Properties of a thermostable β-glucosidase immobilized using tris(hydroxymethyl)phosphine as a highly effective coupling agent

Abstract

A very stable b-glucosidase (EC 3.2.1.21) was immobilized to polyacrylamide-magnetite beads, aminopropyl silica, and chitosan using tris(hydroxymethyl)phosphine (THP) or glutaraldehyde as the coupling reagent. The use of THP on chitosan resulted in greater than 90% yields with respect to free enzyme activity compared with only 60% observed when using the more conventional glutaraldehyde coupling reagent. THP-immobilized enzyme also lost activity more slowly than both glutaraldehyde-immobilized and the free enzyme when incubated at 90°C. Repetitive assays of THP and glutaraldehyde-immobilized enzyme also showed that THP was more able to retain active enzyme on the silica-based support. The pH optimum and Km app were unchanged with respect to free enzyme.