Extraction, Isolation, Purification And Partial Characteristics Of Polyphenol Oxidase From Camellia Sinensis

Polyphenol oxidase (Polyphenol Oxidase, PPO) plays a very important role in physiology metabolism of Camellia sinensis and the processing of tea products. However, the researches about single tea PPO isozymes are little. In this study, the fresh leaves of Camellia sinensis cultivars Longjing NO.43with the best adaptability of processing green tea, were as the experimental materials. Through the performance of ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography, two kinds of PPO isoenzymes were isolated and purified, and enzymology properties of PPO I and PPOⅢ also were studied.The main results are as follows:(1) Crude PPOs were extracted from the fresh leaves of Camellia sinensis cultivars Longjing NO.43by different single factor conditions. Base on,assaying the enzyme activity and comparing with the isozyme bands in the non-denatured gel electrophoresis, the optimal method of extracting crude PPO was selected. The results showed that1:2of the leaf-water weight ratio, adding pre-cooled citric acid-phosphate buffer (0.15mol/L pH7.2) containing5%PVP (w/v), grinding in ice bath, leaching12h in4℃overnight, centrifuging at9000r/min for35min, and taking the supernatant and then filtering it, were to extract the crude PPO with the highest activity and five obvious isozyme bands.(2) The crude PPO prepared by the optimal extraction conditions, was precipited by30%-80%ammonium sulfate which the concertration of the PPO precipitation was obtained in advance, to remove part of miscellaneous protein. The precipited fraction was concentrated by desalting and ultrafiltrating, and purified by an ion exchange chromatography of DEAE Sepharose CL-6B and a gel filtration chromatography of Sephndex G-150. Finally, three isozymes (PPO Ⅰ, PPO Ⅱ,PPOⅢ) was obtained. The molecular weights of PPO Ⅰ, PPO Ⅱ and PPOⅢ are38kDa,48kDa and80kDa, respectively. The purifition multiples of PPO Ⅰ and PPO Ⅱ were14.41times,1.08times and1.30times, respectively; and their specific activities were7177.0U/mg,537.45U/mg and648.22U/mg, respectively. (3) The results of their enzymology characteristics showed that the optimum pH values of PPO Ⅰ and PPOⅢ were5.6and6.8, respectively; and both of them retained more than60%of the relative enzyme activity within the pH rang of5.6-7.2. The optimum temperatures of PPO Ⅰand PPOⅢ were30℃and35℃, respectively; and both of them remained relative high activity within the rang of25℃-40℃, but inactivated at over60℃. Sodium sulfite, cysteine and ascorbic acid all inhibited the activities of PPO Ⅰ and PPOⅢ, but the role of cysteine was relatively more efficient. Only low concentration of Cu2+would increase the activities of both PPO Ⅰ and PPO Ⅲ. When using catechol as the substrate, PPOⅢ had stronger affinity to the substrate than that of PPO Ⅰ. The Km value of PPO Ⅰ and PPOⅢwere91.34mmol/L and51.65mmol/L, respectively.