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
-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 di®raction and transmission electron microscopy. The e®ects of di®erent operational conditions of direct
-amylase binding on MNPs in the presence of CDI were investigated by using the shaking method. Fourier transform infrared spectroscopy was used to con¯rm the success of immobilization. The optimum conditions and catalytic properties of immobilized
-amylase were also evaluated. The e±ciency of immobilization and the residual activity of the immobilized
-amylase were dependent on the mass ratio of MNPs: CDI:
-amylase and the immobilization temperature. The optimum pH for the free and immobilized amylase was 6. The free and immobilized
-amylase showed maximum activity at 20
C and 35
C, respectively. The immobilized
-amylase was more thermostable than the free one. The retained activity for free
-amylase after 19 storage days was 57.7% whereas it was 100% for the immobilized
-amylase. In repeated batch experiments, the immobilized
-amylase retained a residual activity of 45% after 11 repeated uses. The Km and Vmax values for the immobilized enzyme were larger than those of the free enzyme. The immobilization of
-amylase on MNPs using CDI improves its stability and reusability.

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