| By comparison of enzyme immobilization yield and catalytic performance of immobilized enzyme, aluminum pillared interlayered clay calcined at 773K was selected out to be used as the most appropriate support for immobilization of Horseradish Peroxidase among montmorillonite and its inorganic or organic modified samples. Thus, the immobilization conditions of HRP on the material such as dose of support, pH value, ionic strength and immobilization time were investigated. The support and immobilized enzyme were characterized by way of DTA, XRD, FTIR, N2-adsorption and desorption. The mechanism of enzyme immobilization was discussed. The catalytic performance of the immobilized HRP applied in the treatment of phenolic wastewater and enzymatic reaction mechanism were also studied in detail. The results showed that Al-PILC was a promising material used as the support for enzyme immobilization. Enzyme could be efficiently attached to the support with high immobilization yield under simple conditions. Compared with the soluble enzyme, HRP immobilized on Al-PILC had better storage stability and broader appropriate operating pH range, Moreover, immobilized HRP could be reused. When initial concentrations of phenol was 250mg/L, as well as the mass ratio of PEG/phenol and the molar ratio of hydrogen peroxide/phenol were 0.4 and 1.5, respectively, Phenol could be removed over 95% of initial value after three hours retention time. The reaction mechanism of oxidation of phenolic compound catalyzed by HRP could be considered as ping-pong kinetics. Free enzyme exhibited higher affinity to substrate than immobilized enzyme. However, the maximum reaction velocity of oxidation of phenol catalyzed by bound HRP was greater than that obtained from free HRP. The two shape of enzyme took on similar difficulty in terms of phenolic wastewater treatment.
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