RU2712528C1 - Method for producing collagenase preparation in gel based on food chitosan and chitosan succinate - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and solves the problem of increasing the rate of enzymatic reaction and increasing the effectiveness of using the preparation based on collagenase and chitosan, including when reacting on solid surfaces. Presented task is solved by a method for preparing a collagenase preparation in a gel based on food chitosan or chitosan succinate, involving immobilization of collagenase in a buffer solution on a chitosan matrix, incubating at room temperature with periodic mixing, washing the formed precipitate with buffer, characterized by that immobilization is carried out on a chitosan food matrix with a molecular weight less than 100 kDa or chitosan succinate, buffer used is 0.05 M acetate buffer with pH 5.8 or 0.05 M glycine buffer with pH 9.0, incubation is carried out for 2 hours.

EFFECT: method is proposed for preparing collagenase preparation in gel based on food chitosan and chitosan succinate.

1 cl, 3 dwg, 1 ex

Description

The invention relates to biotechnology and can be used in the pharmaceutical industry, medical practice and veterinary medicine.

Numerous methods are known for immobilizing various enzymes, including collagenase. Collagenases (EC 3.4.22.32) belong to a small group of matrix metalloproteases, are highly specific enzymes that can cause hydrolytic cleavage of collagen molecules. Unlike mammalian collagenases, which split the collagen helix in one area, bacterial collagenases break the bonds of several nodes along the helix. The bacterium Clostridium histolyticum synthesizes seven types of collagenases that have been studied to date. These enzymes are among the most effective for the breakdown of collagen. Due to the high catalytic ability of collagenase, Clostridium histolyticum is actively used to isolate specific types of cells from concomitant connective tissue.

Collagenases are used as a softener in the food industry, as it is collagen that is responsible for the strength of red meat products. Collagenases can be used in cosmetology to ensure a uniform change in skin color when acquiring a tan. However, the most common area of application of these enzymes is medicine. Collagenases are used to treat burns and ulcers, to eliminate scar tissue and play an important role in transplantation [Patent RU 2265052, IPC C12N 9/48, C12N 9/64, publ. 11/27/2005; Daboor, S. Budge, A. Ghaly, S. Brooks, D. Dave Extraction and Purification of Collagenase Enzymes: A Critical Review // American Journal of Biochemistry and Biotechnology. - 2010 .-- V. 6 (4). - P. 239-263; Susagawara R., Harper E. Publication and characterization of three forms of collagenase from Clostridium histolyticum // Biochemistry. - 1984. - V. 23. - P. 5175-5182].

Known enzyme preparation of collase (Collasum), which is a mixture of two isoenzymes of serine collagenolytic protease A and C, with necrolytic activity, as well as fibrinolytic and thrombolytic properties. However, a mixture of these two proteases is not able to completely hydrolyze many polypeptide substrates, for example, collagen fibers are only partially hydrolyzed, and it is problematic to clean wounds containing damaged collagen fibers [Patent RU 2365623, IPC C12N 9/48, A61K 38/48 A61P 17/02, publ. 08/27/2009].

Native enzymes are rapidly inactivated when introduced into the wound, therefore it is advisable to use immobilized enzymes. Numerous methods are known for immobilizing various enzymes, including collagenase. The main disadvantage of these methods is the use of predominantly synthetic polymers, which cannot be used in the food and medical industries.

Known examples of use in the form of carriers for immobilization of chitosan. Chitosan is an aminopolysaccharide of animal origin derived from chitin of arthropod shells. Chitosan has valuable properties, thanks to which it is a promising carrier. It is characterized by non-toxicity, non-antigenicity, has a high sorption ability, is compatible with most drugs, exhibits antibacterial and antifungal effects, is biocompatible with human tissues, affects the regeneration of damaged skin, is biodegradable in a natural metabolic way. Chitosan is widely used in biotechnology, pharmacology and is the main component of many medicines and cosmetic preparations. Biocompatibility with living tissues, closeness in functional qualities to the components of the dermis in vivo, bioinertness, biodegradability, biocidal and other properties of chitosan determine the prospects of developing new materials for medical and biological purposes based on it [Gamzazade A.I. Derivatives of chitin / chitosan of a controlled structure as potential new biomaterials // Dis. n - Moscow. - 2005 .-- 363 s; Scriabin K.G. Chitin and Chitosan: Preparation, Properties, and Use / Ed. K.G. Scriabin, G.A. Vikhoreva, V.P. Varlamova. - M .: Nauka, 2002 .-- 368 s; Patent RU 2424812, IPC A61K 31/722, A61K 38/47, A61P 17/02, publ. 07/27/2011].

