title: Purification and characterization of polyphenol oxidase from cauliflower (Brassica oleracea L.).
abstract: Polyphenol oxidase (PPO) of cauliflower was purified to 282-fold with a recovery rate of 8.1%, using phloroglucinol as a substrate. The enzyme appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The estimated molecular weight of the enzyme was 60 and 54 kDa by SDS-PAGE and gel filtration, respectively. The purified enzyme, called phloroglucinol oxidase (PhO), oxidized phloroglucinol (K(m) = 3.3 mM) and phloroglucinolcarboxylic acid. The enzyme also had peroxidase (POD) activity. At the final step, the activity of purified cauliflower POD was 110-fold with a recovery rate of 3.2%. The PhO and POD showed the highest activity at pH 8.0 and 4.0 and were stable in the pH range of 3.0-11.0 and 5.0-8.0 at 5 °C for 20 h, respectively. The optimum temperature was 55 °C for PhO and 20 °C for POD. The most effective inhibitor for PhO was sodium diethyldithiocarbamate at 10 mM (IC(50) = 0.64 and K(i) = 0.15 mM), and the most effective inhibitor for POD was potassium cyanide at 1.0 mM (IC(50) = 0.03 and K(i) = 29 μM).
authors.affiliation: Laboratory of Food Science, Faculty of Agriculture, Saga University, Saga, Japan. faidah83@yahoo.com
authors.forename: Andi Nur Faidah
authors.initials: AN
authors.lastname: Rahman
authors.forename: Mayumi
authors.initials: M
authors.lastname: Ohta
authors.forename: Kazuya
authors.initials: K
authors.lastname: Nakatani
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Hayashi
authors.forename: Shuji
authors.initials: S
authors.lastname: Fujita
mesh_terms: D001937:Brassica / Q000201:enzymology*; D004156:Catechol Oxidase / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010696:Phloroglucinol / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D010696:Phloroglucinol; D004156:Catechol Oxidase