The strain streptomyces hygroscopicus 7 - producer proteolytic enzyme hygromycin

 

(57) Abstract:

The invention relates to biotechnology and can be applied in the leather industry and veterinary medicine. Strain the received selection by under the influence of mitomycin C and stored at the research Institute of agricultural Microbiology at number ARRIAM-481 and in the group alipanah microorganisms. The level of productivity of strain 7 is increased and ranges from 120-170 PE/ml

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

The objective of this technical solution was to obtain a new strain with higher performance to obtain hygromycin, which can be used in veterinary practice for the treatment of dyspepsia animals in combination with antibiotics as growth biostimulant animals and birds, with the addition of it in the feed, in the leather industry for dehairing and bating of raw hides, as well as for protein hydrolysis of different origin.

Hygromycin can be used in medicine when nonspecific respiratory diseases (rasla lizirovania and rejection scab, lysis of purulent surgical fibrinous masses and diseases.

During solution was selected strain with high ability to synthesize hygromycin.

A method of obtaining a proteolytic enzyme hygromycin using as a producer of culture of Actinomyces hygroscopicus (SU, author's certificate N 663715, CL C 12 D 9/14, C 12 D 13/10. Published 25.05.79. Bulletin No. 19). However, the proteolytic activity of Actinomyces hygroscopicus in a specified way reaches only 35 PE/ml. the Disadvantage of this technical solution is low proteolytic activity in the culture fluid.

The closest to the technological nature of the invention is a strain of Streptomyces lavendulae PMBC S-910 - producer of proteolytic enzyme activity in the culture fluid by Kunitz 40-97 PE/ml, which is chosen for the prototype. (SU, author's certificate N 1735364 class. C 12 N 9/52, 1/20, Patent USSR N 1001862, CL C 12 N 9/48, 1983). The lack of strain is low proteolytic activity.

The proposed strain Sreptomyces hygroscopicus 7 obtained by the method of selection for the effects of mitomycin C at a dose of 100 μg/ml Strain produces up to 170 PE/ml of culture fluid. The strain according to the invention is stored in Nauch microorganisms.

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

Strain peptonized milk, gelatin liquefies, does not form hydrogen sulfide, nitrates does not recover. As a source of carbon assimilates glucose, Inositol; weakly increases on the environment arabinose. On potato forms active growth. The proposed strain as well as the prototype, does not form a soluble melanoidin pigments on all agar and liquid media used for cultivation.

Distinctive features of the proposed strain from the known (prototype)

The proposed strain unlike the prototype Streptomyces lavendulae PMBC S-910 refers to the species Streptomyces hygroscopicus and all of the morphological and cultural characteristics different from the prototype.

In the medium with corn extract and starch (N 21/12) at 21 days of growth at 251oC forms a colony 8-9 mm flat with pleats in the center and shyrokoradiuk jagged edge. Aerial mycelium velvety, moderately developed, on the edge of the colony is partially reduced in the form of a fringe of snow-white color (d-3). Substrate mycelium buckskin (1). The medium is not colored. On the organic environment N 2 (Gause) colonies are convex, diameter 8-11 mm with knob in the center and flat izraza is painted.

On ovsanna agar N 61 colonies large 13-16 mm, convex with shyrokoradiuk rough edges. Aerial mycelium velvety, richly sorulari dark ash (4), on the edge of a colony of white color. Substrate mycelium pale sand (3). The medium is not colored.

On agar of čapek with starch forms a sparse growth of colonies of small, 2-3 mm wide, flat, with rugged shyrokoradiuk edge. The aerial mycelium of powdery, smoky (l 1), abundantly sorulari covers the colony completely. Substrate mycelium gray (4). The medium is not colored.

On agar medium of Tresner forms a meager growth, colonies 2-3 mm, convex with tubercle in the centre and jagged edge. The aerial mycelium of powdery, white color (l 3), respirology. Substrate mycelium yellowish brown (d-4). The medium is not colored.