Collagenase and chitosan were used in one preparation to create a foot balm [Patent RU 2112500, IPC A61K 7, publ. 06/10/1998], where they were used as a mixture of components. It was noted that the tolerance of the balm is satisfactory, irritating and allergenic effects were absent. Under the influence of the balm in patients, perspiration of the legs, hyperkeratosis and callosity of the feet decreased, an unpleasant odor (deodorizing effect) disappeared. He well shot the phenomena of inflammation and peeling, contributed to the healing of cracks, scuffs and microerosion.

The technical result of the claimed invention is to increase the speed of the enzymatic reaction and increase the efficiency of use of the drug based on collagenase and chitosan, including when carrying out the reaction on solid surfaces.

In our work, we used as carriers for immobilization of food chitosan with a molecular mass of less than 100 kDa and its derivative, chitosan succinate [Neklyudov AD, Ivankin AN Ecological basis of biotechnology. - M: GOU VPO MGUL, 2006, S. 164-180], and collagenase was immobilized by the method of incorporation into the gel, so that the enzymes become more stable and have a prolonged effect.

The technical result is achieved in that in a method for producing a collagenase preparation in a gel based on food chitosan or chitosan succinate, comprising immobilizing collagenase in a buffer solution to a food chitosan matrix with a molecular weight of less than 100 kDa or chitosan succinate in a ratio of 20 ml of an enzyme solution in a concentration of 1 mg / ml per 1 g of carrier; 0.05 M acetate buffer with a pH of 5.8 or 0.05 M glycine buffer with a pH of 9.0 is used as a buffer solution for immobilization; incubation at room temperature with periodic stirring for 2 hours, washing the precipitate with 50 mM Tris-HCl buffer (pH 7.5) until there is no protein in the washings.

The use of chitosan with the indicated molecular weight combined with the use of 0.05 M acetate buffer made it possible to obtain a gel-like preparation. A gel-like preparation, unlike a powder one, allows carrying out enzymatic reactions not only in solutions, but also on solid substrates, which in turn facilitates the application of the drug to the wound or burn area, and increases the efficiency.

In FIG. 1 is a diagram of protein content values (in mg per 1 g of carrier) in immobilized collagenase preparations; FIG. 2 is a diagram of the values of total activity (in units per 1 ml of solution), in FIG. 3 is a diagram of specific activity values (in units per 1 mg of protein in a sample), where 1 is collagenase immobilized on chitosan succinate using an acetate buffer; 2 - collagenase immobilized on food chitosan using acetate buffer; 3 - collagenase immobilized on chitosan succinate using glycine buffer; 4 - collagenase immobilized on food chitosan using glycine buffer.

An example implementation of the method.

Collagenase from Clostridium histolyticum from Sigma-Aldrich company was chosen as an object; the azocasein from Sigma-Aldrich company was used as a substrate for hydrolysis. Two types of chitosan (Bioprogress CJSC) were used as carriers for immobilization: food chitosan with a molecular mass of less than 100 kDa and chitosan succinate.

Collagenase immobilization on a chitosan matrix was carried out by incorporation into a gel. To 1 g of chitosan was added 20 ml of an enzyme solution (at a concentration of 1 mg / ml), incubated for 2 hours. After incubation, the precipitate formed (in the form of a gel) was washed with 50 mM Tris-HCl buffer (pH 7.5) until there was no protein in the washings (control was performed on an SF-2000 spectrophotometer at λ = 280 nm).

The protein content in immobilized collagenase preparations was determined by the Lowry method [Lowry O.N., Rosebrough N.J., Faar A.L., Randall R.J. Protein measurement with folin-phenol reagent // J. Biol. Chem. - 1951. - V. 193. - P. 265-275]. Determination of the proteolytic activity of the enzyme was carried out on a substrate azocasein (Fluka). 200 μl of Tris-HCl buffer (pH 7.5), 800 μl of azocasein (0.5% in 50 mM Tris-HCl buffer, pH 7.5) were added to 50 mg of the sample and incubated for 2 hours at 37 ° C. Next, 800 μl of TCA (5%) was added, incubated for 10 minutes at -4 ° C, then centrifuged for 3 min at 13000 rpm to remove non-hydrolyzed azocasein. To 1200 μl of the supernatant was added 240 μl of 3% NaOH to neutralize the acid, after which the optical density of the experimental sample was measured at 410 nm in 1 cm cuvette. The control sample contained 800 μl of azocasein, 800 μl of TCA, 50 mg of sample, and 200 μl of Tris-HCl buffer. The amount of enzyme was taken as a unit of catalytic activity, which under experimental conditions hydrolyzes 1 μM of the substrate in 1 min. The specific proteolytic activity of collagenase was calculated by the formula:

