Covalent Immobilization of Thiol Proteinases on Chitosan †

: Plant enzymes such as ficin (EC 3.4.22.3), papain (EC 3.4.22.2) and bromelain (EC 3.4.22.4) are obtained from tropical plants. These biocatalysts belong to thiol proteases, in the active center of which cysteine is contained. Ficin, papain and bromelain have a wide substrate specificity, which provides a demand for their use in various industries. Enzymes in the free state are less commonly used; immobilized biocatalysts are the preferred form. The aim of this work was to determine the optimal concentration of a crosslinking agent in the covalent immobilization of ficin, papain and bromelain on a chitosan matrix. Ficin, papain and bromelain (Sigma) were chosen as objects of study. An acid-soluble chitosan (350 kDa, Bioprogress CJSC) was used as an immobilization carrier. The concentration range of glutaraldehyde (crosslinking agent) ranged from 1 to 25%. Suitable concentrations of glutaraldehyde for covalent immobilization were identified by 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). It was shown that for covalent immobilization of ficin and bromelain on a chitosan matrix, it is most promising to use 10% glutaraldehyde. For immobilization of papain on chitosan by covalent means, the concentration of glutaraldehyde equal to 20% is optimal.

The use of these enzymes in medicine is widely known; however, in addition, they can be used in the leather industry to soften leather products, when purifying wastewater and when removing rust from metals [3,4].
It is known that as a result of immobilization, enzymes acquire the advantages of heterogeneous catalysts: they can be removed from the reaction mixture by simple filtration, thus it becomes possible to transfer many periodic enzymatic processes to a continuous mode using columns or flow-through apparatus with immobilized enzymes. Immobilized enzymes, on the whole, turned out to be much more resistant to external influences than native (soluble) enzymes. They are more durable and thousands to tens of thousands of times more stable than free enzymes [5].
Chitosan is an aminopolysaccharide composed of glucosamine and N-acetylglucosamine polymers. Chitosan has the following properties important for practical use: biocompatibility, film formation, bioadhesiveness, polyfunctionality and hydrophilicity, most of which are associated with its cationic nature, which is unique among polysaccharides and natural polymers [6].
The aim of this work was to determine the optimal concentration of a crosslinking agent in the covalent immobilization of ficin, papain and bromelain on a chitosan matrix.

Methods
Ficin, papain and bromelain (Sigma-Aldrich, Darmstadt, Germany) were chosen as objects of study. Azocasein (Sigma-Aldrich, Darmstadt, Germany) was used as a substrate for hydrolysis. High-molecular weight chitosan (350 kDa, Bioprogress CJSC) was used as a carrier for immobilization.
To 900 mg of chitosan, 18 mL of enzyme (ficin, papain, bromelain) solution and 10 mL of glutaraldehyde (1, 2.5, 5, 10, 15, 20 and 25%) were added, incubated with periodic stirring for 1 h. The suspension was centrifuged at 1.500× g for 10 min. After the end of the incubation, the formed precipitate was washed with 50 mM Tris-HCl buffer (pH 7.5) until there was no protein in the washings (control was carried out on a spectrophotometer at λ = 280 nm).
Protein content in immobilized enzymes was determined by the Lowry method [7].

Results and Discussion
The largest amount of protein in immobilized samples (in mg per g of carrier) was observed during covalent immobilization of ficin and papain on a chitosan matrix using glutaraldehyde with a 25% concentration while binding bromelain-at a concentration of 5%, 10% and 25% (Figure 1). High values of the total activity (in units per ml of solution) of ficin were observed during its immobilization on chitosan using glutaraldehyde with a 10% concentration. When creating immobilized enzymes based on papain and chitosan, the highest activity was detected applying 20% glutaraldehyde. High activity of bromelain immobilized on a chitosan matrix was detected when using glutaraldehyde with 5%, 10% and 20% concentrations (Figure 2). The highest specific activity was shown by ficin and bromelain, immobilized by covalent binding on a chitosan matrix, using glutaraldehyde with a 10% concentration. When developing biocatalysts based on papain and chitosan, the highest specific activity was observed when 20% glutaraldehyde was applied (Figure 3).

Conclusions
It was shown that for covalent immobilization of ficin and bromelain on a chitosan matrix, it is most promising to use 10% glutaraldehyde. For immobilization of papain on chitosan by covalent means, the concentration of glutaraldehyde equal to 20% is optimal.
Author Contributions: Authors have equal contributions in the preparation and writing of the article.
Funding: This work was financially supported in the form of a grant from the President of the Russian Federation for state support to young Russian scientists-doctors of sciences (MD-1982.2020.4. Agreement 075-15-2020-325).