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

• Published in: Applied biochemistry and biotechnology 2016-11, Vol.180 (5), p.954
• Main Authors: Akhond, M, Pashangeh, Kh, Karbalaei-heidari, H R, Absalan, G
• Format: Article
• Language: English
• Subjects: Biotechnology; Enzyme kinetics; Iron oxides; Nanoparticles; Silica
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-016-2145-1

The potential of the modified magnetic nanoparticles for covalent immobilization of porcine pancreatic [alpha]-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 [alpha]-amylase by glutaraldehyde. The optimum enzyme concentration and incubation time for immobilization reaction were 150 mg and 4 h, respectively. Upon this immobilization, the [alpha]-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 2.5 mM for free and immobilized enzymes, respectively. The Vmax values for the free and immobilized enzymes were calculated as 1.75 and 1.03 [mu]mol mg-1 min-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 9 cycles while retaining 68 % of its initial activity.