The Research On Preparation Of Magnetic Nanoparticales And Catalyse Character Of Immobilized Lipase

Magnetic polymer microspheres were new functional polymer materials developed in recent years, which have broad application prospect in the biomedical, molecular boilogy, separation engineering, cellular and bio-engineering and other fields. Magnetic polymer was used as enzyme immobilization carrier provides the theoretical and experimental basis in the immobilized enzyme application for reducing lipase cost, and the problems of biodiesel production were redused. Compared with other magnetic materials, Fe3O4 have good oxidation stability and low toxicity. And different magnetic properties were obtained because of its different chemical composition and structure. Magnetic microspheres were used for the magnetic core. As enzyme im-mobilization carriers, magnetic polymers can be magnetized in an external magnetic field and then directional movement, so it can be separated from the reaction system; when the external magnetic field was withdrawed, the superparamagnetic nanoparticles can be resuspended to the reaction system, with good dispersion properties and manipulation.In this paper, the Fe3O4 magnetic fluid was prepared by modified coprecipitation, and then composite magnetic Fe3O4-SiO2 carriers were got by the sol-gel. The magnetic composite carriers observed by SEM showed that carriers were ballshaped, singlewell dispersed, the average size of about 300nm or so. Silane coupling agent, which was detected by FT-IR spectro-meter, was used for adding amino to magnetic Fe3O4-SiO2 surface.Conditions of immobiling lipase by magnetic Fe3O4-SiO2 composite were studied, the optimal conditions got from single factor experiments were:the amount of enzyme was 8mL, concentration of glutaraldehyde was 8%,25℃,4h. The amount of enzyme, con-centration of glutaraldehyde and immobilized temperature were selected to do Box-Behnken experimental to further optimize the conditions, and experimental data was statistical analysis with the Design-Expert 7 software, the mathematical model was describing the relationship between activity and the expeimental conditions. The optimal conditions of immobilized showed by the model are:the amount of enzyme was 8.30mL, 8.20% glutaraldehyde concentration,23.88℃, then got the maximum activity 3440.74 U/g. Three sets of repeated immobilization test was studied under the conditions defined in the above, the results obtained the average fixed lipase activity was 3430±1.55U/g, and the difference between model prediction was 0.28%, indicating the mo-del consistent with the experimental results.The enzymatic properties of immobilized enzyme was explored. Optimum temperature, thermal stabilities of immobilized enzyme were compared with the free enzyme. The immobilized enzyme were reused. The results showed that the Km for the free enzyme 3.817mmol, Vm was 0.041μmol/min·L, and which of immobilized enzyme were 5.714mmol and 0.087μmol/min·L, respectively. Compared with the free lipase, the optimum temperature, thermal stability, optimum pH value, pH stability of immobilized enzyme without much change, but the thermal stability and pH stability were increased, indicating operation of the immobilized increased the stability of free lipase. Immobilized lipase were reused 8 times, the activity recovery is still 36.4%.