RU2152432C1 - Strain streptomyces hygroscopicus-7 as producer of proteolytic enzyme gigrolytin - Google Patents

Abstract

FIELD: biotechnology, microbiology, enzymes. SUBSTANCE: strain is obtained by selection methods by effect of mitomycin C and deposited in Science-Research Institute of Agriculture microbiology at number VNIISHM-481 in the group of epiphyte microorganisms. The level of productivity of strain is increased and varies from 120 to 170 PU/ml. EFFECT: strain indicated above, increased yield of enzyme.

Description

 The present invention relates to the medical and microbiological industry and can be used to obtain proteolytic enzymes.

 The objective of this technical solution was to obtain a new strain with higher productivity for hygrolitin, which can be used in veterinary practice for the treatment of animal dyspepsia in combination with antibiotics, as a biostimulator of animal and bird growth when added to feed, in the leather industry for dehydration and softening leather raw materials, as well as for hydrolysis of protein of various origins.

 Hygrolitin can be used in medicine for non-specific diseases of the respiratory system (for thinning sputum and purulent-necrotic masses), for the prevention of pleural empyema, in burn and purulent surgery for lysis and rejection of the scab, lysis of purulent-surgical masses and fibrinous diseases.

 During the solution, a strain was selected with high ability to synthesize hygrolithin.

 A known method of producing a proteolytic enzyme gigrolitin using Actinomyces hygroscopicus as a producer of culture (SU, copyright certificate N 663715, class C 12 D 9/14, C 12 D 13/10. Published May 25, 79 Bulletin N 19). However, the proteolytic activity of Actinomyces hygroscopicus in this method reaches only 35 PE / ml. The disadvantage of this technical solution is the low proteolytic activity of the culture fluid.

 The closest in technological essence to the invention is a strain of Streptomyces lavendulae VKPM S-910 - producer of proteolytic enzymes with a culture fluid activity according to Kunitz 40-97 PE / ml, which we have chosen for the prototype. (SU, copyright certificate N 1735364 class C 12 N 9/52, 1/20, USSR Patent N 1001862, class C 12 N 9/48, 1983). The disadvantage of the strain is low proteolytic activity.

 The proposed strain Sreptomyces hygroscopicus 7 obtained by selection when exposed to mitomycin C at a dose of 100 μg / ml The strain produces up to 170 PE / ml of culture fluid. The strain according to the invention is stored in the Research Institute of Agricultural Microbiology under the number "VNIISCHM-481" in the group of epiphytic microorganisms.

 The essential features of the claimed strain of Streptomyces hygroscopicus 7, common with the prototype.

 The strain peptones milk, dilutes gelatin, does not form hydrogen sulfide, does not restore nitrates. As a carbon source, it metabolizes glucose, inositol; weakly grows on the environment with arabinose. On potato forms active growth. The proposed strain, as well as the prototype, does not form soluble melanoid pigments on all agarized and liquid media used for cultivation.

Distinctive features of the proposed strain from the known (prototype)
The proposed strain, in contrast to the prototype Streptomyces lavendulae VKPM S-910, belongs to the species Streptomyces hygroscopicus and differs from the prototype in all morphological and cultural characters.

On a medium with corn extract and starch (N 21/12) on 21 days of growth at 25 ± 1 ^o C forms colonies of 8-9 mm flat with folds in the center and a wide-cut rugged edge. The aerial mycelium is velvety, moderately developed, and partially reduced along the edge of the colony in the form of a border of a snow-white color (d 3). The substrate mycelium is bulanic (to 1). The medium is not colored. On organic medium N 2 (Gauze), the colonies are convex, with a diameter of 8-11 mm, with a tubercle in the center and a flat, rugged edge. The aerial mycelium is velvety, snow-white (d 3). Yellowish-brown substrate mycelium (d 4). The medium is not colored.

 On oat agar N 61, colonies are large 13-16 mm, convex with a wide-growing uneven edge. The aerial mycelium is velvety, abundantly sporulating, dark ash (at 4), on the edge of a colony of a snow-white color. The substrate mycelium is pale sand (to 3). The medium is not colored.

 On agar Chapek with starch forms sparse growth, the colonies are small, 2-3 mm, flat, with a rugged, wide-growing edge. Mealy aerial mealy, smoky (l 1), plentifully sporulating, covers the colony completely. Gray substrate mycelium (in 4). The medium is not colored.

 On an agar medium Tresner forms sparse growth, colonies of 2-3 mm, convex with a tubercle in the center and a rugged edge. Aerial mycelium powdery, snow-white (d 3), non-sporulating. Yellowish-brown substrate mycelium (d 4). The medium is not colored.

 In contrast to the prototype, it does not grow on a medium with fructose, absorbs xylose well, and grows moderately on a medium with sucrose. The inventive strain exceeds the level of biosynthesis of the proteolytic complex of enzymes by 75% prototype (the claimed strain 120-170 PE / ml; prototype - 40-97 PE / ml).

