PURIFICATION AND SOME PROPERTIES OF CHLOROGENIC ACID OXIDASE FROM APPLE (MALUS-PUMILA)

Chlorogenic acid oxidase was extensively purified to homogeneity from apple flesh (Malus pumila cv. Fuji). The enzyme was purified 470-fold, with a total yield close to 70% from the plastid fraction by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography. The molecular weight was determined to be 65,000 by both SDS-PAGE and gel filtration chromatography. The optimum pH for the enzyme activity was around 4.0, and the enzyme was stable in the range of pH 6-8. The pI obtained by isoelectrofocusing was 5.4, and the N-terminal amino acid sequence was N-Asp-Pro-Leu-Ala-Pro-Pro-. The reaction rate of the purified enzyme was much larger for chlorogenic acid than for other o-diphenols such as (+)-catechin, (-)-epicatechin and 4-methyleatechol, and the enzyme lacked both cresolase activity and p-diphenol oxidase activity. The K(m) value for the enzyme was found to be 122 muM toward chlorogenic acid. The purified enzyme had far less thermal stability than the enzyme of the plastid fraction. Diethyldithiocarbamate, sodium azide, o-phenanthroline and sodium fluoride markedly inhibited the enzyme activity.