Preparation of chitosan beads for trypsin immobilization

Abstract

The present study deals with the potential application of chitosan macrobeads for immobilization of trypsin. Macrobeads were prepared by precipitation and their mechanical properties were improved by covalent crosslinking using glutaraldehyde. Scanning electron microscopy studies showed that the beads size varied between 2.5 mm and 3.5 mm depending on the chitosan content and molar excess of glutaraldehyde. Trypsin (EC.3.4.21.4) was chosen as model enzyme for immobilization on the chitosan gel beads pre-activated with glutaraldehyde. Catalytic properties and kinetic parameters of free and immobilized trypsin were determined and the impact of various technological parameters on enzyme activity was investigated. The obtained results showed that the optimal pH and temperature for the hydrolytic reaction catalyzed by immobilized trypsin for low molecular weight substrates were pH 8.0 and 50oC, respectively. The effect of substrate concentration was also studied and the values of the constants thus determined were: Km=1,54 mM/L and Vmax – 3.3 μM/min.