RU2610669C1 - Method for production of proteolytic preparation for medical use - Google Patents

Abstract

FIELD: medicine, pharmacy.

SUBSTANCE: invention refers to biotechnology. A method for extraction of proteolytic enzymes from the digestive organs of fish is proposed. The method includes tissue homogenizing in a buffer, selection of enzymes in the presence of ammonium sulfate, at that, nucleic acids and lipid-protein complexes are removed from the extract prior to protein precipitation in ammonium sulfate by treatment with a polyethyleneimine solution, added to a final concentration from 0.1 to 0.01 by weight %. Residual pyrogens are separated on a hydrophobic sorbent Sferotsell C-80 with further repeated desalination of the extract. The enzyme solution is sterilized by filtration through a membrane with pore size of 0.2 microns, followed by production of lyophilized formulation of the enzyme preparation.

EFFECT: invention allows to obtain proteolytic enzymes with increased collagenolytic activity.

6 cl, 1 dwg, 1 tbl, 7 ex

Description

A method of obtaining a proteolytic drug for medical use

The invention relates to biotechnology, namely, to obtain a complex of proteolytic enzymes with a wide spectrum of proteolytic activity, capable of hydrolyzing protein substrates. The invention may find application in the manufacture of medicines.

Known use in medicine for cleansing wounds of preparations based on microbial collagenase secreted by producer strains of Clostridium histolyticum. Microbial collagenase has high specificity for the main protein of the collagen connective tissue. However, high specificity, for the purpose of cleansing wounds and treating keloid scars, is a drawback. This is due to the fact that the intercellular matrix and connective tissue contains not only collagen fibers, it is a complex network including, in addition to collagen, elastin and adhesive proteins. Another important component of the intercellular matrix are proteoglycans, the axial molecule of which is hyaluronic acid, to which proteins are attached as bristles to the brush, which in turn are connected to many glycosaminoglycans.

Dead tissue in a burn wound is represented mainly by coagulated proteins of the dermis (collagen, elastin, etc.), as well as damaged components of the extracellular matrix. On the surface of the wounds may be fibrinous plaque. The visible effects of wound cleansing under the action of proteolytic enzymes are often the result of the breakdown of fibrin, and not collagen. In the treatment of keloid scars, the main task of enzyme preparations is the destruction of improperly assembled collagen fibrils and proteoglycans to oligomers and individual monomers, which are partially used to assemble a new, more ordered connective tissue.

Based on this, it can be assumed that in medical applications, it may be more effective not drugs with a narrow specific effect (microbial collagenases), but complexes of proteolytic enzymes with a wider range of substrates.

By a complex of proteolytic enzymes we understand the totality of individual digestive system enzymes with proteolytic action, which are released together in the technological process.

Such enzyme complexes include serine proteases isolated from marine invertebrates (crab collagenase) and digestive fish serine proteases.

Unlike true collagenases, serine proteases of invertebrates and fish cleave all three polypeptide chains of tropocollagen, and the resulting peptides undergo further hydrolysis up to individual amino acids, which are either included in the processes of anabolism or are rapidly excreted without causing intoxication.

The most widely developed and are analogues of the proposed method methods for producing crab collagenase from hepatopancreas of crabs. The effectiveness of crab collagenase for cleansing wounds and treating keloid scars has been established and described in the patent literature (US 4801451, US 4963491, US 5945102 and US 6030612, as well as RU 2008353, RU 2039819, RU 226505, RU 2365623).

The main problem in the industrial production of preparations based on crab collagenase is the instability of the supply of raw materials and the long shelf life of the raw materials before delivery to the processing plant, which often leads to abundant contamination of the raw materials by microorganisms and the accumulation of microbial endotoxins (the chemical structure of which is lipopolysaccharides) that can cause a pyrogenic effect and allergies. This complicates the industrial isolation of the complex of proteolytic enzymes and reduces its quality, and the use of such drugs in patients is accompanied by frequent allergic reactions.

As is known from the prior art, complexes of proteolytic enzymes with crab-like N-terminal amino acid sequences can be isolated from the digestive organs of fish. A study of the substrate specificity of these proteolytic enzyme complexes shows that, unlike mammalian digestive proteases, they are able to hydrolyze a wider range of substrates. including collagen of various types, which, due to the characteristics of the amino acid sequence and spatial structure, resistant to most proteases and available for hydrolysis only by specific enzymes - collagenases. The source of the complex of proteolytic enzymes is waste from the food production of fish - offal (digestive organs), which usually goes to waste and for disposal. Compared to crab collagenase, this source is much more accessible and cheaper (Zwilling R., et al., 1975, FEBS Lett. 60, 247-9; Gran GA, et al., 1981, Methods Enzymol 80, 722- 734; Reeck GR and H. Neurath. 1972, Biochem. 11: 503-510, OA Klimova, et al., 1990, BBRC, v. 166, N3, 1411-1420).

For medical use, it is most preferable to use complexes of proteolytic enzymes isolated from the digestive organs of fish, since they have serine proteases that are active against deep hydrolysis of collagen and other connective tissue proteins. As shown in the dissertation "Soloviev MM Characterization of the digestive enzymes of fish in Lake Chany in the early stages of ontogenesis. / Thesis for the degree of Ph.D.: Novosibirsk. - 2011. ”at all stages of the development of peaceful and predatory fish, the group of serine proteinases (up to 70-80%) makes the largest contribution to the activity of alkaline proteinases. The proportion of cysteine proteinases and metalloproteases is much smaller and does not exceed 10-12%. In non-predatory fish, 4–6 isoforms were detected, in predatory fish, 7–8 isoforms of alkaline proteases.

The well-known "Method of isolating a complex of proteolytic enzymes from seafood" by homogenization of raw materials and the deposition of enzymes, characterized in that, in order to expand the raw material base and increase the yield of the target product, pyloric flounders are used as raw materials, and the deposition of enzymes is carried out with acetone at a homogenate ratio: acetone 1: 2 (2.3). Moreover, the isolated complex is a valuable set of enzymes, in particular alkaline, neutral and acidic proteinases, aminopeptidases and carboxypeptidases. The isolation method consists in the fact that appendages isolated from flounder are homogenized and quickly mixed with two volumes of acetone cooled to -15 ° C. After 20 minutes, the material is squeezed from acetone and dried at + 20 ° C. The specific activity is 0.76 for the casein substrate. The proteolytic activity of the isolated enzymes in the neutral and slightly acidic region exceeds the activity of the enzyme isolated from crab hepatopancreas (SU 1030409 C12N 9/64. "Method for isolating the proteolytic enzyme complex from seafood." MV Kolodzeiskaya, SA Kudinov, L.M. Epstein, VN Akulin. Declared March 12, 82, published July 23, 83).

