Synthesis Of Magnetic Nano-Spherical Polyelectrolyte Brush And Immobilized Enzyme Research

Enzyme is a kind of biocatalytic function macromolecule substances,which can catalyze the specific substrate,having high efficiency,specificity,mildness and other features.Enzyme activity is greatly affected by the environment and it is difficult to widely used in industrialization.The immobilized enzyme overcomes the shortcomings of the free enzyme,showing a series of advantages such as high storage stability,easy separation and recovery,repeated use,continuous operation controlling,simple process and so on.However,conventional methods(such as chemical immobilization)immobilized enzymes have resulted in a great loss of enzyme activity.Polyelectrolyte brushes have a good interaction with protein molecules and do not affect the structure of proteins.So it has a good application prospect in the immobilization of the enzyme.In this paper,the protease was immobilized by magnetic Nanometer spherical polyelectrolyte brush(MSPB),and the enzymatic properties of MSPB immobilized enzyme were investigated for the first timeThe main work of this paper are as follows:1.Preparation of magnetic nano-spherical polyelectrolyte brush(MSPB):Magnetic polystyrene latices(MPL)coating Fe304 were prepared by fine emulsion polymerization.Then,benzoin acrylate(BA)was used as photoinitiator,acrylic acid as raw material,MSPB was prepared by photoemulsion polymerization.The influence of the amount of BA,the amount of acrylic acid and the reaction time on the chain length was investigated.MSPB was characterized by thermogravimetry(TG),scanning electron microscopy(SEM)and FTIR Spectrascopy.The results showed that the optimum reaction conditions were BA:styrene molar ratio 0.1:1,acrylic acid/styrene molar ratio 0.9:1,time 2.0 h.MPL particle size was 152nm,MSPB chain length was 160.5nm,MSPB magnetic content was 30%.2.Immobilization conditions of MSPB immobilizing the protease:The conditions of MSPB immobilized protease were investigated and optimized by MSPB as carrier.The effects of pH,ionic strength and time on enzyme activity were investigated.The results show that the best immobilized pH was 6.0,the best ionic strength was 50mM NaCl,and the best immobilization time was 16h at 4?.The adsorbed amount of MSPB was 42.9mg/g,and the immobilized activity was 1.85×104U/g.3.The enzymatic properties of MSPB fixed protease were studied.The effects of pH and reaction temperature on the enzyme activity of MSPB immobilized enzyme were investigated and compared with the free enzyme.The optimal enzymatic reaction PH of MSPB immobilized enzyme was 9.0,the optimum reaction temperature was 60 ?,the Michaelis-Menten constant Km and the catalytic efficiency constant Kcat were 7.72×103?g·mL-1 and 2.81min-1,respectively.While the optimal reaction pH of free enzyme was 9.0,the optimum reaction temperature was 50 ? and the catalytic efficiency constant Kcat was 1.18×104?g·mL-1 and 2.33min-1,respectively.The immobilized enzyme showed a decrease in Km of immobilized enzyme,The affinity of the matrix was enhanced,and the catalytic efficiency constant Kcat was increased.It was found that the stability of the enzyme was improved after immobilization,but the acid and alkali resistance and thermal stability were decreased.By changing the conditions of use,the enzyme activity could still be 57%after using 7 times of immobilized enzyme.