RU2437936C1 - Method of trypsin immobilisation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: trypsin is immobilised by mixing a polymer carrier presented by vinyliden fluoride and hexafluoropropylene copolymer, fluoroelast, vinyliden fluoride and perfluoromethylvinyl ester copolymer, and a cross-linking agent. The enzyme amount varies within 0.0025 to 0.25 g in 1 g of the carrier, and the cross-linking agent 1,4-bis(2-mercaptobenzthiazolyl methylene-piperazine is taken in the amount 0.01-0.04 g per 1 g of the carrier. The ingredients are mixed either in roll mills, or in a mixer at temperature 37°C. The prepared immobilised enzyme shows high proteolytic activity of a native preparation and dispersion activity necessary for producing high-grade cell cultures.

EFFECT: immobilisation increases a shelf life to 3 years, leads to enzyme stability and enables its reuse.

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Description

The invention relates to biotechnology, engineering enzymology and can be used in the production of biological products. Polymers are increasingly used in medicine, in the bio-industry, in the food industry, in particular in the immobilization of enzymes. Under the immobilization of enzymes refers to the attachment of their molecules to the surface of the carrier through physical or chemical interactions.

The use of enzyme preparations in technological processes and its use in medicine and veterinary medicine for therapeutic purposes is associated with irrational waste of the enzyme used for a specific purpose.

The processed material is contaminated with the enzyme preparation, and the problem of obtaining new forms of reusable preparation, that is, prolonged action, is very relevant. The method of immobilizing enzymes on a polymer carrier allowed us to develop a technology for producing an enzyme (using trypsin as an example) of a prolonged action, which reduces the volume of the drug’s use and facilitates storage conditions while maintaining high activity.

The invention relates to biotechnology, engineering enzymology and can be used in scientific research, in the biological industry when using polymer materials in the process of immobilization of enzymes. There are various methods of immobilization of the enzyme / 1, 2 /. Immobilization can be carried out: physical or chemical adsorption, covalent attachment to soluble and insoluble polymers, crosslinking of their protein molecules, incorporation into gels, into membranes. It is known that the enzyme is included in the structure of the polyacrylamide gel, and the enzyme is attached to polystyrene / 1, 2 /. Immobilized trypsin is obtained by binding the enzyme to polysilicic acid, to serum albumin using glutaraldehyde, and to immobilizing using titanium metal powder. The disadvantage of such methods is the low strength of the enzyme with the carrier. The enzyme activity of the enzyme depends on a number of factors — the conformation of their protein molecules, external and internal inhibition, steric hindrance, storage stability and temperature increase, changes in the pH of the medium and other factors.

A known method of obtaining an immobilized enzyme (peroxidase) on alginate fiber, which is unstable and has low activity / 3 /. The specified method for producing an immobilized enzyme involves treating a polymer carrier based on a modified polystyrene with an aqueous enzyme solution (horseradish peroxidase in the presence of sodium bicarbonate at 0-4 ° C with a process duration of 20 hours). The disadvantages of the method are the low specific activity of the immobilized enzyme (peroxidase), the duration and multi-stage process of immobilization. To increase the specific activity of the drug, a silica carrier is modified with 2-3% dextran / 3 /. The disadvantage of this technical solution is the fragility of the material, easily subjected to abrasive erosion, silica matrices are hydrolytically unstable at alkaline pH values, adsorption properties lead to undesirable effects of non-specific sorption of substrates.

The known method / 4 / immobilization by treating the enzyme (catalase) with immunoglobulin and polystyrene, which leads to the stability of the enzyme and the possibility of its use for repeated use. A method for covalently coupling enzymes to the surface of a carrier is to chemically modify the enzyme with an analogue of a monomer. The disadvantage is the use of toxic reagents (for example, acrylic acid chloride). When modified by intramolecular crosslinking of the enzyme with bifunctional reagents, additional purification of the enzyme, allergological and toxicological studies / 4 / are required.

There is a method for producing immobilized enzymes using a complexation reaction with polyiodides and molecular iodine. The activity of enzymes after immobilization is 70-87% of the control and lasts up to 1 month / 5 /.

A known method of immobilizing enzymes (alkaline phosphatase, peroxidase, papain, a-chymotrypsin), which consists in the fact that the organic carrier is a chemisorption fiber based on a copolymer of acrylonitrile with 2.5 vinylpyridine (VION AN-1 brand) is treated with aqueous solutions of various enzymes. The disadvantage of this method is the low activity of immobilized enzymes, which is 45-75%. The half-inactivation time is 14-16 days / 6 /.

