Preparation Of Functionalized Magnetic Nanomaterials And Their Application For Enzyme Immobilization

With the advantages of the large surface area, high binding capacity, good biocompatibility and easy manipulation by an external magnetic force, MNPs have been considered to be an extractive substrate for developing an efficient enzymatic reactor. Special emphasis in our works will be placed on the preparation of functionalized magnetic nanoparticles (MNPs) with simplified synthetic approaches, surface modification and stabilization of the MNPs, and their application for enzyme immobilizaiton. Thus, we prepared a series of intelligent immobilized enzyme system on the functionalized MNPs to resolve the problems such as low enzyme loading capacity, functionality limitations, and so on. This paper carried out the following aspects of research:(1) The preparation and application of GO-CS-Fe3O4 magnetite nanoparticles for lipase immobilizationBased on the solvothermal technology, three dimensional (3D) magnetic graphene oxide-chitosan (GO-CS) composites with orderly self-assembled magnetite (Fe3O4) nanoparticles (GO-CS-Fe3O4) were successfully fabricated. The resulting composite combined the features of the high surface area of GO, abundant amino and hydroxyl functional groups of CS, and the magnetic response of magnetic nanoparticles. Amine-functionalized GO-CS-Fe3O4 support was activated by glutaraldehyde or chloroacetate activation procedure. GO-CS-Fe3O4 support possessed positive charge and could be used to immobilize lipase by electrostatic adsorption, GO-CS-Fe3O4-GA was used to immobilize lipase by covalent bonding, and GO-CS-Fe3O4-IDA-Cu was used to immobilize lipase by metal-ion affinity interactions. The results confirmed that the immobilization enzyme via metal-ion affinity had the shortest immobilized time and the highest loading amounts of enzyme, and also illustrate that the loading amounts and catalytic ability of enzyme is closely related to the immobilized way.(2) Biocatalytic performance of pH-sensitive magnetic nanoparticles derived from layer-by-layer self-assembly of chitosan with glucoamylaseBased on the characteristics of polycations of chitosan (CS) and glucoamylase (GA), which are oppositely charged, they were successfully alternately deposited onto the surface of aldehyde-modified Fe3O4/CS nanoparticles by using a layer-by-layer self-assembly method to form magnetic carriers to construct multilayer films (designated as Fe3O4@(CS/GA)n). The biocatalysts have the advantages of the pH-sensitive properties of CS, the catalytic activity of glucoamylase and the magnetic response of Fe3O4. This novel strategy would simplify the reaction protocol, improve encapsulation efficiency and catalytic activity which offered new potential applications in biotechnology.(3) Reversible immobilization of glucoamylase onto magnetic polystyrene beads with multifunctional groupsThe functional magnetic polystyrene microspheres were prepared by impregnation and subsequent precipitation of ferric and ferrous ions into the functionalized polystyrene particles. Three kinds of end-functional magnetic polystyrene microspheres with carboxyl, imidazolyl and sulphydryl groups were obtained,named as Fe3O4@PS-DEA-IDA, Fe3O4@PS-DEA-imidazole and Fe3O4@PS-DEA-TSC. The resultant magnetic polystyrene microspheres presented excellent good monodispersity, abundant functional groups, fast magnetic response and regeneration of supports. Finally, the functional magnetic polystyrene microspheres were used for the reversible immobilization of glucoamylase via metal-affinity adsorption. The results indicated that the obtained immobilized glucoamylase presented excellent binding capacity, pH endurance and temperature endurance, applicability, and reusability. By contrast, these results show that the enzyme loading content is closely related to the content and chelating ability of metal chelating groups on the functional magnetic microspheres.(4) Preparation of mesoporous magnetic colloidal nanocrystal clusters (MCNCs) for enzyme immobilizationBy controlling the assembly process of the primary magnetic colloidal nanocrystals through solvothermal routes for the fabrication of mesoporous carboxyl-functionalized magnetic colloidal nanocrystal clusters with tunable size and high magnetization were developed. The nanocrystals were assembled into interior porous clusters by using iron (Ⅲ) chloride hexahydrate as a precursor, ethylene glycol as a reducing agent, ammonium acetate as a porogen and EDTA-2Na as a surface modification agent. Moreover, the particle sizes can be simply controlled by varying the relative concentrations of EDTA-2Na. To evaluate the capability of the mesoporous MCNCs for enzyme immobilization, the carboxyl-functionalized mesoporous MCNCs were functionalized via different amination reagents, and were used to immobilize enzyme. When the pore size of MCNCs is a good match for the dimensions of enzyme molecules, which is beneficial for catalytic properties. These immobilized glucoamylase exhibited excellent thermal stability and reusability because of their useful magnetic properties and unique microstructure.(5) Synthesis of magnetic thermosensitive microcontainers for enzyme immobilizationWe present a new approach for the fabrication of magnetic thermoresponsive polymer microcapsules with mobile cores. At first, the superparamagnetic Fe3O4 nanoparticles were fabricated by the solvothermal process, and then a silica layer was coated on the surface of Fe3O4 nanoparticles through a sol-gel process. Finally, a thermoresponsive shell of PNIPAM was deposited onto the surface of the core/shell magnetic microspheres by a distillation precipitation polymerization, and then the silica template was selectively etched to form the magneic polymeric microcontainer. The polymeric microcapsules have magnetic-sensitive, temperature-sensitive behaviors, regular and permeable porous shell.The results indicate that the adsorption/release of lipase from the microcontainers can be controlled by the environmental temperature and magnetic force.