The main disadvantage of this method is the use of large quantities of organic solvent - acetone, which requires compliance with special conditions in the workplace. Another disadvantage is the presence of ballast proteins and lipopolysaccharides in the preparation, which prevents the use of such an enzyme for medical purposes.

A number of technical solutions are known in which the isolation of proteolytic enzymes from fish is described, but the resulting product is also characterized by the presence of ballast proteins and lipopolysaccharides, so the isolated proteolytic enzymes are suitable only for use in the food and leather industries.

In particular, the “Method for the preparation of a proteolytic enzyme preparation from internal organs” is known. This invention relates to biotechnology, in particular to the production of an enzyme preparation, and is used in the manufacture of food products from low-ripening fishing facilities of the Volga-Caspian region. The method involves grinding raw materials. The crushed raw materials are subjected to autolysis in the presence of water at a water module of 1: 0.5 at a pH of 6.2 ± 0.2 for 1-5 hours. After autolysis, a liquid autolysate containing proteolytic enzymes is separated by centrifugation and subjected to purification with isopropyl alcohol at a hydromodule of 3: 1 for 30-40 minutes. The resulting liquid enzyme preparation is spray-dried at a temperature of 150-180 ° C for 5-8 seconds. EFFECT: invention allows to increase the yield of a proteolytic preparation and increase its activity (RU No. 2288951 C12N 9/64 "Method for the production of a proteolytic enzyme preparation from internal organs of fish", publ. 10.12.2006).

There is also a method of producing an enzyme preparation from fish raw materials. This invention relates to biotechnology, in particular to methods for the extraction of proteolytic enzymes from fish stock, and is intended for use in the food industry. The method involves grinding the raw material, isolating the enzyme by extraction in the presence of an alkali metal chloride solution, separating the precipitate, two ultrafiltration stages with crushed ice added to the liquid fraction and freeze-drying the concentrate. In the first stage, ultrafiltration is carried out on a membrane with a transmission limit of 50 kDa, and in the second stage on a membrane with a transmission limit of 15 kDa. The invention improves the yield of proteolytic enzymes and their activity by 3-5 times in comparison with the known analogues (RU No. 2352634 C12N9 / 00 "Method for producing an enzyme preparation from fish raw materials", published on 04/20/2009).

The closest technical solution to the claimed invention is the following sequence of steps for obtaining a complex of proteolytic enzymes from fish tissues (Daboor SM, Budge SM, Chaly A.E., et al. Extraction and Purification of Collagenase Enzymes: A Critical Review. // American Journal of Biochemistry and Biotechnology 6 (4): 239-263,2010.):

- Homogenization of raw materials in 0.05 M Tris-HCl buffer with the addition of 0.005 M calcium chloride at pH 7.4;

- Centrifugation of the homogenate at 25,000 × g for 30 min at + 4 ° C and collecting the supernatant;

- Repeated extraction of the precipitate in 0.05 M Tris-HCl buffer with the addition of 0.005 M calcium chloride at pH 7.4, followed by centrifugation of the homogenate at 25000 × g for 30 min and collecting the supernatant;

- Combining supernatants and centrifugation at 40,000 × g for 30 min at + 4 ° C;

- Filtering the resulting supernatant through a 0.2 μm pore membrane;

- Protein fractionation by reprecipitation in a solution of ammonium sulfate at saturation in the range of 40-80%;

- Isolation of ammonium sulfate precipitate of the fractionated enzyme by centrifugation;

- Dissolution of sediment in the buffer;

- Desalination by passing the solution through hollow fibers with a pore size of 10 kDa;

- Secondary purification of the complex of proteolytic enzymes using ion exchange chromatography.

Most proteolytic enzymes are located in tissue cells either in an inactive form or in lysosomes, otherwise they would digest the cells themselves. Therefore, in order to extract them, it is necessary to destroy the cells. Homogenization is the destruction of cells and lysosomes by mechanical, temperature or chemical factors. The rate and amount of the release of enzymes into the extraction solution depends on the degree of destruction (grinding of the cells). Typically, the extraction takes from a few hours to 2-3 days. Another procedure for the extraction of proteolytic enzymes is the autolysis used in most of the above patent patents. Autolysis of tissues occurs at elevated temperatures. During it, accelerated transport of proteases through lysosomal barriers with their subsequent activation and destruction of cellular and lysosomal barriers under the action of active proteases is observed. Autolysis is also accompanied by side effects, which include autohydrolysis of the enzymes themselves and the accumulation of microbial biomass in autolysed tissues, which leads to a sharp increase in the content of LPS and other microbial toxins.

Centrifugation and membrane filtration are technological procedures aimed at removing insoluble tissue particles, insoluble lipid-protein complexes and other insoluble particles from the homogenizate.

Protein fractionation in ammonium sulfate is a primary purification procedure for the isolation of proteins. The salting out of proteins follows two mechanisms: sulfate ions make the protein molecule more compact, and therefore less soluble, sulfate ions bind free water. One SO ₄ ^2- ion has 13-14 H ² O molecules only in the first hydrate layer and, possibly, more in the second. It is believed that at a concentration of 5.4 mol, ammonium sulfate binds all free water - this is 100% saturation. The advantages of ammonium sulfate is that it has approximately the same solubility in the temperature range of 0- + 30 degrees Celsius and that in a concentration above 0.5 M it stabilizes proteins against denaturation. Typically, individual proteins lose their solubility and precipitate at a certain degree of saturation of the solution with ammonium sulfate. When several individual proteins are isolated at once, as in our case, they are precipitated in a rather wide range of saturation of ammonium sulfate. A purified complex of proteolytic enzymes isolated from fish is usually planted in the saturation region of ammonium sulfate from 40 to 80%. The precipitate is separated from the solution of ammonium sulfate by precipitation in a centrifuge. However, the presence of nucleic acids and lipid-protein complexes in the solution degrades the selectivity of fractionation and leads to the coprecipitation of the target complex of proteolytic enzymes in all fractions of the ammonium sulfate precipitate.

After dissolving the precipitate of the protein fraction in the buffer, the solution contains a significant amount of ammonium sulfate, which will interfere with further purification of the complex of proteolytic enzymes. The solution is subjected to desalination. Typically, this procedure is carried out by ultrafiltration through a membrane with a specific pore size. However, in our case, the appearance of a fine precipitate of the lipid-protein complex leads to rapid clogging of the pores and a sharp inhibition of the process. Therefore, for desalination, it is necessary to use tangential filtration. During tangential filtration, the solution flow is directed parallel to the membrane surface, which allows continuous washing off of the pore-clogging sediment. A variety of tangential filtration are hollow fiber apparatuses and various membrane modules for tangential filtration.

