Kinetic studies and analytical application of cholesterol oxidase and peroxidase immobilized to synthetic polymer

A method for individual and simultaneous covalent immobilization of cholesterol oxidase and peroxidase to copolymer of acrylonitrile with acrylamide is described. The effect of immobilization on the catalytic properties of the covalently bound enzymes was studied. The immobilized enzymes showed no change in pH optima and an increase in temperature optima, activation energy, and Km, compared to data received from experiments with soluble enzymes. A small glass column packed with immobilized multienzyme complex was used to develop a method for manual determination of cholesterol in foodstuffs (e.g., in mayonnaise “Olinease”). The method was characterized by high analytical precision (coefficient of variation = 2.67%). The results show high correlation with those obtained by the Kageyama method (r=0.986). The method is economical (the enzyme-carrier conjugate may be used more than 300 times), precise, easy to perform, and less time-consuming than the manual methods utilizing soluble enzymes. The established manual method can be proposed for cholesterol determination in foodstuffs.