RU2795425C1 - Method for obtaining hybrid preparation of papain and carboxymethylcellulose in the form of a dense solution - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method for producing a hybrid preparation of papain and carboxymethylcellulose in the form of a thick solution is characterized by the following: it includes immobilization of papain carried out by complexation of papain and sodium salt of carboxymethylcellulose, which is used as a carrier for complexation, which is carried out by adding 10 ml of a solution of papain at a concentration of 20 mg/ml, obtained by dissolving in a buffer containing cysteine at a concentration of 0.04 M to prevent the processes of oxidation of the active site of papain, to 1 g of sodium salt of carboxymethyl cellulose, previously dissolved in 10 ml of buffer, is incubated for 2 hours at room temperature with constant stirring at a speed of 250 rpm, as a buffer solution for immobilization, 0.05 M borate buffer is used with the addition of 0.1 M KCl with pH 8.0 or 0.05 M phosphate buffer with pH 5.8–7.5, formed in the process after incubation, the preparation in the form of a thick solution is washed by dialysis using a cellophane bag 34 mm wide, with a capacity of 3.7 ml per 10 mm of length, with a pore diameter of 25 kDa, on the assumption that 400 ml of 50 mM Tris-HCl buffer with pH 7.5 are used to wash 21 ml of the resulting preparation, dialysis is carried out for 8 hours, after which the buffer is changed to a portion of the buffer in a volume of 400 ml and dialysis is continued for another 16 hours until there is no free papain in the washing solution.

EFFECT: invention allows to develop a method for producing a hybrid medicinal product based on immobilized papain and carboxymethylcellulose in the form of a thick solution with an absolute viscosity of 200–220 MPa×s, including only immobilized (stabilized) papain.

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Description

The invention relates to biotechnology and can be used in the food and pharmaceutical industries, medical practice, cosmetology and research purposes. The invention can be used to create drugs in the form of a thick solution for wound healing and anti-burn purposes, recommended to prevent the formation of scars and scars.

et al. Immobilization of papain: A review // International Journal of Biological Macromolecules. - 2021. V. 188. - P. 94-113]. Папаин используется в различных отраслях промышленности, таких как пивоваренная, кожевенная, рыбная и мясная, в процессах производства лекарств и биологически активных пептидов. В пищевой промышленности папаин используют в составе тонизирующих напитков, соков, сиропов, желе; в пивоваренном производстве и виноделии фермент применяется для осветления растворов и увеличения сроков хранения продуктов. Известно его применение для лечения аллергии, заживления язв, ожогов и травм, снижения токсичности или побочного действия лекарств, а также для удаления некротических и гнойных тканей из ложа раны [Н.A. Shouket, I. Ameen, О. Tursunov et al. Study on industrial applications of papain: A succinct review // IOP Conf. Series: Earth and Environmental Science. - 2020. V. 614. - Р. 7].Papain (EC 3.4.22.2) is a proteolytic enzyme isolated from papaya (Carcia Papaya). Its structure is a single polypeptide chain folded into two domains of approximately the same size, but with conformational differences. This chain consists of 212 amino acids with a molecular weight of 23 kDa, includes 7 cysteine residues, one of which is part of the active site of the enzyme along with histidine [Holyavka M., Pankova S., Koroleva V. et al. Influence of uv radiation on molecular structure and catalytic activity of free and immobilized bromelain, ficin and papain // Journal of Photochemistry dnd Photobiology B: Biology. - 2019. V. 201. - P. 111681]. Papain works more efficiently in environments with pH values close to 6.0-7.0, but it manages to maintain its activity in a wide range of pH and temperature values, which increases its scope [VG Tacias-Pascacioa, R. Morellon-Sterling,

et al. Immobilization of papain: A review // International Journal of Biological Macromolecules. - 2021. V. 188. - P. 94-113]. Papain is used in various industries such as brewing, leather, fish and meat, in the production of drugs and biologically active peptides. In the food industry, papain is used as part of tonic drinks, juices, syrups, jelly; in brewing and winemaking, the enzyme is used to clarify solutions and increase the shelf life of products. Its use is known for the treatment of allergies, healing of ulcers, burns and injuries, reducing toxicity or side effects of drugs, as well as for removing necrotic and purulent tissues from the wound bed [H.A. Shouket, I. Ameen, O. Tursunov et al. Study on industrial applications of papain: A succinct review // IOP Conf. Series: Earth and Environmental Science. - 2020. V. 614. - R. 7].

