Development of the hybrid biocatalysts based on ficin and papain complexes with chitosan sulfate and the study of their structural features
Abstract
Ficin and papain are proteolytic enzymes of plant origin used in biomedicine and industry. Soluble forms of enzymes have several disadvantages, such as rapid inactivation, microbial degradation, autolysis, etc. Immobilization of enzymes on polysaccharides can exclude these problems.
In connection with the above, the purpose of the study was to develop a technique for the complexation of ficin and papain with sulfate chitosan with different molecular weights and to study their structural features.
Sulfate chitosan with different molecular weights (200, 350, and 600 kDa) was successfully obtained. A technique for the complexation of ficin and papain with sulfate chitosan has been proposed. The protein content in the immobilized formulations of ficin and papain was estimated by the Lowry method; the protease activity of the samples was evaluated on the substrate azocasein.
It was shown by in silico experiments that the interaction of ficin and papain with sulfate chitosan is due to electrostatic and hydrophobic interactions, as well as the formation of hydrogen bonds between the components. Bonds and interactions with sulfate chitosan are also formed with the participation of amino acid residues forming the active site of the enzymes (Cys25 and His162 for ficin, Cys25 and His159 for papain) and near-locating ones.
It was found that the optimal ratio of protein content (mg per g of carrier), total activity (in units per mL of solution) is achieved for ficin and papain complexes with sulfate chitosan with a molecular weight of 600 kDa.
The proposed approach has broad prospects for practical use in the field of proteinase immobilization due to the prediction of the structural features of the biocatalyst based on in silico simulations that do not require the use of expensive reagents.
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References
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