SU1693048A1 - Method of bilirubin oxidase preparation - Google Patents

Abstract

The invention relates to biotechnology, in particular, to a method for producing a bilirubin oxidase enzyme catalyzing the oxidation of a bile pigment, bilirubin. To increase the yield of the enzyme, improve its quality and stability, the mucous membrane of the small intestine of pigs is used as a raw material, which is homogenized in the presence of ephedrine in an amount of 0.02-0.04% by weight of the raw material, followed by three times precipitation with acetone, salting out with 6-8 M aqueous a solution of sodium chloride at 4-6 ° C for 10-12 hours and after dissolving the precipitate by dialysis against water. After drying, the target product is mixed with 0.1-0.2% glycine. The yield of the target product is 0.8-1.2% by weight of the raw material, which is 16-24 times higher than the yield of the enzyme in the known method, while the amount of cleavable bilirubin in the blood increases by 30-45%, and the stability of the enzyme increases by 8, 5 times when stored at low temperatures. 1 hp f-ly.

Description

The invention relates to biotechnology, in particular to a method for producing a bilirubin oxidase enzyme catalyzing the oxidation of a bile pigment, bilirubin.

A known method for producing bilirubin oxidase by aerobic fermentation of strains of the fungus Trachyderma tsunodae cl. Basidiomyces on a nutrient medium containing sources of nitrogen, carbon and mineral salts at pH 8–9 and 28–45 ° C for 72–96 h followed by purification of the culture fluid by ion exchange resin chromatography and affinity chromatography 1.

The disadvantage of this method is its duration (about 6 days), as well as the fact that the resulting enzyme cleaves no more than 60% of bilirubin, which does not guarantee the accuracy of the results of quantitative determination of non-protein nitrogen and other components in the blood and urine.

The known method of producing bilirubin oxidase by homogenizing the raw material - the fruits of higher plants of the families of nightshade and banana, mainly bananas or eggplants, in an aqueous medium, followed by filtering the homogenate, precipitating the filtrate with acetone, further dissolving the precipitate in water, transferring

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precipitation from 70% aqueous alcohol and drying the desired product 2.

The disadvantages of this method are the use of food raw materials, low enzyme yield (0.08-0.1% of the initial weight of the raw material), its low ability to break down bilirubin (60-65% of the content in the analyzed sample) even at high concentrations (0.15-0.3 mg / ml) and low stability of bilipinokinase during storage (not more than 20 days at 4 ° C).

Closest to the present invention is a method for producing bilirubin oxidase from rat intestinal homogenate, based on extracting the enzyme with water, followed by purification by fractionation with mineral salts and organic solvents 3.

The disadvantage of the method lies in the low activity of the enzyme, expressed in its ability to break down only 40-50% of bilirubin contained in body fluids, as well as in the instability of bilirubine oxidase (the activity remains for 1-2 weeks in a powdery state and 2-4 h in aqueous solution).

The aim of the invention is to increase the yield of the enzyme, improving its quality and stability.

The method of obtaining bilirubin oxidase involves homogenizing the raw material in an aqueous medium, filtering the homogenate, treating the filtrate with acetone, further dissolving the precipitate in water and drying, using the mucous membrane of the small intestine of pigs as the raw material, homogenizing it in the presence of ephedrine in an amount of 0.02-0 , 04% by weight of the raw material, precipitation with acetone is carried out three times at 0 ° C, after dissolving the precipitate in water, the resulting solution is treated with an equal volume of 6-8 M aqueous NaCI solution at 4-6 ° C for 10-12 h followed by dialysis of the solution against water, and after drying the target product is mixed with glycine in an amount of 0.1-0.2% by weight of the target product.

Example 1.1 kg of the mucous membrane of the small intestine of pigs is crushed on the top, 2 l of water is added to the mincemeat, in which 0.2 g of the inducer ephedrine (0.02% by weight of the raw material) is first dissolved, the mixture is homogenized for 1 h, then filtered; the filtrate is poured into 3 liters of acetone cooled to 0 ° C (3 volumes by weight of the raw material), stirred, kept at 0 ° C for 4 hours, the precipitate is separated, dissolved in 1 liter of water, the solution is poured into 3 liters of acetone cooled to 0 ° C, after 4 h, the precipitate is separated, the operation is repeated one more time, the precipitate is dissolved in 1 l of water (1: 1 ratio of the mass of the feedstock and water), filtered,

Filtrate was added ras and a volume of 6 M NaCI aqueous solution and the mixture was kept at 40 ° C for 10 hours, the precipitate was filtered off, dissolved in 100 ml of water, dialyzed against water at 4 ° C for 16 hours, the dialysate was dried

by sublimation, 8 g of bilirubin oxidase powder is obtained (yield 0.8% of the initial weight of the raw material), to which 8 mg of glycine (0.1% by weight of the target product) is added. When high blood is added to the sample

5 content of bilirubin (more than 20 mmol / l) enzyme at a concentration of 0.05 mg / ml 85% bilirubin is cleaved. Bilirubin oxidase retains activity at 4 ° C for 2-4 months.

