Immobilization Of Horseradish Peroxidase On The Difunctional Of Epoxy Carrier And Amino Carrier And Catalytic Oxidation Properties Of Immobilized Enzyme

Enzyme is a kind of highly efficient, highly specific biological catalyst. With thedevelopment of science and technology, enzyme is used widely. However, the poor stability,sensitive to high temperatures, organic solvents and extreme pH, the use of enzymes islimited. The emergence of enzyme immobilization technology opens up a new prospect forthe application of enzyme. Study and application of enzyme immobilization are concernedin the industrial production, chemical analysis, medical, environmental engineering, andmany other field.Horseradish peroxidase （HRP） is a kind of glycoprotein compound enzyme containingprotoheme prosthetic group （iron protoporphyrin）, and possessing special catalytic oxidationproperties. It can catalyze oxidation the aromatic compounds under relatively broad condition.Especially, the catalytic effect is better for the oxidation reaction of phenol and anilinecompounds in water system. Industrial waste water containing phenol has serious harm to theecological balance of water environment and human health. Immobilized horseradishperoxidase used to treat phenolic wastewater is an important research subject inenvironmental protection and enzyme engineering field. Researching and preparingimmobilized horseradish peroxidase carrier with good performance, and applied to catalyticoxidation of wastewater containing phenol degradation process, is an important environmentalmanagement and environmental protection technology.In this paper, based on molecular designing, two kinds of novel difunctional epoxycarrier are prepared, and the properties of immobilized horseradish peroxidase andimmobilization mechanism are studied deeply. The immobilization of horseradish peroxidaseused amino carrier is carried out, and enzymology properties are studied. The immobilized horseradish peroxidase is used for catalytic oxidation of phenol, and the catalytic oxidationproperties are researched. This topic has significant scientific significance in the enzymeimmobilization field, and important reference value for the enzyme catalysis technologyapplication in environmental engineering.Firstly, the glycidylmethacrylate （GMA） is grafted on silica surface using the "graftingfrom" method, and grafted particles PGMA/SiO₂are prepared. By ring-opening reaction ofethylenediamine （EDA） and epoxy groups, ethylenediamine are bonded on the particlesurface, resulting in preparation of the difunctional composite carrier EDA-PGMA/SiO₂.The immobilization of horseradish peroxidase on the difunctional on composite carrierEDA-PGMA/SiO₂is carried out using covalent coupling method. The effect of twosecondary valence bond forces （electrostatic interaction and hydrophobic interaction） in theimmobilization process of HRP is studied focused, and mechanism is researched. Theresults show that the zeta potential keeps positive value in larger range because of theprotonation effect of amino groups on the surface of EDA-PGMA/SiO₂composite carrier.The electrostatic interaction between EDA-PGMA/SiO₂and horseradish peroxidase has asignificant role to promote the enzyme immobilization when pH of8.5. When bonding rateof ethylenediamine is30%, the immobilized enzyme coupling ratio and the specific activityhas the highest,57.85%and0.257U g^-1·min^-1, respectively. Hydrophobic interaction canproduce significant effect on immobilized horseradish peroxidase using chemical method.When the grafted PGMA/SiO₂particles as the carrier, adding NaCl electrolyte in solutioncan effectively promote the hydrophobic interactions between enzyme protein and thecarrier, and enhance coupling rate and specific activity of immobilized enzyme.Secondly, graft particles PGMA/SiO₂is chemically modified using sodium4-hydroxybenzenesulfonate （SHBS）, and anionic difunctional composite carrierSHBS-PGMA/SiO₂is prepared. The immobilization of horseradish peroxidase on thedifunctional on composite carrier SHBS-PGMA/SiO₂is carried out using covalent couplingmethod. The effect of electrostatic interaction in the immobilization process of HRP isstudied focused, and mechanism is researched. The results show that the zeta potential keeps negative value in larger range because of abundant sulfonic acid ions on the surfaceof SHBS-PGMA/SiO₂composite carrier. The electrostatic interaction betweenSHBS-PGMA/SiO₂and horseradish peroxidase has a significant role to promote theenzyme immobilization when pH of6.0. When the bonding rate of SHBS is18%, theimmobilized enzyme coupling ratio and the specific activity has the highest,63.64%and0.27U g^-1·min^-1, respectively.Catalytic oxidation experiments show that, the catalytic activity of two kinds of epoxycarrier immobilized horseradish peroxidase is not high. So, the amino carrier（diethylenetriamine modified crosslinked polyacrylonitrile microsphere, DETA-CPAN）modified using glutaraldehyde as coupling agent, is used to immobilize horseradishperoxidase. The optimal enzyme immobilization conditions are investigated. The resultsindicate that the optimal immobilized conditions as follows: the temperature is30℃,immobilization time is8h, glutaraldehyde concentration is0.02mol L-1, pH value is8.0,and the appropriate amount of enzyme solution is10ml. In optimal conditions, the couplingratio of HRP and the specific activity of immobilized enzyme can reach41.96%and0.32U g^-1·min^-1, respectively.Finally, the enzymatic properties of immobilized enzyme prepared at the optimalconditions are studied. The catalytic oxidation performance to phenol is investigated. Theenzymology properties results show that the optimal pH of the immobilized enzyme is7.0,and the immobilized enzyme has good pH stability. The optimum temperature is30°C, andthe immobilized enzyme remains higher activity within20°C to50℃. The apparentmichaelis constant of free enzyme and immobilized enzyme are1.35×10-3mol L-1,4.05×10-2mol L-1, respectively. This indicates that the affinity between immobilized enzymeand the substrate is lower than that of free affinity between enzyme and substrates. Theexperimental results of catalytic oxidation degradation of phenol show that the catalyticeffect is the best as the ratio of oxidant H₂O₂and substrate phenol of1:1. The removal rateof phenol can reach28.57%at temperature of25°C and time of2h when a small amount（0.5g） of immobilized enzyme used. The removal rate of phenol increases with the increase of enzyme amount. The removal rate of phenol can reach56%when2g of immobilizedenzyme used. The immobilized enzyme still has high enzyme activity after5cycle use andthe immobilized enzyme can be used in recirculation.