A Research On Modification Of Magnetic Nano Materials And Immobilization Of Lipase

Lipase(E.C.3.1.1.3 )is a kind of triglycerides hydrolase. Due to its strong ability of catalyzing, it is widely applied in industry. However, free lipase is unrecyclable and nonrenewable, which causes serious waste during industrial production. Currently, industrial synthesis technology of the ferroferric oxide nanoparticles is quite mature. With certain modification, the ferroferric oxide nanoparticles can be used to immobilizing lipase. In additional magnetic field, separation is accelerated. The recycling leads to effective cost-saving.This thesis takes the industrial ferroferric oxide nanoparticles as magnetic material, and aminopropyltriethoxysilane(APTES)and hexadecyltrimethoxysilane as coupling agents. Fe₃O₄-APTES amino magnetic nano carrier and Fe₃O₄ -hexadecyl hydrophobic nano carrier are synthesized by jointing. With the tetraethyl orthosilicate (TEOS)and aminopropyltriethoxysilane(APTES)as monomers, Fe₃O₄/SiO₂–APTES amino magnetic nano carrier and MNC amino magnetic composite carrier are produced by first embedding then reattaching arm and copolymerization embedding. With the chitosan as raw material, and glutaraldehyde as double function cross-linking agent, Fe₃O₄/CS magnetic composite carrier is prepared by means of inverse-phase suspend cross-linking technology.A characterization analysis is made by using Transmission Electron Microscope (TEM), Fourier transform infrared spectrometer(FT-IR), X-ray diffraction(XRD) and vibrating specimen magnetometer(VSM) respectively. The result shows that Fe₃O₄-APTES and Fe₃O₄–hexadecyl carrier have orbicularstructure structure of 20nm partical diameter with extremely good dispersion. Meanwhile, modified perssads exist on the surface without magnetic changes. The saturation magnetization are respectively 47.59emu/g and 45.74emu/g, processing superparamagnetism; the partical diameter of Fe₃O₄/SiO₂–APTES is larger, but still within the nano category. It has amino groups and good dispersion, but the magnetism is decreased. Fe₃O₄/CS and MNC have relatively bigger partical diameter, good dispersion. modified perssads also exist. Their saturation magnetizations are significantly decreased to 10.76emu/g,15.16emu/g respectively, but still process superparamagnetism.Initial mobilization of CRL is conducted after activating the five enzyme carriers mentioned above. The results indicate that the immobilized efficiency is highest with MNC. The enzyme activity is also the best. Based on this indication, we choose MNC as the research target, by optimizing the immobilized conditions. The effects of the amount of lipase added, immobilized time and immobilized temperature on immobilized efficiency and enzyme activity are studied by single factor experiment analysis. An optimum condition is hence concluded. With the reaction system being pH 7.0, the optimum immobilizing condition is as follows: 75mg/g of the amount of lipase added, 1.5h of mechanical rabbling, 20℃of the temperature, the immobilized efficiency is 63.8% and the enzyme activity is 32.48KU/g.Using the immobilized enzyme under the optimum condition as the sample, we chose olive oil emulsion as mode substrate. A study is conducted on the optimum condition of enzymatic reaction to ensure the performance parameter of CRL. The result shows the optimum catalytic temperature is 45℃and pH7.0. After catalyzing the hydrolysis substrate for 8 times, the remainder activity is 83.7%, with a good reusability. Under the condition of 4M urea concentration, the apparent enzyme activity of substrate reaches as high as 78.1%, which indicates a strong contravariance. Compared with free lipase, immobilized lipase optimal temperature increased by 5℃, the optimal pH increased by 0.5 unit, temperature and degeneration agent of urea tolerance got significantly improved, and it has high reusablity, which provide reference for future industrial application.