Covalent immobilization of alpha-amylase onto poly(2-hydroxyethyl methacrylate) and poly(styrene-2-hydroxyethyl methacrylate) microspheres and the effect of Ca2+ ions on the enzyme activity

alpha-Amylase (1,4-alpha-D-glucan-glucanohydrolase; EC 3.2.1.1, Type VI-B f rom porcine pancreas, extra pure 29 units mg(-1)) was covalently immobilize d on poly (2-hydroxyethyl methacrylate), p(HEMA), and poly (styrene-2-hydro xyethyl methacrylate), p(St-HEMA) microspheres, which were activated by usi ng epichlorohydrin (ECH). The properties of the immobilized enzyme were inv estigated and compared with those of the free enzyme. For the assays carrie d out at 25 degrees C and pH 6.9, the relative activities were found to be 61.7 and 67.0% for ECH-activated P(HEMA) and P(St-HEMA) bound enzymes, resp ectively. The maximum activities were obtained at lower pH values and highe r temperatures upon immobilization compared to free enzyme. Kinetic paramet ers were calculated as 2.51, 22.4 and 6.62 g dm(-3) for K-m and 1.67 x 10(- 3), 1.63 x 10(-3) and 1.35 x 10(-3) g dm(-3) min(-1) for V-max in the case of free, P(HEMA) and P(St-HEMA) bound enzymes, respectively. Enzyme activit y was found to be ca. 38.9% for ECH-activated P(HEMA) bound enzyme after st orage for 1 month. On the other hand, free enzyme lost its activity complet ely in 20 days. Immobilization, storage stability and repeated use capabili ty experiments that were carried out in the presence of Ca2+ ions demonstra ted higher stability. The enzymes immobilized in the presence of Ca2+ ions retained 90.7 and 80.0% of their original activities even after 30 days for ECH-activated P(HEMA) and P(St-HEMA) systems, respectively. In repeated ba tch experiments, 20 uses in 3 days, in the absence of Ca2+ ions, retention of 79% of the original enzyme activities was observed for ECH-activated P(H EMA) immobilized enzymes. On addition of Ca2+ ions to the assay medium, 90. 0 and 80.0% of retention was observed for ECH-activated P(HEMA) and P(St-HE MA) systems, respectively. (C) 1999 Published by Elsevier Science Ltd. All rights reserved.