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

Immobilization glucoamylase onto plain and a six-carbon spacer arm (i.e., h examethylene diamine, HMDA) attached poly(2-hydroxyethylmethacrylate-ethyle n col dimethacrylate) [poly(HEMA-EGDMA] microspheres was studied. The micro spheres were prepared by suspension polymerization and the spacer arm was a ttached covalently by the reaction of carbonyl groups of poly(HEMA-EGDMA). Glucoamylase was then covalently immobilized either on the plain of microsp heres via CNBr activation or on the spacer arm-attached microspheres via CN Br activation and/or using carbodiimide (CDI) as a coupling agent. Incorpor ation of the spacer arm resulted an increase in the apparent activity of th e immobilized enzyme with respect to enzyme immobilized on the plain of the microspheres. The activity yield of the immobilized glucoamylase on the sp acer arm-attached poly(HEMA-EGDMA) microspheres was 63% for CDI coupling an d 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 coupl ing. The Km values for the immobilized glucoamylase preparations ton the sp acer arm-attached microspheres) via CDI coupling 0.9% dextrin (w/v) and CNB r 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 rat e 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 glucoamy lase. (C) 2001 John Wiley & Sons, Inc.