RU2771183C1 - Method for producing ficin preparation in gel based on carboxymethylcellulose - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology. A method is proposed for obtaining a heterogeneous preparation in a gel based on ficin and carboxymethyl cellulose, including the immobilization of an enzyme preparation with an enzyme concentration of 3 mg/ml solution in 0.05 Μ borate buffer with 0.1 Μ KCl in the composition, pH 9.0 in a ratio of 20 ml of the enzyme solution per 1 g of sodium salt carboxymethyl cellulose with a molecular weight of ~ 90 kDa; incubation at room temperature with occasional stirring, and washing with 0.05 Μ Tris-HCl buffer pH 7.5 until there is no protein in the washings.EFFECT: invention provides an extension of the arsenal of carriers for obtaining immobilized ficin, which has greater activity and provides enzymatic reactions in solution and on a solid substrate.1 cl, 3 dwg, 1 ex

Description

The invention relates to biotechnology and can be used in the chemical and pharmaceutical industry, medical practice, cosmetology and research purposes. The invention can be used in the creation of medicinal preparations in the form of gels for wound healing and anti-burn purposes, as well as for preventing the formation of scars.

Ficin (EC 3.4.22.3) is a proteolytic enzyme of plant origin from the class of hydrolases, catalyzing the hydrolysis of peptides, amides and esters. Ficin is isolated from fruits, stems and leaves of tropical plants of the genus Ficus. Its molecular weight is 23 kDa, it is active in the pH range of 6.5-9.5, the temperature optimum of activity is in the range of 60-65°C, the enzyme is inactivated at 80°C.

СЕ. et al. Novel in-office peroxide-free tooth-whitening gels: bleaching effectiveness, enamel surface alterations, and cell viability // Scientific Reports. - 2020. V.10. - Р. 8].Ficin is used in many areas of the national economy, for example, in the production of active peptides from inexpensive proteins, to obtain antibodies by specific hydrolysis of peptide bonds, to improve the organoleptic or functional properties of food products, in the brewing, pharmaceutical and cheese-making industries. The enzyme has the ability to break down fibrinogen and therefore can act as an anticoagulant [Hamed Μ. B., El-Badry M. O., Kandil E. I., Borai I. H., Fahmy AS A contradictory action of procoagulant ficin by a fibrinolytic serine protease from Egyptian Ficus carica latex // Biotechnology Reports. - 2020. - V. 27 - P. 8]. In addition, it has properties such as anti-inflammatory, antithrombotic, anticancer, immunomodulatory [Ribeiro JS, Barboza ADS,

SE. et al. Novel in-office peroxide-free tooth-whitening gels: bleaching effectiveness, enamel surface alterations, and cell viability // Scientific Reports. - 2020. V.10. - R. 8].

Barbosa О., Zidoune M.N., Fernandez-Lafuente R. Immobilization/Stabilization of Ficin Extract on Glutaraldehyde-Activated Agarose Beads. Variables That Control the Final Stability and Activity in Protein Hydrolyses // Catalysts. - 2018. - V. 8. - P. 8].Enzymes in general, and cysteine proteases in particular, are highly efficient biocatalysts capable of operating under mild reaction conditions, with high selectivity and low environmental and physiological toxicity. Native enzymes are highly susceptible to changing environmental conditions, so it is advisable to immobilize them. Immobilization of the enzyme can facilitate its reuse and thus make the process more cost effective. Moreover, stability, resistance to chemicals or inhibitors is improved, and the purity of the enzyme is increased [Siar E.-H.,

Barbosa O., Zidoune MN, Fernandez-Lafuente R. Immobilization/Stabilization of Ficin Extract on Glutaraldehyde-Activated Agarose Beads. Variables That Control the Final Stability and Activity in Protein Hydrolyses // Catalysts. - 2018. - V. 8. - P. 8].

A known method for producing an immobilized enzyme preparation based on ficin, polysaccharides modified with vinyl monomers, and hyaluronic acid [Patent RU 2744457 C1, IPC A61K 38/00, A61K 38/46, A61K 47/36, C12N 11/08, A61P 17/02 , publ. 03/09/2021. Bull. No. 7]. The method includes dissolving ficin in an aqueous solution of low molecular weight hyaluronic acid 300 kDa, or medium molecular weight hyaluronic acid 500 kDa, or high molecular weight hyaluronic acid 800 kDa in the ratio of 10 mg of ficin per 2 ml of an aqueous solution of low molecular weight hyaluronic acid 300 kDa, or medium molecular weight hyaluronic acid 500 kDa, or high molecular weight hyaluronic acid 800 kDa at a concentration of 1.5%, while stirring until complete dissolution at room temperature; then ficin is immobilized by adding to the resulting mixture a carboxymethyl cellulose graft copolymer with a molecular weight of 50-100 kDa (CMC) or chitosan (ChTS) with a molecular weight of 50-100 kDa with N-vinylimidazole (VI) or Ν,N-dimethylaminoethyl methacrylate (DMAEMA) ) in an amount of 100 to 290 mg to obtain a liquid preparation or from 300 to 500 mg to obtain a gel. The disadvantages of the method include the impossibility of obtaining solid or powder preparations by this method.

