Mass Spectrum Identification And Enzymatic Properties Of Polyphenol Oxidase Isozyme From Camellia Sinensis Cv. Longjing43

Polyphenol oxidase (PPO) has always been the hot spot of domestic and foreign research over the years, which plays a key role in the formation of tea quality. However the research of single PPO isoenzyme is lacking currently. This topic had used tea variety called Longj ing43as researching material, during the processing of congtained ammonium sulfate precipitation, ion exchange chromatography and gel chromatography etc, the research had obtained3single PPO isozymes (PPO I, PPO Ⅱ, PPO Ⅲ).Then aiming at PPO II, the research has studied its enzymatic properties and carried out the preliminary identification by mass spectrometry analysis.The main researching results were as follows:1The acquisition of tea tree PPO isoenzymesExperiment showed that during the processing steps of buffer extraction, grade of ammonium sulfate precipitation, DEAE Sepharose CL-6B ion exchange chromatography, Sephadex G-150gel filtration chromatography, the tea tree PPO isoenzymes (PPO Ⅰ, PPO Ⅱ,PPOⅢ) can be effectively purified; the specific activity were964.63,2325.39,2406.78U/mg, and molecular weight were35,53,85kDa, respectively.2The mass spectrum identificationAiming at PPO isoenzymes (PPO Ⅰ,PPO Ⅱ,PPOⅢ),through the mass spectrometry analysis, has retrieved four similar substances:cytoplasmic ribosome protein, lactoylglutathione lyase, ribulose1,5-bishosphate carboxylase/large subunit protein and methionine oxysynthase; The first two test results are for PPO I, and the last two are for PPO Ⅱ and PPOⅢ, respectively.Two varieties of similar materials were obtained by detecting PPO I:one variety was similar to the cytoplasmic ribosome protein of Camellia sinensis with the molecular weight of17267Da, pI10.26; Another is similar to the fragments derived from lactoylglutathione lyase of rice with the molecular weight of32875Da, pI5.51.PPO Ⅱ was similar to the large subunit protein of ribulosel,5-bishosphate carboxylase/oxygenase existed in Franklinia alatamaha with the molecular weight of53050Da, pI6.13, and it was promised to be related to the photosynthesis in plant.PPO Ⅲ was similar to the methionine synthase presented in Camellia sinensis with the molecular weight of85180Da, pI6.14. It was promised to be related to methionine and folic acid circulation which regulate the physiological process of the organism. 3The characterization of Polyphenol OxidaseUsing the spectrophotometric technology and catechol as substrate in studying the enzymatic properties of tea PPO isoenzymes at various inhibitors, temperatures, pH, Cu2+concentration etal. The results showed that:The optimum pH of PPO Ⅱ was found to be7.2; the Kinetic parameters of enzymatic browning reaction were Km=13.05mmol/L, Vmax=0.019OD460/min; Inhibitors of ascorbic acid, sodium sulfite and L-cysteine on PPO Ⅱ isozymes are inhibited, and with increasing concentration of inhibitors, the residual activity decreased; residual enzyme activity; The activity of PPO Ⅱ decreased with the concentration of Cu2+rising first, when the concentration of Cu2+rises up to0.25х10-7mol/L, the enzyme activity has reached the highest, then The activity of PPO Ⅱ disappeared when the Cu2+concentration reaches2х10-7mol/L.