Ion exchange chromatography is an efficient way to separate individual proteins. Proteins bind to the sorbent and elute from it in accordance with its charge. Ion exchange chromatography allows you to separate the target proteins from the accompanying ones. However, when a complex of proteolytic enzymes is isolated from the digestive organs of fish, ion-exchange chromatography becomes ineffective, again due to the presence of a protein-lipid complex. The protein-lipid complex irreversibly sits on the sorbent and binds a significant amount of proteins of the complex of proteolytic enzymes. As a result, their yield from ion exchange chromatography does not exceed 10%.

The disadvantage of this known method is that the proposed method does not take into account the peculiarities of fish tissue. They contain a large number of lipids, ballast and hydrophobic proteins, as well as nucleic acids and other polyanionic molecules, which are prone to the formation of lipid-protein complexes that impede the release of highly purified enzymes. Lipids and nucleic acids are able to co-precipitate with enzymes. The formation of lipid-protein and polyelectrolyte complexes complicates ultrafiltration and membrane filtration, and also leads to non-specific irreversible binding of large quantities of target enzymes to the sorbent at the technological stages of ion exchange chromatography. As a result, significant disadvantages of the scheme adopted as a prototype are the low yield of the final product - (less than 10% of the content in the raw material) and low collagenolytic activity.

The lipid-protein complex refers to the complexes formed in the extract as a result of the binding of phospholipids to proteins due to ionic and hydrophobic interactions, which lose their solubility in the process of isolation of the target enzymes and fall out in the form of insoluble complexes and micelles.

They are able to capture and precipitate target enzymes, which leads to a decrease in the yield of the target product.

An object of the invention is to provide an effective method for producing a purified complex of proteolytic enzymes for medical use and to expand the arsenal of methods for producing a purified complex of proteolytic enzymes.

The technical result, which provides a solution to the problem, is to obtain a complex of proteolytic enzymes containing alkaline, neutral and acidic proteinases, with increased collagenolytic activity due to increased specific activity with respect to deep hydrolysis of collagen and gelatin, as well as the breakdown of hemoglobin, casein, and decreased pyrogenicity ( the ability of a substance to cause an increase in body temperature), simultaneously with an increase in the quantitative yield of the target products. It was found that in addition to the above substrates, the complex of proteolytic enzymes is able to cleave chitosan, an analogue of glycosaminoglycans of connective tissue, to a lesser extent.

The specified technical result is achieved by including in the process of obtaining a complex of proteolytic enzymes at the optimal stages of separation of nucleic acids and lipid-protein complexes by treating the extract with polyethyleneimine, as well as chromatographic purification on a hydrophobic sorbent, which allows timely removal of lipopolysaccharides responsible for the enzyme pyrogenicity. The lack of pyrogenicity significantly improves the quality of the drug compared with analogues in medical use.

The essence of the invention lies in the fact that the method of isolating a complex of proteolytic enzymes from the digestive organs of fish includes ... sequentially homogenizing tissues in a buffer, separating the crushed tissues by centrifugation, filtering the supernatant through a membrane, salting out proteins in ammonium sulfate, separating the ammonium sulfate precipitate by centrifugation, dissolving the precipitate in the buffer , desalting the extract by diafiltration, purification by ion-exchange chromatography on DEAE-cellulose, characterized in that before precipitation of the solution of proteins in ammonium sulfate, nucleic acids and lipid-protein complexes are removed from the extract by treatment with a solution of polyethyleneimine, which is added to a final concentration of polyethyleneimine in the extract from 0.1 to 0.01 weight. %, and after ion-exchange chromatography, residual pyrogens are separated on the hydrophobic sorbent Spherocell S-80, sterilizing filtration through a 0.2 μm membrane and re-desalting the extract by diafiltration, followed by lyophilization of the finished product.

Preferably, to obtain the finished dosage form after separation of the residual pyrogens, a cryoprotectant is introduced, for example, a physiologically acceptable buffer, mono- or disaccharide, in an amount of from 1 to 10% by weight. %, and / or medical biopolymer, in an amount of from 0.1 to 10% by weight. %

Preferably, the homogenization is carried out gradually by adding 0.05 M Tris buffer with the addition of 0.005 M calcium chloride CaCl ₂ pH 7.4. followed by extraction in the cold.

In some particular cases of the method, the desalination of the extract by diafiltration is carried out by passing the solution through hollow fibers with a pore size of 10 kDa, in other cases, the desalination of the extract by diafiltration is performed on the tangential ultrafiltration module.

Preferably, the mono- or disaccharide is selected from the group: mannitol, lactose or trehalose, and the medical biopolymer from the group of dextrans, polyvinylpyrralidones, polyvinyl alcohols.

The drawing shows an electrophoresis drawing.

EXAMPLES OF PRODUCING A COMPLEX OF PROTEOLYTIC ENZYMES

Example 1

As the feedstock used 400 g of the digestive organs of African catfish. The organs were washed from the blood with water and homogenized the tissues in a buffer, for which they were disintegrated in a homogenizer (10 min), gradually adding 0.05 M Tris buffer with 0.005 M calcium chloride CaCl ₂ pH 7.4 (800 ml). To separate the crushed tissues (homogenate) by centrifugation, extraction was performed in the cold for 24 hours. The total volume of the extract was 1200 ml. The homogenate was centrifuged at 25,000 rpm, + 4 ° C for 30 minutes. The upper layer of lipids was thrown back.

The precipitate was re-homogenized in 0.05 M Tris buffer with the addition of 0.005 M calcium chloride CaCl ₂ pH 7.4 (200 ml), repeated extraction in the cold during the day. The total volume of the extract was 300 ml. The extract (homogenate) for separation of crushed tissues was centrifuged at 25,000 rpm, + 4 ° C for 30 minutes. The upper lipid layer and sediment were thrown back.

Both supernatants were combined and received 1200 ml of solution. The resulting solution was filtered through a membrane with a pore size of 0.2 μm.

The solution was triturated with a 0.1 M sodium hydroxide solution to a pH of 8.0. Separately, a solution containing 5 wt. % polyethyleneimine. While stirring in 1200 ml of the supernatant solution, 24 ml of a solution of polyethyleneimine (PEI) was added to a final concentration of polyethyleneimine in a solution of 0.1 weight. % The final concentration of polyethylenimine in solution (C _{PEI con} ) is calculated by the formula 1:

where V _PEI is the volume of the added solution of polyethyleneimine in milliliters,

With _PEI - the concentration of the taken PEI solution in weight percent,

V _p-pa is the volume of supernatant solution in milliliters.

The final concentration of polyethyleneimine will be (24 * 5) / 1224≈0.1 (weight percent of polyethyleneimine in the extract).

A nucleic acid precipitate formed with PEI. The solution was stirred for another 40 minutes at room temperature to enlarge the precipitate.

The precipitate (nucleic acids) was isolated in a centrifuge at 12,000 rpm for 25 minutes. The precipitate of PEI and nucleic acids had practically no proteolytic activity. They threw him away.

