Fabrication of Fe3O4@SiO2@PDA-Ni2+ nanoparticles for one-step affinity immobilization and purification of His-tagged glucose dehydrogenase

Abstract

To avoid the additional purification procedures required before immobilization, the Fe3O4@SiO2 @ polydopamine (PDA)-Ni2+ magnetic nanoparticles (MNPs) were prepared to achieve the selective purification and immobilization of the His-tagged glucose dehydrogenase (GluDH) from the crude cell lysate. The size and morphology of the prepared nanoparticles were ascertained by Fourier transform infrared spectroscopy (FT-IR), Scanning electron microscopy (SEM), and X-ray diffraction (XRD). The processes of His-tagged GluDH purification and immobilization on Fe3O4@SiO2@PDA-Ni2+ were investigated. The results indicated that the optimal immobilization circumstances were achieved. After mixing at a protein-carrier ratio of 0.13 in Tris-HCl (0.2 M pH 8.0) and incubating for 60 min at 30 °C, the activity recovery and protein loading were 44.27 % and 26.33 μg mg−1, respectively. The immobilized GluDH had better tolerance to thermal and pH than the free enzyme, and only 43.58 % of the initial catalytic activity was lost after 10 cycles. Though the Vmax decreased after immobilization, the Km value for immobilized GluDH was 1.38-fold higher than the free GluDH. Therefore, compared with the commercial Ni-NTA resin, Fe3O4@SiO2@PDA-Ni2+ MNPs displayed greater specific affinity to His-tagged GluDH.