Paramagnetic Functionalized Mesoporous Materials For Immobilization Of Penicillin G Acylase

Paramagnetic functionalized mesoporous materials, synthesized by combination of paramagnetic Fe3O4nanoparticles with mesoporous materials through chemical bonds, have several advantanges, such as good biocompatibility, high stability, low toxicity, easy magnetic separation and plenty of accessible functional groups on the surfaces, and thus have promising applications in the fields of catalysis, biotechnology or biomedicine, magnetic resonance imaging, data storage and environmental protection.In our works, a series of paramagnetic functionalized mesoporous materials were synthesized for immobilization of penicillin G acylase (PGA), and their textural properties and the performance of immobilized PGA were investigated. Paramagnetic functionalized mesoporous materials are easily recycled by an external magnetic field, which overcomes the obstacle of difficult separation because of smaller particle sizes of mesoporous materials. The valuable conclusions were drawn as follows:1. Paramagnetic epoxy-functionalized mesostructured cellular foams (PEMCFs) were synthesized by grafting L-cysteine modified Fe3O4nanoparticles on the outer surface of y-glycidoxypropyltrimethoxysilane (GPTMS) modified MCFs. The particle size of Fe3O4nanoparticles was controlled larger than the window size of MCFs, which decreased effect of the preparation process on the textural properties of PEMCFs as little as possible. L-cysteine modified Fe3O4nanoparticles were covalently grafted stably on the outer surface of epoxy-functionalized MCFs. PEMCFs had ordered mesoporous structures, high surface area of469m2/g, large pore volume of1.6cm3/g, large pore sizes of Dw=15.1nm and Dc=29.2nm, and paramagnetism of Ms=14emu/g. PEMCFs was used as the support for immobilization of PGA and PGA was mainly immobilized covalently on the inner surface of PEMCFs, in which high initial activity of8800U/g and high operational stability of94.5%of the initial activity after recycled for10times were achieved.2. In order to increase the enzyme activity, paramagnetic aldehyde-functionalized mesostructured cellular foams (PAMCFs) were synthesized by grafting-aminopropyltriethoxysilane (APTES) modified Fe3O4nanoparticles on the outer surface of MCFs functionalized by y-trimethoxysilylpropanal (TMSP) with short-chain groups. The particle size of Fe3O4nanoparticles was also controlled larger than the window size of MCFs. PAMCFs with the content of Fe3O4of15wt%had ordered mesoporous structures, high surface area of469m2/g, large pore volume of1.73cm3/g, large pore sizes of Dw=15.0nm and Dc=29.5nm, and paramagnetism of Ms=11emu/g. APTES modified Fe3O4nanoparticles were grafted covalently on the outer surface of aldehyde-functionalized MCFs. PAMCFs was used as the support for immobilization of PGA and PGA was mainly immobilized covalently on the inner surface of PAMCFs. PGA/PAMCFs-15showed higher initial activity of9563U/g and the operational stability of89.1%of the initial activity after recycled for10times. Effects of PGA concentration, reaction temperature and reaction pH value on the performance of immobilized PGA were investigated. The enzyme loading and the initial activity increased with increasing PGA concentration. When PGA concentration reached at1.96mg/mL, both the enzyme loading and the initial activity achieved at the maximums. The enzyme activity increased with an increase in reaction temperature. When the reaction temperature reached at45℃, the enzyme activity achieved at the maximum and then decreased with further increasing reaction temperature. The enzyme activity also increased with increasing reaction pH value. The enzyme activity kept at the maximum when the pH value ranged from7.8to10.0, and then decreased rapidly with further increasing reaction pH value.3. Oleic acid modified Fe3O4nanoparticles were prepared by co-precipitation method. Aldehyde-functionalized mesoporous silica-Fe3O4nanocomposites with aldehyde groups on the surface were prepared by co-condensation of tetraethylorthosilicate (TEOS) and y-trimethoxysilylpropanal (TMSP) in the presence of triblock copolymer Pluronic P123, NaCl and Fe3O4in neutral solution. The amount of aldehyde groups was adjusted by changing the molar ratio of TEOS and TMSP in the synthetic solution. When there was no addition of TMSP, the structure of nanocomposites was vesicle. With addition of TMSP, the structure of nanocomposites partially changed to cannular. When addition of TMSP increased toa certain content, all nanocomposites were cannular structure and Fe3O4nanoparticles were embedded among the cannular mesoporous silica layers. Fe3O4/CHO-SiO2-0.15(the molar ratio of TMSP to all silicon species was0.15) had high surface area of368m2/g, large pore volume of0.7cm3/g, large pore sizes of Dw=7.4nm and Dc=11.3nm, and paramagnetism of Ms=35.1emu/g. The nanocomposites were used as supports for immobilization of PGA. The intial activity of PGA/Fe3O4/CHO-SiO2-0.15was6231U/g and the operational stability was91.0%of the initial activity after recycled for10times.4. Fe3O4microspheres were prepared by the solvothermal method. After Fe3O4microspheres were treated with hydrochloric acid, paramagnetic aldehyde-functionalized Fe3O4@CHO-mSiO2core-shell microspheres were prepared by co-condensation of TEOS and TMSP in the NaCl-ethanol-water solution. With addition of TMSP, the thickness of SiO2shell reduced due to the steric hindrance effect of TMSP. Fe3O4@CHO-mSiO2-0.15(the molar ratio of TMSP to all silicon species was0.15) had surface area of117m2/g, pore volume of0.18cm3/g, pore sizes of Dw=6.3nm and Dc=9.1nm, and paramagnetism of Ms=44.7emu/g. Fe3O4@CHO-mSiO2-0.15was used as the support for immobilization of PGA. With addition of TMSP, the shell thickness of core-shell microsphere became thinner, and the enzyme loading and the intial activity reduced. PGA/Fe3O4@CHO-mSiO2-0.15had the intial activity of1887U/g and the operational stability was83.7%of the initial activity after recycled for10times.