Ammonium sulfate was added to the solution collected after PEI treatment to 80% saturation. A precipitate was formed in the refrigerator during the day. The precipitate was isolated by centrifugation at 12,000 rpm, + 4 ° C for 20 minutes. Protein precipitate was dissolved in 0.05 M Tris buffer. The solution was desalted by diafiltration on hollow fibers with a pore size of 10 kDa.

Conducted by ion exchange chromatography on DEAE-cellulose. A column containing 80 ml of a suspension of DEAE-cellulose sorbent was pre-equilibrated with 0.01 M Tris buffer. A desalted protein solution was applied to the column. The sorbed proteins were eluted using a gradient of sodium chloride 0.05-1.0 M NaCl in 0.01 M Tris buffer, pH 8.0. The elution rate is 20-40 ml / hour. The protein content was monitored using a UV detector at a wavelength of 280 nm. Upon elution, three fractions of the protein having activity were collected. All active fractions were combined. The combined solution was desalted by diafiltration in a tangential ultrafiltration unit.

Chromatography of the resulting solution was carried out on a hydrophobic sorbent to separate residual pyrogens. 50 ml of the hydrophobic sorbent Spherocell C-80 containing hydrophobic C8-C10 ligands (a sorbent based on a macroporous cellulose matrix modified with octyl / decyl glycidyl ether) were placed in a column. The column was equilibrated with 0.01 M phosphate buffer supplemented with 0.05 M NaCl, pH 8.0. The solution was passed through a sorbent column at a rate of 20-40 ml / hour. The enzyme yield was monitored by a UV detector by the optical density of the solution. The collected solution was desalted by diafiltration in a tangential ultrafiltration unit and concentrated to a protein content of 4 mg / ml (according to Lowry). The pH of the resulting solution was adjusted to 7.4. Then spent sterilizing filtration through a 0.2 μm membrane.

Mannitol monosaccharide was introduced into the resulting solution to a concentration of 3.2% and polyglucin polymer (dextran) to a concentration of 0.1%. The solution was sterilized by filtration through a 0.2 μm pore membrane. The solution was poured into sterile vials at 1.25 ml / vial.

Vials were frozen to -40 ° C for 2-10 hours and lyophilized for 24 hours according to the standard program. After lyophilization, the bottles were hermetically sealed.

Got a purified complex of proteolytic enzymes with collagen activity of 1500-2500 μg of substrate / min × mg of enzyme, which can be used for medical purposes. In 1 vial 5 mg of purified complex of proteolytic enzymes. A total of 120 bottles were received.

Electrophoresis of the isolated complex of proteolytic enzymes in a polyacrylamide gel was performed. After staining the gel plate with Coomassie Blue dye, a large set of protein bands with different masses is visible. The most intensively colored bands correspond to masses 25 ± 2 and 36 ± 3 kDa and less intense in the region of 45 kDa.

The specific collagenolytic activity of the proteolytic enzyme complex isolated in the examples was determined by the rate of enzymatic hydrolysis of collagen by staining the released amino groups with trinitrobenzenesulfonic acid (TNBS). Activity was determined in micrograms of hydrolyzed substrate by 1 milligram of enzyme per minute.

The TNBS method is known from the technical literature: Practical protein chemistry: Per. from English / Ed. A. Darbre. - M .: Mir, 1989 .-- 623 p.

Example 2

As the feedstock used 400 g of the digestive organs of pikes. The organs were washed from the blood with water and homogenized the tissues in a buffer, for which they were disintegrated in a homogenizer (10 min), gradually adding 0.05 M Tris buffer including 1 M sodium chloride NaCl and 0.005 M calcium chloride CaCl ₂ pH 7.4 (800 ml). To separate the crushed tissues (homogenate) by centrifugation, extraction was performed in the cold for 24 hours. The total volume of the extract was 1200 ml. The homogenate was centrifuged at 25,000 rpm, + 4 ° C for 30 minutes. The upper layer of lipids was thrown back.

The pellet was re-homogenized in 0.05 M Tris buffer supplemented with 0.005 M calcium chloride CaCl ₂ pH 7.4 (200 ml). spent repeated extraction spent in the cold during the day. The total volume of the extract was 300 ml. The extract (homogenate) for separation of crushed tissues was centrifuged at 25,000 rpm, + 4 ° C for 30 minutes. The upper lipid layer and sediment were thrown back.

Both supernatants were combined and received 1200 ml of solution. The resulting solution was filtered through a membrane with a pore size of 0.2 μm.

The solution was triturated with a 0.1 M sodium hydroxide solution to a pH of 8.0. While stirring in 1200 ml of the supernatant solution, 6 ml of a solution of polyethyleneimine (PEI) was added to a final concentration of polyethyleneimine in a solution of 0.025 weight. %

The final concentration of polyethyleneimine was (6 * 5) / 1206≈0.025 (weight percent of polyethyleneimine in the extract).

A nucleic acid precipitate formed with PEI. The solution was stirred for another 40 minutes at room temperature to enlarge the precipitate.

The precipitate (nucleic acids) was isolated in a centrifuge at 12,000 rpm for 25 minutes. The precipitate of PEI and nucleic acids had practically no proteolytic activity. They threw him away.

Ammonium sulfate was added to the solution collected by the PEI treatment to 80% saturation. A precipitate was formed in the refrigerator during the day. The precipitate was isolated by centrifugation at 12,000 rpm, + 4 ° C for 20 minutes. Protein precipitate was dissolved in 0.05 M Tris buffer. The solution was desalted by diafiltration on hollow fibers with a pore size of 10 kDa.

Conducted by ion exchange chromatography on DEAE-cellulose. A column containing 80 ml of a suspension of DEAE-cellulose sorbent was pre-equilibrated with 0.01 M Tris buffer. A desalted protein solution was applied to the column. The sorbed proteins were eluted using a gradient of sodium chloride 0.05-1.0 M NaCl in 0.01 M Tris buffer, pH 8.0. The elution rate is 20-40 ml / hour. The protein content was monitored using a UV detector at a wavelength of 280 nm. Upon elution, three fractions of the protein having activity were collected. All active fractions were combined. The combined solution was desalted by diafiltration in a tangential ultrafiltration unit.

Chromatography of the resulting solution was carried out on a hydrophobic sorbent to separate residual pyrogens. 50 ml of the hydrophobic sorbent Spherocell C-80 containing hydrophobic ligands C8-C10 were placed in the column. The column was equilibrated with 0.01 M phosphate buffer supplemented with 0.05 M NaCl, pH 8.0. The solution was passed through a sorbent column at a rate of 20-40 ml / hour. The enzyme yield was monitored by a UV detector by the optical density of the solution. The collected solution was desalted by diafiltration in a tangential ultrafiltration unit and concentrated to a protein content of 2 mg / ml (according to Lowry). The pH of the resulting solution was adjusted to 7.4. Then spent sterilizing filtration through a 0.2 μm membrane.

