title: 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 150 mg and 4 h, 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 Kmand Vmax. The Km values were found as 4 and 2.5 mM for free and immobilized enzymes, respectively. The Vmaxvalues for the free and immobilized enzymes were calculated as 1.75 and 1.03 μmol mg-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 9 cycles while retaining 68 % of its initial activity.
authors.affiliation: Professor Massoumi Laboratory, Department of Chemistry, Faculty of Sciences, Shiraz University, Shiraz, 71454, Iran.
authors.forename: M
authors.initials: M
authors.lastname: Akhond
authors.affiliation: Professor Massoumi Laboratory, Department of Chemistry, Faculty of Sciences, Shiraz University, Shiraz, 71454, Iran.
authors.forename: Kh
authors.initials: K
authors.lastname: Pashangeh
authors.affiliation: Department of Biology, Faculty of Sciences, Shiraz University, Shiraz, 71454, Iran.
authors.forename: H R
authors.initials: HR
authors.lastname: Karbalaei-Heidari
authors.affiliation: Professor Massoumi Laboratory, Department of Chemistry, Faculty of Sciences, Shiraz University, Shiraz, 71454, Iran. gubsulun@yahoo.com.
authors.forename: G
authors.initials: G
authors.lastname: Absalan
mesh_terms: D000588:Amines / Q000737:chemistry*; D000818:Animals; D055162:Biocatalysis / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D058185:Magnetite Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D008670:Metals / Q000494:pharmacology; D010179:Pancreas / Q000201:enzymology*; D059027:Recycling; D012995:Solubility; D017550:Spectroscopy, Fourier Transform Infrared; D034421:Sus scrofa; D013696:Temperature; D013997:Time Factors; D014961:X-Ray Diffraction; D000516:alpha-Amylases / Q000378:metabolism*
chemical_list: D000588:Amines; D004800:Enzymes, Immobilized; D007477:Ions; D058185:Magnetite Nanoparticles; D008670:Metals; D000516:alpha-Amylases
keywords: Covalent Immobilization; Enzyme activity; Enzyme stability; Magnetic nanoparticles; Porcine pancrease α-amylase