Isolation And Identification Of Polyphenol Oxidase Isozymes From Camellia Sinensis And Its The Enzymatic Synthesis Of Theaflavins

Polyphenol Oxidase(PPO) is widespread exist in the nature, including Cu2+ and the main enzyme as oxidordeuctase, it plays an important role in the physiology metabolism of Camellia sinensis and tea processing. Many researches focus on tea PPO as a whole, about its cellular localization, physiology, genetics, enzyme characteristics, screening and the use of an enzyme source, extraction and separation, enzymatic oxidation synthesis Theaflavins and enzyme immobilization, and made significant research results. But in the isozymes Isolation and identification of PPO component aspects Theaflavins, which failed to make a breakthrough. Study on integrated series of modern isolation and purification technology, to be identified by separating Camellia sinensis cultivars Zheng he da bai fresh leaves PPO isozymes and proven its enzymatic properties, based on in-depth study of its directional effect enzymatic synthesis Theaflavins. In order to provide a scientific basis of separate the tea PPO isozymes preparation and directed enzymatic synthesis Theaflavins. The main results are follows:1 Using the fresh leaves of Camellia sinensis cultivars Zheng he da bai of a bud and two leaves as material, The PPO was purified by acetone powder, homogenized extraction buffer, ammonium sulfate fractionation, dialysis, Q-Sepharose Fast Flow strong anion exchange chromatography, Sephadex G-75 gel column chromatography, ultrafiltration membrane desalination obtaining a PPO isozymes PPO1 and PPO2,The Specific activity of PPO1 was 10850.00 U/mg,which exhibited a purification fold of 22.03,and PPO2 was 24102.31U/mg,which exhibited a purification fold of 48.94.2 By MALDI-TOF/TOF mass spectrometry, Found PPO1 molecular weight 85089 Da, isoelectric point pI of 6.1, and similarity Methionine synthase isozyme fragment matches the amino acid sequence of each peptide YLFAGVDGR, AGINVIQIDEAALR, YGAGIGPGVYDIHSPP, LQEELDIDVLVHGEPER; PPO2 molecular weight of 42531.7 Da, isoelectric point pI of 5.49, and similarity Phosphoglyc-erate kinase enzyme amino acid sequence of each peptide fragment matches have VDLNVPLDDNLNITDDTR, YSLKPLVPR, LASLADLYVNDAFGTAHR.3 The enzyme properties of the PPO1 and PPO2, PPO1 substrate binding capacity gallic acid> EGC> catechol> EGCG> tyrosine, PPO2 was the focus of gallic acid> EGCG> phthalic diol> EGC> tyrosine; Optimum temperature were 30℃,38℃ respectively, exceed 45℃ the two PPO isozymes are inactivated accelerated; Optimum pH were 5.5 and 6.0 respectively, pH value range of 4.5～6.5, there are able to maintain more than 40% relative activity of the original enzyme; Thermal stability PPO1 more than PPO2 stronger; Ba2+, Fe3+ had no effect on the two isozymes, and Cu2+, Al3+ inhibited its, Mg2+ to PPO1 inhibited, while the PPO2 performance promotion, Ca2+ at 10mmol/L concentration, the two PPO isozyme promoted; Inhibitor of sodium sulfite, ascorbic acid and cysteine of the two isoforms has strong inhibitory enzyme, EDTA, oxalic acid and citric acid on performance PPO1 inhibition, while the PPO2 facilitative.4 Enzymatic synthesis of theaflavins, PPO1 enzymatic synthesis product is predominantly single simple theaflavin component TF, and the enzymatic synthesis PPO2 TF, TF-3-G, TF-3’-G and TFDG four kinds of tea flavin component, while at pH 5-6, at a temperature of about 35℃, higher enzymatic synthesis of Two Isoenzymes Theaflavins efficiency simply by changing the substrate ester catechins and catechins The content and the proportion of the synthetic product can theaflavins composition and content of the regulation, where the enzymatic synthesis Theaflavins of TF concentration of 0.973 11.807μg/mL.