Lactose disaccharide was added to the resulting solution to a concentration of 10% and g polymer polyvinylpyrralidone (grade 17 PF collidone) to a concentration of 2.5%. The solution was sterilized by filtration through a 0.2 μm pore membrane. The solution was poured into sterile vials at 2 ml / vial.

Vials were frozen to -40 ° C for 2-10 hours and lyophilized for 24 hours according to the standard program. After lyophilization, the bottles were hermetically sealed.

Received a purified complex of proteolytic enzymes with collagen activity of 1200-2000 μg of substrate / min × mg of enzyme, which can be used for medical purposes. In 1 bottle 4 mg of purified complex of proteolytic enzymes. A total of 125 bottles were received.

Electrophoresis of the isolated complex of proteolytic enzymes in a polyacrylamide gel was performed. After staining the gel plate with Coomassie Blue dye, a large set of protein bands with different masses is visible. The most intensively colored bands correspond to masses 25 ± 2 and 36 ± 3 kDa and less intense in the region of 45 kDa.

Example 3

400 g of the digestive organs of the flounder were washed from the blood with water. The tissues were homogenized in the buffer, for which the tissues were disintegrated in the homogenizer (10 min for 5 times), gradually adding 0.05 M Tris buffer, including 3 M sodium chloride NaCl, 0.005 M calcium chloride CaCl ₂ , pH 7.4, 0.02 % sodium azide NaN ₃ . (800 ml). Extraction was carried out in the refrigerator for three days. The total volume of the extract is about 1200 ml. Spent the separation of crushed tissues by centrifugation. The extract was centrifuged at 14,000 rpm, + 4 ° C for 20 minutes. The upper lipid layer and sediment were thrown back.

Received 800 ml of solution. The resulting solution was filtered through a membrane with a pore size of 0.2 μm.

The solution was triturated with a 0.1 M sodium hydroxide solution to a pH of 8.0. While stirring, in 800 ml of a supernatant solution, 1.6 ml of a solution of polyethyleneimine (PEI) was added to a final concentration of polyethyleneimine in a solution of 0.01 weight. %

The final concentration of polyethyleneimine was (1.6 * 5) / 801.6≈0.01 (weight percent of polyethyleneimine in the extract).

A nucleic acid precipitate formed with PEI. The solution was stirred for another 25 minutes at room temperature to enlarge the precipitate.

The precipitate (nucleic acids) was isolated in a centrifuge at 12,000 rpm for 25 minutes. The precipitate of PEI and nucleic acids had practically no proteolytic activity. They threw him away.

Ammonium sulfate was added to the solution collected by the PEI treatment to 80% saturation. A precipitate was formed in the refrigerator during the day. The precipitate was isolated by centrifugation at 12,000 rpm, + 4 ° C for 20 minutes. Protein precipitate was dissolved in 0.05 M Tris buffer. The solution was desalted by diafiltration on hollow fibers with a pore size of 10 kDa.

Conducted by ion exchange chromatography on DEAE-cellulose. A column containing 80 ml of a suspension of DEAE-cellulose sorbent was pre-equilibrated with 0.01 M Tris buffer. A desalted protein solution was applied to the column. The sorbed proteins were eluted using a gradient of sodium chloride 0.05-1.0 M NaCl in 0.01 M Tris buffer, pH 8.0. The elution rate is 20-40 ml / hour. The protein content was monitored using a UV detector at a wavelength of 280 nm. Upon elution, three fractions of the protein having activity were collected. All active fractions were combined. The combined solution was desalted by diafiltration in a tangential ultrafiltration unit.

Chromatography of the resulting solution was carried out on a hydrophobic sorbent to separate residual pyrogens. 50 ml of the hydrophobic sorbent Spherocell C-80 containing hydrophobic ligands C8-C10 were placed in the column. The column was equilibrated with 0.01 M phosphate buffer supplemented with 0.05 M NaCl, pH 8.0. The solution was passed through a sorbent column at a rate of 20-40 ml / hour. The enzyme yield was monitored by a UV detector by the optical density of the solution. The collected solution was desalted by diafiltration in a tangential ultrafiltration unit and concentrated to a protein content of 2 mg / ml (according to Lowry). The pH of the resulting solution was adjusted to 7.4. Then spent sterilizing filtration through a 0.2 μm membrane.

Phosphate buffer up to 0.1 M and a polymer polyvinylpyrralidone (grade 17 PF collidone) were introduced into the resulting solution to a concentration of 10%. The solution was sterilized by filtration through a 0.2 μm pore membrane. The solution was poured into sterile vials at 2 ml / vial.

Vials were frozen to -40 ° C for 2-10 hours and lyophilized for 24 hours according to the standard program. After lyophilization, the bottles were hermetically sealed.

Got a purified complex of proteolytic enzymes with collagen activity of 1500-2000 μg of substrate / min × mg of enzyme, which can be used for medical purposes. In 1 bottle 4 mg of purified complex of proteolytic enzymes. A total of 150 bottles were received.

Polyacrylamide gel electrophoresis was performed on an isolated complex of proteolytic enzymes. After staining the gel plate with Coomassie blue dye, the bands corresponding to the masses of 25 and 36 kDa and less intense in the region of 45 kDa are most intensely colored (Fig. 1).

Example 4

400 g of the internal organs of piranha were washed from the blood with water. Homogenized the tissues in the buffer. Tissues were disintegrated in the homogenizer, gradually adding 0.05 M Tris buffer, 0.005 M CaCl ₂ , pH 7.4 with 0.02% sodium azide, NaN ₃ . (800 ml). Extraction was carried out in the refrigerator during the day. The total volume of the extract is about 1200 ml. Separation of crushed tissues by centrifugation. The extract was centrifuged at 14,000 rpm, + 4 ° C for 20 minutes. The upper lipid layer and sediment were thrown back.

Received 600 ml of solution. The resulting solution was filtered through a membrane with a pore size of 0.2 μm.

The solution was triturated with a 0.1 M sodium hydroxide solution to a pH of 8.0. While stirring in 600 ml of the supernatant solution, 3 ml of a solution of polyethyleneimine (PEI) was added to a final concentration of polyethyleneimine in a solution of 0.025 weight. %

The final concentration of polyethyleneimine was (3 * 5) / 603≈0.025 (weight percent of polyethyleneimine in the extract).

A nucleic acid precipitate formed with PEI. The solution was stirred for another 30 minutes at room temperature to enlarge the precipitate.

The precipitate (nucleic acids) was isolated in a centrifuge at 12,000 rpm for 25 minutes. The precipitate of PEI and nucleic acids had practically no proteolytic activity. They threw him away.

