title: Purification and characterization of polyphenol oxidase from rape flower.
abstract: The purification and partial enzymology characteristics of polyphenol oxidase (PPO) from rape flower were studied. After preliminary treatments, the crude enzyme solution was in turn purified with ammonium sulfate, dialysis, and Sephadex G-75 gel chromatography. The optimal conditions and stability of PPO were examined at different pH values and temperatures. Subsequently, PPO was also characterized by substrate (catechol) concentrations, inhibitors, kinetic parameters, and molecular weight. Results showed that the optimal pH for PPO activity was 5.5 in the presence of catechol and that PPO was relatively stable at pH 3.5-5.5. PPO was moderately stable at temperatures from 60 to 70 °C, whereas it was easily denatured at 80-90 °C. Ethylenediaminetetraacetic acid, sodium chloride, and calcium chloride had little inhibitive effects on PPO, whereas citric acid, sodium sulfite, and ascorbic acid had strongly inhibitive effects. The Michaelis-Menten constant (K(m)) and maximal reaction velocity (V(max)) of PPO were 0.767 mol/L and 0.519 Ab/min/mL of the crude PPO solution, respectively. PPO was finally purified to homogeneity with a purification factor of 4.41-fold and a recovery of 12.41%. Its molecular weight was 60.4 kDa, indicating that the PPO is a dimer. The data obtained in this research may help to prevent the enzymatic browning of rape flower during its storage and processing.
authors.affiliation: School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009, China. sunhanjv@163.com
authors.forename: Han-Ju
authors.initials: HJ
authors.lastname: Sun
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.forename: Xue-Ming
authors.initials: XM
authors.lastname: Tao
authors.forename: Juan
authors.initials: J
authors.lastname: Shi
authors.forename: Mei-Ying
authors.initials: MY
authors.lastname: Huang
authors.forename: Zhe
authors.initials: Z
authors.lastname: Chen
mesh_terms: D029741:Brassica rapa / Q000737:chemistry / Q000201:enzymology*; D004156:Catechol Oxidase / Q000737:chemistry* / Q000302:isolation & purification*; D019281:Dimerization; D004795:Enzyme Stability; D035264:Flowers / Q000737:chemistry / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D010940:Plant Proteins; D004156:Catechol Oxidase