0 PRI mme R 2. 1 kg of the mucous membrane of the small intestine of pigs is crushed on top, 2 l of water is added to the mincemeat, in which 0.3 g of inducer ephedrine (0.3% by weight of the raw material) is dissolved beforehand,

5, the mixture is homogenized for 1 hour, then filtered, the filtrate is poured into 3 liters of acetone cooled to 0 ° C (3 volumes by weight of the raw material), stirred, kept at 0 ° C for 4 hours, the precipitate is separated, dissolved in 1 liter of water,

0, the solution is poured into 3 l of acetone cooled to 0 ° C, after 4 h the precipitate is separated, the operation is repeated one more time, the precipitate is dissolved in 1 l of water (the ratio of the mass of the feedstock and water is 1: 1), filtered,

5 an equal volume of M NaCl aqueous solution is added to the filtrate, the mixture is kept at 5 ° C for 11 h, the precipitate is separated by filtration, dissolved in 100 ml of water, dialysed against water at 4 ° C for 16 h, the dialysate is dried

0 by sublimation, and 10 g of bilirubin oxidase powder is obtained (yield 1% of the initial weight of raw material), to which 15 mg of glycine (0.15% by weight of the target product) is added. When a blood enzyme is added to a sample with a high bilirubin content (> 20 mmol / l) at a concentration of 0.075 mg / ml, 90% of bilirubin is cleaved. Bilirubin oxidase retains activity at 4 ° C for 2-4 months.

0 EXAMPLE 3. 1 kg of the mucous membrane of the small intestine of pigs is crushed on top, 2 l of water is added to the mincemeat, in which 0.4 g of inducer ephedrine (0.04% by weight of the raw material) is dissolved beforehand,

5, the mixture is homogenized for 1 hour, then filtered, the filtrate is poured into 3 liters of acetone cooled to 0 ° C (3 volumes by weight of the raw material), stirred, kept at 0 ° C for 4 hours, the precipitate is separated, dissolved in 1 liter of water, the solution is poured into 3 liters of acetone, cooled to 0 ° C after 4 hours, the precipitate is separated, the operation is repeated once more, the precipitate is dissolved in 1 l of water (the ratio of the mass of the raw material and water is 1: 1), filtered, and the filtrate is added equal to a volume of 8M aqueous solution of NaCl, the mixture is kept at 6 ° C for 12 h, the precipitate is separated by filtration, dissolved in 100 ml of water, dialyzed against water. s at 4 ° C for 16 hours, the dialysate was freeze dried to give 12 g of PS bilirubinoksida- powder (yield: 1.2% of initial feedstock weight) to which was added 10 ml of glycine (0.2% by weight of the desired product). When a high bilirubin content (more than 20 mmol / l) enzyme is added to the sample at a concentration of 0.1 mg / ml, 95% bilirubin is cleaved. Bilirubin oxidase retains its activity at 4 ° C for 2-4 months.

When this set of features changes, a positive effect is not achieved, which is given by the following comparative examples.

Example 4. 1kg of pig liver is ground on top, 2 l of water is added to the mincemeat, in which 0.2 g of ephedrine is previously dissolved, and then proceed as in example 1. 9 g of powder is obtained that does not have the ability to break down bilirubin into blood samples even at a concentration of 0.1 mg / ml.

Example 5. 1 kg of cattle spleen is crushed on a choke, 2 l of water is added to the mincemeat, in which 0.2 g of ephedrine is dissolved beforehand, and then proceeded as in example 1. 8 g of powder are obtained, adding it to a blood sample at a concentration of 0.1 ml / ml does not cleave bilirubin.

Example 6. 1 kg of the mucous membrane of the small intestine of pigs is crushed on top, 2 l of water is added to the mincemeat, in which 0.1 g of ephedrine (0.1% by weight of the raw material) is first dissolved, and then proceed as in Example 1 0.7 g of bilirubin oxidase powder is obtained (yield 0.07% by weight of the starting material), which cleaves 55% of bilirubin in blood samples at a concentration of 0.15 mg / ml.