Morphological and cultural characteristics
On a medium with corn extract and starch (N 21/12) on 21 days of growth at 25 ± 1 ^o C forms colonies of 8-9 mm flat with folds in the center and a wide-cut rugged edge. The aerial mycelium is velvety, moderately developed, and partially reduced along the edge of the colony in the form of a border of a snow-white color (d 3). The substrate mycelium is bulanic (to 1). The medium is not colored. On organic medium N 2 (Gauze), the colonies are convex, with a diameter of 8-11 mm, with a tubercle in the center and a flat, rugged edge. The aerial mycelium is velvety, snow-white (d 3). Yellowish-brown substrate mycelium (d 4). The medium is not colored.

 On oat agar N 61, colonies are large 13-16 mm, convex with a wide-growing uneven edge. The aerial mycelium is velvety, plentifully sporulating, dark ash (at 4), on the edge of a colony of a snow-white color. The substrate mycelium is pale sand (to 3). The medium is not colored.

 On agar Chapek with starch (N 84) forms a sparse growth, the colonies are small, 2-3 mm, flat, with a rugged wide-growing edge. Mealy aerial mealy, smoky (l 1), plentifully sporulating, covers the colony completely. Gray substrate mycelium (in 4). The medium is not colored.

 On agar medium Tresner (N 64) forms scanty growth, colonies 2-3 mm, convex with a tubercle in the center and a rugged edge. Aerial mycelium powdery, snow-white (d 3), non-deformers. Yellowish-brown substrate mycelium (d 4). The medium is not colored.

Physiological and biochemical characteristics
The strain pentonizes milk, hydrolyzes starch, completely dilutes gelatin on the 9th day of growth at 28 ^o C, does not form H ₂ S, grows moderately on fiber, forms a copious growth of grayish color, does not restore nitrates to nitrites, does not form melanoid pigments . The reaction to sucrose inversion is negative.

 On the Pridhema-Gottlieb medium, it grows well in the presence of glucose, maltose, xylose, moderately grows on a medium with Na-citric acid, inositol, sucrose, sorbitol, weakly grows with arabinose, lactose, ramnose, galactose, mannitol. Does not grow on a medium with fructose and sodium acetate.

The optimum temperature for growth and development is 28 ^o C, the minimum growth temperature is 20-25 ^o C, withstands a maximum temperature of 32 ^o C.

Storage conditions of the claimed strain
The strain is stored on agar 21/12 with starch and corn extract at room temperature for 3 months.

The composition of the medium 21/12:
1. Corn extract 5 g (dry weight)
2. (NH ₄ ) ₂ HPO ₄ 4 g
3. KH ₂ PO ₄ 2 g
4. MgSO ₄ • 7H ₂ O 0.25 g
5. CaCO ³ 1 g
6. Instant starch 20 g
7. Agar-agar 25 g
8. Tap water up to 1 l
pH of the medium before sterilization 7.3
Sterilization for 40 minutes at 0.8 ati.

Antagonistic signs
Antagonistic properties in relation to other microorganisms were not observed.

 The inventive strain is not toxic and does not apply to microorganisms with pathogenic properties.

 When deep fermentation of the claimed strain accumulates in the culture fluid a proteolytic enzyme - hygrolithin.

Hygrolithin Formation Conditions
To obtain hygrolitin, the strain is cultivated for 72-96 hours on a shaker (speed 220-250 min ^-1 ) in Erlenmeyr flasks with a capacity of 750 ml, containing 50 ml of medium of the following composition,%:
protein-vitamin concentrate (BVK) - 3.0
cornmeal - 2.0
hydrol or green molasses - 6.0-7.0
chalk - 1.2
monosubstituted potassium phosphate - 0.02
sunflower oil - 1,0
tap water - the rest
The flasks were inoculated with 5 ml of inoculum obtained by culturing in Erlenmeira flasks with a capacity of 750 ml (28 ± 1 ^o C, 220-250 rpm) containing 100 ml of medium of the following composition,%. soy flour - 1.5, corn extract - 0.2, (NH ₄ ) ₂ SO ₄ - 0.2, NaCl - 0.5, CaCO ₃ - 0.3, glucose - 2%, BVK - 1.0. The cultivation period of the seed 48 hours

For the preparation of seed, a culture grown in test tubes with a medium of 21/12 was used. The culture was grown for 14 days at 28 ^o C.

 The proteolytic activity of strain 120-170 PE / ml (determination method: Laskowsky M., Preparation and assay of enzymes. 2. Chymotrypsinogenes and chymotrypsins. Methods Enzymol., 1955, v. 2, p. 8-26).

Claims (1)

 The strain Streptomyces hygroscopicus 7 is a producer of the proteolytic enzyme gigrolitina.