Isolation, Purification And Characterizations Of Polyphenol Oxidase Isozyme I-1 From Camellia Sinensis Cv. Longjing 43

Polyphenol oxidase(EC 1.10.3.1, PPO) is a oxidoreductase widely existed in plants, and also is a key enzyme in tea processing. Many studies have been carried out on Camellia sinensis PPO, but mostly on crude enzyme mixed with many kinds of isozymes, and there is lack of in-depth investigation on single isozyme. An isozyme PPO I-1 was isolated and characterized from C. sinensis cv. Longjing 43 in this study.The PPO were extracted from a bud and one or two leaves of C. sinensis cv. Longjing 43 by buffer homogenate method, and purified by ammonium sulfate and DEAE-Sepharose CL-6B anion exchange chromatography, and identified by native-electrophoresis analysis. Finally, PPO isozmy I-1were obatained in a electrophoretic purity.The enzymatic characteristics of PPO I-1(such as substrate specificity, Michaelis constant, optima pH, optima temperature, thermal stability, effects of inhibitors and metal ions) were detected, and compared with those of crude PPO. The results showed that the optima substrates of PPO I-1 and crude enzyme both were catechol, and their responding Michaelis constants were 73.50 mmo/L and 77.30 mmol/L. The enzymatic activities presented a peak value in acidity and alkaline conditions, respectively, The activities of PPO I-1 showed two peak values at pH 5.6 and pH 8.8, while those of crude PPO at pH 5.2 and pH 8.4. The optima temperatures of PPO I-1 and crude PPO both were 35 °C. The enzymatic activities of PPO I-1 and crude PPO both were stable at 35 °C, and their relative activeities both retained more than 60.00% after 6 h inocubation; when innocubated at 45 °C, the enzymatic activities of PPO I-1 and crude PPO both were declined rapidly, and their relative activeities both retained less than 6.37% after 6 h inocubation; the relative activeities of PPO I-1 and crude PPO both retained less than 30.00% after 1 h inocubation under 55-75 °C; The thermal stability of PPO I-1 was higher than that of crude PPO, and its enzymatic activity was declined slower.The inhibitition abilities of these inhibitors to PPO I-1 were Na2SO3> L- cysteine > Vc > citric acid, and their corresponding relative activities for the highest inhibitor concentrarion was 3.19%、5.72%、23.88% and 77.69%, respectively; the inhibitition abilities of these inhibitors to crude PPO were Na2SO3> Vc > L- cysteine > citric acid, and their corresponding relative activity for the highest inhibitor concentration was 2.90%, 10.94% and 24.59% and 88.76%, respectively. The inhibition abitlity of EDTA-2Na was weak, the relative enzyme activities of PPO I-1 and crude PPO were 92.93% and 77.16% under 100.00 mmol/L EDTA-2Na, respectively. The effects of metal ions were complex with the increase of their concentration; Cu2+ promoted the PPO activity at low concentrations, while inhibited it at high concentration; Mg2+ inhibited the enzymatic activity of PPO I-1, but showed inhibitition effects at low concentrations and promotion effects at high concentration for the activity of crude PPO; Fe3+ and Al3+ both promoted the enzymatic activity of PPO I-1 and crude PPO; the effects of Na+ on the PPO activities were not significant.The optimum conditions for the catalytic formation of theaflavins for both PPO I-1 and crude PPO were 1.50 mg/mL of tea polyphenols, 37 ℃ of temperature, 1 h of reaction time, and the optimum pH 4.0 and 4.4 for PPO I-1 and crude PPO, respectively. The theaflavin monomer content from high to low was TFDG, TF-3?-G, TF-3-G and TF, and the change trend of theaflavin monomer and the total theaflavins content were consistent with each other. The catalytic ability of PPO I-1 was stronger than crude enzyme, PPO I-1 could catalyze the synthesis of TF-3-G, TF-3?-G and TFDG at low substrate concentration, and synthetised more the monomer content and total content of theaflavins in than those of crude PPO under different conditions.PPO isozyme I-1 was purified from C. sinensis cv. Longjing 43, and its enzymatic characteristics were analyzed when compared with crude PPO, which provides a theoretical basis for the production of PPO and the breeding of tea varieties.