Isolation, Purification, Characterization And Modification Of Groups Of Polyphenol Oxidase From Sweet Potato Leaves

Polyphenol Oxidase (EC1.10.3.1d) is a ubiquitous copper metalloprotein.It is widely distributed in plants,animals and microorganisms.PPO can catalyze oxidization of phenolic compounds to produce the quinones and finally polymerized into the brown pigment.Browning of fruits and vegetables is mainly due to the PPO,which results in the loss of appearance quality,nutrient value and economic value.In addition,PPO has relationship with the plant disease resistance,plant photosynthesis,design and color formation and ethylene generation. Sweet potato leaves as a cold resistant to poor green vegetables has attracted widespread attention.It contains rich mineral,vitamin and so on,it can improve human disease-resistant ability and have fall blood sugar, anti-tumor effects. Currently, polyphenol oxidase from sweet potato leaves purification have not been reported. Sweet potato leaves was used as raw material in this paper. The research on the polyphenol oxidase will provide the use value of the sweet potato leaves and improve the economic benefits.The main results were as follows:In order to obtain purified polyphenol oxidase from sweet potato leaves for the study of its enzymatic properties,ammonium sulfate precipitation,ion exchange chromatography on DEAE-Sepharose column and Superdex-200 gel filtration were used to purify polyphenol oxidase from sweet potato leaves.The specific activity of purified PPO was 50075.43U/mg,which exhibited a purification fold of 597.99 and the recovery rate is 0.48%.The relative molecular weight of the polyphenol oxidase from sweet potato leaves was 239.90 kD, and the weight of subunit was 64.4 kD. The result shows that the enzyme consists of two identical subunits. The optimal pH and temperature were 6.5 and 35℃,respectively,and the polyphenol oxidase was stable in 25-55℃and pH 6-7. The apparent Km values of the enzyme with different concentrations of catechol as the substrate was 0.0445mol/L.Among several mental ions, Ca2+,Hg2+ are strongly inhibited the activity, Pb2+,Co2,Fe2+can activate the enzyme activity, K+,Mn2+,Li+,Zn2+,Mg2+,Ba2+have little effect on activity of the PPO from sweet potato leaves.Organic solvents such as methanol,ethanol and isopropanol could inhibit the activity of this enzyme and the order according to the inhibitory effect from strong to weak was isopropanol,ethanol and methanol.Low concentration of sodium sulfite and ascorbic acid had strong inhibition to PPO, critic acid was the lower inhibitor and the most ineffective inhibitor was EDTA,urea has little effect on activity of the enzyme.By modificating the function groups of the enzyme, the results show that DTT and BD have a strong inhibitory effect to the PPO activity from sweet potato leaves, PCMB and PMSF can activate the enzyme activity,While BrAc,Maleic anhydride,SUAN,PCMB and PMSF have little effect on activity of the enzyme.All of these suggested that Arg residues and disulfid bond might be essential function groups of the PPO.