SU201249A1 - METHOD FOR OBTAINING ENZYME PREPARATIONS - Google Patents

Description

There are known methods for producing enzyme preparations by cultivating microorganisms producing them on a nutrient medium containing nitrogen sources, with the addition of other nutrients, the carbohydrate and mineral salts necessary for growth, at a temperature of 20-35 ° C under aerobic conditions, followed by the release of enzyme preparations from biomass one of the known methods, for example salting out with ammonium sulfate, adsorption, drying the culture fluid.

According to the proposed method, preparations with high activity are obtained containing proteolytic enzymes capable of spreading keratin in combination with laminarinase, chitinase, elastase and lipase. For this purpose, bacteria of the genus Cytofaga (NCIB 9497) are used as producing microorganisms.

The proposed method is as follows.

Bacteria culture of the Cytofaga genus (Cytofaga) "NCIB 9497 is grown on nutrient media by surface method or deep in aerobic conditions.

are parallel, the ends are rounded, divided, and sometimes paired with each other. Gram is stained after incubation on nutrient agar for three days at 20 ° C. Gram - negative, short, straight sticks with parallel sides, rounded ends, outer appearance of color - uneven stripes.

A similar outland species is when grown on agar containing peptone and glucose. Okrashchapie uniformly after cultivation on blood agar.

Growth on various environments. On agar (four days cultivation at 20 ° C) is light yellow, translucent, rounded, convex, completely smooth, shiny, 1-1.5 mm in diameter, with a pungent smell.

On agar, peptone and glucose (four-day cultivation at 20 ° C) - light yellow, translucent, rounded, convex, completely smooth, glossy, slimy, 1-2.5 mm E in diameter, with a pungent smell.

On the blood agar (four days cultivation at 20 ° С) - grayish-yellow, translucent, rounded, convex, completely smooth, glossy, 1-1.5 mm in diameter, with a pungent smell.

good sticky, thick and viscous deposits. Weak growth also noted

supports

.V- j "% -„,

On water containing peptone (two-day cultivation at 20 and 30 ° C) - the growth is insignificant, turbidity is very weak and non-uniform.

On water containing peptone and glucose {two-day cultivation at 20 and 30 ° C) - moderate growth, weak, non-uniform turbidity.

The relationship between growth and temperature. No growth at 5 or 10 ° C. Insufficient growth at 37 ° C. There is no sensitivity to penicillin and streptomycin. Sensitivity to chloramphenicol and hydrotetracycline. Does not cause the formation of acid or gaseous products as a result of exposure to glucose, maltose, mannitol or starch at 20 or 30 ° C. There is no reaction in the medium according to Hyug and Leifson containing glucose.

Litmus milk is petopicized, and the indicator is restored at 20 ° C and non-tonized at 30 ° C. Gelatinu thins at 20 ° C. Kovac oxidase is positive at 20 and 30 ° C. No indole formation occurs, methyl red is negative, acemethylcarbinol is not produced, ammonia is released from peptone at temperatures of 20 and 30 ° C. Citrate is utilized as a carbon source in the form of ash at 20 and 30 ° C. The formation of lipase or lecithinase on agar containing chicken egg yolk is not observed at 20 or 30 ° C.

Nutrient media used for cultivation mainly contain a nitrogen source with the addition of carbohydrate and / or nutrient salts, if necessary. Nitrogen sources include malt extract, meat extract, peptone, corn or grain locking fluid, however, the most suitable nitrogen source is mycelium, derived as a waste in the production of antibiotics. It should be noted that the source of nitrogen can fully satisfy the need of the grown organism Cytophage "NCIB 9497" in carbon or in other nutrient salts without adding them. Thus, for example, this microorganism can develop on a medium consisting only of meat extract and peptone. In some cases, the addition of carbohydrates, such as glucose, maltose, is necessary.

Elements that may also be necessary include magnesium, iron, and zinc.

Cytology NCIB 9497 is cultivated at a temperature of 20-35 ° C (the optimum temperature is 26 ° C), the pH of the medium is in the range of 5-8, preferably 5.5-6.

The resulting culture fluid can either be used directly, or the enzyme complex can be extracted from it by one of the known methods. Thus, for example, the culture liquid is dried or clarified (by means of centrifugation), and then dried (by freezing method), thus obtaining a crude preparation. To obtain a purified preparation, the latter is precipitated with acetone or salting out with ammonium sulfate, or by adsorption on kaolin.

The resulting preparation contains a complex of the following enzymes: protease, laminarine, chitinase, keratinase, elastase, and lipase. This complex of enzymes can cause the disintegration of the mycelium in various fungal organisms, such as mold of the genus Penicillium and Cephalosporium.

0 The ability to cause the breakdown of fungal mycelium allows the use of the above enzyme complex to destroy mycelium waste after the production of antibiotics, especially cephalosporin products.

5 The soluble material thus obtained can be used as a nutrient medium for the subsequent fermentation processes. The extent to which the enzyme complex acts on the fungal mycelium depends on the species of mycelium in each particular case.

It was noted keratinase action of a complex of enzymes, which acts, apparently. Not. hair.

