Development of a method for the sorption immobilization of bromelain on N-maleoylchitosan and the study of the structural features of the obtained complex
Abstract
Bromelain is a proteolytic enzyme that has found application in medicine and industry. However, the use of free forms of enzymes has its drawbacks, such as their instability to changing environmental conditions, labor intensity and complexity of separation from the initial reagents and reaction products after the completion of the process. This problem can be solved by immobilizing bromelain on polysaccharides.
In connection with the above, the aim of the study was to develop a technique for the sorption immobilization of bromelain on N-maleoylchitosan with different molecular weights and to study the structural features of the resulting complexes.
The synthesis of N-maleoylchitosan with different molecular weights and degrees of substitution was carried out. A technique for carrying out sorption immobilization of bromelain on N-maleoylchitosan has been proposed. The interaction between the carrier and the enzyme was studied by IR spectroscopy and flexible molecular docking, the functional groups of the polysaccharide and amino acids of the surface of bromelain globules involved in the formation of the complex were identified. The protein content in the immobilized preparations of bromelain was measured by the modified Lowry method; the protease activity of the samples was determined on the azocasein as substrate.
It is shown that the interaction of bromelain and N-maleoylchitosan is due to electrostatic and hydrophobic interactions, as well as the formation of hydrogen bonds between the components. Bonds and interactions with N-maleoylchitosan are formed, among other things, with the participation of amino acid residues that make up the active cite of the enzyme (Cys26 and His158), which affects the proteolytic activity of the samples obtained.
It has been established that 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) is achieved when N-maleoylchitosan with a molecular weight of 200 kDa is used as a carrier matrix. .
The resulting preparations of bromelain immobilized on N-maleoylchitosan can become the basis for solving a number of problems in biomedicine that arise when creating preparations of bactericidal, decongestant, antithrombic, and antiviral effects. Due to the combination of relatively inexpensive components, the method proposed by us is available for Russian laboratories and is promising for further application in industry.
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References
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