Binary immobilization of Candida rugosa lipase on chitosan

Industrial application of lipase requires efficient methods to immobilize the enzyme, yielding a biocatalyst with high activity and stability compared to free lipase. In the present study, lipase was immobilized to chitosan beads utilizing its amino and the hydroxyl groups, which is called “binary immobilization”. Lipase was first immobilized to chitosan beads by activating its hydroxyl groups with carbodiimide followed by cross-linking more lipase to the amino groups with glutaraldehyde. Under optimum conditions, the binary method of immobilization yielded a higher protein loading of 287.2 μg/g-chitosan and an activity of 13.8 U/g-chitosan as compared to the immobilized lipase prepared by activation and by cross-linking. Broader pH tolerance and higher heat stability could be achieved by this method. Immobilized lipase retained 74% residual activity after ten hydrolysis cycles and 67% after 7 days of storage. Kinetic parameters Vmax and Km and the energy of activation (Ea) were determined for the immobilized lipase.