Covalent Immobilization of a α-Amylase onto Poly(methyl methacrylate-2-hydroxyethyl methacrylate) Microspheres and the Effect of Ca2+ Ions on the Enzyme Activity

α-Amylase was covalently immobilized onto poly(methyl methacrylate-2-hydroxyethyl methacrylate) microspheres, which were activated by using either epichlorohydrin (ECH) or cyanuric chloride (C₃N₃Cl₃). 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 73.0% and 90.8% for epichlorohydrin and cyanuric chloride bound enzymes, respectively. Upon immobilization, the maximum activities were obtained at lower pH values and higher temperatures as compared 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 × 10⁻³ gL⁻¹ min⁻¹ 2.89 × 10⁻⁴ gL⁻¹ min⁻¹ and 1.89 × 10⁻³ gL⁻¹ min⁻¹ for V_max for free, epichlorohydrin and cyanuric chloride bound enzymes, respectively. Enzyme activities were found to 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 lost 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 enzymes 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 the absence of Ca²⁺ ions retentions of 79.2% and 77.1% of the original enzyme activities were observed for ECH and C₃N₃Cl₃ immobilized enzymes, respectively, whereas, in the case of addition of Ca²⁺ ions to the assay medium, these values were enhanced to 95.3% and 92.2%.