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alpha-Amylase (1,4-alpha-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 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, 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 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 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. (C) 1999 Published by Elsevier Science Ltd. All rights reserved. |
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