Immobilization Of Horseradish Peroxidase And Its Applications On The Catalytic Oxidation Of Wastewater Containing Phenol

Horseradish peroxidase(HRP,EC 1.11.1.7)is a kind of relatively stable glycoprotein composite oxidase,which containing a dark brown iron protoporphyrin agon.When hydrogen peroxide was used as hydrogen donor,horseradish peroxidase could catalyze a variety of phenolic compounds,and it is widely used in the treatment of the wastewater containing phenol.In this paper,horseradish peroxidase was immobilized on the polyacrylonitrile beads by covalent crosslinking with glutaraldehyde,the enzymatic properties of immobilized horseradish peroxidase and immobilization mechanism were also studied deeply.In order to explore the biocatalytic performance of the immobilized horseradish peroxidase,the HRP-immobilized beads were used in the oxidative degradation of phenol in water.In chapter two,modified PAN-based beads,as carriers,were used for the horseradish peroxidase by covalent crosslinking with glutaraldehyde,and the initial and immobilized beads were characterized by SEM/EDS and FTIR.The optimal enzyme immobilization conditions are investigated and showed as follows:glutaraldehyde concentration is 10%;the appropriate concentration of horseradish peroxidase is 1 mg/mL;immobilization time is 20h,pH value is 7.0.Under this optimal conditions,the maximum amount and the retained activity of immobilized HRP can reach 20mg/gbeads and 70%,respectively.In chapter three,we investigated the enzymatic properties of free and immobilized horseradish peroxidase.Results are as follows:the optimal pH of free and immobilized enzyme are 6.0,7.0,respectively;Optimum temperature have no obvious changes,both are 25 ?;The affinity between immobilized enzyme and the substrate is lower than that of free between free enzyme and substrates.In addition,when the concentration ratio of hydrogen peroxide to phenol is 1:1,the oxidation removal efficiency of phenol is highest by using immobilized enzyme.The experimental results show that HPR-immobilized beads can be highly effective in removal of phenol,even completely oxidation remove phenol from wastewater.What's more,after immobilized,the immobilized horseradish peroxidase appears better operating stability and storage stability than free enzyme.In chapter four,the biological safety of the immobilized enzymatic oxidation of phenol was evaluated by MTT methods.Experimental results show that the treated phenol solutions gave an improved relative survival rate of cells compared to corresponding phenol solutions.In addition,the results showed that the phenol could deactivate/block the HeLa cells in the S phase,and ultimately resulting in cell apoptosis,this may because actions of phenol could lead to cell cycle protein(cyclin E)denaturation.Finally,the FFT-base protein docking method was used to simulate the binding of phenol and Cyclin E.The docking data showed that phenol could bind on the cyclin E with relative binding energy-124.43 kJ/mol,and this binding mainly relied on the hydrogen bond.