A biocatalyst is known in which an enzyme (s) are included in a polymer gel matrix based on polyvinyl alcohol (PVA). In this case, the preparation is dispersed in an aqueous PVA solution, followed by freezing the resulting dispersion at -5 ... -40 ° C for 4-48 hours and further thawing. This technical solution is the closest to the declared and taken as a prototype. A biocatalyst with a trypsin-based filler crosslinked in the presence of a synthetic polymer of N-vinylpyrrolidone with vinylamine contains 2.5 wt.% Trypsin and 11 wt.% PVA. The enzymatic activity of the obtained biocatalyst during the hydrolysis of n-nitroanilide N ^a -benzoyl-DL-arginine (measured in 0.3 M Na-phosphate buffer, pH 8.0; 25 ° С) is 81% of the activity of the initial trypsin preparation per 1 mg protein. In a granular form, the biocatalyst consists of a filler based on particles of cross-linked trypsin with a content of 14 wt.% And PVA - 13.6 wt.%. The enzymatic activity of the obtained biocatalyst during the hydrolysis of n-nitroanilide N ^a -benzoyl-DL-arginine (measured in 0.3 M Na-phosphate buffer, pH 8.0; 25 ° С) is 75% of the activity of the initial trypsin preparation per 1 mg protein / 7 /. This technical solution is of interest, but technologically difficult and time-consuming to perform, requires the use of a cryogel, freezing the resulting dispersion. In granular form, the activity of the drug is 75% of the initial activity of the enzyme 111.

A method is proposed for immobilizing an enzyme (using trypsin as an example), which involves the use of both organic and inorganic carriers in the trypsin immobilization. Silicon dioxide (aerosil, white soot) was used from inorganic carriers, and fluorine-containing polymers were used from organic ones. The former did not give an effect, and the use of a polymeric carrier (fluorine-containing), inert to various media and resistant to temperature effects, contributes to the immobilization of the enzyme. The technical result of the proposed method is that they use a polymer carrier to obtain an immobilized enzyme with high activity. The method is as follows.

Example 1

In this work, we studied the preparation of prolonged-action enzyme preparations by immobilization of the trypsin proteolytic enzyme on a polymer carrier. Fluorine-containing polymers — a copolymer of vinylidene fluoride and hexafluoropropylene (SKF-26) —was used as a carrier; fluoroelast A1201 (SKF-26 V); copolymer of vinylidene fluoride and perfluoromethylvinyl ether (SKF-260 VRT).

The rubbers SKF-26 and SKF-260 VRT and A1201 were expanded at a temperature of 37 ° C, the trypsin incorporation into the polymer structure was carried out gradually when mixing on rollers (or mixers such as Banbury, Intermix). The amount of the enzyme ranged from 0.0025 to 0.25 g per 1 g of the polymer, without a crosslinking agent and with a crosslinking agent of the piperazine class in an amount of 0.04 g - compound 1.4 bis (2-mercaptobenziazolylmethylene) piperazine of the formula:

Immobilized samples were obtained in the form of plates with a thickness of about 1 mm. The starting polymers and immobilized trypsin samples on a support were compared by swelling in distilled water (Table 1).

At a concentration of immobilized trypsin of 0.0025-0.025 g in the polymer, the smallest swelling was observed for rubber SKF-26, at a concentration of 0.25 g, swelling increases in all polymers. The study of immobilized trypsin during swelling in an aqueous medium showed that the pH is practically at the control level before the introduction of trypsin into the polymer matrix. A crosslinking agent in the amount of 0.04 g per 1 g of the carrier stabilizes the composition with an enzyme content of 0.0025-0.025 g, which leads to the lowest conductivity of their solutions.

The conductivity of the solution with the introduction of 0.25 g of the enzyme after 96 hours of swelling in the composition based on SKF-26, A1201 is 100 and 80 9 / LS, respectively, and in the composition based on SKF-260 VRT - 22 mS.

Example 2

The claimed immobilized and control trypsin was evaluated by proteolytic and dispersing activity using a 0.25% trypsin solution, which was determined by dispersing quail (PE) and chicken embryos (CE) tissues for 30 minutes (Table 2). To prepare enzyme solutions, buffer solutions with a pH of 7.2-7.4 are used, which is necessary when culturing cells. Upon receipt of the primary cell culture, chicken embryos of 8-10 days old and quails of 8-9 days old are used. Under aseptic conditions, the crushed embryo tissue is placed in a flat-bottomed flask with a capacity of 1-1.5 L and filled with a warm (37 ° C) 0.18-0.25% trypsin solution at the rate of 800 ml of trypsin per 400-500 carcasses of embryos. The trypsinization time of quail embryos was 20 minutes (with a single trypsinization) or 2 times 10 minutes (with a double trypsinization) and 35 minutes with chicken embryos. The resulting suspension is filtered through a 2-layer gauze filter into a centrifuge bottle, in which cattle serum (cattle) is poured in advance based on the concentration in the suspension of 2-3%. The filtered cell suspension is centrifuged for 10-15 minutes at 1200 rpm. After centrifugation, trypsin is drained, and medium containing 10% cattle serum is added to the cell pellet. The cell suspension is collected in one vessel, thoroughly mixed under flares through a 3-4 layer filter. The cell suspension is introduced into a growth medium containing 10% serum of cattle and antibiotics (penicillin - 100 units / ml, streptomycin - 100 μg / ml) and nystatin (50 μg / ml), then packaged in a culture dish.

