Preparation And Properties Of Magnetic Nano-Core-shell Structured Immobilized Enzymes

Fe₃O₄ magnetic nanoparticles have been used as an enzyme carrier due to easy separation and high specific surface area.However,the disadvantages of easy agglomeration and oxidation limit their application.In addition,although the enzyme immobilization on the surface of the carrier can improve the stability of the enzyme,the carrier cannot directly protect the enzyme from external extreme conditions,making the enzyme easily inactivated.In this study,Si O2 was used as a core-shell material to coat magnetic nanoparticles by sol-gel method to prepare Fe₃O₄@Si O2core-shell magnetic nanospheres with good dispersibility,oxidation resistance and easy recovery.The immobilized catalase was prepared using the Fe₃O₄@Si O2 coreshell magnetic nanospheres as a carrier.By means of molecular self-assembly technology,the nanocoating of organic-inorganic polymer materials is formed on the surface of the immobilized enzyme,an enzyme shielding protection strategy is established,and the catalytic performance of the enzyme is significantly improved.The specific research content is as follows:?1?Synthesis of Fe₃O₄ Magnetic Nanoparticles and Preparation of Core-shell CarriersThe Fe₃O₄ magnetic nanoparticles were prepared by direct coprecipitation,semi reduced coprecipitation and solvothermal method.The Fe₃O₄ magnetic nanoparticles were coated by sol-gel method,and the tetramethoxysilane?TMOS?concentration and the ammonia concentration required for the silica-coated Fe₃O₄ magnetic nanoparticles were optimized through a single-factor experiment.The prepared Fe₃O₄ magnetic nanoparticles and Fe₃O₄@Si O2 were characterized.The results showed that the Fe₃O₄ magnetic nanoparticle prepared by hot solvent method had spherical shape and good homogeneity compared with other methods;the optimal condition for preparing Fe₃O₄@Si O2 composite particles is 0.5 mol/L ammonia,and 0.06 mo/L TMOS.The characterization results showed that the prepared Fe₃O₄ magnetic nanoparticles were superparamagnetic,and Si O2 was evenly coated on the surface of Fe₃O₄ magnetic nanoparticles.?2?Preparation,Properties and Characterization of Core-Shell Magnetic Nano Immobilized Catalase Containing Silica NanoparticlesA core-shell magnetic immobilized catalase with a silica nanocoating was prepared,and the immobilized enzyme coated with silica gel was characterized,and various catalytic properties of the immobilized enzyme were determined.The optimized preparation conditions were as follows: carrier concentration was 4 mg/ml,enzyme concentration was 0.7 mg/ml,glutaraldehyde concentration was 0.1%,glutaraldehyde treatment time was 10 min,immobilized enzyme time was 50 min.Under the optimized conditions,the enzyme activity recovery increased from 28% to85%.The characterization results showed that catalase had been successfully immobilized on the Fe₃O₄@Si O2,and a layer of silica nanocoating has formed on the surface of the immobilized enzyme.The immobilized enzyme exhibited strong paramagnetic properties.The core-shell magnetic immobilized enzyme containing silica nanocoating was more p H-stability,thermostability,denaturant tolerance and storage stability than immobilized enzyme without the silica nanocoating and free enzyme.The residual activity of immobilized enzyme without the silica nanocoating retained only 27.84% of the original enzyme activity after repeated use 9 times,while the immobilized enzyme with silica nanocoating still retained 46% of the original enzyme activity.?3?Preparation of core-shell structure-immobilized catalase particles with metalorganic film nanocoating and dual enzyme co-immobilizationThe single factor method is used to optimize number of metal-organic film on immobilized enzymes.Metal-organic film was formed by self-assembly of Fe3+ and tannic acid on the surface of immobilized catalase particles with core-shell structure and on the surface of co-immobilized enzymes?alpha-amylase and glucoamylase?.The immobilized enzyme was characterized and its catalytic performance was studied.The results showed that catalase,?-amylase and glucoamylase were successfully immobilized on Fe₃O₄@Si O2 support.A layer of metal-organic film nanocoating was formed on the surface of all immobilized enzymes.When the number of coating layers is three,the immobilized enzyme exhibited the highest activity recovery.When alpha-amylase and glucoamylase were co-immobilized at a mass ratio of 2:3,their ability to catalyze the hydrolysis of starch was 26% higher than that of single-amylase,and 34% higher than that of monosaccharidase.Moreover,the thermal stability,p H stability,and reusability of immobilized enzymes with metal-organic film have been significantly improved.