Purification and Characterization of Polyphenol Oxidase from Japanese Butterbur (Petasites japonicus)

Abstract

　Polyphenol oxidase (o-diphenol: oxygen oxidoreductase, EC 1.10.3.1, PPO) in Japanese butterbur was purified about～151-fold with a recovery rate of 11.3% by acetone fractionation, ion exchange chromatography, hydrophobic chromatography, and gel filtration. The purified enzyme appeared as a single band on PAGE and SDS-PAGE. The molecular weight of the enzyme was estimated to be about 26,000 and 25,000 by gel filtration and SDS-PAGE, respectively. The purified enzyme quickly oxidized chlorogenic acid and (－) -epicatechin. The Km values of the enzyme were 0.14 mM for chlorogenic acid (pH 5_.0, 30℃) and 0.7 mM for (－) -epicatechin (pH 8.0, 30℃). The optimum pHs were 5.0 and 8.0 for chlorogenic acid oxidizing (ChO) and (－) -epicatechin oxidizing (EpO) activities, respectively. In the pH range from 4.0 to 9.0, both ChO and EpO activities were stable at 4℃ for 22 h. The optimum temperature of both activities was found at 30℃. Both activities were 50%-inactivated after heat treatment at 60℃ for 10 min. Both activities were strongly inhibited by _L-ascorbic acid and _L-cysteine at 5 mM