title: Purification and characterization of polyphenol oxidase from garland chrysanthemum (Chrysanthemum coronarium L.).
abstract: Polyphenol oxidase (PPO) of garland chrysanthemum (Chrysanthemum coronarium L.) was purified approximately 32-fold with a recovery rate of 16% by ammonium sulfate 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 47000 and 45000 by gel filtration and SDS-PAGE, respectively. The purified enzyme quickly oxidized chlorogenic acid and (-)-epicatechin. The K(m) value (Michaelis constant) of the enzyme was 2.0 mM for chlorogenic acid (pH 4.0, 30 degrees C) and 10.0 mM for (-)-epicatechin (pH 8.0, 40 degrees C). The optimum pH was 4.0 for chlorogenic acid oxidase (ChO) and 8.0 for (-)-epicatechin oxidase (EpO). In the pH range from 5 to 11, their activities were quite stable at 5 degrees C for 22 h. The optimum temperatures of ChO and EpO activities were 30 and 40 degrees C, respectively. Both activities were stable at up to 50 degrees C after heat treatment for 30 min. The purified enzyme was strongly inhibited by l-ascorbic acid and l-cysteine at 1 mM.
authors.affiliation: Laboratory of Food Science, Department of Applied Biological Sciences, Faculty of Agriculture, Saga University, Saga 840-8502, Japan.
authors.forename: Eline
authors.initials: E
authors.lastname: Nkya
authors.forename: Chie
authors.initials: C
authors.lastname: Kouno
authors.forename: Ying-Jie
authors.initials: YJ
authors.lastname: Li
authors.forename: Chang-Peng
authors.initials: CP
authors.lastname: Yang
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Hayashi
authors.forename: Shuji
authors.initials: S
authors.lastname: Fujita
mesh_terms: D002392:Catechin / Q000378:metabolism; D004156:Catechol Oxidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002726:Chlorogenic Acid / Q000378:metabolism; D002845:Chromatography; D031202:Chrysanthemum / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010084:Oxidation-Reduction; D013696:Temperature
chemical_list: D002726:Chlorogenic Acid; D002392:Catechin; D004156:Catechol Oxidase