Preparation And Characterization Of Magnetic Silica Support And Application In Immobilization Of Enzyme

The technology of immobilization enzyme has been widely used in biochemistry bioengineering, medicine and food industry. The key of the technology of immobilization enzyme is to choose the appropriate support materials. Compared to the normal supports, magnetic materials have some new advantages in immobilization of enzyme. In this article amino modified and epoxy modified magnetic silica supports were prepared and characterized, and applied in immobilization of trypsin and lipase. The properties of immobilized enzyme were further investigated.Fe3O4 magnetic nanoparticles (MNP) were prepared by co-precipitation of Fe²⁺ and Fe3+ in ammonia solution, and the average diameter of MNP was about 27nm. The core-shell type magnetic silica (MS) was synthesized by hydrolyzing of triethoxysilane (TEOS) in the methanol solution of MNP. Theγ-APTS as the coupling agent was further to react with MS, and the amino-modified magnetic silica support (AMS) was prepared. The ATR-FTIR showed that there were two peaks around 2887 cm-1, which indicated the vibration of C-H bond ,and amino group was successfully modified on the surface of AMS. The crystal structures of MNP, MS and AMSl were magnetite conformed by XRD. The amounts of the amino group on the surfaces of AMS were measured by back titration and there were 0.1454 mmol amino groups on per gram AMS supports. The magnetic properties of MNP, MS and AMS were measured by VSM. The Mr of these three supports were 69.1,57.0,23.7 emu/g, but they had no superparamagnetic properties. Trypsin was immobilized on AMS. The optimum immobilization conditions for trypsin was pH value 7.5 and the amount of glutaraldehyde 100 ul (1%). The optimum pH value of immobilized trypsin was improved 0.5 pH compared with that of free trypsin; the residual activity of AMS bound trypsin still retained 76% of its original activity after being reused 8 times. epoxy modified magnetic silica supports are probable kind of new materials to immobilization enzyme. Epoxy modified magnetic silica support (EMS) was prepared by a silica coupling agent GPTMS reaction with MNP in catalyzing of fluoride ion. The ATR-FTIR confirms that there are two feature peaks in 1255 cm-1,904 cm-1 indicated the existing of epoxy groups. The crystal structure of EMS was magnetite measured by XRD. The amount of the epoxy groups on the surface of EMS was measured by titration of sodium thiosulphate, and there were 0.1200 mmol epoxy groups on per gram EMS. The magnetic properties of EMS were measured by VSM. The Mr of EMS were 45 emu/g, but it had no superparamagnetic properties. Lipase was immobilized on EMS. The optimum immobilization conditions for lipase was pH 8.0 and the temperature 25℃. The optimum pH value of immobilized lipase was improved 1 pH compared with that of free lipase; The thermal stability of EMS bound lipase was improved 40% compared to free enzyme at 60℃; The residual activity of EMS bound lipase still retained 70% of its original activity after being reused 9 times.In order to confirm that the amino group of the enzyme can open the ring of epoxy on EMS and covalent coupled on EMS, lipase was directly immobilized on MS, AMS and EMS under the conditions of 25℃,PBS of pH 8.0 respectively. The coupling efficiency, recovery of activity and relative activity of MS, AMS and EMS bound lipases were investigated respectively. The results showed that the couping efficiency of the lipase immobilized on EMS was up to 90%, and recovery of activity was up to 60%, the relative activity EMS bound lipase was up to 66%. These three immobilized enzyme property indexes of EMS bound lipase were much better than that of the other MS and AMS bound lipase.These results illustrated that EMS is kind of good support to immobilize lipase.