Study On Preparation Of Magnetic Composite Carriers And Their Application For Immobilized Lipase

Enzyme immobilization technology was a bio-engineering technology that developed since 1960.Enzyme immobilization technology was to make extensive and effective use has been an important tool.The performance of immobilized enzyme depended on the properties and immobilized method of the enzyme carrier material. This thesis systematically summarized the synthesis methods of the magnetic polymer material,the progress of methods and carriers of immobilized enzyme,and then three kinds of novel magnetic carriers were designed and prepared.They were employed in immobilizing lipase.The main contents for this thesis are listed as below:1.Fe₃O₄ nanopaticles with superparamagnetism were synthesized by the chemical co-precipitation.The average diameter and saturation magnetization were found to be 15 nm and 72.3 emu/g respectively.2.Oleic acid was used to modify the Fe₃O₄ nanoparticles to improve their compatibility with monomers.Magnetic carriers with active epoxy groups were developed.They were synthesized by the suspension copolymerization of glycidyl methacrylate,ethylene glycol dimethacrylate and styrene in the presence of oleic acid-coated Fe₃O₄ nanopaticles.The effect of the amount of OA- Fe₃O₄added on the magnetic properties of microspheres was studied,and structures and properties were characterized.The average diameter and satuation magnetization were found to be 15μm and 10.5 emu/g carriers.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.Lipases were covalently immobilized on the magnetic microspheres.The immobilization conditions were optimized;the themal stability,organic solvents stability and reusability of the immobilized lipase were also investigated.The results indicated that immobilized lipase that the themal stability and organic solvents stability were improved;the immobilized lipase could be separated,recovered and reused easily and rapidly.3.Fe₃O₄/SiO_x compound particles were synthesized by coating Fe₃O₄ nanoparticles with tetraethyl orthosilicate after hydrolysis and condensation.The Fe₃O₄/SiO_x compound particles were modified by 7-aminopropyltriethoxysilane,the amino groups reacted with chloroacetyl chloride,and reactive groups were introduced onto the surface of the Fe₃O₄SiO_x compound particles.Then,magnetic Fe₃O₄/SiO_x-g-P(GMA) carriers were prepared by atom transfer radical polymerization, and their structures and properties were characterized.The Fe₃O₄/SiO_x-g-P(GMA) carriers held smaller diameter(100nm),higher saturation magnetization(19.6 emu/g), and showed superior magnetic responsibility.Lipase was covalently immobilized on the carriers.The resulting immobilized lipase had better resistence 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.4.Magnetite(Fe₃O₄) nanoparticles were modified by vinyltriethoxysilicane,thus improved their compatibility with monomers,and introduced reactive groups onto the surface of the nanoparticles,and then 2-hydroxyethyl methacrylate and diallyl dimethyl ammonium chloride were grafted onto the surface of modified-Fe₃O₄ nanoparticles by surface-initiated radical polymerization onto coated Fe₃O₄ nanoparticles.The obtained magnetic Fe₃O₄/P(HEMA-DMDAAC) carriers were actived by epichlorohydrin to introduce reactive epoxy groups.The structure and properties of Fe₃O₄/P(HEMA-DMDAAC) carriers were characterized.The average diameter and saturation magnetization were found to be 50-200 ran and 38.3 emu/g carriers.There was no hysteresis in the magnetization with both remanence and coercivity being zero.The carriers remained dispersive and superparamagnetic,and could be separated form the reaction medium rapidly and easily.The carriers were employed in immobilizing lipase under mild conditions for large amount of reactive epoxy groups and positive electrical charges on the surface.The immobilized lipase exhibited better themal and store stability comparing with free lipase,and could be easily separated and reused several times.