CS252604B1 - Bacterial strain Bacillus subtilis CCM 3855 - Google Patents

Abstract

The present invention relates to a bacterial strain of Bacillus subtilis CCM 3855, without thermo-resistant exocellular protease synthesizing spores. The advantage of the mutant strain compared to the strain and the longer sporulating strains of Bacillus used so far is that it does not produce thermosensitive spores during 28 hours at liquid cultures in liquid or solid media. The production of exocellular proteases by the strain is the same or somewhat higher than that of the original production strain.

Description

The present invention relates to a novel bacterial strain Bacillus subtilis CCM 3855 free of thermoresistant spores and synthesizing exocellular proteases. Characteristic features of the new strain are the ability to grow and produce proteases in the same liquid gray media as the starting strain, but in which they do not form thermoresistant spores within 48 hours at 28 ° C. They do not create spores within 48 hours or in a special sporulation of the medium.

Is it known that in the production of exocellular enzymes, including? proteases, using bacilli, the product is contaminated with thermoresistant spores of the production strain. This represents a certain disadvantage both from a hygienic and a technological point of view, since the demands on the subsequent sterilization of the fermentation plant are increasing. At the same time, the risk of contamination of other fermentations and products with thermoresistant spores of Bacillus subtilis increases.

In contrast, most asporogenic mutant bacilli are incapable of forming exocellular proteases.

These disadvantages are overcome by the bacterial strain Bacillus subtilis without thermoresistant spores and synthesizing exocellular proteases. After mutagenic treatment and sowing of the casein-containing solid agar nutrient broth, both protease-forming ability and altered colony shape, which usually indicates a loss of sporulovic ability, were observed simultaneously in individual colonies. This procedure resulted in a mutant synthesizing exocellular protease without thermoresistant spores.

The strain was obtained after treatment of the germinated spores with Methyl Ethane Methanesulfonate as follows: Spores of the starting strain 115 (AO 201 63Θ, deposited in the US collection of microorganisms under the number CCM 3,622) were activated in 0.1 M phosphate buffer pH 7.2 by heating to 70 ° C for 15 minutes and germinated in a mineral salt solution of pH 7.2 supplemented with glucose (5 mg / mL) and yeast extract (0.2 mg / mL) for 1 hour at 35 ° C. The germinated culture was transferred to phosphate buffer and mutagenized with 0.1 M ethyl methanesulfonate with shaking for 17 hours at 30 ° C. The survival rate of germinated spores was 1%.

The mutagenized culture was centrifuged, washed with phosphate buffer and sown on casein agar after resuspension in phosphate buffer. Colonies differing from the parent strain either morphologically or by the size of the undigested casein zone were subjected to more detailed examination. The strains obtained were lyophilized in 10% lactin.

The characteristics of the new bacterial strain 115/13 are given below. A sample of this mutant is deposited in the Czechoslovak Collection of Microorganisms in Brno under the number CCM / 3,855.

a) Morphological and growth characteristics

Both the starting strain 115 and the 115/13 mutants are thin gram positive rods, growing in a 5 mg / mL glucose mineral salt medium mostly alone or in pairs. The specific growth rate of both in this medium at 30 ° C is in the range of 0.43 to 0.55 h ^-1 (generation time 1.3 to 1.6 h. When sowing on nutrient agar Difco) strain 115 grows. in the form of smooth colonies with slightly irregular edges and mutent in the form of large low colonies, in the middle of shiny, with grooves radially diverging from the center.

b) Protease forming ability

The starting sporulating strain 115 and the mutant derived therefrom were cultured with shaking at 28 ° C for 5 hours in medium containing 5% sweeten, 1% dried kvs, 2% Laktino milk and 5% calcium carbonate (default pH 7.5). After 48 hours, the cultures were centrifuged and proteolytic activity was determined in the supernatants at pH 7.2 with 1% casein substrate. Proteolytic activity is expressed in tyrosine units (TU / mL).

strain 1. pokus 2. pokus 3. pokus 115 59.5 33.7 26.7 115/13 45.5 51.8 33.4

c) Ability to form thermoresistant spores

Cultures grown from 8 hours at 30 ° C in mineral medium with 1% glucose with 0.2 mg / ml yeast extract were centrifuged and resuspended to an optical density corresponding to 1 mg / mL dry matter in sporulation medium of the following composition (MgSO 4. 0.5 mM; Κ ^ ΗΡΟ ^ mM; CaCl 3 0.3 mM; sodium acetate 5 mM; sodium succinate 2 mM; pH 7.2 µ,

The cultures were then incubated for 48 hours at 28 ° C with shaking. The content of thermoresistant spores was determined by inoculating samples inactivated for 15 minutes at 75 ° C on Nutrient agar -7 *

Difco. The number of colonies grown after dilution of 0 to 10 was determined after 24 hours. The parent culture (115) showed the presence of 2.3 x 10 ⁰ thermoresistant spores in 1 mL.

No thermoresistant spores were detected in the mutant culture. Neither microscopic examination of mutants in cells revealed spores. In an experiment in production sweetening medium (see b), the parental strain produced 2.9 x 10 ° thermoresistant spores in 1 ml (from 1.7 χ 10 ⁹ cells / ml) after 48 hours. The mutant did not create thermoresistant disputes. The mutant of the invention can be used for the industrial production of exocellular proteases useful, for example, for the preparation of washing powders, as well as for the hydrolysis and removal of various proteinaceous materials.

Claims (1)

SUBJECT OF THE INVENTION The bacterial strain Bacillus subtilis CCM 3,855 without thermoresistant spores synthesizing exocellular proteases.