CS252600B1 - Bacterial strain Bacillus subtilis CCM 3853 - Google Patents

Abstract

The solution concerns a bacterial strain of Bacillus subtilis without thermoresistant spores synthesizing exocellular proteases, deposited in the Czechoslovak Collection of Microorganisms in Brno under the number CCM 3 853. The advantage of the mutant strain is that when cultivated in liquid or solid media, it does not form thermoresistant spores within 48 hours at 28 °C. The bacterial strain Bacillus subtilis can be used for the industrial production of exocellular proteases, which can be used, for example, for the preparation of washing powders and for the hydrolysis and removal of various protein materials.

Description

The present invention relates to a novel bacterial strain Bacillus substilis CCM 3,853 free of thermoresistant spores and synthesizing exocellular protease. Characteristic features of the new strain are the ability to grow and produce proteases in the same liquid nutrient media as the parent strain, but do not form thermoresistant spores within 48 hours at 28 ° C. They do not create disputes within 48 hours or in a special sporulation medium.

It is known that in the production of exocellular enzymes, including proteases, by bacilli, the product is contaminated by thermoresistant spores of the production strain. This presents a 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 mutants of the bacilli are incapable of forming exocellular proteases.

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

The strain was obtained following the treatment of the germinated spores with the mutagen of ethyl methanesulfonate as follows: spores of the starting strain 115 (AO 201 638, deposited in the collection of microorganisms under the number CCM 3,622) were activated in 0.1 M phosphate buffer pH 7.2 70 ° C for 15 minutes and germinated in a solution of mineral salts 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, after resuspension in phosphate buffer, plated on casein agar. 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 115/6 are given below. A sample of this mutant is deposited in the Czechoslovak Collection of Microorganisms in Brno under the number CCM / 3,853.

a) Morphological and growth characteristics

Both the starting strain 115 and the 115/6 mutant are thin Gram positive rods, growing in a medium with 5 mg / ml mineral salt and glucose mostly alone or in pairs. The specific growth rate of both in this medium at 30 ° C is in the range of 0.43-0.55 h -1 (generation time 1.3-1.6 h). When sowing on Nutrient agar Difco, strain 115 grows in the form of smooth colonies with slightly irregular edges and the mutant in the form of large low colonies, shiny in the middle, with grooves diverging from the center.

b) Protease forming ability

The starting sporulating strain 115 and the mutant derived therefrom were cultured with shaking for 48 hours at 28 ° C in a medium containing 5% sweeteners, 1% dried yeast, 2% Laktino milk powder 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 and 1% casein substrate. Proteolytic activity is expressed in tyrosine units (TU / ml).

strain

115

115/6

1st attempt 59,5 51,0

2nd attempt 33.7 39.0

3rd attempt 26.7 28.2

(c) Ability to form thermoresistant disputes

Cultures grown in 30 hours at 30 ° C in mineral medium with 1% glucose and 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 .7 I 4 O 0.5 mM; K i HPO 10 10 mM; CaCl 3 0.3 mM; sodium acetate 5 mM; sodium succinate 2 mM; pH 7.2.

The cultures were then incubated for 48 hours at 28 ° C with shaking. Content thermoresistant spores was determined vyočkováním samples inactivated for 15 minutes at 75 ° C on Nutrient Agar Difco number of colonies formed after dilution 0-10 ^-? was detected after 24 hours. In parental culture (115), the presence of 2.3χ10 ⁴ θ thermoresistant spores was detected 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 2.9x10 thermoresistant spores after 48 hours

Q in 1 ml (from 1.7x10 6 cells / ml). 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)

The bacterial strain Bacillus subtilis CCM 3,853 without thermoresistant spores synthesizing exocellular proteases.