Unlike the prototype does not grow on medium containing fructose, well metabolizes xylose, moderately growing on medium containing sucrose. The inventive strain exceeds the level of biosynthesis of proteolytic enzyme complex for 75% of the prototype (the claimed strain 120-170 PE/ml; prototype - 40-97 PE/ml).

Morphological and cultural characteristics

In the medium with corn extract and starch (N 21/12) for 21 days rodusky mycelium velvety, developed moderately, on the edge of the colony is partially reduced in the form of a fringe of snow-white color (d-3). Substrate mycelium buckskin (1). The medium is not colored. On the organic environment N 2 (Gause) colonies are convex, diameter 8-11 mm with knob in the center and flat jagged edge. Aerial mycelium velvety, white (d-3). Substrate mycelium yellowish brown (d-4). The medium is not colored.

On ovsanna agar N 61 colonies large 13-16 mm, convex with shyrokoradiuk rough edges. Aerial mycelium velvety, richly sorulari, dark ash (4), on the edge of a colony of white color. Substrate mycelium pale sand (3). The medium is not colored.

On agar of čapek with starch (N 84) forms a sparse growth of colonies of small, 2-3 mm wide, flat, with rugged shyrokoradiuk edge. The aerial mycelium of powdery, smoky (l 1), abundantly sorulari covers the colony completely. Substrate mycelium gray (4). The medium is not colored.

On agar medium of Tresner (N 64) forms a meager growth, colonies 2-3 mm, convex with tubercle in the centre and jagged edge. The aerial mycelium of powdery, white color (l 3), respirometry. Substrate mycelium yellowish brown (d-4). The medium is not colored.

Physio is on Tue 9th days of growth at 28oC, H2S does not form on the tissue grows moderately, on the potato produces abundant growth grayish color, does not restore the nitrate to nitrite, melanoidin pigments are not formed. Response to inversion of sucrose negative.

On the environment Pridham-Gottlieb grows well in the presence of glucose, maltose, xylose, grows moderately in the medium with Na-citrate, Inositol, sucrose, sorbitol, weakly increases with arabinose, lactose, Ramezay, galactose, mannitol. Does not grow on medium containing fructose and acetic acid sodium.

The temperature optimum for growth and development of the 28oC, the minimum temperature for growth of 20-25oC, can withstand a maximum temperature of 32oC.

Storage conditions of the proposed 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. (NH4)2HPO44 grams

3. KH2PO42 g

4. MgSO47H2O 0.25 g

5. CaCO31 g

6. The soluble starch 20 g

7. Agar-agar 25 g

8. Tap water to 1 liter

the pH of the Medium prior to sterilization 7,3

Sterilization 40 min to 0.8 MPa.

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

In submerged fermentation declare strain accumulates in the culture liquid of the proteolytic enzyme action - hygromycin.

Conditions of education hygromycin

To obtain hygromycin strain cultured for 72-96 h on a rocking chair (speed 220-250 min-1in flasks of Erlenmeyer with a capacity of 750 ml, containing 50 ml of medium of the following composition, %:

protein-vitamin concentrate (PVC) - 3,0

corn flour - 2,0

gidrol or green syrup - 6,0-7,0

chalk - 1,2

potassium phosphate one-deputizing - 0.02

sunflower oil - 1,0

water tap - rest

Bulb inoculant 5 ml of inoculum, obtained by cultivation in flasks of Erlenmeyer with a capacity of 750 ml (281oC, 220-250 rpm) containing 100 ml of medium of the following composition, %. soy flour - 1,5, corn extract to 0.2, (NH4)2SO4to 0.2, NaCl and 0.5, CaCO3to 0.3, glucose 2%, and the BVK is 1.0. The period of growing seed 48 hours

For preparation of inoculum used cultures grown in test tubes with the medium 21/12. The culture was grown in flow. , Preparation and assay of enzymes. 2. Chymotrypsinogenes and chymotrypsins. Methods Enzymol., 1955, v.2, p. 8-26).

The strain Streptomyces hygroscopicus 7 - producer proteolytic enzyme hygromycin.

 

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