R. Morellon-Sterling et al. Bioactive peptides from Hsheries residues: A review of use of papain in proteolysis reactions // International Journal of Biological Macromolecules. - 2021. V. 184. - P. 415-428].At present, a direction with the use of enzymes as industrial biocatalysts is developing at a high pace. Enzymes are almost ideal catalysts due to their high activity under “mild” conditions, selectivity (reducing the likelihood of by-product formation) and specificity (allowing to avoid modification of molecules similar in structure to the substrate) [Kholyavka M.G., Artyukhov V.G. Immobilized biological systems: biophysical aspects and practical application // textbook, Voronezh: Publishing house of VSU. - 2017. - S. 261]. However, enzymes are relatively unstable, prone to inhibition by various compounds. These problems can be overcome by various methods of their immobilization and complexation [VG Tacias-Pascacio, D.

R. Morellon-Sterling et al. Bioactive peptides from Hsheries residues: A review of the use of papain in proteolysis reactions // International Journal of Biological Macromolecules. - 2021. V. 184. - P. 415-428].

Biopolymers are promising carriers for the immobilization of enzymes and the formation of stable complexes with them. Carboxymethylcellulose (CMC) is an anionic, water-soluble derivative of cellulose. Due to the easy and cheap synthesis process, the abundance of raw materials, characteristic surface properties, mechanical strength, adjustable hydrophilicity, viscosity, CMC has found wide application in the biomedical, pharmaceutical, textile, construction, food, cosmetics, paper and oil industries. For example, in biomedicine, CMC is widely used in wound dressings, in the manufacture of 3D scaffolds for biocompatible implants, artificial organs, as well as in antimicrobial and antioxidant drugs. Due to their biocompatibility, biodegradability, binding ability with respect to biologically active compounds such as proteins, peptides and enzymes, hydrogels, films and other hybrid materials based on CMC have aroused high interest in pharmaceuticals, especially for drug delivery, emulsification and stabilization [M.S. Rahman, M.S. Hasan, A.S. Nitai et al. Recent Developments of Carboxymethyl Cellulose // Polymers. - 2021. V. 13. - P. 48]. In the food industry, CMC acts as a suspending agent, thickener, protective colloid, moisturizer and can be used to regulate the crystallization processes of some other components [Patent RU 2334762 C2, IPC S08B 11/12, S08B 15/00, S08B 1/08, S08B 1 /10, publ. 27.09.2008, Bull. No. 27].

There is a way to obtain a heterogeneous drug based on papain with regenerative properties [Patent RU 2677873 C2, IPC A61K 38/48, A61K 47/30, A61R 17/02, publ. 01/22/2019, Bull. No. 3], which includes processing the matrix of ion-exchange fibers VION AN-1 or VION KN-1 with a solution of papain, incubation, characterized in that 0.2 M acetate buffer (pH 4.5-5.5) is used for immobilization on VION KN-1 ) or 0.05 M borate buffer with the addition of KCl (pH 9.0-9.5), and for immobilization on VION AN-1 - 0.05 M tris-glycine (pH 9.0) or 0.05 M glycine (pH 10.0) buffer at the rate of 20 ml of an enzyme solution at a concentration of 5 mg/ml per 1 g of fibers, incubation is carried out for 24 h at room temperature, the precipitate formed is washed with the buffer used during immobilization until there is no protein in the washing water.

A known method of obtaining a heterogeneous biocatalyst based on papain immobilized on ion exchange resins [Patent RU 2768742 C1, IPC C12N 11/04, C12N 11/10, publ. 03/24/2022, Bull. No. 9], including adsorption immobilization of papain in a buffer solution onto an ion-exchange resin matrix in a ratio of 20 ml of papain solution at a concentration of 5 mg / ml per 1 g of carrier, incubation at room temperature with occasional stirring, washing of the precipitate formed with buffer until it is absent in the washings protein waters, characterized in that immobilization is carried out on a matrix of air-dry ion-exchange resin AV-16-GS, and 0.05 M phosphate buffer, pH 11.0 is used as a buffer solution for immobilization, incubation is carried out for 2 hours, washing formed precipitate spend 0.05 M Tris-HCl buffer, pH 7.5.