There are a large number of carriers used to immobilize enzymes. Biopolymers are of great interest. Carboxymethylcellulose (CMC) is a polysaccharide that is a chemical modification of cellulose. CMC is non-toxic to humans, water soluble. The wide range of applications of CMC includes: the healing of chronic wounds (foot ulcers, diabetics) and the healing of acute wounds such as abrasions, first and second degree burns. In addition, CMC wound dressings are known for their flexibility, ability to absorb exudate, promote angiogenesis and autolytic wound debridement. It has provided the basis for many drugs and materials implanted or installed in the human body.

Erythropoietin preparations and a method of topical application for healing skin wounds are proposed [Patent RU 2465003 C2, IPC A61K 38/18, A61K 9/10, A61K 47/38, A61P 17/02, publ. 10/27/2012. Bull. No. 30]. The invention relates to the field of medicine and the pharmaceutical industry and is a gel-like or viscous composition suitable for local and local healing of wounds on injured skin, containing erythropoietin (EPO) and at least one gel-forming polysaccharide, amenable to swelling, at a concentration of 0.4-4% wt ./wt., selected from one or more than one member of the group consisting of: hydroxyethylcellulose, hydroxymethylcellulose, carboxyethylcellulose, carboxymethylcellulose, which can be obtained by mixing EPO in lyophilized or suspended form with a pre-swollen polysaccharide having a viscosity of less than 5000 mPa s , and initiating full swelling, where the fully swollen polysaccharide has a viscosity of 20,000-60,000 mPas, and EPO is present at a concentration of 100-500 IU/g of the gel composition, where said mixing of EPO is achieved by diffusion of EPO in the gel for at least 24 h The invention provides stabilization of the active o compounds and its uniform release, as well as effective treatment in case of trauma or pathologically induced skin damage.

A known method of obtaining a gel based on carboxymethylcellulose [Patent RU 2352584 C1, IPC SW 15/04, A61L 15/60, SW 15/00, SW 37/00, publ. 20.04.2009. Bull. No. 11]. The invention relates to a technology for producing gel solutions based on modified carboxymethyl cellulose (CMC) and can be used in the chemical industry, for example, in the production of drilling fluids or in medicine, for example, as a means of preventing intraoperative drying of the peritoneum and the formation of postoperative adhesions during operations on organs, having a serous coating. In the method for obtaining a gel based on carboxymethylcellulose, the initial product carboxymethylcellulose in sodium form (Na-CMC) is first heat-treated at 120–140°C for 5–30 min, and then dissolved in water at 18–25°C to form a more viscous 0.02– 4.5% high viscosity gel solution. EFFECT: simplification of the technological process for obtaining a gel based on modified carboxymethyl cellulose, reduction of its cost. In addition, obtaining solutions with a higher viscosity makes it possible to significantly expand the scope of their application, for example, in operations on the abdominal organs.

A method has been developed for producing an anti-adhesion film material based on carboxymethyl cellulose [Patent RU 2629842 C1, IPC A61L 17/10, A61L 31/04, A61L 31/14, A61P 41/00, C08J 5/18, publ. 09/04/2017. Bull. No. 25]. The invention relates to the field of medicine and chemical technology of macromolecular compounds, and in particular to a method for producing anti-adhesion film material, including the dissolution of a polymer, which is a mixture of carboxymethylcellulose and hydroxyethylcellulose in a ratio of 8:2 to 3:7, in water in the presence of a structuring agent - glutaric acid in an amount of 10-50% by weight of polymers, drying at 18-25°C and heat treatment in air at 98-105°C for 180-360 min. EFFECT: invention provides a longer stay of the film in the area of postoperative recovery and an increase in the anti-adhesion effect, as well as simplification and increase in the safety of the technological process and a decrease in the heat treatment temperature.

