Study of bromelain and carboxymethyl cellulose surface interaction features in the process of their adsorption immobilisation
Abstract
Adsorption immobilisation is a common approach to stabilise the catalytic activity of enzymes. It involves the attachment of their macromolecules to the surface of a carrier due to weak physical interactions and hydrogen bonds. According to modern concepts, this process is the ‘gentlest’ immobilisation technique in terms of the structure of the enzyme globule. Despite this fact, the activity of the immobilised preparation is often lower compared to the native enzyme. To understand the reasons, it is necessary to study the specific features of interactions within the enzyme-carrier system and to identify the types of interactions occurring between its components. When studying the structure of enzymes, it is critical to determine the nature and qualitative composition of amino acid residues on the surface of the enzyme macromolecule that interact with the carrier. If catalytically important residues are involved in the process, there may be a significant decrease in the enzyme activity. In this regard, the aim of the work was to study the features of interaction between bromelain, which is a cysteine protease, and carboxymethyl cellulose by desorption of the enzyme from the formed complex under different conditions (in the presence of ammonium sulphate or surfactant Triton X-100, including at different temperatures), as well as by flexible molecular docking. The studied substances are promising components for the production of biocatalysts for food or biomedical applications, so the study of their interaction features will expand the applications of bromelain. We determined that incubation of bromelain immobilised on carboxymethyl cellulose in solutions of ammonium sulphate with a concentration of 32 mM or higher and Triton X-100 with a concentration of 100 mM or higher resulted in the destruction of the complex and the desorption of the enzyme. It confirms that non-covalent interactions contribute to the formation of immobilised preparation. When we increased the incubation temperature of the complex above 60 °C, we also observed the release of the enzyme from the preparation, indicating the formation of hydrogen bonds between the enzyme and the carrier. In silico study confirmed the formation of these types of bonds and interactions. It was determined that the amino acid residues forming the active site of bromelain also formed bonds with the carrier.
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