Immobilization of Cholesterol Oxidase in Chitosan Magnetite Material for Biosensor Application

Cholesterol oxidase, a bio-catalyst that can catabolize cholesterol, has proven applications in medicine. Here, a support material was used to enhance the characteristics of the enzyme. Magnetite (Fe₃O₄) is widely used as an enzyme support; however, the interaction between the enzyme and the support should be capped with another material, such as chitosan biopolymer-based material. In this study, chitosan-magnetite materials were synthesized by mixing both compounds and activating with glutaraldehyde. The materials were then characterized by Fourier Transform Infrared (FTIR) Spectroscopy. The enzyme kinetic parameters were studied by following the cholesterol oxidation reaction using high-performance liquid chromatography (HPLC) and comparing the results between the free and the immobilized enzyme. The substrate concentration was 2.5 mg/mL. The effect of enzyme concentration was tested using different concentrations of enzyme (0.5, 1, and 2 mg/mL) to determine the best operating conditions. The best conditions for the oxidation reaction were immobilized enzyme at a 2 mg/mL concentration. Enzyme immobilization significantly decreased the optimum substrate concentration to 0.1 mg/mL.