The Studies On Resin Immobilize Polyphenol Oxidase And It Catalysis Tea Polyphenol Formating Theaflavins

Immobilized enzyme technology is an advance bioengineering, it was rised at 1960s,and has developmented greatly 21st century. At enzyme immobilizing, immobilized carrier has significantly effection in activity of immobilization enzymed and efficiency of enzyme immobilized.This research consider comprehensively activity and application of immobilization enzyme, using resin as carrier immobilizes polyphenol oxidase, and researching on immobilization polyphenol oxidase, as well as using it catalytic oxidative tea polyphenol formation theaflavins.1 Accord to activity and efficiency of enzyme immobilized, researched different aperture, materials and polarity of macroporous adsorption resin and ion exchange resin.Experimental results show that cation exchange resins better than macroporous adsorption resin and anion exchange resin in immobilizes polyphenol oxidase action.Among of cation exchange resins, D152 resin is the best.2 Researched the optimum action conditions of D152 resin carrier immobilized polyphenol oxidase,obtained the optimum action conditions:10.00g pretreatmented D152 resin,adding in 50ml 0.2mg/ml polyphenol oxidase solution, 120 rotates per minute adsorptive-immobilized 2 hours at 4℃,then adding in 0.080ml 25% glutaraldehyde solution crosslinking action 1 hour at 4℃120 rotates per minute.3 Researched the properties of immobiliztion polyphenol oxidase.(1)The Km of immobilization polyphenol oxidase is 0.113mg/ml, it has increased 17.6% compare with SPPO;(2)The half-life of polyphenol oxidase from 17.2 days increased to 52.5 days,increasing to 300 precent; (3)Immobilized polyphenol oxidase has well stability of reuse,reusing 20 times,then activity retentives 40%; (4)The optimum pH of polyphenol oxidase moves to alkali one unit, The optimum temperature increases 5℃;(5)The stability in organic solvent of immobilizatio n polyphenol oxidase better than solution polyphenol oxidase;(6)The resistance of metal ions hasn't significantly change about immobilization polyphenol oxidase and solution polyphenol oxidase;(7)The immobilization polyphenol oxidase column has well stability.4 Applied immobilization polyphenol oxidase to preparing theaflavins.Using orthogonal test treatment considering the main effect factors of IPPO catalytics TP forming TFs as temperature(X1),pH(X2), contents of substrate(X3),action time(X4) and speed of adding oxygen(X5).Using the experiment results anlysis obtaining polynomial regression equation: Y=-0.4366+0.0186X1+0.1065X2-0.1121X3 +3.6350X5-0.0002X1*X1-0.0034X3*X3+0.0003X4*X4+0.4025X5*X5-0.0034X1*X2+0.0034X1*X3-0.0623X1*X5+0.0149X2*X3-0.0012X3*X4-0.0328X4*X5,R2=0.9998.By rhe regression equation, obtaining the optimum experiment conditions as X1=64.6℃, X2=6.57, X3=11.53mg/ml, X4=59.7min, X5=0.10L/min,the theoretical value of Y is 1.03mg/ml. Design experiment as the optimum conditions,getting the observation value of Y is (0.90±0.06)mg/ml,,the accuracy of authentication is 87.46%.