Mesoporous Silica Magnetic Composite Microspheres: Preparation And Laccase Immobilization

Mesoporous silica magnetic composite material is an excellent biologicalenzyme immobilization carrier, due to its tunable pore size, high specific surface area,large pore volume and good magnetic response performance, which is beneficial toseparation and recovery of the composite carrier material in a magnetic field. Laccaseis a polyphenol oxidase, and possesses important applications in the field of drugtargeting delivery and enzyme immobilization. Laccase immobilized on themesoporous silica magnetic composite material can be recycled and reused after beingseparated in an applied magnetic field.In this thesis, the synthetic procedure of the mesoporous SiO₂magneticmicrospheres is as follows: the mesoporous silica shell was coated on the home-madeβ-FeOOH/SiO₂microspheres and the home-made β-FeOOH hollow microspheres byhydrolysisand condensation of tetraethoxysilane （TEOS） in the presence of a cationicsurfactant （CTAB）. The mesoporous silica magnetic composite microspheres wereobtained through calcining the composite microspheres at500℃for5h to remove thetemplate, finally calcinated in reducing atmosphere （5%H2/95%Ar （V/V）） at350℃for2h and at400℃for4h. Laccase was immobilized on the mesoporousSiO₂/Fe₃O₄/SiO₂microspheres and the mesoporous SiO₂/Fe₃O₄hollow microspheresby the physical adsorption method. The effect of reaction factors, such as, solution pH,temperature, the source of iron and silicon source concentrationon the microstructureof composite microsphereswas investigated. The adsorption behavior of laccaseimmobilization on composite microspheres were observed. The stability ofimmobilized laccase was also investigated.The results show that the coating thickness of mesoporous SiO₂/Fe₃O₄hollowmicrospheres is about50nm and average pore size of4.3nm when the concentrationof TEOS was0.053mol/L and the concentration of CTAB was0.049mol/L. Both thesurface area （772.79m²/g） and the pore volume （0.83cm³/g） of the mesoporousSiO₂/Fe₃O₄hollow microsphereas decrease to390.15m²/g and0.66cm³/g afterimmobilizing laccase respectively. At3h, the immobilization amount increases to thelargest value280.7mg/g. The immobilization amount of per gram mesoporous silicain mesoporous SiO₂/Fe₃O₄microspheres is689.24mg/g, which is much larger thanthat of MCM-41. The adsorption rate constant （k₂=0.00342g/（mg·h）） is also larger than that of MCM-41. At50℃, the the activity of free laccase reaches the maximumvalue at pH4, whereas the activity of mesoporous SiO₂/Fe₃O₄hollow microspheresachieves the maximum at pH4.5. With the increasing temperature of the reactionsystem, the laccase immobilized on the mesoporous SiO₂/Fe₃O₄magneticmicrosphere can keep a higher activity and better stability than free laccsase at thecondition of pH=3～7and60℃. The Michaelis constant of the immobilized laccase（K_m=1.046mmol/L） is less than that of free laccase, which indicates that the affinitybetween the immobilized laccase and the substnate declines slightly. The removal rateof2,4-DCP reacted with immobilized laccase is more than81.64%at6h when the2,4-DCP concentration of10mg/L. Immobilized laccase has excellent catalyticactivity.