Separation of an isoenzyme of polyphenol oxidase from Duranta plumieri by expanded bed chromatography.
Aqueous extracts of seeds of Duranta plumieri were found to be rich in polyphenol oxidase activity. The anion-exchange chromatography of the crude extract on Streamline DEAE resolved the activity into three fractions. The major fraction (77% of the total activity) was further purified by treating it with concanavalin A-agarose in the batch mode. The enzyme preparation eluting with alpha-methylmannoside showed a single band on SDS-PAGE. The minimum molecular weight corresponded to 14,000 Da. The K(m) and V(max) of this isoenzyme were found to be 7.1 mM and 73.5 U ml(-1) min(-1) respectively. The k(cat) of this isoenzyme was calculated to be 8235 s(-1). The isoenzyme also showed the phenomenon of latency and the activity could be enhanced by 196% on heating it at 55 degrees C for 30 min.
Roy
Ipsita
I
Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi, 110 016, India.
Sharma
Shweta
S
Gupta
Munishwar Nath
MN
D004156:Catechol Oxidase / Q000302:isolation & purification*; D002847:Chromatography, Agarose; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003208:Concanavalin A; D007527:Isoenzymes / Q000302:isolation & purification*; D007700:Kinetics; D037121:Plant Lectins; D012639:Seeds / Q000737:chemistry; D012685:Sepharose
D007527:Isoenzymes; D037121:Plant Lectins; D003208:Concanavalin A; D012685:Sepharose; D004156:Catechol Oxidase