| Polyphenol oxidase(PPO)is widely distributed in microorganism,plant, animal and human body,it has some important physiological functions in living creatures.In this paper,fresh mulberry leaf was studied with modern isolation technology and advanced methods of analysis and testing to extract,isolate and purify the mulberry leaf PPO;spectrophotometric analysis method and scanning electron microscopy were used to determine its structure,enzymatic properties and inhibitory effects of different inhibitors on it;regulatory mechanism of the PPO activity and immobilization were also discussed,in order to empolder the mulberry leaf PPO and increases its economic value.Main results were:The optimal extraction technics for mulberry leaf PPO were ascertained, 1.0%Tween-80,pH 6.0,extraction time were 2h,the ratio of solid to solvent were 1:2.The purification processes for mulberry leaf PPO were discussed,the classification deposition was disposed using 30%and 80%saturation of ammonium sulfate to isolate the impurity protein from PPO and increase the PPO yield.The Sephadex G-75(16×800mm)column,velocity of flow in elution was 15mL/h to further purify the crude PPO.First elution peak value was mulberry leaf PPO.The mulberry leaf PPO structure,molecular weight and enzymatic properties were determined,the enzyme was fibrous protein observed by scanning electron microscopy;its molecular weigh was 47Kd;the optimum substrate was pyrogallic acid,its Km value was 0.093mol/L;and its optimum pH and temperature were 7.0 and 40℃respectively;with the same concentrations of different inhibitors,the total inhibitory effects were:ascorbic acid＞sodium hydrogen sulfite＞citric acid＞magnesium chloride＞sodium chloride.The immobilization of mulberry leaf PPO were researched,4%sodium alginate,0.2%glutaraldehyde,0.2mol/L CaCl2 used to immobilize the PPO, with an immobilization time of 2h and crosslinking time of 4h,could make the immobilized PPO activity reach its maximum.After its immobilization,the optimum pH was 6.0,and the enzyme showed a good stability between the pH from 5.0 to 7.0;the optimum temperature was 50℃for the enzyme,and the enzyme became less susceptible to the temperature,the temperature did not more affect to the enzymatic activity. |