Ammonium sulfate was added to the solution collected by the PEI treatment to 80% saturation. A precipitate was formed in the refrigerator during the day. The precipitate was isolated by centrifugation at 12,000 rpm, + 4 ° C for 20 minutes. Protein precipitate was dissolved in 0.05 M Tris buffer. The solution was desalted by diafiltration on hollow fibers with a pore size of 10 kDa.

Conducted by ion exchange chromatography on DEAE-cellulose. A column containing 80 ml of a suspension of DEAE-cellulose sorbent was pre-equilibrated with 0.01 M Tris buffer. A desalted protein solution was applied to the column. The sorbed proteins were eluted using a gradient of sodium chloride 0.05-1.0 M NaCl in 0.01 M Tris buffer, pH 8.0. The elution rate is 20-40 ml / hour. The protein content was monitored using a UV detector at a wavelength of 280 nm. Upon elution, three fractions of the protein having activity were collected. All active fractions were combined. The combined solution was desalted by diafiltration in a tangential ultrafiltration unit.

Chromatography of the resulting solution was carried out on a hydrophobic sorbent to separate residual pyrogens. 50 ml of the hydrophobic sorbent Spherocell C-80 containing hydrophobic ligands C8-C10 were placed in the column. The column was equilibrated with 0.01 M phosphate buffer supplemented with 0.05 M NaCl, pH 8.0. The solution was passed through a sorbent column at a rate of 20-40 ml / hour. The enzyme yield was monitored by a UV detector by the optical density of the solution. The collected solution was desalted by diafiltration in a tangential ultrafiltration unit and concentrated to a protein content of 2 mg / ml (according to Lowry). The pH of the resulting solution was adjusted to 7.4. Then spent sterilizing filtration through a 0.2 μm membrane.

Tris buffer up to 0.025 M and a polymer polyvinyl alcohol to a concentration of 0.1%, and trehalose disaccharide to a concentration of 4% were introduced into the resulting solution. The solution was sterilized by filtration through a 0.2 μm pore membrane. The solution was poured into sterile vials at 2 ml / vial.

Vials were frozen to -40 ° C for 2-10 hours and lyophilized for 24 hours according to the standard program. After lyophilization, the bottles were hermetically sealed.

Got a purified complex of proteolytic enzymes with collagen activity of 1500-2200 μg of substrate / min × mg of enzyme, which can be used for medical purposes. In 1 bottle 4 mg of purified complex of proteolytic enzymes. A total of 100 bottles were received.

Polyacrylamide gel electrophoresis was performed on an isolated complex of proteolytic enzymes. After staining the gel plate with Coomassie blue dye, the bands corresponding to the masses of 25 and 36 kDa and less intense in the region of 45 kDa are most intensely colored.

Example 5

As a feedstock, 500 g of the digestive organs of a catran shark (Squalus acanthias) were used. The organs were washed from the blood with purified water and disintegrated in a homogenizer (10 min), gradually adding 0.05 M Tris buffer with 1 M NaCl and 0.005 M calcium chloride CaCl ₂ pH 7.4 (1000 ml). Extraction was carried out in the cold. The total volume of the extract was 1500 ml. The homogenate was centrifuged at 25,000 rpm, + 4 ° C for 30 minutes. The upper layer of lipids was thrown back.

Received 1200 ml of solution. The resulting solution was filtered through a membrane with a pore size of 0.2 μm.

The solution was triturated with a 0.1 M sodium hydroxide solution to a pH of 8.0. While stirring in 1200 ml of the supernatant solution, 12 ml of a solution of polyethyleneimine (PEI) was added to a final concentration of polyethyleneimine in a solution of 0.05 weight. %

The final concentration of polyethyleneimine was (12 * 5) / 1212≈0.05 (weight percent of polyethyleneimine in the extract).

A nucleic acid precipitate formed with PEI. The solution was stirred for another 30 minutes at room temperature to enlarge the precipitate.

The precipitate (nucleic acids) was isolated in a centrifuge at 12,000 rpm for 25 minutes. The precipitate of PEI and nucleic acids had practically no proteolytic activity. They threw him away.

Ammonium sulfate was added to the solution collected by the PEI treatment to 80% saturation. A precipitate was formed in the refrigerator during the day. The precipitate was isolated by centrifugation at 12,000 rpm, + 4 ° C for 20 minutes. Protein precipitate was dissolved in 0.05 M Tris buffer. The solution was desalted by diafiltration on hollow fibers with a pore size of 10 kDa.

Conducted by ion exchange chromatography on DEAE-cellulose. A column containing 80 ml of a suspension of DEAE-cellulose sorbent was pre-equilibrated with 0.01 M Tris buffer. A desalted protein solution was applied to the column. The sorbed proteins were eluted using a gradient of sodium chloride 0.05-1.0 M NaCl in 0.01 M Tris buffer, pH 8.0. The elution rate is 20-40 ml / hour. The protein content was monitored using a UV detector at a wavelength of 280 nm. Upon elution, three fractions of the protein having activity were collected. All active fractions were combined. The combined solution was desalted by diafiltration in a tangential ultrafiltration unit.

Chromatography of the resulting solution was carried out on a hydrophobic sorbent to separate residual pyrogens. 50 ml of the hydrophobic sorbent Spherocell C-80 containing hydrophobic ligands C8-C10 were placed in the column. The column was equilibrated with 0.01 M phosphate buffer supplemented with 0.05 M NaCl, pH 8.0. The solution was passed through a sorbent column at a rate of 20-40 ml / hour. The enzyme yield was monitored by a UV detector by the optical density of the solution. The collected solution was desalted by diafiltration in a tangential ultrafiltration unit and concentrated to a protein content of 2.5 mg / ml (according to Lowry). The pH of the resulting solution was adjusted to 7.4. Then spent sterilizing filtration through a 0.2 μm membrane.

Tris buffer up to 0.025 M mannitol monosaccharide was added to the resulting solution to a concentration of 4%. The solution was sterilized by filtration through a 0.2 μm pore membrane. The solution was poured into sterile vials at 2 ml / vial.

Vials were frozen to -40 ° C for 2-10 hours and lyophilized for 24 hours according to the standard program. After lyophilization, the bottles were hermetically sealed.

A purified complex of proteolytic enzymes with collagen activity of 1800-2600 μg of substrate / min × mg of enzyme, which can be used for medical purposes, was obtained. In 1 vial 5 mg of purified complex of proteolytic enzymes. A total of 130 bottles were received.

Polyacrylamide gel electrophoresis was performed on an isolated complex of proteolytic enzymes. After staining the gel plate with Coomassie blue dye, the bands corresponding to the masses of 25 and 36 kDa and less intense in the region of 45 kDa are most intensely colored.

