Immobilization of horseradish peroxidase on chitosan beads for phenol removal

Abstract

This research aims to search for the appropriate chitosan for horseradish peroxidase (HRP) immobilization for phenol removal. The supports used were chitosan membrane, spray dried chitosan, 2%, 3%, 5% (w/v) of dry chitosan beads and 3% (w/v) of wet chitosan beads. Scanning electron microscopy (SEM) showed that 3% (w/v) of both chitosan beads were appropriate to be used for the immobilization of enzymes by covalent linkage using glutaraldehyde. The immobilization parameters such as concentration of coupling agent and coupling time were optimized. It was determined that the optimum conditions for enzyme immobilization were to activate dry support with 1.5% (v/v) of glutaraldehyde for 8 hours and 1.0% (v/v) of glutaraldehyde for 10 hours for wet support. The activated support was then incubated with the enzyme solution for 2 hours at room temperature, using 2.5 units for both supports. After immobilization, the optimum pH and temperature of immobilized HRP was found to be the same as that of the free enzyme (5.5 and at 25oC). The pH stability of free and enzyme immobilized on both dry and wet chitosan beads was 5.5, whereas the thermal stability of immobilized HRP on dry beads was slightly higher than that of the immobilized HRP on wet beads. The performance of enzymes immobilized on both supports was compared. In the first set of the experiment, glutaraldehyde (1.5%, v/v, 8 hours) and HRP solution (10 µg) were allowed to react with the beads sequentially with and without agitation. In another set of experiments, glutaraldehyde and HRP solution were mixed prior to the addition to the supports with and without agitation. It was found that wet chitosan beads incubated with 1.5% (v/v) glutaraldehyde and HRP consecutively gave best immobilized activity at 2 hours incubation. Phenol removal by HRP immobilized on wet chitosan beads was highest at room temperature and at pH 10.0 Reusability of the immobilized enzyme for the removal of phenol after 5 repeated use still retained the ability to remove phenol up to 36%. The HRP immobilized on wet beads showed higher stability than the native enzyme when stored at both 25 and 4°C for 1 month.