The Application Of Magnetic Chitosan Nanoparticle On B-D-Galactosidase Immobilization

(3-D-Galactosidase (EC3.2.1.23), commonly known as lactase, has been received particular interest because it can mediate the trangalactosylation reaction for the preparation of galactooligosaccharides (GOS) as well as the hydrolytic reaction of lactose for the preparation of low-lactose milk. The immobilization of β-D-Galactosidase has been widely studied. However, using magnetic Fe3O4-CS nanoparticle as carrier has rarely been reported yet. Magnetic nanoparticles has broad application in the field of pharmaceutical and enzyme immobilization due to its large specific surface area and easy separation from reaction mixture by magnetic field.The following three aspects have been systematically studied:the preparation and characterization of magnetic Fe3O4-CS nanoparticles, the immobilization of β-D-Galactosidase and its properties, GOS synthesis and lactose hydrolysis using immobilized enzyme.Firstly, Fe3O4nanoparticles were prepared by co-precipitation. The resulting particles exhibited ideal magnetic properties. During the preparation process, the Fe2+/Fe3+ratio was studied and set at1:1, the other conditions are as follows:ferric salt concentration0.12mol/L, agitation speed1000r/min, reaction temperature30℃, reaction pH8, aging temperature80℃, aging time30min. The magnetic Fe3O4-CS nanoparticles were prepared by electrostatic adsorption of chitosan onto the surface of Fe3O4nanoparticles. The nano-composites prepared by dry and wet method both showed excellent dispersion and magnetic properties. Taking convenience into consideration, the following experiments employed the wet method. For preparation of Fe3O4-CS nanoparticles by wet method, the Fe3O4nanoparticles were dispersed in100mL of4mg/mL CS solution, and a solution of tripolyphosphate (50mL,0.5mg/mL) was added simultaneously. The suspension was treated by ultrasonic for30min to ensure the nanoparticles coated evenly. The resultant material was characterized by Transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), vibrating sample magnetometry (VSM) and thermogravimetric analysis (TGA). The results demonstrated that CS has been bound successfully to the magnetic Fe3O4nanoparticles with mass ratio of5.2%. Moreover, the crystal structure has not been changed after the binding of CS.β-D-galactosidase was covalently immobilized onto the nano-composites using glutaraldehyde (GA) as activating agent. The results showed the optimal conditions for (3-D-galactosidase immobilization were immobilized time3h, crosslinking time4h, enzyme concentration0.5mg/mL, GA concentration4%, the initial pH values of GA4and the initial pH of enzyme solution6. The immobilized P-D-galactosidase exhibited stronger thermal and pH stability compared to its free form. The kinetics constant Km for immobilized enzyme is0.2625mmol/L with ONPG as substrate, lower than that for free enzyme (0.3630mmol/L).The immobilized enzyme was applied to GOS synthesis and lactose hydrolysis, and their reaction processes were studied. The optimal conditions for GOS synthesis were reaction temperature60℃, reaction pH4.6, enzyme concentration6U/mL, lactose concentration500mg/mL, and the GOS yield can reach125mg/mL. The adding of glucose and galactose both would inhibit GOS synthesis. The hydrolysis reaction was conducted using5%lactose as substrate, the optimal conditions were reaction temperature50℃, reaction pH4.6, enzyme concentration4U/mL.