In both methods, papain immobilization is carried out by adsorption on insoluble carriers in a ratio of 20 ml of papain solution at a concentration of 5 mg/ml per 1 g of carrier with periodic stirring, which does not allow fully automating the process. In addition, an insoluble form of the enzyme is obtained, which, of course, has its advantages, but does not allow reactions to be carried out on solid substrates.

A known method of obtaining an immobilized enzyme preparation based on papain, hyaluronic acid and polysaccharides modified with vinyl monomers [Patent RU 2750378 C1, IPC A61K 8/66, A61K 31/702, A61K 31/728, A61K 31/717, A61K 31/722, A61P 31/00, A61P 17/00, C12N 11/04 publ. 06/28/2021, Bull. No. 19], including the dissolution of papain in an aqueous solution of low molecular weight hyaluronic acid (300 kDa) or medium molecular weight hyaluronic acid (500 kDa) or high molecular weight hyalu of junic acid (800 kDa) at a ratio of 10 mg of papain 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 papain is immobilized by adding to the resulting mixture a graft copolymer of carboxymethylcellulose (CMC) or chitosan (CTZ) with N-vinylimidazole (VI) or N,N-dimethylaminoethyl methacrylate (DMAEMA) at a polysaccharide molecular weight of 50-100 kDa in an amount of from 100 to 290 mg for a liquid preparation or 300 to 500 mg for a gel.

The disadvantage of this method is the high cost of hyaluronic acid and polysaccharides modified with vinyl monomers. The use of CMC will reduce costs.

A known method of stabilizing proteases for use in cosmetic purposes [US 2011/0177052]. The disadvantage of this method is the use of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, which causes severe skin burns and eye damage, can lead to allergic skin reactions, if inhaled can cause allergies, asthma symptoms or difficulty breathing, which limits the use of the drug in cosmetology.

As a prototype served as a method of obtaining a heterogeneous preparation of papain in a gel based on food chitosan or chitosan succinate [Patent RU 2712690 C1, IPC C12N 11/04, C12N 11/10, publ. 01/30/2020, Bull. No. 4], including the immobilization of papain in a buffer solution on a chitosan matrix in a ratio of 20 ml of an enzyme solution at a concentration of 1 mg/ml per 1 g of carrier; incubation at room temperature with occasional stirring; washing the precipitate formed with 50 mM Tris-HCl buffer (pH 7.5) until there is no protein in the washings, characterized in that the immobilization is carried out on a matrix of food chitosan with a molecular weight of less than 100 kDa or chitosan succinate; as a buffer solution for immobilization use 0.05 M glycine buffer pH 10.0 or 0.05 M acetate buffer pH 5.8; incubation is carried out for 2 hours.

Unlike the prototype, our method allows you to get immobilized papain in a different form, i.e. not in the form of a gel, but in the form of a thick solution, including only immobilized (stabilized) papain and completely washed out of its unstabilized (non-immobilized) form. The claimed invention is intended to expand the number of polysaccharides used to stabilize papain preparations. In addition, carboxymethyl cellulose is a water-soluble carrier, which makes it possible to work with it in a wide range of pH values.

The technical result of the claimed invention consists in the development of a method for obtaining a hybrid preparation based on immobilized papain and carboxymethyl cellulose in the form of a thick solution with an absolute viscosity of 200-220 MPa × s, including only immobilized (stabilized) papain and completely washed out from its unstabilized (non-immobilized) form, with In this case, during immobilization, the active site of papain is protected from oxidation by the addition of cysteine at a concentration of 0.04M.

