Research On Extraction, Purification And Immobilization Of Polyphenol Oxidase From Mulberry Leaf

The fresh mulberry leaf was studied in this paper, and this research has fixed and separated out the polyphenol oxidase in it. To reveal the mulberry leaf polyphenol oxidase properties, further development and the utilization of mulberry leaf PPO laid a preliminary basis. The main research contents and conclusions are as follows:Analyzing on homogenate extraction, ultrasonic wave assisted extraction and acetone extraction, it determine the optimal method as acetone extraction method. According to orthogonal concluded that the extraction process conditions is that:the extracting time is3hours, the addition of liquid materials is1:20and the extracting pH is6.5.The determination of purification process for mulberry leaf polyphenol oxidase has been obtained.40%to80%ammonium sulfate is used to precipitation the Polyphenol oxidase. After dialysised and enrichmented, the pure mulberry leaf polyphenol oxidase was separated by using medium separation purification system. Through to the protease SDS-PEAG linear equation of calculated the molecular weight of39.5kd. The determination of immobilized technology for mulberry leaf polyphenol oxidase has been obtained.This paper screen alginate as polyphenol oxidase immobilized of carrier, and through the orthogonal experiment we get the best conditions for PPO fixed is that:5%of sodium alginate in1.5%alcium chloride fixed2.5h, then use0.2%glutaraldehyde crosslinking3h. finally the highest recovery rate of enzymes live is85.6%. Further adopts magnetic Fe3O4with sodium alginate composite modification of carrier, optimized conditions after optimum technological conditions have changed much,and7.84%of the enzyme live be increased.Analyzing the mulberry leaf polyphenol oxidase inhibitors on its inhibition, gain the conclusion that the best inhibitors for ascorbic acid, when ascorbic acid concentration is1.5g/L, inhibition rate has reached97%. Analyzing on immobilized enzyme physical and chemical character,we find that the sensitive degree of temperature and pH reduced after mulberry leaf polyphenol oxidase immobilized by carrier. The optimum temperature of immobilized enzyme is40℃, more than10℃by free enzymes. The optimal pH is8, direction to move alkaline one unit; Km value from the3.27x10-3mol/L of free enzymes increase to7.46x10=3mol/L of immobilized enzyme, so the substrate with immobilized enzyme affinity reduced. Mulberry leaf polyphenol oxidase are immobilized, which enzymes living can kept the original enzymes living27.5%after six times used.