Preparation Of Magnetic Nanoparticles And Research On Immobilization Of Marine Lipase ADM47601

Lipase （glycerol ester hydrolase, Lipase EC3.1.1.3）, an important biologicalcatalyst, which can catalyze a variety of reactions and is widely used in the food,chemical and pharmaceutical industry. The catalytic technology of free lipase is verymature, but taking into account the issues of can not be reused, difficult to achieve aprocess of continuous. Magnetic nanoparticles were new functional materials, havegradually developed after1970s, which have broad application prospect. As theproperties of superparamagnetic, high saturation magnetization, anisotropy, highdispersion and biocompatibility properties, Magnetic nanoparticles were widely usedin various fields of biotechnology, such as enzyme immobilization, drug targeting anddrug transport, magnetic fluid, contrast media, immunoassay, cell separation,biosensors, separation and purification of proteins and nucleic acids, and cancerdiagnosis. The technology of magnetic nanoparticles immobilized enzyme not onlymakes the enzyme be reused and achieve continuous process, but also make effectiveseparation of enzyme and substrate. So magnetic nanoparticle used immobilizedenzyme has a broad application prospect.This thesis prepared amino nanomicrospheres, and carried out the study ofmagnetic nanomicrospheres immobilized marine lipase ADM47601. It laid thefoundation for the development of new magnetic nanoparticles and the application ofmarine lipase ADM47601. The main contents for this thesis were listed as belows:1. Magnetic Fe₃O₄nanoparticles （MNP） were synthesized by chemicalco-precipitation method, after a series of characterization, The results showed that:The average diameter and saturation magnetization were found to be7.6nm and47.87emu/g respectively. And the synthetized magnetic Fe₃O₄nanoparticles showedsuperparamagnetism.2. This thesis took magnetic Fe₃O₄nanoparticles as nucleus,3-aminopropyl triethoxysiliane （APTES） as coupling agents, and the amino groups were introducedonto the surface through two different strategies:（1） Magnetic Fe₃O₄@SiO₂-NH₂nano microspheres were prepared via two steps. Firstly, magnetic Fe₃O₄nanoparticleswere modified by tetraethyl orthosilicate （TEOS）, and magnetic Fe₃O₄@SiO₂compound particles with silicon hydroxyl on the surface were prepared. Secondly,The Fe₃O₄@SiO₂compound particles were modified by3-aminopropyltriethoxysiliane （APTES）, and the amino groups were introduced on the surface,formed Fe₃O₄@SiO₂-NH₂nano microspheres.（2） Fe₃O₄@NH₂nano microsphereswith amino groups were prepared by coating Fe₃O₄nanoparticles with3-aminopropyltriethoxysiliane （APTES） as coupling agents. The two products were preparedthrough the characterization analysis made by using Transmission ElectronMicroscope （TEM）, Fourier transform infrared spectrometer （FT-IR）, X-raydiffraction （XRD）, superconducting quantum interference device （SQUID）andthermogravimetric analyses （TGA） respectively. The results showed that: The averagediameters of the synthetic materials were about8nm. All of the two particlesexhibited good crystallization performance, apparent superparamagnetism; Thesaturation magnetization and amino content of Fe₃O₄@SiO₂-NH₂and Fe₃O₄@NH₂were19.5emu/g and36.3emu/g,20%and3%, respectively. MagneticFe₃O₄@SiO₂-NH₂nano microspheres had better particle size uniformity and higheramino content compared with Fe₃O₄@NH₂nanoparticles. So, MagneticFe₃O₄@SiO₂-NH₂nano microspheres were selected to immobilize lipase.3. Magnetic Fe₃O₄@SiO₂-NH₂nano microspheres were used as the carrier toimmobilize the marine lipase ADM47601and the immobilizing conditions wereoptimized. The optimal conditions of magnetic nanomicrospheres Fe₃O₄@SiO₂-NH₂immobilized marine lipase ADM47601were: the concentration of protein was0.125mg/ml,30℃, pH8.0,10h, then got the maximum activity recovery of immobilizedmarine lipase87.7%, and the adsorption quantity of protein reached23.7mg/g, by thetime.4. Using the immobilized enzyme under the optimum condition as the sample, wechoose p-NitroPhenyl-Laurate（pNPL） as substrate. A study was conducted on theoptimum condition of enzymatic reaction, The result showed the optimum catalytictemperature was40℃and pH8.0of immobilized lipase. Immobilized lipase couldretain42%activity after60℃for1h, compared with10%of free lipase. Theactivity of the immobilized and free lipase showed most stable in the pH9.0buffer under30℃for24h, immobilized enzyme had94%residual activity, compared with56%of free lipase. Immobilized lipase could retain81%activity under25℃for30days, compared with59%of free lipase, the storage half-time of the immobilizedenzyme and free lipase were99days and39days under25℃in pH9.0buffer,respectively. Immobilized lipase were reused8times, the activity was still44%, theoperating half-time was7times. The Michaelis-Menten kinetic constants wereevaluated for the immobilized and freely soluble lipase, they were0.79mg/ml and0.23mg/ml, respectively.