Purification and Characterization of Polyphenol Oxidase from Edible Yam (Dioscorea opposita Thunb.)

抄録

Soluble polyphenol oxidase of edible yam (Dioscorea opposita Thunb.) was purified approximately 203-fold with a recovery rate of 15% by ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography and gel filtration using dopamine as a substrate. The purified enzyme appeared as a single band on native PAGE and SDS-PAGE. The molecular weight of the enzyme was estimated to be approximately 42kDa and 44kDa using gel filtration and SDS-PAGE, respectively. The purified enzyme quickly oxidized dopamine. The apparent K_m value (Michaelis constant) of the enzyme was 1.5mM for dopamine (pH 7.0, 30°C). The optimum pH was 7.0 for dopamine oxidase. In the pH range from 6 to 10, the activity was quite stable at 5°C for 22h. The optimum temperature of enzyme activity was 25-30°C. The activity was stable up to 50°C after heat treatment for 20min. The browning reaction by the enzyme was completely inhibited by 1mM L-ascorbic acid, which reduced o-quinone to dopamine. The reaction was also completely inhibited by 1mM L-cysteine, which is a known quinone coupler. About 35% inhibition of edible yam PPO was observed using citric acid and acetic acid at 10mM in 0.1M citrate/ 0.2M sodium phosphate buffer (pH 7). In consideration of the observed results, L-ascorbic acid, L-cysteine, acetic acid, and citric acid are expected to be used as effective inhibitors of enzymatic browning in edible yam.