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

α-Amylase (1,4-α-d-glucan-glucanohydrolase; EC 3.2.1.1, Type VI-B from porcine pancreas, extra pure 29 units mg−1) was covalently immobilized on poly (2-hydroxyethyl methacrylate), p(HEMA), and poly (styrene- 2- hydroxyethyl methacrylate), p(St-HEMA) microspheres, which were activated by using epichlorohydrin (ECH). The properties of the immobilized enzyme were investigated and compared with those of the free enzyme. For the assays carried out at 25°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, respectively. The maximum activities were obtained at lower pH values and higher temperatures upon immobilization compared to free enzyme. Kinetic parameters were calculated as 2.51, 22.4 and 6.62 g dm−3 for Km and 1.67×10−3, 1.63×10−3 and 1.35×10−3 g dm−3 min−1 for Vmax in the case of free, P(HEMA) and P(St-HEMA) bound enzymes, respectively. Enzyme activity was found to be ca. 38.9% for ECH-activated P(HEMA) bound enzyme after storage for 1 month. On the other hand, free enzyme lost its activity completely in 20 days. Immobilization, storage stability and repeated use capability experiments that were carried out in the presence of Ca2+ ions demonstrated 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 batch 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(HEMA) 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-HEMA) systems, respectively.