Immobilized alcalase alkaline protease on the magnetic chitosan nanoparticles used for soy protein isolate hydrolysis

Abstract

An efficient immobilization of Alcalase 2.4L alkaline protease has been developed by using chitosan-coated magnetic nanoparticles as support via glutaraldehyde cross-linking reaction. The Fe3O4 nanoparticles, Fe3O4-chitosan, and immobilized Alcalase 2.4L alkaline protease were characterized by X-ray diffraction, transmission electron microscope, Fourier transform infrared spectroscopy, electron spin resonance, and vibrating sample magnetometry. Results showed that the binding of chitosan and Alcalase 2.4L alkaline protease on Fe3O4 through cross-linking was successful. In addition, the Alcalase 2.4L alkaline protease immobilized with chitosan-coated magnetic nanoparticles enhanced the activity, the optimum reaction temperature and pH value for the immobilized enzyme were 55 °C and 10, respectively, compared with the free enzyme, and the optimal temperature and pH profile range were considerably broadened. Similarly, the thermal stability was enhanced by immobilization, and the kinetic parameters of free and immobilized Alcalase 2.4L alkaline protease were determined. Then, from our hydrolysis experiments, we found that immobilized Alcalase 2.4L alkaline protease uses Fe3O4-chitosan had a greatest hydrolytic activity, and the DH of soy protein isolate (SPI) can reach to 18.38 %, against 17.50 % with the free enzyme after 140 min. Furthermore, the immobilized Alcalase 2.4L alkaline protease could maintain about 86 % of its original activity after ten consecutive operations. Thus, Fe3O4-chitosan immobilized Alcalase 2.4L alkaline protease a good candidate for the continuous hydrolysis of SPI.