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Purification and Characterization of Polyphenol Oxidase from Rape Flower

Author:
Sun, Han-Ju, Wang, Jing, Tao, Xue-Ming, Shi, Juan, Huang, Mei-Ying, Chen, Zhe
Source:
Journal of agricultural and food chemistry 2012 v.60 no.3 pp. 823-829
ISSN:
1520-5118
Subject:
EDTA (chelating agent), ammonium sulfate, ascorbic acid, calcium chloride, catechol, catechol oxidase, citric acid, enzymatic browning, flowers, molecular weight, pH, sodium chloride, sodium sulfite, temperature
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 (Kₘ) and maximal reaction velocity (Vₘₐₓ) 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.
Agid:
313811