Cross-linking Assisted Immobilization Of Peroxidase For The Decoloration Of Dyes

1.Horseradish peroxidase was successfully immobilized on a ZnO nanowires/macroporous SiO₂ composite support through in-situ cross-linking method using adsorbed epoxy compounds as the cross-linker.The effect of various conditions was optimized on the immobilization including the concentration and isoelectric point of HRP,medium pH,structure of cross-linkers and crosslinking time.Among three epoxy compounds diethylene glycol diglycidyl ether?DDE?was the best cross-linker,with which the loading amount of HRP with pI of 5.3 reached as high as 118.1mg/g and specific activity was up to 14.9 U/mg-support.The catalytic performance of immobilized HRP to decolorize anthraquinone dye from aqueous solution was explored by using Reactive Blue19 and Acid Voilet 109 as model substrates.The results indicated that the immobilized HRP exhibited high decolorization efficiency and good reusability.A complete decolorization of these two dyes has been realized within 2³ hours by using this new biocatalysis system.2.A ZnO nanowires/macroporous SiO₂ composite was used as support to immobilize horseradish per-oxidase?HRP?by in-situ cross-linking method.Using diethylene glycol diglycidyl ether?DDE?as a long-chained cross-linker,it was adsorbed on the surface of ZnO nanowires before reaction with HRPs,the resulted composite was quite different from the traditional crosslinking enzyme aggregates?CLEAs?on both structure and catalytic performance.The immobilized HRP showed high activity in the decolorization of azo dyes.The effect of various conditions such as the loading amount of HRP,solution pH,temperature,contact time and concentration of dye were optimized on the decolorization.The decolorization percentage of Acid Blue 113 and Acid black 10 BX reached as high as 95.4% and 90.3%,respectively.The immobilized HRP gave the highest decolorization rate under dye concentration as 50 mg/L and reaction time of 35 min.The immobilized HRP exhibited much better resistance to temperature and pH inactivation than free HRP.The storage stability and reusability were greatly improved through the immobilization,from the decolorization of Acid blue 113 it was found that 80.4% of initial efficiency retained after incubation at 4 ?C for 60 days,and that 79.4% of decolorization efficiency retained after 12 cycles reuse.3.Chloroperoxidase?CPO?and horseradish peroxidase?HRP?were successfully coimmobilized on a ZnO nanowires/macroporous SiO₂ composite support through in-situ crosslinking method.Using a 1/1 mixing of CPO and HRP in buffer solution,the loading amount of enzymes reached 132.4 mg/g-support,and the total specific activity was up to 15.7 U/mg-support.Enzyme activity assays showed that the co-immobilized enzymes have catalytic advantages over the immobilized CPO or HRP.In the decolorization of azo dyes the co-immobilized enzymes exhibited higher catalytic activities under wide range of pH,temperature and H2O₂ concentration.Under the optimum conditions the decolorization of Direct Black 38 and Acid Blue 120 exceed92%.A complete decolorization of these two dyes has been realized within 3 hours.The storage stability and reusability were also greatly improved through the co-immobilization.The coimmobilized CPO and HRP have been proved to be a robust and stable biocatalyst which can be used in dye-containing waste water treatment.