Example 6

As the raw material used 500 g of the digestive organs of a stingray sea fox (Raja clavata). The organs were washed from the blood with purified water and disintegrated in a homogenizer (10 min), gradually adding 1 M NaCl and 0.005 M calcium chloride CaCl ₂ , pH 7.4 (1000 ml). Extraction was carried out in the cold. The total volume of the extract was 1500 ml. The homogenate was centrifuged at 25,000 rpm, + 4 ° C for 30 minutes. The upper layer of lipids was thrown back.

Received 1200 ml of solution. The resulting solution was filtered through a membrane with a pore size of 0.2 μm.

The solution was triturated with a 0.1 M sodium hydroxide solution to a pH of 8.0. While stirring in 1200 ml of the supernatant solution, 4 ml of a solution of polyethyleneimine (PEI) was added to a final concentration of polyethyleneimine in a solution of 0.017 weight. %

The final concentration of polyethyleneimine was (4 * 5) / 1204≈0.017 (weight percent of polyethyleneimine in the extract).

A nucleic acid precipitate formed with PEI. The solution was stirred for another 40 minutes at room temperature to enlarge the precipitate.

The precipitate (nucleic acids) was isolated in a centrifuge at 12,000 rpm for 25 minutes. The precipitate of PEI and nucleic acids had practically no proteolytic activity. They threw him away.

Ammonium sulfate was added to the solution collected by the PEI treatment to 80% saturation. A precipitate was formed in the refrigerator during the day. The precipitate was isolated by centrifugation at 12,000 rpm, + 4 ° C for 20 minutes. Protein precipitate was dissolved in 0.05 M Tris buffer. The solution was desalted by diafiltration on hollow fibers with a pore size of 10 kDa.

Conducted by ion exchange chromatography on DEAE-cellulose. A column containing 80 ml of a suspension of DEAE-cellulose sorbent was pre-equilibrated with 0.01 M Tris buffer. A desalted protein solution was applied to the column. The sorbed proteins were eluted using a gradient of sodium chloride 0.05-1.0 M NaCl in 0.01 M Tris buffer, pH 8.0. The elution rate is 20-40 ml / hour. The protein content was monitored using a UV detector at a wavelength of 280 nm. Upon elution, three fractions of the protein having activity were collected. All active fractions were combined. The combined solution was desalted by diafiltration in a tangential ultrafiltration unit.

Chromatography of the resulting solution was carried out on a hydrophobic sorbent to separate residual pyrogens. 50 ml of the hydrophobic sorbent Spherocell C-80 containing hydrophobic C ₈ -C ₁₀ ligands were placed in the column. The column was equilibrated with 0.01 M phosphate buffer supplemented with 0.05 M NaCl, pH 8.0. The solution was passed through a sorbent column at a rate of 20-40 ml / hour. The enzyme yield was monitored by a UV detector by the optical density of the solution. The collected solution was desalted by diafiltration in a tangential ultrafiltration unit and concentrated to a protein content of 2.5 mg / ml (according to Lowry). The pH of the resulting solution was adjusted to 8.0. Then spent sterilizing filtration through a 0.2 μm membrane.

Tris buffer up to 0.025 M mannitol monosaccharide was added to the resulting solution to a concentration of 4%. The solution was sterilized by filtration through a 0.2 μm pore membrane. The solution was poured into sterile vials at 2 ml / vial.

Vials were frozen to -40 ° C for 2-10 hours and lyophilized for 24 hours according to the standard program. After lyophilization, the bottles were hermetically sealed.

A purified complex of proteolytic enzymes with collagen activity of 1800-2600 μg of substrate / min × mg of enzyme, which can be used for medical purposes, was obtained. In 1 vial 5 mg of purified complex of proteolytic enzymes. A total of 120 bottles were received.

Polyacrylamide gel electrophoresis was performed on an isolated complex of proteolytic enzymes. After staining the gel plate with Coomassie blue dye, the bands corresponding to the masses of 25 and 36 kDa and less intense in the region of 45 kDa are most intensely colored.

COMPARISON OF THE RECEIVED COMPLEX OF PROTEOLYTIC ENZYMES WITH KNOWN ANALOGUES

Comparative specific activity was determined for the obtained complex of proteolytic enzymes in comparison with the commercial preparation of crab collagenase and the commercial serine protease chymotrypsin. High molecular weight photo gelatin was used as a substrate. The enzymatic hydrolysis reaction was carried out at + 37 ° C. To obtain comparable results, the test drugs were dissolved in the same protein concentration. Protein concentration was normalized by calibration curves for optical density at 280 nm. To determine the activity used the concentration of enzymes 0.25, 0.1, 0.05, 0.025 mg / ml. The activity of the enzyme depends on the ionic strength, the nature of the salts present and the pH of the medium. In deionized water, proteolytic enzymes are not very active. To create a different acid-base balance of the medium, 0.2 M phosphate-citric acid buffer was used.

The results are presented in the table. The specific activity of the isolated complex of proteolytic enzymes in the pH range 7.0–9.2 practically coincides with the specific activity of the complex of enzymes isolated from crab hepatopancreas. Chymotrypsin showed low activity in relation to the substrate.

The table shows the specific activity of proteolytic enzymes in μg of substrate * min / mg of enzyme.

It is known that for each enzyme there is an optimal pH range at which its enzymatic activity is maximum. To establish the pH optimum of action, universal buffers are usually used that are effective over a wide pH range. We used phosphate citric acid buffer. Unfortunately, it turned out that the anion of citric acid (citrate) reduces the activity of proteolytic enzymes. Nevertheless, firstly, it was possible to establish that the pH optimum for the complex of proteolytic enzymes lies in the pH range 7.0–9.0, and secondly, the complex of proteolytic enzymes isolated from the digestive organs of fish has a close specific activity and the pH optimum is characteristics of crab collagenase

Table

EXAMPLE OF APPLICATION OF THE RECEIVED COMPLEX OF PROTEOLYTIC ENZYMES

The therapeutic effect of the drug - the effect of cleansing wounds and scab rejection was investigated on a model of thermal burns in male rats. The experiments were performed on white outbred rats, males - 60 pieces. Animals were divided into groups using the randomization method using body weight as the main criterion. In a comparative study of the effect of the complex on the dynamics of rejection of the necrotic scab, 5 rats were used each. In the study of specific activity, the experimental and control groups consisted of 10 rats (burns). Groups of 5 rats were used in the study group of the locally irritating effect of the complex.

Animals from the control and experimental groups were anesthetized with diethyl ether and a thermal burn of a 2 cm ² metal plate heated to 200 ° C with an exposure of 10 seconds was applied to the depilated back skin in the sternum-lumbar region. Experimental therapy was carried out with a complex of proteolytic enzymes obtained according to examples 1-6 and a crab collagenase comparison drug when diluted with water.

