Extraction, Purification And Characteristics Of PPO From Agaricus Bisporus

Enzymatic browning is the major feature for postharvest Agaricus bisporus browning, in which polyphenol oxidase is the key enzyme. According to respective different of dissolving degrees, molecule size and electric charge, polyphenol oxidase was isolated from Agaricus bisporus and purified by using extraction with phosphate buffer, centrifugation at low temperature, precipitation with ammonium sulfate, and separation by column chromatography with DEAE-Sephadex anion column chromatography (DE-52), Sephadex (G-100) filtration. SDS-PAGE showed the protein as a single polypeptide that PPO was purified to electrophoretic homogeneity. The specific activity, final purification fold and ratio of recovering protein are 566.57U·mg-1,103.20 and 0.47%, respectively. Its molecular weight is about 27.27kDa, the final quality is 5.21mg.To effectively control enzymatic browning of Agaricus bisporus, Polyphenol oxidase activities were spectrophotometrically determined at 450nm with catechol as substrate at diferent pH value, substrate concentrations, temperatures and enzyme inhibitors and so on. The results showed that the optimum pH value and substrate concentration were 6.8 and 0.06mol·L-1. The Michaelis constant of pyrocatechol catalyzed was Km=0.1072 mol·L-1. The optimum temperature was 20℃. L-sodium sulfite was stong PPO activity inhibitor, when its concentration was 0.5mmol·L-1, the PPO activity was completely inhibited. Ascorbic acid and acetic acid had strong restrained effects, too. When their concentrations were 0.20% and 0.15%, their inhibition ratios were 90% and 83.7%, respectively.