Immobilized Metal Ion Affinity Nanospheres For Alpha-Amylase Immobilization

Abstract

Immobilized metal chelate affinity chromatography (IMAC) support was practiced for alpha-amylase immobilization. Poly(hydroxyethylmethacrylate-methacryloylamidotryptophan)-Ni2+ [p(HEMA-MAT)-Ni2+] nanospheres, average diameter 100 nm, were produced by surfactant free emulsion polymerization. Characterizations of p(HEMA-MAT)-Ni2+ nanospheres were carried out by Fourier transform infrared (FTIR) spectroscopy and scanning electron microscope (SEM). In addition, average particle size, size distribution, and surface charge were specified. The amount of N-methacryloylamidotryptophan (MAT) incorporated to polymer was determined as 1.95 mmol/g polymers by using nitrogen stoichiometry. The specific surface areas of poly(hydroxyethylmethacrylate) [p(HEMA)] and p(HEMA-MAT) nanospheres were calculated as 1856 m(2)/g and 1914 m(2)/g, respectively. Protein adsorption increased with increasing initial protein concentration and maximum alpha-amylase adsorption on p(HEMA-MAT)-Ni2+ nanospheres was observed at pH 4.0. Both free and immobilized alpha-amylase showed pH optimum at pH 7.0. It was determined that the immobilized alpha-amylase had better thermostability than the free one. Immobilization of the enzyme did not significantly change the kinetic parameters. The storage stability of alpha-amylase increased upon immobilization. It was also observed that p(HEMA-MAT)-Ni2+ nanospheres can be repeatedly used for alpha-amylase immobilization.