On the model of thermal burns in rats, earlier clinically significant signs of collagenolytic properties of the studied complex were established - marginal, incomplete and complete rejection of the necrotic scab, comparable with those in the treatment of burn wounds of rat skin with crab collagenase. The positive effect of the complex of proteolytic enzymes was reflected in the active epithelization of the surface of the wound defect with less pronounced reactive changes in the stratified squamous epithelium at the edges of the wound compared to the control group, in which less pronounced regeneration processes were observed with a tendency to pseudoepithelial hyperplasia at the edges of the wound.

The complex of proteolytic enzymes obtained in accordance with the claimed method, in particular, according to examples 1-6, does not have a local irritant effect on the skin of rats with 21 days of application.

At the same time, it has a regenerating, analgesic and deodorizing effect.

Determination of the authenticity and purity of a complex of proteolytic enzymes by electrophoresis

To determine the authenticity (correspondence to molecular weight) and purity (absence of extraneous impurities), the method of vertical electrophoresis in polyacrylamide gel is used under native and reducing conditions, followed by visualization with a Coomassie staining solution. Used system for electrophoresis "Mini-PROTEAN 3". Electrophoresis was carried out under the following conditions: concentration of the separation gel 4–20%, concentration of the concentration gel 4%, current strength 25 mA, voltage during protein separation 240 V, duration 50 min, medium temperature +7 deg. Celsius. The test solution of the preparation in 0.5 M Tris-HCl buffer (((hydroxymethyl) aminomethane) pH 6.8 in the amount of 2-7 μl was added to the track wells, so that the load of the substance on the gel track was about 2.5 μg.

At the end of electrophoresis, the gel was fixed in an alcoholic solution of acetic acid, then stained with Coomassie colloidal dye for 40-60 minutes on a shaker, then the dye was drained and the gel was washed with water until the background disappeared. To evaluate the results of electrophoresis, a mixture of calibration proteins with molecular masses of 18 kDa, 25 kDa, 35 kDa, 45 kDa, 66 kDa, 116 kDa is introduced into one of the tracks.

In the drawing, lane 1 of the electrophoresis pattern contains calibration proteins. Lanes 2 and 3 contain characteristic protein bands of a complex of proteolytic enzymes under reducing conditions. The load on lane 2 was 3 μl of the solution; the load on lane 3 was 5 μl of the solution.

The complex of proteolytic enzymes gives specific colored bands in the region of 23-28 kDa, corresponding to elastases, in the region of 32-37 kDa, corresponding to trypsin-like enzymes, in the region of 40-50 kDa, corresponding to chymotrypsin-like enzymes. Depending on the execution procedure and the degree of autolysis, the colored bands in these regions can be either single or split into doublets and triplets of isoenzymes.

The sequence of bands obtained in electrophoresis characteristic of a complex of proteolytic enzymes is different from the sequence of bands in electrophoresis of crab collagenase and individual preparations of proteolytic enzymes, such as trypsin, chymotrypsin, etc. For the complex of proteolytic enzymes, there are no differences in the pattern of electrophoresis under non-reducing and reducing conditions.

Native enzymes and proteins are very often found in the form of dimers, tetramers and octamers. In order to separate them in the electrophoresis procedure to individual proteins, special conditions are created, usually by adding a reducing agent, mercaptoethanol, which breaks the disulfide bonds between the proteins.

Thus, the production of a complex of proteolytic enzymes containing alkaline, neutral and acidic proteinases with increased collagenolytic activity is achieved due to an increase in specific activity with respect to deep hydrolysis of collagen and gelatin, as well as the breakdown of hemoglobin, casein, and a decrease in pyrogenicity (the ability of a substance to increase body temperature) , while increasing the quantitative yield of the target products. In addition to the above substrates, the complex of proteolytic enzymes is able to cleave chitosan to a lesser extent.

The advantages of the proposed method are due to the inclusion in the process of obtaining a complex of proteolytic enzymes at the optimal stages of the separation of nucleic acids and lipid-protein complexes by treating the extract with polyethyleneimine, as well as the chromatographic purification operation on a hydrophobic sorbent, which allows timely removal of the lipopolysaccharides responsible for pyrogenicity from the secreted enzyme. The lack of pyrogenicity significantly improves the quality of the drug compared with analogues in medical use

The invention allows to obtain a proteolytic drug that can be used in medicine, in cosmetics and in biotechnology, in medicine when cleaning infected wounds from necrotic tissues, burn wounds, frostbite, to accelerate the rejection of scabs and necrotic ulcers, in the treatment of hernia of the intervertebral disc of the spine, treatment of keloid scars, for the treatment of postoperative adhesions in the abdominal organs; in cosmetics to clean problem skin; in biotechnology to obtain and isolate cell cultures of human and animal tissues.

Claims (6)

1. A method of isolating a complex of proteolytic enzymes from the digestive organs of fish, including sequentially homogenizing tissues in a buffer, separating the crushed tissues by centrifugation, filtering the supernatant through a membrane, salting out proteins in ammonium sulfate, isolating the ammonium sulfate precipitate by centrifugation, dissolving the precipitate in a buffer, desalting the diafiltrate extract purification by ion exchange chromatography on DEAE-cellulose, characterized in that prior to the deposition of proteins in ammonium sulfate nucleus new acids and lipid-protein complexes are removed from the extract by treatment with a solution of polyethyleneimine, which is added to a final concentration of polyethyleneimine in the extract from 0.1 to 0.01 wt.%, and after ion exchange chromatography, residual pyrogens are separated on a hydrophobic sorbent Spherocell C-80, sterilizing filtration through a membrane with a pore size of 0.2 μm and repeated desalination of the extract by diafiltration, followed by preparation of the finished form by lyophilization. 2. The method according to p. 1, characterized in that to obtain the finished dosage form after separation of the residual pyrogens, a cryoprotectant is introduced, for example, a physiologically acceptable buffer, mono- or disaccharide, in an amount of from 1 to 10 wt.%, And / or a medical biopolymer, in an amount of from 0.1 to 10 wt.%. 3. The method according to p. 2, characterized in that the homogenization is carried out with the slow addition of 0.05 M Tris buffer with the addition of 0.005 M calcium chloride CaCl ₂ pH 7.4. followed by extraction in the cold. 4. The method according to any one of paragraphs. 1-3, characterized in that the desalination of the extract by diafiltration is carried out by passing the solution through hollow fibers with a pore size of 10 kDa. 5. The method according to any one of paragraphs. 1-3, characterized in that the desalination of the extract by diafiltration is carried out on a tangential ultrafiltration module. 6. The method according to any one of paragraphs. 1-3, characterized in that the mono- or disaccharide is selected from the group: mannitol, lactose or trehalose, and the medical biopolymer from the group of dextrans, polyvinylpyrralidones, polyvinyl alcohols.

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