Preparation Of Functionalized Magnetic Carriers And Research For Immobilization Lipase With The Carriers

Since 1960s, enzymes immobilization technology developed rapidly, as an important part of immobilized enzymes, the structures and properties of carrier materials affect the activities and operation stabilities of the immobilized enzymes greatly. The design and preparation of carrier materials will be one of the most significant researches for the development of immobilized enzymes in future. This thesis systematacially summarized the progress of methods and carriers of immobilized enzymes, especially for the development of magnetic carriers, and then three kinds of novel reactive magnetic carriers were designed and prepared, they were employed in immobilizing lipase. The main contents for this thesis are listed as belows.1. Fe₃O₄ nanoparticles with superparamagnetism were synthesized by the chemical co-precipitation, the average diameter and saturation magnetization were found to be 15nm and 58.9emu/g respectively.2. Fe₃O₄/SiO_x compound particles with silicon hydroxyl on the surface.were prepared by coating Fe₃O₄ nanoparticles with tetraethyl orthosilicate (TEOS) after hydrolysis and condensation. Effect of the amount of tetraethyl orthosilicate added on the Fe₃O₄/SiO_x compound particles was investigated. Then, the Fe₃O₄/SiO_x compound particles were modified by y-aminopropyltriethoxysilane (APTS), and the amino groups were introduced onto the surface, formed Fe₃O₄/SiO_x-APTS carriers, and their structures and properties were characterized. The Fe₃O₄/SiO_x-APTS carrier holds smaller diameter (30-50nm), higher saturation magnetization (28.3emu/g carrier), and shows superior magnetic responsibility. Lipase was covalently immobilized on this carrier by using glutaraldehyde as bifunctional agent through two different strategies. The immobilization conditions were optimized, the themal stability and reusability of the immobilized lipase were also investigated. The results idicated that immobilized lipase by two strategies was successfully, the themal stability was improved, and the immobilized lipase could be separated, recovered and reused easily and rapidly. By comparison, FSA-2 has more advantages in immobilization of lipase.3. Oleic acid was used to modify the Fe₃O₄ nanoparticles to improve their compatibility with monomers. Magnetic porous carrier with active epoxy groups was developed. They were firstly synthesized by the suspension copolymerization of glycidyl methacrylate (GMA), ethylene glycol dimethacrylate (EGDMA) and vinyl acetate (VAc) in the presence of oleic acid-coated nano-magnetite (Fe₃O₄) and porogen. Effect of the amount of OA-Fe₃O₄ added on the magnetic properties of microspheres was studied, and microspheres' structures and properties were characterized. The magnetic microspheres exhibit the apparent morphology with porous surface, the average diameter and saturation magnetization were found to be 80um and 7.61emu/g carrier. There was no hysteresis in the magnetization with both remanence and coercivity being zero, and the microspheres can be separated from the reaction medium rapidly and easily in a magnetic field. Lipase was covalently immobilized on the magnetic microspheres. The resulting immobilized lipase had better resistance to pH and temperature inactivation in comparison to free lipase, the adaptive pH and temperature ranges of lipase were widened, and it exhibited good thermal stability and reusability. The resistance of immobilized lipase against urea denaturation was also improved. The K_m value for immobilized was higher than that of the free lipase.The immobilized lipase could be separated from the reaction medium rapidly and easily in a magnetic field.4. Magnetic Fe₃O₄/P(GMA-MATAC) compound carrier was prepared via two steps. Firstly, magnetite (Fe₃O₄) nanoparticles were modified by Vinyltriethoxysilicane (VTES), thus improved their compatibility with monomers, and introduced reactive groups onto the nanoparticles surface. Secondly, Glycidyl methacrylate (GMA) and Methacryloxyethyl trimethyl ammonium chloride (MATAC) were grafted onto the surface of modified-Fe₃O₄ nanoparticles by surface-initiated radical polymerization to coat Fe₃O₄ nanoparticles. Effect of the amount of Vinyltriethoxysilicane and initiator added on the properties of carriers was investigated, and carriers' structures and properties were characterized. The average diameter and saturation magnetization were found to be 200-400nm and 27.3emu/g carrier. There was no hysteresis in the magnetization with both remanence and coercivity being zero. The carriers remained dispersive and superparamagnetic, and could be separated from the reaction medium rapidly and easily in a magnetic field. The carriers have large amount of positive electrical charges and reactive epoxy groups on the surface, and were employed in immobilizing lipase under mild conditions. The immobilized lipase had better thermal stability comparing with free lipase, and could be easily separated and reused.