title: Purification and characterisation of polyphenol oxidase from leaves of Cleome gynandra L.
abstract: A polyphenol oxidase was purified and characterised from leaves of the common spiderflower. Purification sequentially with ammonium sulphate, dialysis, DEAE-Sepharose ion-exchange chromatography and Sephadex G-75 gel filtration chromatography resulted in 37.8-fold enrichment in the specific activity and 44.3% recovery of the total activity. Purified PPO is a monomeric protein of 52.6kDa revealed by Coomassie and active staining and Western blot. It was optimally active at pH 8.0 and 60°C, and stable from pH 3.0 to 9.0 and below 60°C. It displayed enzymatic activity towards monophenols, diphenols and triphenols, especially towards diphenols, and substrate specificity towards methylated and methoxylated substrates. Its activity was slightly increased by 0.1% SDS, heavily inhibited by Hg(2+) and Pb(2+), and completely inhibited by 1.0mM of ascorbic acid, l-cysteine, β-mercaptoethanol, sodium diethyldithiocarbamate and thiourea, and by 10mM of dithioerythritol, sodium metabisulphite and sodium sulphite.
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China; Department of Food Engineering (Bioengineering), Xuzhou Institute of Technology, Xuzhou 221008, China.
authors.forename: Zhao-Jian
authors.initials: ZJ
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
authors.forename: Jian-Bing
authors.initials: JB
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China. Electronic address: xiaoxg@cau.edu.cn.
authors.forename: Xing-Guo
authors.initials: XG
authors.lastname: Xiao
keywords: Cleome gynandra L.; Diphenolase; Monophenolase; Polyphenol oxidase; Triphenolase