Isolation, Purification And Characterization Of Polyphenoloxidase From Camellia Sinensis Var. Assamica

The most important factor of affecting black tea quality is PPO activity(Polyphenol oxidase) which contributes to the oxidizing reaction of tea polyphenols that can form TFs and TRs. In this study, two isozymes of PPO were extracted, isolated and purified from the fresh leaves of Camellia sinensis var. assaminca cv. Fengqing, and their enzymatic characteristics were comprehensively investigated. The main results are as follows:(1) According to their enzyme activity and Native-PAGE electrophoresis, the extracted conditions of Fengqing PPO were optimized. Taking appropriate amount of fresh leaves in the mortar, added precooling citric acid buffer(0.1 mol/L, p H 7.2) by the 1:3(W/V) ratio of material to liquid and 5%(g/m L) PVP, and grinding in ice-bath. The grinded leaf solution was extracted for 12 h in 4 °C, and then was centrifugated by 9000 r/min, 4 °C for 30 min, and the supernatant was the crude PPO.(2) Through the processes of the 30-80% ammonium sulfate precipitation, the ion exchange chromatography(DEAE Sepharose CL-6B) and gel filtration chromatography(Sephndex G-150), two isozymes PPO was purified from the crude PPO: PPOⅠ, one single protein subunit(approximately 35 k Da), and its purified fold 19.45. PPOⅡ, a dimmer of two protein subunits(66.2-55 k Da), and its purified fold 30.18.(3) The enzymatic characteristics of PPOⅠandⅡwere that their optimum temperatures both were 40 °C. The optimum p H of PPOⅠand PPOⅡ were 5.2 and 5.6, respectively. The optimal Cu2+ concentration of PPOⅠand PPOⅡwere 0.50×10-7 mol/L and 1.00×10-7 mol/L, respectively. The inhibitory effects on PPOⅠand PPOⅡwere ascorbic acid>sodium sulfite>EDTA>cysteine. When the concentration of ascorbic acid was the highest, PPO activity was inhibited 50%. Catechol and pyrogallic acid were the best substrates for PPOs, followed by resorcinol and gallic acid, and hydroquinone is not apt to PPOs. The Km of PPOⅠand PPOⅡwere 4.42 and 3.46 mmol/L for catechol as substrate, respectively, and the Km of PPOⅡwas 3.46 mmol/L for using pyrogallic acid as substrate.