α-Glucosidase immobilization on functionalized Fe3O4 magnetic nanoparticles for screening of enzyme inhibitors

Magnetic nanoparticles (MNPs) are widely used for the immobilization of enzyme owing to the unique properties such as good biocompatibility and rapid separation. Herein, we used Fe3O4 magnetic nanoparticles (Fe3O4 MNPs) as the carrier core with (3-aminopropyl)triethoxysilane (APTES) modification by our approach, in which α-glucosidase was stereoscopically immobilized on the surface of Fe3O4 MNPs via covalent binding. The result of immobilization was characterized by scanning electron microscope (SEM) and fourier transform-infrared spectroscopy (FT-IR). Then we optimized some key parameters of the immobilization reaction, including the ratio of MNPs to enzyme, GA concentration, crosslinking time and immobilization time. Moreover, under the optimal conditions, pH tolerance, thermo stability and reusability of the immobilized enzyme were investigated and compared with the free one. In order to evaluate the change of the affinity of the enzyme to its specific substrate after immobilization, the Michaelis-Menten constant (Km) was also studied. Finally, the immobilized α-glucosidase combining with high performance liquid chromatography-tandem mass spectrometry technique (HPLC-MS/MS) was applied to screen and identify eight inhibitors from Polygonum cuspidatum extract. These results indicated that the established method had the broad prospects for biotechnological applications.