The technical result is achieved by the fact that in the method for producing a hybrid preparation of papain and carboxymethylcellulose in the form of a thick solution, including immobilization of the papain enzyme preparation in a buffer solution with a carrier, incubation at room temperature for 2 hours and washing, according to the invention, papain immobilization is carried out by complexation in a thick solution of sodium carboxymethyl cellulose in a ratio of 10 ml of a solution of papain at a concentration of 20 mg/ml, obtained by dissolving in buffer, per 1 g of sodium carboxymethyl cellulose, previously dissolved in 10 ml of buffer, with constant stirring at a speed of 250 rpm; as a buffer solution for immobilization, 0.05 M borate buffer with the addition of 0.1 M KCl buffer with a pH of 8.0 or 0.05 M phosphate buffer with a pH of 5.8-7.5 containing cysteine at a concentration of 0.04 M is used , the preparation formed during incubation in the form of a thick solution is washed by dialysis using a cellophane bag 34 mm wide, with a capacity of 3.7 ml per 10 mm of length with a pore diameter of 25 kDa against 50 mM Tris-HCl buffer with pH 7.5, from calculation that 400 ml of 50 mM Tris-HCl buffer with pH 7.5 is used to wash 21 ml of the resulting preparation, dialysis is carried out for 8 hours, after which the buffer is changed to a portion of fresh in a volume of 400 ml and dialysis is continued for another 16 hours to the absence of free papain in the washing solution.

Fig. 1. Diagram of protein content values (in mg per 1 g of carrier) in hybrid preparations in the form of a thick solution of papain and carboxymethylcellulose using the following buffers: 1 - 0.05 M acetate, 2 - 0.05 M phosphate, 3 - 0.05 M borate with the addition of 0.1 M KCl, 4 - 0.05 M tris-glycine, 5 - 0.05 M glycine.

Fig. 2. Diagram of the values of the total activity (in units per 1 ml of solution) of hybrid preparations in the form of a thick solution of papain and carboxymethylcellulose using the following buffers: 1 - 0.05 M acetate, 2 - 0.05 M phosphate, 3 - 0.05 M borate with the addition of 0.1 M KCl, 4 - 0.05 M tris-glycine, 5 - 0.05 M glycine.

Fig. Fig. 3. Diagram of specific activity values (in units per 1 mg of protein in the sample) of hybrid preparations in the form of a thick solution using the following buffers: 1 - 0.05 M acetate, 2 - 0.05 M phosphate, 3 - 0.05 M borate with the addition of 0.1 M KCl, 4 - 0.05 M tris-glycine, 5 - 0.05 M glycine.

An example of the implementation of the method.

Papain from Sigma-Aldrich was chosen as the object of study, and azocasein from Sigma-Aldrich served as the substrate for hydrolysis. Sodium carboxymethyl cellulose (Sigma-Aldrich) with a molecular weight of 90 kDa and a degree of substitution of 0.7 was used as a carrier for complexation.

The complexation of papain and carboxymethylcellulose was carried out as follows. To 1 g of sodium salt of carboxymethyl cellulose, previously dissolved in 10 ml of buffer, was added 10 ml of an enzyme solution at a concentration of 20 mg/ml, obtained by dissolving in a buffer containing cysteine at a concentration of 0.04 M to prevent oxidation of the active center of papain; incubated for 2 hours at room temperature with constant stirring at a speed of 250 rpm; as a buffer solution for immobilization, 0.05 M borate buffer with the addition of 0.1 M KCl buffer with a pH of 8.0 or 0.05 M phosphate buffer with a pH of 5.8-7.5 was used; the preparation formed during the incubation in the form of a thick solution was washed by dialysis using a Spectra/Por 6 Standard RC cellophane bag, 34 mm wide, with a capacity of 3.7 ml per 10 mm length, made from regenerated cellulose, with a pore diameter of 25 kDa versus 400 ml of 50 mm Tris-HCl buffer pH 7.5 for 8 hours, after which the buffer was changed to 400 ml of fresh and continue dialysis for another 16 hours until the absence of free papain in the washing solution. For washing 21 ml of the resulting preparation, 800 ml of 50 mM Tris-HCl buffer pH 7.5 was used. After this time, the presence of protein in the wash water was monitored using the SF-2000 spectrophotometer at λ=280 nm.

