Extraction, Purification And Application Of Polyphenol Oxidase From Cassava Leaves

The extraction, purification and immobilization of polyphenol oxidase obtained from Cassava Leaves were studied. The present investigation was also aimed to research its application in the degradation of wastewater containing benzene ring. The results were as follows:The extraction of polyphenol oxidase from Cassava Leaves was studied and the results showed that:effects of homogenate extraction, acetone powder and assisted extraction on polyphenol oxidase extraction were compared and the results indicated that sample pretreatment by acetone powder method followed by assisted extraction obtained the highest yield of polyphenol oxidase. The best extraction condition was:the ratio of phosphate buffer (pH6.6) to leaf material was1:6, and the extraction time was6h.The purification of polyphenol oxidase from Cassava Leaves was obtained and the results indicated that:the polyphenol oxidase was precipitated by ammonium sulfate50%-80%, and dialyzed to remove salts using a dialysis bag of8-14KDa molecular entrapment quantity, and then concentrated by reverse dialysis. Polyphenol oxidase was purified by gel permeation chromatography of Sephadex G-75superfine and ion-exchange chromatography of DEAE Sephadex-A50. The polyphenol oxidase was deter mined by SDS-PAGE, which showed that the molecular mass of the enzyme is66KDa.Optimal conditions for the immobilization of polyphenol oxidase from Cassava Leaves was that three mesoporous silica immobilization carriers namely MCM-41, SBA-15, and SBA-16were used for the immobilization of polyphenol oxidase. It was concluded that the best immobilized conditions were as follows:the load of the enzyme (500mg/mL) on SBA-16(10mg) was3mL, and then immobilized in phosphate buffer pH7.2for3h, after that cross-linked with0.3%glutaraldehyde for2h. The thermal, pH and operation stability of polyphenol oxidase were significantly improved by immobilization. However, the Km value of the immobilized polyphenol oxidase from Cassava Leaves was lower (Km2=0.12135) than that of the free enzyme (Km1=0.13247), which indicated that affinity of the enzyme with substrate decreased.The application of the polyphenol oxidase immobilized by SBA-16in the degradation of wastewater containing benzene ring was also studied. The results sμggested that:the degradation ratios of catechol, congo red, phenol,2,4-dichlorophenol were99.36%(10h),96%(2h),93.41%(12h), and83.6%(7h), respectively.