In-situ Cross-linking Of Peroxidase Based On Nano-composite Support And Catalytic Degradation Of Synthetic Dyes

In this thesis ZnO nanowires/macroporous SiO₂ composite was used as support and an anionic bi-epoxy compound was used as cross-linker,peroxidase including chloroperoxidase（CPO）and horseradish peroxidase（HRP）were immobilized through in-situ cross-linking method.The obtained nano-biocatalysts were used to catalyze the degradation of synthetic azo dyes.1.A novel ZnO nanowires/macroporous SiO₂ composite was used as support to immobilize chloroperoxidase（CPO）by in-situ cross-linking method.An anionic bi-epoxy compounds was synthesized and used as along-chained anionic cross-linker,it was adsorbed on the surface of ZnO nanowires through static interaction before reaction with CPO,creating a new approach to change the structure,property and catalytic performance of the produced cross-linking enzyme aggregates（CLEAs）of CPO.The immobilized CPO showed high activity in the decolorization of three azo dyes.The effect of various conditions such as the loading amount of CPO,solution pH,temperature,and dye concentration were optimized on the decolorization.Under optimized conditions the decolorization percentage of Acid Blue 113,Direct Black 38 and Acid Black 10 BX reached as high as 95.4%,92.3%and 89.1%,respectively.The immobilized CPO exhibited much better thermostability and resistance to pH inactivation than free CPO.The storage stability and reusability were greatly improved through the immobilization.It was found from the decolorization of Acid Blue 113 that 83.6%of initial activity retained after incubation at 4°C for60 days,and that 79.4%of decolorization efficiency retained after 12 cycles reuse.2.Chloroperoxidase（CPO）and horseradish peroxidase（HRP）were co-immobilized on ZnO nanowires/macroporous SiO₂ composite support through in-situ cross-linking method.An anionic bi-epoxy cross-linker was used by adsorption on the surface of ZnO nanowires before cross-linking.The cross-linking was carried out under proper conditions such as solution pH:6.5,reaction temperature:15°C and reaction time:15 h.The loading ratio of CPO and HRP can be adjusted by changing the initial concentration of CPO and HRP.Using a 1/1 mixture of CPO and HRP resulted the co-immobilized enzyme with loading amount of 79.6 mg_CPO/g_support and 52.8 mg_HRP/g_support,and the total specific activity was up to 15.7 U/mg_support.In the decolorization of azo dyes the co-immobilized CPO（60%）/HRP（40%）exhibited higher catalytic activity in very wide ranges of pH,temperature and H₂O₂ concentration.By using this robust biocatalyst complete decolorizations ofAcid Blue 120 and Direct Black 38 have been realized within 3 hours of reaction.The co-immobilized enzyme also showed good stability in 60 days of storage and excellent reusability in20 times of repeat using.