RU2208633C1 - Strain bacillus subtilis p-1 as producer of protease - Google Patents

Abstract

FIELD: biotechnology, biochemistry, microbiology. SUBSTANCE: invention relates for preparing the novel strain Bacillus subtilis P-1 as a producer of protease with high total and specific activity in cultural fluid. The strain is prepared by selection from the strain Bacillus subtilis 534 by multiple reinoculation through nutrient medium with increasing concentration of antibiotic. Size of cells is (7- 1.3) x (0.5) mcm that are distributed separately or as small chains and form internal central spores of oval form. The strain grows on different nutrient media, it is an aerobic, gram-positive microorganism, shows motility, its optimal growth conditions: temperature 37 C, pH value 6.0-7.5. Invention provides high level of synthesis of protease and reduced time of culturing. EFFECT: valuable properties of strain. 1 dwg, 2 tbl, 2 ex

Description

 The invention relates to biotechnology, in particular to the creation of a new strain used to obtain the enzyme - protease. Proteolytic enzymes are widely used in medicine, in the food, agricultural, leather and other industries. Proteolytic enzymes are obtained as products of biosynthetic activity from fungi or bacteria. However, bacteria are preferable to use, because they have a faster cycle of protease development and synthesis compared to fungi. In this regard, the selection of new bacterial strains for proteases is still relevant.

 Closest to the claimed object is a strain of Bacillus subtilis FU-79, registration TsMPMV-1989 (1), one of the most powerful producers of protease. A known strain can produce a protease in an amount of 14.7-30 PE / ml, depending on the nutrient medium, for 38 hours of biosynthesis.

 The disadvantage of this strain is the duration of the cultivation process - 38 hours, as well as the need for its cultivation on environments that include non-standard food components - corn extract, saccharified starch, which increase the cost of nutrient media, make them less standard, and also reduce the specific activity of the protease enzyme in the culture fluid, as additional protein is introduced with them.

 The objective of the invention is the creation of a new strain of Bacillus subtilis, capable of producing protease in industrial volumes on a synthetic nutrient medium.

 The technical result obtained from the use of the new strain is to obtain an enzyme, a protease with high total and specific activity in the culture fluid while reducing the duration of the cultivation process.

 The problem is solved by creating a new strain by selection from the known strain of Bacillus subtilis 534, registration VKMV-1666D (2), capable of rapid growth on simple compositional nutrient media.

 Strain Bacillus subtilis 534 was repeatedly passaged through a nutrient medium containing increasing concentrations of the antibiotic. The resulting population of the antibiotic-resistant variant of the strain was seeded with monospore sieving on an agar medium with casein to isolate colonies with high protease production. As a result of selection, a strain of Bacillus subtilis P1 was isolated, which has high protease production on simple compositional nutrient media.

To confirm the genetic differences between the original Bacillus subtilis 534 strain and the selected Bacillus subtilis P1 strain, the genomic polymorphism of the Bacillus subtilis 534 and P1 strains was analyzed by end-labeling of restriction enzyme fragments with a radioactive label [a- ³² P] DATP followed by separation by high-voltage sequencing genomerizing electrophoresis conditions (drawing). On the radiograph presented in the drawing, you can see a large number of bands common to the strains. At the same time, it is possible to note the presence of a number of bands, allowing to reveal the existing differences between the studied strains.

 To identify differences between the strains in the level of protease production, we studied the hydrolysis zones of hungry agar with casein of individual colonies of the population of the initial strain 534 and the new strain P1. Comparative analysis data are presented in table 1. We also studied the levels of protease production in the culture fluid obtained by growing the strains in a liquid nutrient medium with aeration and mixing. The proteolytic activity of the culture fluid was determined according to the Ansona method in the modification of Petrova I.S. - Vinciukaite M.M. (3). Comparative indicators of the levels of proteolytic activities of the culture fluid of the strains of Bacillus subtilis 534 and P1 in comparison with the prototype are presented in table 1.

 Thus, as a result of selection, a new strain of Bacillus subtilis P1 was obtained, which has a high level of protease synthesis and a shorter duration of the cultivation process, which makes it possible to increase the manufacturability of the enzyme production process using the Bacillus subtilis P1 strain.

 The strain is stored in the form of freeze-dried culture in vacuum ampoules in the scientific laboratory of the state unitary enterprise "Immunopreparat" in Ufa.

