The Decolorization And Degradation Of Wastewater Containing Orange ? Catalyzed By Immobilized Horseradish Peroxidase And Biotoxicity Study

Horseradish peroxidase(HRP;EC 1.11.1.7)is a composite glycoprotein enzyme containing iron protoporphyrin prosthetic group,which could catalyze the degradation of various aromatic compounds in the presence of H2O2.In this research,HRP was immobilized on modified polyacrylonitrile beads and polyvinyl alcohol blended film by covalent crossinglinking and entrapment respectively,which were applied in the disposal of Orange I.In chapter 2,polyacrylonitrile beads were modified with some chemicals firstly.Then HRP was immobilized on their surface by covalent crosslinking with glutaraldehyde,and initial/modified/immobilized beads were characterized by FTIR and SEM/EDS.The optimal immobilization conditions are investigated and the results are as follows:glutaraldehyde concentration is 10%;the appropriate concentration of enzyme is 1mg/mL;immobilization time is 24h;pH value is 7.0.Under the optimal conditions,the maximum amount and the retained activity are 10mg/mL and 67%.Compared to free enzyme,the immobilized enzyme expresses better thermal stability and storage stability.In chapter 3,HRP,polyvinyl alcohol and sodium alginate were mixed to obtain homogeneous solution,and the solution was stretched over a smooth plate glass.Then the glass was immersed into the solution containing 50%sodium nitrate and 1%calcium chloride to form a film,which was immobilized enzyme.The above-mentioned immobilized enzyme was characterized with SEM/EDS.The optimal immobilization conditions are investigated and the results are showed as follows:polyvinyl alcohol concentration is 10%;sodium alginate concentration is 1.2%;the appropriate concentration of enzyme is 2mg/mL;pH value is 7.0.Under the optimal conditions,the maximum amount is 0.23mg/cm2 and the retained activity is 78%.The immobilized enzyme expresses better thermal stability and storage stability when compared to free enzyme.In chapter 4,the application of free enzyme,immobilized enzyme based on modified polyacrylonitrile beads and immobilized enzyme based on polyvinyl alcohol blended film in the decolorization and degradation of Orange I were studied.When the three aforementioned enzyme are applied in the decolorization of Orange 1,the optimum conditions are:pH value are 7.0,6.0 and 6.0 in sequence;H2O2 concentration are 1.0 mM/mM dye,1.2 mM/mM dye and 1.5 mM/mM dye respectively;the enzyme concentration are 0.5mg/mL,0.09g/m and 3.0cm2/mL respectively;contact time are 40min,90min and 120min respectively;the temperature are 25?,30? and 35? in sequence.Through the study of the operation stability of two kinds of immobilized enzyme,we find that the percent decolorization of Orange I still reaches about 30%when the immobilized enzyme has been used eight for both the two kinds of immobilized HRP.L02 cells and lettuce seeds were chosen to test the changes of cytotoxicity and phytotoxicity before and after decolorization of Orange I.The results of biotoxicity tests show that both the cytotoxicity and phytotoxicity of Orange I are reduced,which demonstrates that the decolorization and degradation of Orange I with immobilized HRP has good biological safety.