Efficient Immobilization of Porcine Pancreatic α-Amylase on Amino-Functionalized Magnetite Nanoparticles: Characterization and Stability Evaluation of the Immobilized Enzyme.

Abstract

The potential of the modified magnetic nanoparticles for covalent immobilization of porcine pancreatic α-amylase has been investigated. The synthesis and immobilization processes were simple and fast. The co-precipitation method was used for synthesis of magnetic iron oxide (Fe3O4) nanoparticles (NPs) which were subsequently coated with silica through sol-gel reaction. The amino-functionalized NPs were prepared by treating silica-coated NPs with 3-aminopropyltriethoxysilane followed by covalent immobilization of α-amylase by glutaraldehyde. The optimum enzyme concentration and incubation time for immobilization reaction were 150mg and 4h, respectively. Upon this immobilization, the α-amylase retained more than 50% of its initial specific activity. The optimum pH for maximal catalytic activity of the immobilized enzyme was 6.5 at 45°C. The kinetic studies on the immobilized enzyme and its free counterpart revealed an acceptable change of Km and Vmax. The Km values were found as 4 and 2.5mM for free and immobilized enzymes, respectively. The Vmax values for the free and immobilized enzymes were calculated as 1.75 and 1.03μmolmg-1min-1, in order, when starch was used as the substrate. A quick separation of immobilized amylase from reaction mixture was achieved when a magnetically active support was applied. In comparison to the free enzyme, the immobilized enzyme was thermally stable and was reusable for 9cycles while retaining 68% of its initial activity.