| Compared with the free enzymes, immobilized enzymes have many advantages. Immobilized enzymes can be used repeatedly. The operational stability can be improved. Immobilization enzyme can be separated with substrate and product easily. Purification process can be simple. And the high yield and good quality of product can be got. Immobilized enzymes can be installed in the column or tower, and then react continuously. So they are suitable for production automatically and save labor. Additionally, immobilized enzymes are suitable for multienzymatic reaction. This thesis systematically summarized the immobilized enzyme method, carrier material, magnetic microsphere material and research progresses in domestic and overseas. Taking these into consideration, magnetic composite microspheres coated with chitosan and silica were prepared, and they were applied to immobilized lipase. The mainly content of this thesis is as following:1. The magnetic Fe3O4nanopaticles were prepared through the chemical coprecipitation method. The average diameter of the prepared nanoparticles was10～15nm. The saturation magnetization was70.8emu/g. These nanoparticles were Super paramagnetic. The magnetic Fe3O4nanopaticles were modified by surfactant oleic acid, which improved the compatibility of the Fe3O4magnetic nanoparticles with nonpolar solvent.2. Firstly, the Fe3O4/OA magnetic nanoparticles were dispersed in methylbenzene, and then dispersed in alcohol/water phase. The Fe3O4magnetic nanoparticles were coated with a layer of silica via Stober method, and oleic acid were replaced simultaneously. The average diameter of the prepared Fe3O4/SiO2nanoparticles was20～25nm. The saturation magnetization was39.2emu/g. None of residual magnetism and coercive force was left. These nanoparticles were also super paramagnetic. After that, a layer of chitosan was decorated on the surface of the Fe3O4/SiO2nanoparticles through the ionotrophic gelation method. The ionotrophic gelation method makes use of the effect of ion between chitosan and sodium tripolyphosphate to form the ion network to prepare the microspheres. The saturation magnetization of the chitosan coated Fe3O4/SiO2nanoparticles was30.1emu/g. None of residual magnetism and coercive force was left. These nanoparticles were also super paramagnetic. The modification of the surface of Fe3O4nanoparticle doesn’t cause the phase change of the crystal structre.3. The above prepared magnetic composite nanoparticles were used for immobilizing lipase through glutaraldehyde crosslinking method. The concentration of glutaraldehyde, the pH of the phosphate buffer solution and the immobilized time were optimized. Additionally, the operational stability of the immobilized enzyme was studied. The results showed the optimum condition was as follows:the concentration of the glutaraldehyde was8%, the pH value of the phosphate buffer was7.5, and the immobilization time was10h. The optimum pH of the free enzyme was7.5. The optimum pH of the immobilized enzyme was7.0, which was shift0.5towards the acid direction than the free enzyme. The optimum temperature of the free enzyme was40℃. The optimum temperature of the immobilized enzyme was50℃, which was increased10℃than that of the free enzyme. The activity of the immobilized enzyme maintained at about50%after reusing7times. These conclusions showed the immobilized enzyme have good operational stability, and the prepared magnetic composite microspheres were good enzyme carriers.This paper also studied the inhibition of flavonol A on tyrosinase. Flavonol A was extracted from Tibetan herb medicine Chrysosplenium nudicaule Bunge. The results showed that the inhibition of flavonol A on tyrosinase was competitive reversible inhibition. And from the Lineweaver-Burk plot, we knowed that the maximum reaction velocity Vm was20μmol/min, Michaelis constant Km was0.2mmol/L, the inhibition constant Ki was5μmol/L... |
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