Carbodiimide for Covalent α-Amylase Immobilization onto Magnetic Nanoparticles

Abstract

Covalent cross-linking of enzymes to magnetite (Fe3O4) nanoparticles (MNPs) is one of the useful enzyme immobilization methods which provides repeated use of the catalyst, facilitates enzyme separation from the reaction mixture, and sometimes improves biocatalysts stability. The aim of this study was to immobilize [Formula: see text]-amylase onto MNPs via covalent attachment using carbodiimide (CDI) molecules. MNPs were synthesized by the co-precipitation method. The size and the structure of the particles were characterized by X-ray diffraction and transmission electron microscopy. The effects of different operational conditions of direct [Formula: see text]-amylase binding on MNPs in the presence of CDI were investigated by using the shaking method. Fourier transform infrared spectroscopy was used to confirm the success of immobilization. The optimum conditions and catalytic properties of immobilized [Formula: see text]-amylase were also evaluated. The efficiency of immobilization and the residual activity of the immobilized [Formula: see text]-amylase were dependent on the mass ratio of MNPs: CDI: [Formula: see text]-amylase and the immobilization temperature. The optimum pH for the free and immobilized amylase was 6. The free and immobilized [Formula: see text]-amylase showed maximum activity at 20[Formula: see text]C and 35[Formula: see text]C, respectively. The immobilized [Formula: see text]-amylase was more thermostable than the free one. The retained activity for free [Formula: see text]-amylase after 19 storage days was 57.7% whereas it was 100% for the immobilized [Formula: see text]-amylase. In repeated batch experiments, the immobilized [Formula: see text]-amylase retained a residual activity of 45% after 11 repeated uses. The [Formula: see text] and [Formula: see text] values for the immobilized enzyme were larger than those of the free enzyme. The immobilization of [Formula: see text]-amylase on MNPs using CDI improves its stability and reusability.