Immobilized trypsin on carriers in the form of samples in the calculation of obtaining a 0.25% enzyme solution was chemically sterilized (a solution of Pervomura - (mixture in a ratio of 1: 1-3% hydrogen peroxide solution and 1% formic acid solution)), followed by washing with sterile water or a buffer; or by treatment with an alcohol solution and a sterile buffer solution. Experimental sterile samples were used for working trypsin solutions in a buffer solution similar to the control native trypsin.

The results of cell cultivation after trypsinization with immobilized trypsin and control trypsin are shown in Table 2. The sowing dose was 600-700 thousand per 1 ml.

As can be seen from table 2, the proteolytic activity of immobilized samples was at the control level. Tests of the immobilized enzyme as a dispersing agent showed the possibility of using it for trypsinization of PE and CE tissues.

According to the results of the experiment, the output of cells from a unit of tissue was at the control level. Enzymatic dispersion with immobilized trypsin made it possible to obtain viable cells, which were used in monolayer cultivation of quail and chicken embryo cells. The shelf life of immobilized samples is 3 years (observation period) compared with the native form of the enzyme (2 years).

Example 3

Immobilized trypsin was used repeatedly after washing with sterile buffer solution and distilled water. After using immobilized enzyme five times, the yield of cells from 1 g of tissue and viability practically did not change. Subject to sterility, cell growth was ensured at the control level and higher. Studies have shown the possibility of obtaining an immobilized trypsin enzyme, which can be used repeatedly (table 3).

Table 3 The effect of immobilized trypsin on cell culture with repeated use N Media Name Characteristics PE CE Frequency of use Frequency of use one 2 3 four 5 one 2 3 four 5 one SKF-26 The output of cells with 1 g of tissue million 125 125 124 124 123 150 150 149 148 148 Cell viability,% 97 97 96 96 95 97 97 96 95 95 2 SKF-260 VRT The output of cells with 1 g of tissue million 145 144 144 143 142 160 159 158 158 157 Cell viability,% 96 95 95 94 93 97 96 95 95 94 3 A1201 The output of cells with 1 g of tissue million 125 125 124 124 123 150 150 149 148 148 Cell viability,% 97 97 96 96 95 97 97 96 95 95 four Without carrier The output of cells with 1 g of tissue million 85 - - - - 120 - - - - Cell viability,% 96 - - - - 97 - - - -

The proposed method of immobilization of the enzyme (using trypsin as an example) allows to obtain a preparation with high activity of the initial enzyme, which is 100% compared with the prototype (76-81%) and ensures the retention of the enzyme within the particles of the carrier. The immobilization of the enzyme increases the shelf life up to 3 years (observation period) compared with the original drug. The output of cells and their viability obtained using an immobilized enzyme (trypsin) is preserved with repeated use.

Information sources

1. Immobilized cells and enzymes. Methods / Ed. J. Woodward. - M.: Mir, 1988.

2. Immobilized enzymes. The current state and prospects / 2 t. Ed. I.V. Berezina, V.K. Antonova, K. Martenik. - M.: Moscow State University, 1976.- T. 1 - 296 s.- T. 2.

3. Patent RU 2005784, C12N 11/14, C12N 11/10.

4. Patent RU 2016901, C12 N 11/02, 9108.

5. Patent RU 2157405, C12 N 11/14.

6. Patent RU 2054481, C12 N 11/08.

7. Patent RU 2233327, C12 N 11/0804, 11/18.

Claims (1)

A method of immobilizing trypsin on a polymer carrier, comprising mixing the carrier, which is a copolymer of vinylidene fluoride and hexafluoropropylene (SKF-26); fluoroelast A1201 (SKF-26 V); a copolymer of vinylidene fluoride and perfluoromethylvinyl ether (SKF-260 VRT), trypsin in an amount of 0.0025-0.25 g and a crosslinking agent 1.4 bis (2-mercaptobenzthiazolylmethylene) piperazine 0.01-0.04 g per 1 g of carrier, while the mixture is carried out on rollers or in a mixer at a temperature of 37 ° C.