Silanization Of Amino Group Coated Magnetic Microspheres And Its Application Study On Immobilizing Enzymes

Nano-Magnetic Particles(NMPs) were one of the most promising magnetic materials, which emerged,developed and expanded after the 70's of 20 century.Because of their well magnetic responsibility,small particle size,larger specific surface area and well biocompatibility,they were widely used in every fields of life science,such as cell isolation,targetable drug delivery,immunodetection,purification of protein and nucleic acid and enzyme immobilization,etc.The main content of this article was the preparation and characterization of amino group coated magnetic microspheres by silanization and its application of enzyme immobilization.1.The magnetic Fe₃O₄ nanoparticles were prepared by the improved chemistry coprecipitation method.The average diameter of the Fe₃O₄ nanoparticles was about 6nm-12nm as confirmed by TEM.The dispersivity of Fe₃O₄ nanoparticles was very good. Superparamagnetic particles's aturation magnetization was 62.6 emu/g,XRD patterns of the magnetic nanoparticles showed that the samples exhibit identical cubic inverse spinel structures.We assigned the absorption band at 578cm^-1 and 3421cm^-1 tally with the standard Fe₃O₄ characteristic absorption peaks.2.The magnetic Fe₃O₄/SiO₂ composite nanoparticles were prepared by sol-gel method in ethonal/water solution using Fe₃O₄ nanoparticles as core,ammonia as catalyst and TEOS as precursor.The average diameter of the composite nanoparticles was about 20nm-30nm as confirmed by TEM.The dispersivity of the composite nanoparticles was very good.Superparamagnetic particles' saturation magnetization was 55.2 emu/g.XRD patterns of the magnetic nanoparticles showed that the samples exhibit identical cubic inverse spinel structures.So the low angle peak present in the composite nanoparticles suggested the silica was without much indication of ordering.We assigned the absorption band at 1097cm^-1 and 950 cm^-1 tally with the standard SiO₂ characteristic absorption peaks.3.The amino group coated magnetic microspheres were prepared by silanization, using Magnetic Fe₃O₄/SiO₂ composite nanopartieles as core.The average diameter of the microspheres was about 40nm as confirmed by TEM.Its aturation magnetization was 50 emu/g.It was superparamagnetic and -NH₂ groups coated on the surface. 4.The silanized amino group coated magnetic microspheres were used to immobilize endoglucanase.The reaction condition for the immobilization of enzyme was optimized and the similarities and differences between immobilized enzyme and free enzyme were investigated.The result showed that,the optimum condition for immobilization of endoglucanase were:the glutaraldehyde concentration 3%,the reaction time 6 hours,respectively;the optimal reaction condition for immobilized enzyme was pH 7.0,reaction temperature 60℃,Km value 24.39 mg/mL,enzyme recovery 26.3%.The surplus enzyme activity of immobilized enzyme was 56.1%of incipient enzyme activity after resused for 5 times,it was proved that the amino-silane treated magnetic silica nanospheres could enhance the thermo stability,the pH endurance and the operation times of the enzyme compared to free enzyme.5.The silanized amino group coated magnetic microspheres were used to immobilize papain.The reaction condition for the immobilization of enzyme was optimized and the similarities and differences between immobilized enzyme and free enzyme were investigated.The result showed that,the optimum condition for immobilization of papain were:the ratio of enzyme and carrier 10 mg/g,the glutaraldehyde concentration 5%,the reaction time 4 hours,respectively;the optimal reaction condition for immobilized enzyme was pH 6.8,reaction temperature 70℃,Km value 34.48 mg/mL, enzyme recovery 46.5%.The surplus enzyme activity of immobilized enzyme was 60.4% of incipient enzyme activity after resused for 10 times,it was proved that the amino-silane treated magnetic silica nanospheres could enhance the thermo stability,the pH endurance and the operation times of the enzyme compared to free enzyme.6.In order to obtain nutritious mung bean polypeptide,the immobilized papain was introduced to hydrolyze the mung bean protein and the degree of hydrolysis of mung bean protein was detect.The result showed that the degree of hydrolysis of mung bean protein is 8.5%.After hydrolyzed by immobilized papain,the mung bean protein was improved with regard to its viscosity,solubility,air bubble,emulsibility,emulsification stability.