5 Able to decompose gelatin, casein, affect keratin, and its action proceeds quickly.

This complex can be used: in the production of bakery products; 0 in the manufacture of dishes from cereals (for example, oats nka, corn flakes);

for the accelerated ripening of the meat in order to soften it;

for protection against cooling during zivo5 boiling;

in animal feed production; in the production of protein hydrolysates; for softening of large leather raw materials and dehairing cheeks;

to remove stains by dry cleaning; to remove slippage from textiles;

in medicine.

Example 1. Conical flasks (250 ml) containing 40 ml of the following medium, (%): maltose4

malt extract2,4

peptone1

distilled water - up to 100.

The pH of pH 7 is set on the machine by reciprocating motion with an amplitude of 5 cm, operating at 200 rpm at an ambient temperature of 26 ° C. Two days after the start of incubation, the culture is characterized by a strong lytic effect with respect to the Cephalosnorium acremonium (Brotsu). "IMI -49137. Example 2. Conical flasks (250 ml) containing 100 ml of the following medium,%: minus extract1 peptone1 sodium chloride 0.5. The pH of the medium is adjusted to pH 8 by the addition of sodium hydroxide, boiled for 1 hour, filtered, the medium is adjusted to pH 7.2 by addition of hydrochloric acid, inoculated with a loop by a culture of the microorganism Cytophage "NCIB 9497, and incubated at 26 ° s The contents of the flasks are mixed on a reciprocating apparatus, with an amplitude of 5 cm, at 200 rpm. One day after the start of incubation, the culture is characterized by a strong lytic effect on "K. acremonium. Example 3. Z.L environment of the following composition (sterilized for 30 minutes at a temperature of 120 ° C),%: mycelium "K. acremonium, squeezed raw 6 acidic phosphoric acid potassium secondary 0.07 primary 0.03 magnesium sulphate 7H2O0.05 iron sulphate 7H2O0.001 zinc sulphate 7H200.001 glucose (sterilized separately) 0.25. 60 ml of a well-expanded culture of the cytophage microorganism NCIB 9497 are inoculated. The culture is maintained at a temperature of 26 ° C, stirred at 550 rpm and aerated by passing air at a rate of 3 liters / minute for 20 hours, then 4 hours later - at a rate of 1 l / min and finally 2 hours - with a speed of 0.5 l / min. The culture was clarified by centrifugation and the supernatant was lyophilized. 6.6 g of a cream-colored solid are obtained, which has lytic activity with respect to “K-acremonium and proteolytic activity with respect to casein. Example 4. 1.5 l of a liquid culture grown as indicated in example 3 is centrifuged, the medium is adjusted to pH 6.1 by the addition of acetic acid and then concentrated under vacuum at a temperature below 40 ° C to a volume of 250 liters. The concentrate is slowly added to 2 liters of acetone cooled to + 5 ° C, with vigorous stirring. A light brown product in the amount of 7.4 g is washed with acetone and dried over phosphorus pentoxide. The resulting product is characterized by high activity. Example 5. 2l liquid culture grown as indicated in example 3 is freed from solid impurities by centrifugation and then slowly poured with stirring into 20 liters of acetone, cooled with ice. The product is filtered off, washed with water and dried over phosphorus pentoxide; product weight - 10 g. The product has full activity, drying the original liquid medium. Example 6. Pieces of fresh calfskin are immersed in a 1% solution of an enzyme preparation (prepared as described in Example 5) in a Tris M / 50 buffer at pH 7. Soak for 18 hours at 37 ° C, after which the hair Noah cover is easily removed by gently scraping. Dialogical treatment in a buffer environment. Tris without adding a prepared preparation leaves hair the same as it was before processing. Example 7. Mycelium Trichophyton rubrum (one of the fungi causing fungal disease of the legs) is prepared by cultivation in a vessel adapted to shake for 24 hours at a temperature of 26 ° C in a medium containing peptone and malt extract. The obtained mycelium is filtered and suspended at a level of 0.2% in a 0.2% solution of a previously prepared enzyme preparation (prepared as indicated in Example 5) in a Tris M / 50 buffer medium at a pH of 7. Mycelium is completely dissolved after a three-hour exposure at a temperature of 27 ° C. Example 8. Adsorption of the enzyme preparation on kaolin. 1.65 g of kaolium was added to 600 ml of the fermentation liquid medium prepared as indicated in Example 4, then 180 g of ammonium sulfate was added in separate portions with mechanical stirring until complete dissolution. The kaolin-protein mixture is filtered off, washed with water and then dried at room temperature in a vacuum oven. A product weighing 11.7 g has full activity inherent in the original liquid medium. The subject of the invention is a method for producing enzyme preparations by growing the microorganisms producing them on a nutrient medium containing nitrogen sources, adding other nutrients necessary for growth, such as carbohydrates and mineral salts, under aerobic conditions, followed by isolating the enzyme preparations from the biomass obtained by one of known methods, characterized in that, in order to obtain preparations with high activity, containing proteolytic enzymes capable of cleaving keratin, and Also, la7 mncariasis, chitinase, elastase and lipase, bacteria of the genus Cytofaga К ° «NCIB 9497 are used as producing microorganisms8.