Study On The Separation And Purification And Enzymatic Properties Of Oxidase From Lotus Root Tender Bine

Lotus root tender bine (Lotus sprout) is our country main aquatic vegetables, it with a nutrient-rich, crisp and refreshing, but also has some medicinal value.After picking lotus sprout with exposure prone to enzymatic browning in the air.causing the surface black and soft, not only affect the appearance, but also the flavor and quality will decline.Therefore, the study of enzymatic browning of lotus root with mining mechanism and control measures of great significance for the development of this feature vegetable resources. Lotus sprout was used as raw material in this paper,purification,properties and fuction group of POD,PPO and CAT were studied.The results of our study were follows:1. Purification of POD.PPO and CAT from Lotus sproutPOD was extracted by phosphate buffer from lotus sprout, and the optimal extracting conditions were optimized, pH 7.2, extraction time were 4h, the radio of solid to solvent were 1:2.POD from the fresh Lotus sprout was purified using ammonium sulfate precipitation, ion exchange chromatography on DEAE-52 Fast Flow column and gel filtration chromatography on Sephadex G-75.The purification fold was 32.68,and the recovery was 2.11%.SDS-PAGE analysis show that the molecular weight of POD from Lotus sprout is approximately 42KD.PPO was extracted by phosphate buffer from lotus sprout, and the optimal extracting conditions were optimized, pH 6.0, extraction time were 3h, the radio of solid to solvent were 1:3.PPO from the fresh Lotus sprout was purified using ammonium sulfate precipitation, ion exchange chromatography on DEAE Sephadex A-50 Fast Flow column and gel filtration chromatography on Sephadex G-75.The purification fold was 101.73,and the recovery was 0.35%.SDS-PAGE analysis show that the molecular weight of PPO from Lotus sprout is approximately 62KD.CAT was extracted by phosphate buffer from lotus sprout, and the optimal extracting conditions were optimized, pH 7.2, extraction time were 2h, the radio of solid to solvent were 1:1. CAT from the fresh Lotus sprout was purified using ammonium sulfate precipitation, ion exchange chromatography on DEAE-Sepharose Fast Flow column and gel filtration chromatography on Supdex-200.The purification fold was 102.19,and the recovery was 29.79%.SDS-PAGE analysis show that the molecular weight of CAT from Lotus sprout is approximately 28KD.2. Properties of POD,PPO and CAT from Lotus sprout.(1) Properties of POD from Lotus sproutThe optimum temperature and optimum pH for the enzyme of POD from Lotus sprout was 40? and pH 5.0.Stable conditions for POD activity were 30?50? and pH 5.0?pH 8.0.The apparent Km values of the enzyme with different concentration of H2O2 as the substrate was 36.5mmol/L.Oxalic acid enhanced POD activity at low concentration,significantly inhibited more than 0.01mol/L;SDS,KSCN and carbamide inhibited POD activity;ASA inhibited POD activity completely. Zn2+ and Cu2+ enhanced POD activity completely; Na+,Li+,K+,Mn2+ and Fe2+ partially inhibited POD activity.(2) Properties of PPO from Lotus sprout The optimum temperature and optimum pH for the enzyme of PPO from Lotus sprout was 40? and pH 6.0.Stable conditions for PPO activity were 30?60? and pH 3.0?pH 7.0.The apparent Km values of the enzyme with different concentration of catechol as the substrate was 37.7mmol/L. EDTA and carbamide inhibited PPO activity;ASA,critic acid and cysteine inhibited PPO activity completely. Zn2+,Cu2+,Mg2+,Co2+ and Fe2+ enhanced PPO activity; Na+,Li+,Al3+ and Ca2+ partially inhibited PPO activity.(3) Properties of CAT from Lotus sproutThe optimum temperature and optimum pH for the enzyme of CAT from Lotus sprout was 40? and pH 7.0.Stable conditions for CAT activity were 30?50? and pH 4.0?pH 8.0.The apparent Km values of the enzyme with different concentration of H2O2 as the substrate was 37.7mmol/L. EDTA,KSCN,SDS and oxalic acid inhibited CAT activity; Pb2+ and K+ enhanced CAT activity; Cu2+,Ag+ and Zn2+ partially inhibited CAT activity.3. Function group of POD,PPO and CAT from Lotus sproutThe study of function group of POD from Lotus sprout indicated Serine residue,arginine residue,methionine residue,disulfide bond,histidine residue and sulfydryl might be essential function group of POD,but lysine residue and tyrosine residue might not be.The study of function group of PPO from Lotus sprout indicated Arginine residue and disulfide bond might be essential function group of PPO,but serine residue,lysine residue,methionine residue,histidine residue,sulfydryl and tyrosine residue might not be.The study of function group of CAT from Lotus sprout indicated Disulfide bond might be essential function group of CAT,but arginine residue,serine residue,lysine residue,methionine residue,histidine residue,sulfydryl and tyrosine residue might not be.