AbstractThis paper reports on a novel and efficient β-glucosidase immobilization method using magnetic Fe3O4 nanoparticles as a carrier. Based on response surface methodology, the optimal immobilization conditions obtained were: glutaraldehyde (GA) concentration, 0.20%; enzyme concentration, 50.25 μg/mL; cross-linking time, 2.21 h; and the maximum activity recovery reached 89.35%. The magnetic immobilized enzyme was characterized by Fourier transform infrared spectroscopy (FTIR), transmission electron microscope (TEM), and vibrating sample magnetometer (VSM). FTIR revealed that β-glucosidase was successfully immobilized on the magnetic nanoparticles. TEM showed that enzyme-magnetic nanoparticles possessed nano-scale size distribution. VSM confirmed that the enzyme-magnetic nanoparticles were superparamagnetic. The properties of the immobilized β-glucosidase were improved, and the immobilized β-glucosidase exhibited wider pH and temperature ranges of activation, higher accessibility of the substrate, better thermal stability, and better storage stability than that of the free enzyme. The enzyme-magnetic nanoparticles could be separated magnetically for easy reuse. Immobilization of β-glucosidase onto the magnetic nanoparticles has the potential for industrial application.