The protein content in papain hybrid 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].

Papain protease activity was determined 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 itermeius // FEBS Lett. - 2010 - V. 584 (21), P. 4419-4425]. To 50 mg of the sample, 200 µl of 0.05 M Tris-HCl buffer, pH 7.5, 800 µl of azocasein (0.5% wt. 0.05 M Tris-HCl buffer, pH 7.5) were added and incubated for 2 hours at 37°C. Next, 800 μl of trichloroacetic acid (TCA) (5% wt.) 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 was added 240 μl of NaOH solution (3% wt.) 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 complexed enzyme was added to the control sample last, other operations were performed similarly to the experimental samples).

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

A=D*1000/120/200/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 hybrid preparations of papain and carboxymethylcellulose, we used the following buffers as a medium for complex formation: 0.05 M borate with the addition of 0.1 M KCl with pH 8.0-10.0, 0.05 M acetate with pH 4.0 -5.8, 0.05 M glycine pH 8.6-10.5, 0.05 M tris-glycine pH 8.5-9.0, 0.05 M phosphate pH 5.8-7, 5. The results are shown in FIG. 1-3.

Analysis of the protein content in hybrid preparations showed that the highest amount of papain (in mg per g of carrier) is observed when using 0.05 M acetate buffer pH 4.0 and 0.05 M borate buffer with the addition of 0.1 M KCl pH 10 ,0 (Fig. 1.)

The total activity (in units per ml solution) of papain was higher when using 0.05 M borate buffer with the addition of 0.1 M KCl pH 8.0 (Fig. 2).

Papain preparations obtained using 0.05 M phosphate buffer pH 5.8-7.5 and 0.05 M borate buffer with the addition of 0.1 M KCl pH 8.0 showed the highest specific activity (Fig. 3).

We compared the obtained results on the determination of catalytic activity and protein content for papain preparations in a thick solution of 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 stabilizing papain by incorporating carboxymethylcellulose into a thick solution using 0.05 M borate buffer with the addition of 0.1 M KCl pH 8.0 or 0.05 M phosphate buffer pH 5.8-7.5.

Thus, a method was developed for obtaining a hybrid preparation of papain and carboxymethylcellulose in the form of a thick solution with an absolute viscosity of 200-220 MPa s, including only immobilized (stabilized) papain and completely washed out from its non-stabilized (non-immobilized) form, because after dialysis of 21 ml of the preparation against 800 ml of 50 mM Tris-HCl buffer with pH 7.5, the transition of the enzyme from the carrier phase to the solution is excluded, which makes it possible to carry out reactions, obtaining a product not "contaminated" by the enzyme. In addition, during immobilization, the active site of papain is protected from oxidation by the addition of cysteine at a concentration of 0.04 M.

Claims (1)

A method for producing a hybrid preparation of papain and carboxymethylcellulose in the form of a thick solution, characterized in that it includes the immobilization of papain, which is carried out by complexation of papain and sodium salt of carboxymethylcellulose, which is used as a carrier for complexation, which is carried out by adding 10 ml of a solution of papain at a concentration of 20 mg /ml, obtained by dissolving in a buffer containing cysteine at a concentration of 0.04 M to prevent the processes of oxidation of the active center of papain, to 1 g of sodium carboxymethylcellulose, previously dissolved in 10 ml of buffer, incubated for 2 hours at room temperature with constant stirring with speed of 250 rpm, as a buffer solution for immobilization, 0.05 M borate buffer is used with the addition of 0.1 M KCl with pH 8.0 or 0.05 M phosphate buffer with pH 5.8-7.5, formed in during the incubation process, the preparation in the form of a thick solution is washed by dialysis using a cellophane bag 34 mm wide, with a capacity of 3.7 ml per 10 mm of length, with a pore diameter of 25 kDa, on the basis that 400 ml of 50 mM are used to wash 21 ml of the resulting preparation Tris-HCl buffer with pH 7.5, dialysis is carried out for 8 hours, after which the buffer is changed to a portion of the buffer in a volume of 400 ml and dialysis is continued for another 16 hours until there is no free papain in the washing solution.

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