The prototype was a method for obtaining a heterogeneous enzyme preparation based on ficin, which has wound healing and regenerating properties [Patent RU 2677858 C2, IPC A61K 31/74, A61K 38/46, A61P 17/02, C12N 11/08, publ. 01/10/2019, Bull. No. 1], which includes the immobilization of ficin in a buffer solution on a matrix of ion-exchange fibers VION KN-1 in a ratio of 20 ml of a buffer solution of ficin at a concentration of 1 mg / ml per 1 g of fibers, while 0.05 Μ Tris-HCl buffer with pH 7.0; then carry out incubation for 24 hours at room temperature with occasional stirring; and then the formed precipitate is washed with 0.05 Μ Tris-HCl buffer with pH 7.0 until the absence of ficin in the washings.

The disadvantage of the prototype is to obtain a fibrous means that does not allow enzymatic reactions on solid substrates.

The claimed invention is intended to expand the number of carriers used to obtain immobilized ficin. The use of carboxymethylcellulose for this purpose will make it possible to obtain a biocatalyst with greater activity and simplify the sorption process. The method is simple to perform, does not require preliminary activation of the carrier, and the immobilized ficin retains high activity and stability. In addition, compared with the prototype, the time of incubation of the enzyme and the carrier (ie, immobilization time) is reduced from 24 to 2 hours.

The technical result of the claimed invention consists in the development of a method for immobilizing ficin on a carrier - sodium salt of carboxymethyl cellulose, by inclusion in a gel, which makes it possible to carry out enzymatic reactions at high rates in solutions and on solid substrates, while the enzyme becomes more stable and has a prolonged action, and immobilization time is no more than 2 hours.

The technical result is achieved by the fact that in a method for obtaining a heterogeneous preparation in a gel based on ficin and carboxymethyl cellulose, including the immobilization of the enzyme preparation in a buffer solution in a ratio of 20 ml of the enzyme solution per 1 g of the carrier, incubation at room temperature with occasional stirring, and washing with 0.05 Μ Tris- HCl buffer until there is no protein in the washings; as a buffer solution for immobilization, 0.05 Μ borate buffer with 0.1 Μ KCl in the composition with pH 9.0 is used; incubation is carried out for 2 hours; the formed precipitate was washed with 0.05 M Tris-HCl buffer pH 7.5.

Fig. 1. Diagram of protein content values (in mg per 1 g of carrier) in preparations of ficin included in the gel of sodium carboxymethyl cellulose using the following buffers: 1 - 0.05 Μ acetate, 2 - 0.05 Μ phosphate, 3 - 0.05 Μ borate buffer with 0.1 Μ KCl in the composition, 4 - 0.05 Μ tris-glycine, 5 - 0.05 Μ glycine.

Fig. Fig. 2. Diagram of total activity values (in units per 1 ml of solution) of ficin included in the gel of sodium carboxymethylcellulose salt using the following buffers: 1 - 0.05 Μ acetate, 2 - 0.05 Μ phosphate, composition, 4 - 0.05 Μ tris-glycine, 5 - 0.05 Μ glycine.

Fig. Fig. 3. Diagram of specific activity values (in units per 1 mg of protein in the sample) of ficin included in the gel of sodium carboxymethylcellulose salt using the following buffers: 1 - 0.05 Μ acetate, 2 - 0.05 Μ phosphate, 3 - 0.05 Μ borate buffer with 0.1 Μ KCl in the composition, 4 - 0.05 Μ tris-glycine, 5 - 0.05 Μ glycine.

An example of the implementation of the method

Ficin from Sigma-Aldrich was chosen as the object of study, and azocasein from Sigma-Aldrich served as the substrate for hydrolysis. Carboxymethylcellulose sodium salt with a molecular weight of ~90 kDa from Sigma-Aldrich (catalog number 419273) was used as a carrier for immobilization.

Ficin was immobilized on a carboxymethyl cellulose matrix by incorporation into a gel. To 1 g of sodium salt of carboxymethylcellulose was added 20 ml of enzyme solution at a concentration of 3 mg/ml, incubated for 2 hours at room temperature with occasional stirring. After the end of the incubation, the formed precipitate (in the form of a gel) was washed with 0.05 Μ Tris-HCl buffer (pH 7.5) until there was no protein in the washings (control was carried out on an SF-2000 spectrophotometer at λ=280 nm).

