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

alpha-Amylase was covalently immobilized onto poly(methyl methacry late-2-h ydroxyethyl methacrylate) microspheres, which were activated by using eithe r epichlorohydrin (ECH) or cyanuric chloride (C3N3Cl3). The properties of t he 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 relativ e activities were found to be 73.0 % and 90.8 % for epichlorohydrin and cya nuric chloride bound enzymes, respectively. Upon immobilization, the maximu m activities were obtained at lower pH values and higher temperatures as co mpared with the free enzyme. Kinetic parameters were calculated as 2.51 g/L , 28.54 g/L and 15.50 g/L for K-m and 1.67 x 10(-3) gL(-1) min(-1), 2.89 x 10(-4)gL(-1) min(-1) and 1.89 x 10(-3) gL(-1) min(-1) for V-max for free, e pichlorohydrin and cyanuric chloride bound enzymes, respectively. Enzyme ac tivities were found be ca. 32.7 % for ECH and 41.1 % for C3N3Cl3 activated matrices after storage for one month. On the other hand the free enzyme los t its activity completely within 20 days. Immobilization, storage stability and repeated use capability experiments carried out in the presence of Ca2 + ions demonstrated higher stability in the presence of these ions. The enz ymes immobilized in the presence of Ca2+ ions retained 90.6 % and 90.8 % of the original activities even after 30 days in the case of ECH and C3N3Cl3 activations, respectively. In repeated batch experiments, i.e., 20 uses of the enzyme in 3 days; in th e absence of Ca2+ ions retentions of 79.2 % and 77.1 % of the original enzy me activities were observed for ECH and C3N3Cl3 immobilized enzymes, respec tively, whereas, in the case of addition of Ca2+ ions to the assay medium, these values were enhanced to 95.3 % and 92.2 %.