Effect of deacetylation degree of chitosan as solid support in lipase immobilization by glutaraldehyde crosslink

Abstract

Background: Interaction between -NH2 group of chitosan to -CHO groups of glutaraldehyde is critical in lipase immobilization using chitosan as a matrix and glutaraldehyde as a linker. Since the Deacetylation Degree (DD%) of chitosan represent the number of -NH2 group, it is important to understand the effect of the DD% value of chitosan on the effectiveness of immobilization. Chitosan beads and powder have different accessible -NH2 group, therefore, the influence of the form of chitosan is also interesting to be investigated. Materials and Methods: Three chitosans with different DD prepared with various repetition of deacetylation. This chitosan was set in beads and powder form then cross-linked to lipase (porcine’s pancreas) using glutaraldehyde as across-linking agent. The amount of the immobilized lipase as well as the hydrolytic activity of lipase was observed to determine the effectiveness of the immobilization. The thermal stability and reusability of this immobilized lipase were also investigated. Results: The optimum DD% value of powder chitosan using in lipase immobilization is at 90% DD with the optimum condition by using 1.5% of glutaraldehyde at pH 6.0 with efficiency up to 89%. The optimum DD% of beads chitosan is at 81% DD with the optimum condition by using 0.5% of glutaraldehyde at pH 6 with efficiency up to 53%. The immobilization at optimum condition of powder chitosan capable in retaining 32% of the activity, while the beads chitosan could keep 60% of the activity based on a comparison of the specific activity data. The result is corresponding to the kinetic data, where in beads the KM value is 100 mM higher than that of the free enzyme with 86 mM in KM value. Immobilization of lipase improves the stability of the enzyme with no significant reduction of activity up to five times of reuse and thermal stability up to 50°C. Conclusion: The DD% value of chitosan plays an essential role in lipase immobilization using crosslink method with glutaraldehyde as alinker with medium DD% give the best result. Immobilization of lipase improves the stability of the enzyme leading to reusability and thermal of the enzyme.