared to 33 units / Nm using a known strain.

The inhibitor preparation obtained by chromatography on KM-cellulose was tested for its ability to inhibit fungal proteases. Upon receipt of the enzyme K-acetyl-O-glucosaminidase

3 12425244

from the filtrate of the culinary liquid of the inhibitor per 1 ml of cultured liquid AcmeTOnium 237 by salting out the sulphate of the drug, the volume of the ammonium is increased by adding 10 units. by 20%

Claims (1)

The inhibitor drug obtained 55 by chromatography on KM-cellulose, tested for the ability to inhibit fungal proteases. Upon receipt of the enzyme N-acetyl ^ -O-glucosaminidase 35 40 3 1242524 4 from the filtrate culture fluid inhibitor 1 ml of culture fluid Aschetopgite 237 by salting out the sulfa bones, the yield of the drug increases the amount of ammonium with the addition of 10 units. by 20%.