Example 7. 1 kg of the mucous membrane of the small intestine of pigs is crushed on the top, 2 l of water is added to the mincemeat, in which 0.2 g of phenylalanine (0.02% by weight of the raw material) is previously dissolved, and then proceed as in Example 1. 1 g of bilirubin oxidase powder is obtained (yield 0.1% by weight of the raw material), which cleaves 65% of bilirubin in blood samples at a concentration of 0.2 mg / ml.

Example 8. 1 kg of the mucous membrane of the small intestine of pigs is crushed on the top, 2 l of water is added to the mincemeat, in which the inducer does not dissolve, and then proceed as in Example 1. 1 g of bilirubine oxidase powder is obtained (yield 0.1% of the mass of the raw material), splitting 60% of bilirubin in blood samples at a concentration of 0.3 mg / ml.

Example 9. 1kg of the mucous membrane of the small intestine of pigs is crushed on top, 2 l of water is added to the mincemeat, in which 0.2 g of ephedrine is previously dissolved, the mixture is homogenized for 1 h, then filtered, the filtrate is poured into 3 l of acetone cooled to 0 ° C (3 volumes to the weight of the raw material), stirred, kept at 0 ° C for 4 hours, the precipitate separated, dissolved in 1 liter of water, filtered, added to the filtrate an equal volume of 6 M aqueous solution of NaCI and the mixture was kept at 4 ° C. h. Due to the presence of a significant amount of lipids in the sediment, which the additional As a result of salting out, no sediment is formed, which does not allow to isolate bilirubin oxidase.

Example 10. 1 kg of the mucous membrane of the small intestine of pigs is crushed on top and then proceed as in example 1, except that the filtrate obtained after filtration of the homogenate is treated with ethyl alcohol. 0.5 g of bilirubin oxidase powder is obtained (yield 0.05% of the initial mass of the raw material), which cleaves 35% of bilirubin in blood samples at a concentration of 0.15 mg / ml.

Example 11. 1 kg of the mucous membrane of the small intestine of pigs is crushed on the top and then proceeds as in example 1, except that the precipitate after transplanting from acetone is dissolved in water and dried by sublimation, mine the dialysis stage against water. 18 g of powder is obtained which does not possess bilirubin oxidase activity even at a concentration of 0.3 mg / ml in blood samples.

Example 12. 1 kg of the mucous membrane of the small intestine of pigs is crushed on the top and then proceeds as in Example 1, except that 8 g of glycine (0, 0 mg) is added to 8 g of bilirubine oxidase powder (yield 0.8% by weight of raw material). 05% by weight of the powder). At the same time, the enzyme remains active at 4 ° C for 15 days.

Example 13. 1 kg of the mucous membrane of the small intestine of pigs is crushed on the top and then proceeds as in example 1, except that 8 g of the obtained bilirubin oxidase powder (yield 0.8% of the initial weight of the raw material) is not added to the stabilizer. At the same time, the enzyme remains active at 4 ° C for 8 days.

Example 14. 1 kg of the mucous membrane of the small intestine of pigs is ground on the top and then proceeds as in Example 1, except that 8 g of the obtained bilirubin oxidase powder (yield 0.8% of the initial mass of raw material) is added as a stabilizer 8 mg of glucose (0.1% by weight of the powder). At the same time, the enzyme retains its activity at 4 ° C for 14 days.

The proposed method of obtaining bilirubin oxidase compared with the known method allows to increase the yield of the enzyme by 16-24 times, to increase the amount of cleavable bilirubin in the blood by 30-45% when the concentration of the enzyme added to the blood is 10 times less, and also in 8 , 5 times increase its stability in conditions of low temperatures.

Claims (2)

Claims 1. Method for producing bilirubin oxidase, which involves homogenization of the intestines of mammals in an aqueous medium, filtering the homogenate, salting out, followed by dissolving the precipitate in water and drying, characterized in that, in order to increase the yield of the enzyme, to increase its quality and stability, as raw materials use the mucous membrane of the small intestine of pigs, the homogenization is carried out in the presence of ephedrine in an amount of 0.02-0.04% by weight of the raw material, three times before salting out precipitation with acetone, salting out is carried out with a 6-8 M aqueous solution of sodium chloride at 4-6 ° C for 10-12 hours, and after dissolving the precipitate, dialysis is carried out against water. 2. A method according to claim 1, characterized in that after drying the target product is mixed with 0.1-0.2% glycine.