 The strain of Bacillus subtilis P1 is characterized by the following properties.

 Cultural and morphological properties.

Vegetative stick cells of an ovoid shape with sizes of (7-1.3) x ((0.5) microns, located separately or in small chains. Form internal central spores of an oval shape. In the population of flagella, lofotrichial and peritrichous. Colonies on meat and peptone agar are flat, slightly shiny, white in color with smooth edges. On the meat and peptone broth at a temperature of 37 ^o C after 18-24 hours there is a uniform clouding of the broth. Aerob, gram-positive, has mobility, optimal growth conditions at a temperature of 37 ^o C and pH 6.0-7, 5.

 Biochemical properties.

 It does not grow under anaerobic conditions, hydrolyzes urea and starch, liquefies gelatin. Gives a positive reaction of Voges-Proskauer. Ferments glucose, sucrose, maltose, mannitol, lactose. Reduces nitrates. Produces catalase, a protease. It does not have lecithinase activity. Forms ammonia and hydrogen sulfide, does not form indole.

Antibiotic resistance
The determination of the sensitivity of the strain to antibiotics was studied by the disk diffusion method using standard disks impregnated with antibiotics. The strain was plated on an agar medium and 5 antibiotic discs were placed on a Petri dish. Crops were incubated at a temperature of 37 ^o C for 24 hours. Stable was considered a strain in the absence of growth inhibition by antibiotic.

 The strain is resistant to penicillin, carbenicillin, ampicillin, oxacillin, polymyxin.

Virulence
The culture of B. subtilis P1 strain was tested for virulence. To study virulence, the strain was grown on meat-peptone agar for 24 hours at a temperature of 37 ^o C. The grown biomass was washed with 0.9% sodium chloride solution and bacterial suspensions were prepared with cell concentrations from 0.5 to 5.0 billion / v ml The resulting suspensions were administered to white mongrel mice weighing (16 ± 1) g in two ways: once intraperitoneally and five times orally (one dose daily for 5 days). The test results are presented in table.2.

 Observations of animals were carried out for 5 days after the end of the course of injections or oral administration. During this period, all animals remained active, ate well-fed diets, gained weight, physiological functions and behavioral reactions did not change. The data obtained indicate the safety of the strain Bacillus subtilis P1.

 The strain Bacillus subtilis P1 was used to obtain a proteolytic enzyme.

 The invention is characterized by the following examples.

 Example 1. The strain was grown by periodic homogeneous deep cultivation in an AK-10 fermenter with a volume of nutrient medium - 8 l. To grow a strain of Bacillus subtilis P1, in order to obtain a protease, a synthetic nutrient medium containing mineral salts is used, urea as a nitrogen source, and maltose as a carbon source.

Inoculum was obtained by growing a strain of Bacillus subtilis P1 in vials at a temperature of 37 ^o C for 16-18 hours in a liquid nutrient medium of the same composition as for cultivation. The resulting seed, contribute in the amount of 1% of the volume of the nutrient medium in the fermenter. Fermentation is carried out at a temperature of 37 ^o With aeration for 24 hours. As a result of cultivation, 6 l of enzyme-containing culture fluid with a protease activity of 28-32 PE / ml and specific activity (20.0 ± 2.0) PE / mg of protein were obtained.

 Example 2. The strain was grown by periodic homogeneous deep cultivation in a Bior-100 reactor with a volume of nutrient medium - 70 l. Culture medium and preparation of the uterine strain, as in example 1.

The resulting seed, contribute in the amount of 1% of the volume of the nutrient medium in the reactor "Bior - 100". Fermentation is carried out at a temperature of 37 ^{° C.} with aeration for 24 hours. As a result of cultivation, 60 l of an enzyme-containing culture fluid with a protease activity of 30 ± 2 PE / ml specific activity (20.0 ± 2.0) PE / mg protein were obtained.

Sources of literature
1. A.S. 885253, cl. MKI ³ C 12 N 9/28, 11/30/81.

 2. Nikitenko V.I. Bacterial drug for the prevention and treatment of inflammatory processes and allergic diseases // International application. N 89/09607, WO, 10.19.1989.

 3. Petrova I.S., Vintsiunaite M.M. Determination of the proteolytic activity of enzyme preparations of microbiological origin // Applied Biochemistry and Microbiology. - 1966. - N 2. -C. 322-327.

Claims (1)

 The bacterial strain Bacillus subtilis P-1 is a protease producer.

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