| Chitosan, a natural linear biopolyaminosaccharide is obtained by alkaline deacetylation of chitin, extracted from abandoned shrimp, crab shell, which is the second abundant polysaccharide next to cellulose while chitosan comprises of copolymers of glucosamine and N-acetyl-glucosamine. Chitosan is the uinique natural existence alkaline polysaccharide, with the following advantages:reticulation structure, source rich, cheap, simple preparation, chemical stability after modified, heat resistance, good resistant to acid, alkali and organic reagents, good mechanical properties, and good biological compatibility and adaptability that can be natural degradation and degradation products also non-toxic. Because of the above virtues it can be good carrier for fixing the enzyme. Chitosan has wide application prospects in medicine, food and industrial area, and achieved the reuse.At present, chitosan can be used as immobilized enzyme carriers has the following kinds of forms:microspheres, thin films, nanoparticles, amorpphis, and the most frequently used is the microspheres, which can be used in packing column for affinity adsorption and affinity screening, and the technology of preparation of microspheres are quite mature. Although, there exists some insufficients, for example, the the surface of microspheres are smooth when used inverse suspension crosslinking method, lead to specific surface area for immobilization are very small and low efficiency. Chitosan microspheres without crosslinking are usually made by NaOH ion gel method, was the most frequently used for made microspheres in reported literature for immobilized enzymes. It is porous state on the surface and in the inner part, specific surface area is great, the preparation process do not add any toxic reagents, and because of without any chemical modification it can keep the most of the amino and hydroxyl in chitosan themself, but the diameter of microspheres are too long which obtained by the syringe hand knock, the spherical particle size are heterogeneity, and time-consuming, force-consuming, not cross-linked microspheres will dissolve in acid. It is necessary to modify and create the existing system method for the preparation of microsphers so as to expand the chitosan for enzyme immobilization of scope, improve the efficiency of immobilized enzyme.Studies are included in the thesis:1. The chitosan as raw materials, glutaraldehyde as crosslinking agent, we successfully prepared uniform chitosan microspheres by the method of inverse suspended. Investigated the operation conditions on the synthesis influence on the performance of the microspheres. Furthermore, examination of IR spectra, scanning electron microscope and electron microscope characterization of its structure. Because of the synthesis of chitosan microspheres brought out smooth surface, and the area of microspheres can be used in the immobilized enzymes is lesser, we bring in the acid etching processing and a second crosslinking to the synthetic chitosan microspheres to solve these problems, in addition, investigation of BSA and lipase immobilized efficiency. The results show:the good resistant to acid and alkali, high mechanical strength of chitosan microspheres can be prepared by reverse emulsification crosslinking. Acid treatment of chitosan microspheres made the surface of microspheres changed to be rough, and the immobilization efficiency of BSA and enzyme were increased.2. Silicon dioxide as the porogenic agent, control of the particles of silica particle size added, with sodium hydroxide dissolved the added silica through the inverse suspension method, we can successfully preparaed hole-diameter-controlled porous chitosan. Characterize its structure by IR spectrum, scanning electron microscope and electron microscope. Investigaed its physical and chemical properties, optimize the process of the various factors affecting the hole of the porous microspheres. The results show that:the silica can be successful made hole, microspheres specific surface area greatly improved. The concentration of sodium hydroxide and the temperature of dissolved process play a important effect on preparation of hole.3. Combine reverse phase suspension method with sol-gel method can make microspheres easily. First, use the inverse suspension method to get chitosan oil-in-water emulsions particles, then quickly poured the emulsions particles into the high concentration of condensate fluid containing sodium hydroxide, hence we can get high porous chitosan gel microspheres. Characterized its structure by IR spectrum, scanning electron microscope and electron microscope. Investigate its physical and chemical properties, optimize the process of the various factors affecting the process of the porous microspheres. And compared the catalysis efficiency of immobilized enzyme between syringe systems with emulsion-gel method. The results show that this emulsion-gel method is used to the preparation of the microspheres is size controllable, concentration controllable, and catalysis efficiency of immobilized enzyme is higher than syringe method. This method can replace the injector manual method for preparation of non crosslinking gel microspheres, and can realize the industrialization production, used in the immobilized enzyme field. |