The protein content in immobilized ficin preparations was determined by the Lowry method [Lowry O.H., 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 protease activity of ficin was carried out on the substrate azocasein (Sigma, USA) [Sabirova A.R., Rudakova N.L., Balaban N.P., Ilyinskaya O.N., Demidyuk I.V., Kostrov S.V., Rudenskaya G.N., Sharipova M.R. A novel secreted metzincin metalloproteinase from Bacillus intermedius // FEBS Lett. - 2010 - V. 584 (21), P. 4419-425]. To 50 mg of the immobilized sample, 200 µL of 0.05 µ Tris-HCl buffer, pH 7.5, 800 µL of azocasein (0.5% in 0.05 µ Tris-HCl buffer, pH 7.5) were added and incubated for 2 hours at 37°C. Next, 800 μl of trichloroacetic acid (TCA) (5%) was added, incubated for 10 minutes at 4°C, then centrifuged for 3 minutes at 11700 g to remove non-hydrolyzed azocasein. To 1200 μl of the supernatant, 240 μl of 3% NaOH was added to neutralize the acid, after which the optical density of the test sample was measured at 410 nm in a 10 mm cuvette. The control sample contained 800 μl of azocasein, 800 μl of TCA, 50 mg of the sample, and 200 μl of buffer (the immobilized enzyme was added last to the control sample, the remaining operations were performed similarly to the experimental samples).

The unit of protease activity was the amount of ficin, which hydrolyzes 1 μM of azocasein in 1 min under experimental conditions. Specific protease activity was calculated by the formula:

A \u003d D * l 000/120/200 / C,

where A is the protease activity of the drug, µM/min per 1 mg of protein,

D is the optical density of the solution at 410 nm,

C - protein concentration in the sample, mg / ml, measured by the Lowry method,

120 - incubation time in minutes,

200 - sample volume, µl,

1000 - coefficient for conversion to µM.

All experimental studies were carried out in at least 8 replicates. Statistical processing of the obtained results was carried out at a significance level of 5% using Student's t-test.

To obtain heterogeneous biocatalysts based on ficin immobilized by incorporation into a gel based on the sodium salt of carboxymethyl cellulose, we used the following buffers as an immobilization medium: 0.05 Μ borate buffer with 0.1 Μ KCl in the composition with pH 8.0–10.0, 0.05 Μ acetate buffer with pH 4.0 -5.8, 0.05 Μ glycine with pH 8.6-10.5, 0.05 Μ tris-glycine with pH 8.5-9.0, 0.05 Μ phosphate with pH 5.8-7.5. The results are shown in FIG. 1-3.

Analysis of the protein content in heterogeneous preparations showed that the largest amount of ficin (in mg per g of carrier), immobilized by incorporation into a gel based on sodium carboxymethylcellulose, is observed when using 0.05 M acetate buffer with pH 4.0, 4.5, 5.8, and 0.05 M borate buffer with 0.1 Μ KCl in composition with pH 8.0 (Fig. 1).

The total activity (in units per ml of solution) of ficin immobilized by inclusion in a gel based on the sodium salt of carboxymethyl cellulose was higher when using 0.05 M borate buffer with 0.1 M KCl in the composition with pH 9.0 and 10.0 (Fig. 2).

The highest specific activity was shown by ficin preparations immobilized by incorporation into a gel based on the sodium salt of carboxymethyl cellulose using 0.05 M phosphate buffer with pH 5.8 (Fig. 3).

The method of immobilization by incorporating ficin into a gel has its own advantages. Thanks to immobilization, the enzyme is protected from adverse environmental conditions, its stability increases, the biocatalyst can be given various configurations, and its targeted delivery to the body is possible.

We compared the results obtained by determining the catalytic activity and protein content for ficin preparations included in the gel of sodium carboxymethyl cellulose. 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 obtained by immobilizing ficin by incorporating sodium carboxymethyl cellulose into the gel using 0.05 Μ borate buffer with 0.1 Μ KCl in composition with pH 9.0.

Thus, a procedure was developed for obtaining a heterogeneous biocatalyst based on ficin immobilized by incorporating sodium carboxymethyl cellulose into the gel.

Claims (1)

A method for obtaining a heterogeneous preparation in a gel based on ficin and carboxymethyl cellulose, including immobilization of the enzyme preparation in a buffer solution at a ratio of 20 ml of the enzyme solution per 1 g of the carrier, incubation at room temperature with occasional stirring, and washing with 0.05 Μ Tris-HCl buffer until it is absent from the wash water protein, characterized in that sodium salt of carboxymethyl cellulose with a molecular weight of ~ 90 kDa is used as a carrier, the concentration of the enzyme solution is 3 mg/ml; as a buffer solution for immobilization, 0.05 Μ borate buffer with 0.1 Μ KCl in the composition with pH 9.0 is used; incubation is carried out for 2 hours; the formed precipitate was washed with 0.05 M Tris-HCl buffer pH 7.5.

Images