PA = D * 1000/120/200 / Cp,

where PA is the proteolytic activity, μM / min per 1 mg of protein, D is the optical density of the sample at 410 nm, Cp is the protein concentration in the sample, mg / ml, measured by the Lowry method, 120 is the incubation time in minutes, 200 is the sample volume , 1000 - recalculation in microns.

Statistical processing of the results was carried out at a significance level of 5% using t-student criterion.

At the first stage of the experiments, to obtain heterogeneous biocatalysts based on collagenase immobilized by incorporation of chitosan and food chitosan into a gel based on succinate, we used 0.05 M acetate buffer solutions with pH ranging from 4.0 to 5.8 and 0.05 M glycine buffer solutions as an immobilization medium with a pH in the range of 8.6 to 10.5. The results for the most suitable buffer systems are shown in FIG. 1-3.

Analysis of the protein content in heterogeneous preparations showed that the largest amount of collagenase (in mg per g of carrier) immobilized by incorporation into food gel based on food chitosan was observed using a glycine buffer with a pH of 8.6. When immobilized on chitosan succinate, the amount of collagenase was higher when using glycine buffer with pH 8.6, 9.5, 10.0, 10.5 and acetate buffer with pH 4.0, 5.5 (Fig. 1).

The total activity (in units per ml of solution) of collagenase immobilized by incorporation into food gel based on food chitosan turned out to be higher when using glycine buffer with pH 8.6-10.0 and acetate buffer with pH 5.8. When immobilized on a chitosan succinate matrix, the total collagenase activity was higher when using glycine buffer with a pH of 9.0, 10.5, and acetate buffer with a pH range of 5.5-5.8 (Fig. 2).

The highest specific activity was shown by collagenase preparations immobilized by incorporation into a gel based on food chitosan and chitosan succinate using 0.05 M glycine buffer with pH 9.0 and 0.05 M acetate buffer with pH 5.8 (Fig. 3).

The method of immobilization by incorporating collagenase into the gel has several advantages over other methods of binding the enzyme to the carrier matrix. Thanks to this method of immobilization, the enzyme is not actually attached to the carrier, therefore, there are no steric obstacles, the drug is protected from many adverse environmental factors, including from inactivation due to bacterial infection, it is also possible to create immobilized preparations of any geometric configuration. We compared the results obtained for determining the catalytic activity and protein content for collagenase preparations immobilized on chitosan succinate and food chitosan matrices. The optimal ratio of protein content (mg per g of carrier), total activity (in units per ml of solution) and specific activity (in units per mg of protein) was revealed when collagenase was immobilized by incorporation into a gel based on food chitosan or chitosan succinate when used as an immobilization medium 0.05 M glycine buffer with a pH of 9.0 or 0.05 M acetate buffer with a pH of 5.8. When using drugs in medicine or analytical practice, a compromise is needed between high activity and catalyst stability.

From the above material, it follows that among the immobilization options we tested for creating heterogeneous preparations based on collagenase and chitosan, the most promising is the inclusion of the enzyme in a gel based on chitosan succinate or food chitosan when using 0.05 M glycine buffer with pH 9.0 or 0.05 M acetate buffer with pH 5.8.

Thus, a technique was developed for producing a heterogeneous biocatalyst based on collagenase immobilized on a matrix of food chitosan or chitosan succinate by incorporation into a gel.

Claims (1)

A method of producing a collagenase preparation in a gel based on food chitosan or chitosan succinate, comprising immobilizing collagenase in a buffer solution to a food chitosan matrix with a molecular weight of less than 100 kDa or chitosan succinate in a ratio of 20 ml of an enzyme solution in a concentration of 1 mg / ml per 1 g of carrier; 0.05 M acetate buffer with a pH of 5.8 or 0.05 M glycine buffer with a pH of 9.0 is used as a buffer solution for immobilization; incubation at room temperature with periodic stirring for 2 hours, washing the precipitate with 50 mM Tris-HCl buffer (pH 7.5) until there is no protein in the washings.

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