SU1060678A1 - Strain bacillus subtilis g-38 producing exo-betan-acetylglucoseaminidase - Google Patents

Abstract

The strain Bacillus subtilis G-38 (collection of the Central Museum of Industrial Microorganisms of the Institute of Scientific Research Institute of Genetics, collectible, measures IMPM B-2471) is the producer of exo-L-acetylglucosaminidase.

Description

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The invention relates to the microbiological industry, in particular to the preparation of a new strain used for the biosynthesis of the enzyme exo- (E-M-acetylglucosaminidase.

There are known producer strains of exo-E acetylglucosaminidase, such as Bacillus subtilis B., Bacillus center. Bacillus megatherium. Bacillus lichemiformig ClJ ,.

The disadvantage of these producers is a relatively low level of ec 3o- / i-N-acetyl glucose amide activity.

The closest to the technical essence and the achieved positive effect to the proposed is the strain of Bacillus subtilis G-19, which, under deep cultivation, has exo- / 3-M-acetylglucosaminidase activity by colorimetric method and is 13 honey / ml 2

The disadvantage of the strain is that it is characterized by low enzymatic activity during deep cultivation, the culture itself is poorly stable, the culture is not used in the production of the enzyme preparation, exo -N-jb-acetylglucosaminidase. Strain 0 Bacillus subtilis G-19 is known as a producer of endo- and exo-acetylgluco-3-amide,

The purpose of the invention is to obtain a new highly purified strain - pro-, g-ducent, which preserves the stability of biosynthesis during deep cultivation.

To obtain such a producer strain exo-pR-acetylglucosaminidase, various microorganisms capable of biosynthesis of this enzyme have been isolated from nature.

A highly active strain on the biosynthesis of exo- / K-acetylglucosaminidase 5 was selected from the strains isolated and identified as Bacillus subtilis G-38.

The resulting new strain of Bacillus subtilis G-38, the producer of SKSo-p-N-acetylglucosaminidase xr anits, in the 50th Central Museum of Industrial Organisms of the Institute of Scientific Research Institute for Genetics under the collection number: CMPM B-2471,

The culture of Bacillus subtilis G-38 55 has the following morphological and cultural-physiological properties. The size of the cells of a single-day culture on a m sopetonic ulon of 1.6-2.7 microns by 0.3-0.5 microns. The 0-year cells are highly variable in size. Grown on a medium with glucose or on a karofel medium in smears under a microcopy, they are represented as

thick large sticks, usually arranged in chains. The sticks are stained evenly, moving with peretrihov flagella, gram-positive, forming spores without capsules. The spores are usually extracentral, less often located in the center. They are cylindrical or ellipsoidal, sizes 1.1-1.3 microns by 0.5-0.7 microns. Spores Usually germinate equatorially by breaking the membrane along the equator.

After one-day incubation in a thermostat at 37 ° C, colonies of dya are formed on m-septon agar. meter 2-7 mm. They are greyish, opaque, folded, in the central part of the surface of the colony of the vertebrate of whitish-gray color.

On slices of potatoes, the growth of the culture is rather abundant, the film is dry, thick, wrinkled with oblong folds, grayish-white.

On m soeptonic broth, the growth is especially characteristic, the surface of the broth is covered with a thick wrinkled yellowish gray film, and the broth itself remains transparent.

Bacillus subtilis G-38 culture is strictly aerobic type with an optimal growth temperature of 37 ° C at a pH of 6.4-6.6. The Bacilus subtilis G-38 producer for growth assimilates glucose, sucrose, maltose and glucosamine to form acids without liberating gas, liquefies gelatin, hydrolyzes starch. Nitrates restores to nitrites. On the environment of Clarke forms acetylmethylcarbonol.

Example 1. For the cultivation of a strain of Bacillus subtilis G-38, a nutrient medium of the following composition is used, g / l of water:

Starch45.0

Kalium sulphate 3,3 Magnesium sulphate 0.17 Sodium chloride 0.3 Cucumber extract 26.0 Ammonium phosphate 5.0 Manganese sulphate 1.0 Calcium carbonate 0.13 Nutrient medium of the indicated composition is poured into 100 ml volumes in each of 750 ml flasks and sterilized at 121 ° C for 30 minutes. Sterile nutrient medium with a pH of 6.5 is inoculated with Bacillus subtilis G-38 culture grown on potato slices. Cultivation of the producer wire on rocking chairs (150-180 swings / min at 37 ° C). At 8–12 h of growth of the exo- / -N-acetylglucosaminidase activity, the average in the culture fluid is 16 honey / ml. Actuality of acz / z-K-acetylglucosaminide

determine colimetrically by the color intensity of the formed. p-nitrophenyl in the enzymatic cleavage of the synthetic substrate p-nitrophenyl-p-N-acetylglucosaminide.

Example 2. Cultivation of the Bacillus ubtilis G-38 strain in fermentative ycTaHoikax of the Biolophit type.

For cultivation of this producer in fermentation plants of the Biolofit type with a capacity of 20 liters with a volume of 10 liters, a nutrient medium is used, the composition of which is similar to Example 1. The components of the nutrient medium in Biolofit are sterilized at 120 ° C. Sterile nutrient medium (pH 6.5) is cooled to 37-40 ° C and

Sowed with Bacillus subtilis G-38 spore culture. Cultivation was carried out at 37 ° C, aeration 5 L, stirring 300 rpm. The maximum activity in the culture fluid was determined at 7-12 hours of growth of the Bacillus subtilis G-38 strain. Optimal activity in the culture fluid is 22 honey / ml.

When using a new strain

0 Bacillus subtilis G-38 producer of exo-pH-acetylglucosaminidase creates the opportunity to increase the activity of this enzyme by 69%.

Economic efficiency with

5 widespread production of this strain is 20 thousand rubles. in year.

Claims (1)

Strain Bacillus Subtilis G-38 (57) The strain Bacillus subtilis G-38 (collection of the Central Museum of Industrial Microorganisms of the Institute VNIIgenetika, collection number PMPM V-2471) is a producer of exo-β-N-acetylglucose nidase.