Purification and Characterization of Polyphenol Oxidase from Japanese Radish (Raphanus sativus L.) Root

Abstract

　Polyphenol oxidase (PPO) was purified from the Japanese radish root by ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, and gel filtration using phloroglucinol as a substrate. The enzyme was purified about 192-fold with a recovery rate of 15%. The purified enzyme appeared as a single band on SDS-PAGE. The molecular weight of the purified PPO was estimated to be about 44 kDa by gel filtration and 45.7 kDa by SDS-PAGE. The purified enzyme quickly oxidized phloroglucinol (1, 3, 5-trihydroxybenzene) with a K_m of 2 mM. The enzyme also oxidized 1, 2, 3-trihydroxybenzenes, such as pyrogallol and gallic acid; however, it did not oxidize o-diphenols, such as chlorogenic acid and dopamine. Peroxidase (POD) activity was also present in the purified enzyme preparation with the final preparation having a purification and recovery rate of 259-fold and 20%, respectively. The optimum pH for the PPO and POD activities was 8.0 and 5.0, respectively, and the measured activities were stable at 5℃ for 20 h in the pH ranges of 3.0～10.0 and 3.0～11.0, respectively. Both enzyme activities were stable up to 50℃ after heat treatment for 10 min and were strongly inhibited by treatment with sodium diethyldithiocarbamate, potassium cyanide, L-ascorbic acid, chlorogenic acid, and hydroquinone at a final concentration of 10 mM.