SU377159A1 - METHOD OF OBTAINING PROTEAS INHIBITOR - Google Patents

Description

one

A known method for producing a protease inhibitor by autolysis of crushed animal raw materials, separating the cake, extracting, basifying, treating the extract with ammonium sulfate, heating, basifying the solution, filtering, drying.

However, the known method does not provide for obtaining a protease inhibitor in a single technological cycle with heparin and peptone and, in addition, the protease inhibitor is not sufficiently active.

To increase the activity of the protease inhibitor while at the same time obtaining heparin and peptone from protein waste, after extracting the inhibitor, the extract after autolysis is treated with water and before heating the precipitate is treated with 0.65 n. The hydrochloric acid solution is treated with sodium chloride, followed by treatment of the precipitate with potassium acetate, then the precipitate after heparin extraction is cooled, treated with water and peptone is recovered in a known manner.

Example. 10 kg of frozen lungs of cattle are ground on a cutter and kept at room temperature for 17 hours. Then add 3 volumes of water cooled to 8-12 ° and mix for 2 hours.

The lung tissue (cake) is separated by centrifugation, washed with a small amount (about 0.5 volume) of water at pH 7.0, and used to isolate heparin.

Isolation of the protease inhibitor.

The extract and the washings were combined by the addition of 5N. caustic soda solution to establish a pH of 7.0 and salted out ballast proteins with ammonium sulfate at the rate of 113 g / l. The precipitate is separated by sut filtering.

beta and emit, and from the filtrate salted out the raw inhibitor ammonium sulfate at the rate of 510 g / l. After settling for 10-12 hours at 5 ° C, the supernatant is siphoned and the sediment is separated

filtering on suction filter. Then, the precipitate containing the protease inhibitor is dissolved in 3.5 volumes of 0.65 n. hydrochloric acid solution, heated at 80 ° C for 4 min, then rapidly cooled to room temperature

temperature and centrifuged. The protein precipitate is washed with 2 volumes of water. The washings are combined with the basic solution and the addition of 5N. caustic soda solution to establish a pH of 7.5. The protease inhibitor is precipitated with ammonium sulfate at a rate of 600 g / l, separated by filtration on a Buchner funnel, dissolved in 10 volumes of water, desalted by dialysis and lyophilized. The output of the protease inhibitor activity 650-

700 U out of 1 kg of the lungs, 320 mg of PM are obtained after removing the trypsin inhibitor and washing it is extracted with 1.5 volumes of 0.7 mol. a solution of iatra chloride at pH 9-9.5 and a temperature of 55-60 ° for 1.5 hours. Then the temperature of the mixture is raised to 100 ° C to coagulate the ballast proteins, cooled to 35-40 ° C and filtered. The precipitate is used to produce peptone. The resulting filtrate is adjusted to pH 2.5 by adding 17% hydrochloric acid. The precipitated heparin crude hydrochloride precipitate is separated by centrifugation and suspended in 4 volumes of a 15% sodium chloride solution. A 20% calcium chloride solution (pH 9–9.5) and a temperature of 70–75 ° C was added to the suspension, the mixture was heated to 95 ° C, coagulated ballast was separated by filtration, the solution was cooled to 30–35 ° C, and acidic hydrochloric acid to pH 2.5. The precipitate was separated by centrifugation. The solution is adjusted to pH 8.2-8.3 by adding 5 p. Of sodium hydroxide solution and heparin is precipitated with 1.5 volumes of ethanol.

After 12-15 hours, the supernatant was removed by siphoning, and the precipitate was separated by centrifugation, dissolved in 10 volumes of water at pH 9-9.5 and a temperature of 60-70 ° C. A 30% solution of hydrogen peroxide for decolorization is then added to the volume of the mixture, cooled to room temperature, filtered. Up to two moles of potassium acetate and acetic acid to pH 5.5 are added to the transparent filtrate, heated to 55-60 ° C and rapidly cooled to 5-8 ° C, and then kept at this temperature for 10-12 hours. The precipitated precipitate is separated by centrifugation, dissolved in 10 volumes of water, treated with KU-2 ion-exchange resin in H-form under static conditions, and after separation of the resin from the solution, heparin is precipitated at pH 6.0-6.5 (set by adding sodium hydroxide) equal volume of alcohol. Yield: 1 kg of lungs - 11700 units. with an activity of 96 units / mg.

Getting peptone.

After removal of heparin, lung tissue residues are rapidly cooled to 15–18 ° C with water, lowering the pH to 6.0–6.5. The residue is suspended with two volumes of water and heated to 40 ° C. Phosphoric acid is added to the heated mixture so that the initial pH of the mixture is 1.7-1.8. 8% of the pork stomach mucosa is added to the mixture and kept at 45 ° C for 18-23 hours until the tissue is completely digested. After the end of the hydrolysis, the clear liquid is boiled with the addition of calcium hydroxide, and the solution is adjusted to a pH of 8.2-8.5. The hot solution is separated from the precipitate and, at 50 ° C, 1% of the pancreas of slaughter animals is added.

The trypsin hydrolysis is carried out at 50 ° C, pH 8.2-8.5, until complete digestion of the protein is determined by the qualitative reaction to the protein in the presence of 20% trichloroacetic acid.

At the end of proteolysis, the lysate is brought to a boil, then the pH is adjusted to 6.0-7.0 by the addition of phosphoric acid and filtered while hot through the beta cloth.

The clear solution is dried by known methods, for example on a spray dryer.

Peptone yield - 50 g from 1 kg of lungs.

Subject invention

The method of producing a protease inhibitor by autolysis of crushed animal raw materials, separating the cake, extracting, alkalinizing, treating the extract with ammonium sulfate, heating, alkalinizing the solution, filtering, drying, characterized in that, in order to increase the yield of the target product while producing autolysis extract is treated with water and before heating the precipitate is treated with 0.65 in. solution of hydrochloric acid, cake is treated with sodium chloride

After the precipitate is treated with potassium acetate, the precipitate after heparin extraction is cooled, treated with water and peptone is separated in a known manner.