Preparation Of Solid And Hollow Magnetic Polymer Supports And Their Application For Lipase Immobilization

Lipase (triacylglycerol ester hydrolases, EC3.1.1.3) is a kind of ubiquitous enzymes with various biological activities, including enantioselective hydrolysis and esterification, chiral resolution, synthesis of enantioenriched monomers and macromolecules for polymerization, and other enzymatic reactions. However, the stability of free enzymes is pretty terrible, and their activity, broad applicability and reusability are still needed to be proved. Thus, the technology of enzyme immobilization, which could perform enzyme reuse and prove the stability of enzyme, was developed to overcome these inconveniences. This dissertation reviews the studies of enzyme immobilization, the application of magnetic polymer materials for enzyme immobilization and the preparation of magnetic polymer materials. Thus, we prepared four kinds of magnetic polymer supports for lipase immobilization and studied the properties of immobilized lipase. The main content of this dissertation is list as follows:(1) Preparation of superparamagnetic sodium alginate nanospheres for covalent immobilization of lipaseSuperparamagnetic sodium alginate (SA) nanospheres with diameter around25-30nm were prepared with a water-in-oil emulsion method. The resultant magnetic SA nanopspheres was activated with glutaraldehyde and epichlorohydrin to form nanoscale supports. Candida Rugosa Lipase (CRL), hereby chosen as a model enzyme, was covalently immobilized on the resultant magnetic support. The structure and magnetic behavior of the magnetic nanoparticle were confirmed by transmission electron microscopy(TEM), Fourier transform infrared spectroscopy(FT-IR), and vibrating sample magnetometer(VSM). Then, it was verified that the supports we prepared has obvious advantages:small size, excellent dispersibility and high magnetization (51.4emu/g). The immobilized CRL (ICRL) we prepared displayed excellent stability, pH and temperature tolerance, kenetics behaviour and reusability.(2) Preparation of superparamagnetic Fe3O4@alginate/chitosan nanospheres by self-assembly and their immobilization for lipaseSuperparamagnetic alginate nanospheres with diameter of50nm were prepared by self-assembly of alginate in the solution containing Ca2+; and then superparamagnetic alginate/chitosan nanospheres, which could adsorb lipase directly, were obtained with a following assembly of chitosan based on the electrostatic interaction between alginate and chitosan. Subsequently, oxydic poly (ethylene glycol)(PEG) was used to functionalize the magnetic alginate/chitosan nanospheres. Thus, the magnetic nanospheres with aldehyde group and brush-like structure were formed. With various kinds of characterization, it was verified that the magnetic alginate/chitosan nanospheres held small diameters (around60nm) and displayed superparamagnetism with high saturation magnetization. The candida rugosa lipase (CRL), meanwhile, was immobilized onto the magnetic alginate/chitosan nanospheres non-functionalized or functionalized by electrostatic adsorption and covalent bonding, respectively. Afterwards, the immobilized CRL (ICRL) was coated with covering layers made up of alginate and chitosan by a layer-by-layer (LBL) assembly process. Studying the properties of ICRL such as activity, kinetic behaviors, stability and reusability, the ICRL prepared by covalent bonding was proved to be more excellent than that prepared by electrostatic adsorption. Additionally, coating ICRL with covering layers showed good effect on improving the stability of ICRL.(3) Building on size-controllable hollow nanospheres with superparamagnetism derived from solid Fe3O4nanospheres:preparation, characterization and application for lipase immobilizationMonodisperse porous hollow nanospheres with superparamagnetism were prepared via a hydrothermal reaction based on solid Fe3O4nanospheres. By investigating the effect of carbon precursors, the kinds and amounts of structure direction agents and the reaction time on the formation of hollow spheres, it was proposed that the main formation mechanism of hollow spheres is a gas-bubble-assisted Ostwald ripening process. Additionally, it is found that the diameter of hollow spheres and the size of hollow core could be adjusted by changing the above factors. The resultant hollow spheres were characterized by means of Brunauer-Emmett-Teller gas sorptometry, transmission electron microscopy, scanning electron microscopy, X-ray diffraction, Fourier-transform infrared spectrophotometer, Thermogravimetric analysis and vibrating sample magnetometer. It is verified that the resultant hollow spheres are porous and have high saturation magnetization. For further application, these hollow spheres were utilized to immobilize CRL and they showed excellent immobilization capacities compared with the solid microspheres. Finally, the resultant ICRL also displayed good applicability and reusability.(4) Preparation of hollow magnetic Fe3O4/P(GMA-DVB-St) microspheres and their application for lipase immobilizationOn the basis of hollow magnetic nanoparticles, the monomer GMA and St, the cross-linker DVB were selected to be polymerized to form hollow magnetic Fe3O4/P(GMA-DVB-St) microspheres. It was verified that this hollow microspheres had an especial structures consisting of mesoporous shell and hollow core, which would improve its ability for lipase immobilization. Additionally, there were expoxy groups on the surface of these microspheres due to the existence of GMA, thus, these microspheres could react with lipase directly. Then, we studied the properties of immobilized CRL (ICRL). The ICRL showed excellent stability and reusability. Copared with the ICRL prepared by hollow magnetic nanospheres without polymeric modification, the ICRL prepared by hollow magnetic polymer microspheres displayed more excellent properties.