SU1068479A1 - Strain bacillus subtilis 81-og production neutral protease - Google Patents

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The invention relates to microbiological production and production of strains producing neutral t.protease, and preparations obtained on the basis of the enzyme neutral protease are used in food prokkleleneni i in medicine, tanning industry, in animal husbandry and YAR. A known strain of you.subtlEis 2D3 with neutral protease activity / equal. 52.5 units / ml, determined by the splitting of a casein, which corresponds to 12-13 units / ml according to the Leleyn-Folgrdrd method. Strain you. The subtif 203 is of only scientific interest, as the level of activity of the neutral protease synthesized by this strain is 10–15 p lower compared to the strains using 1–1 mi in production G13. The closest in technical essence and the achieved positive effect is the Baci6P subtitls 103 strain, which forms a neutral protease with an activity of 6070 units / ml according to the Leleyn-Folgard method. The strain is characterized by a high viscosity of the culture fluid and sensitivity to phages 21. A disadvantage of the known strains is relatively low level of neutral protease aKTHB.HqcTH in production, high viscosity of the culture fluid, which makes it difficult to filter, dry and, therefore, limit the use of these strains only for preparing a crude preparation protosubtilin GZH. . The disadvantage of the strain Bac.subtiCls 103, in addition to these qualities, is the instability of the activity of the neutral protease, associated with its sudden decrease. The aim of the invention is to obtain a producer of a neutral protease with a high and stable enzyme activity under production conditions, as well as a low viscosity of the culture fluid. A new strain of Bac.subtiPls v1-OG is obtained by genetic-selection methods from the strain of you.subtle is ARRI Genetics-44-producer of oL-amylase. Under the conditions of production in 63 m fermenters, a sporoben strain to produce a neutral protease with an activity of up to 100-118 units / ml (Leleyn-Volgard method), superior in activity to the strain You produce. subtifis 103. The table gives a comparative analysis of the level of activity of the Neutral Protease, the fermentation time and the volume of the drain obtained from an experimental test of ShTamm - New You. subtieis 81-or you. subtieis 103 in the Omutninsky chemical conditions.

The benefits of shaksh you. subti- jia 81-og. Compared to the Stage Bac.subtiPis 103: a higher level of neutral protease activity (by 46% on average), a shorter fermentation time, which allows, under production conditions, to save electricity / a larger amount of culture liquid drains,

In other words, a higher yield of the target product, resistance to infection.

60 Strain Bac.subti is 81-OG characterized by low viscosity of the culture fluid, which distinguishes it from known strains - producers of neutral protease - and so

65 expands the possibilities of its use for obtaining purified preparations. On the basis of purified preparations of neutral protease, drugs of medical purpose can be obtained. The strain was tested under the conditions of the Omutninsky chemical plant and is characterized by resistance to phages prevalent in this plant, and extraneous microflora. During the test period, not a single case of the activity of Erms & is observed, which suggests that you are. subtlpis 81-OH HC only exceeds the level of activity of the neutral proteinase known producers of this enzyme, but is more technological in its properties. The strain Bac.subtiPis 81-OG-producer of the neutral protease is stored in the Central Museum of Promoted Microorganisms VNIIgenetic and has the registration number CMPM B-2574. The biological characteristics of the strain Bac.subtifis 81-OG. And an example of the use of the strain in production conditions in fermenters with a capacity of 63 m. Cultural and morphological recognition of st11mma Bac.subtitls 81-OG. Morphology. The cells of the strain are Gram-positive, spore-forming pgshochka in size 2.54, 5-0.7 microns. The dispute size is 1.0-1.2 "0.6-0.7 microns. Cultural and physiological | signs of the strain Bac.subtlEls 81-OG M co-peptone agar (MPA). After 48 h of growth, the culture forms dense, leathery gray colonies. The middle part of the colony is darker, the margins are lighter (whitish). The surface of the colony is small. . M co-peptone broth (ffflB). Pr cultivated in MPV for 24 hours with no shaking growth without turbidity, a dry film is formed on the surface. When shaken on a rocking chair, the medium is muted. standing film is formed. Agarization environment Hottinger. After 48 h of growth, the culture forms a yellowish serm, round flat colonies with white lobed edges. The diameter of the colonies of 3.54, 5 mm. The surface of the colonies is rough wool, mat. On synthetic medium with mineral nitrogen (Spipisen medium), the culture grows in the form of small, round, grayish-white colonies with a diameter of 1-2 mm. The surface of the colonies is dull. On potato slices, the culture forms a wrinkled, thinly folded yellowish film. When stored in a refrigerator at 2-4 ° C, the film does not lick for a month. Gelatinous thins. Starch hydrolyzes. Peptonized milk. Relation to carbon sources. Glucose, lactose, fructose, sucrose-digests. The storage of the strain is carried out in a lyofilfio dried state in glass glasses. An example of the use of the strain Bac.subtlfls 81-EXG for the production of a neutric protease under production conditions. Seed material is obtained by growing the strain .subt1 (is 81-GO in flasks in the form of a film in a liquid nutrient medium of the following composition,%: starch 10; Hz monium citrate 2-s 3 3; KHyPO4 0.03; itlHfO 0.15; CaSO 0.14; FeSO4 0.002J MnSO4 0.000125, - CuSO4 0.00075; h-aminobenzoic acid 0.001; water 86.2. PH 6.0-6.3. Temperature. The resulting seed in an amount of 0.01% the volume of the medium in the fermenter is seeded with an apparatus of 63 m volume with 30 m3 of fermentation medium of the following composition,%; saccharified starch 13; CaCO5, 0.05; KCC 0.15; CaSRO / O; MgSO4 0.05; (NH J-HPO - 1.2; corn 3KCTpaKt 0.8; foam-.-Extinguisher piiol 0.05. pH 6.6. Fermentation is carried out with aeration for 44 hours. Neutral, protease activity in the culture fluid is 306 units / mtG (Leleyn-Volgard method). The culture fluid is dried on a spray dryer 1 (, e "Preparation yield The amount of the GKH from the culture liquid is 63.5%. G The expected annual economic effect from the use of the strain under the conditions of the Omutninsky Chemical Plant is 128 thousand rubles.

Claims (1)

(54> strain Bacillus jultllu · 81.-HUMAN PRODUCER OF NEUTRAL PROTEASE. (5-7) Strain Baciffus subtifis 81-OH - (collection of the Central Museum of Industrial Microorganisms of the Institute of the All-Union Scientific Research Institute of Genetics and Selection of Industrial Organizations, collection number CIMM B -2574) producer of a neutral protease.