Immobilization of a thermostable α-amylase onto reactive membranes: kinetics characterization and application to continuous starch hydrolysis

Epoxy groups containing porous membranes were prepared by UV-initiated photopolymerisation of hydroxyethylmethacrylate (HEMA) and glycidyl methacrylate (GMA). Epoxy supports could provide multipoint covalent attachment of enzymes, therefore, to stabilize their three-dimensional structure. α-Amylase was immobilized onto the poly(HEMA-GMA-1-3) membranes by means of the amide linkage formation between the amino groups of α-amylase and the epoxy groups of the support. The α-amylase immobilization capacity of the membranes was increased as the GMA ratio increased in the membrane structure. The retained activity of the immobilized α-amylase was 76% with poly(HEMA-GMA-2) membrane. The decrease in activity of the immobilized α-amylase could be considered to be due to reduced conformational flexibility of the immobilized α-amylase molecules for binding its large substrate, starch, as a result of the covalent immobilization. The immobilized α-amylase has more resistance to temperature inactivation than that of the free form. The optimum pH value of α-amylase was not affected by the immobilization reaction, but the pH profile was broadened for the immobilized enzyme. Kinetic parameters were determined for immobilized α-amylase as well as for the free enzyme. The values of the Michaelis constant Km of α-amylase, were significantly larger (ca. 2.3 times) upon immobilization, indicating decreased affinity of the enzyme for its substrate, whereas Vmax was smaller for immobilized α-amylase. In a 120 h continuous operation at 35 °C, only 4% of immobilized α-amylase activity was lost. The operational inactivation rate constant (kopi) of the immobilized α-amylase with 2% starch was 8.06×10−6 min−1.