Immobilization of glucoamylase on the plain and on the spacer arm-attached poly(HEMA-EGDMA) microspheres

Abstract

Immobilization glucoamylase onto plain and a six-carbon spacer arm (i.e., hexamethylene diamine, HMDA) attached poly(2-hydroxyethylmethacrylate-ethylen col dimethacrylate) [poly(HEMA-EGDMA] microspheres was studied. The microspheres were prepared by suspension polymerization and the spacer arm was attached covalently by the reaction of carbonyl groups of poly(HEMA-EGDMA). Glucoamylase was then covalently immobilized either on the plain of microspheres via CNBr activation or on the spacer arm-attached microspheres via CNBr activation and/or using carbodiimide (CDI) as a coupling agent. Incorporation of the spacer arm resulted an increase in the apparent activity of the immobilized enzyme with respect to enzyme immobilized on the plain of the microspheres. The activity yield of the immobilized glucoamylase on the spacer arm-attached poly(HEMA-EGDMA) microspheres was 63% for CDI coupling and 82% for CNBr coupling. This was 44% for the enzyme, which was immobilized on the plain of the unmodified poly(HEMA-EGDMA microspheres via CNBr coupling. The Km values for the immobilized glucoamylase preparations ton the spacer arm-attached microspheres) via CDI coupling 0.9% dextrin (w/v) and CNBr coupling 0.6% dextrin (w/v) were higher than that of the free enzyme 0.2% dextrin (w/v). The temperature profiles were broader for both immobilized preparations than that of the free enzyme. The operational inactivation rate constants (k(iop)) of immobilized enzymes were found to be 1.42 x 10(-5) min(-1) for CNBr coupled and 3.23 x 10(-5) min(-1) for CDI coupled glucoamylase. (C) 2001 John Wiley & Sons, Inc.