Study Of Purification、Characterisation And Inhibitory Kinetics Of Polyphenol Oxidase From Honeysuckle

Fresh honeysuckle moisture content is about80%. Honeysuckle always bringabout serious browning and the decreases of chlorogenic acid content in storage anddrying process, this phenomenon serious causes the reducing of the active ingredientcontent and affecting its use value. In this paper，fresh honeysuckle was studied withmodem isolation technology and advanced methods of analysis and testing to extract，isolate and purify the honeysuckle PPO；spectrophotometric analysis and doublereciprocal plot method were used to determine its enzymatic properties, physical andchemical properties and inhibitory effects of different inhibitors on it. The mainresults are as follows：The results showed that it is more effective using PBS buffer to isolatehoneysuckle PPO than using acetone by comparative study. By using single factor andresponse surface methodology, the optimal extraction parameters were pH8.0and6.3:1(mL/g) of liquid-to-material ratio for1.7h with1g/100mL PVPP, resulting inPPO specific activity of667.56U/mg. The purification processes for honeysuckle PPOwere discussed，the classification deposition was disposed using40﹪saturation ofammonium sulfate to isolate the impurity protein from PPO and increase the PPOyield, fllowed by dialysis desalting and Sephadex G-100gel column chromatography,finally gained a single band by SDS-PAGE, and molecular weigh was43000D．The optimum substrate of honeysuckle PPO was chlorogenic acid, and KmandVmaxwere0.0059mol/L and4.5393OD/min, the optimum concentration was0.005mol/L; The optimum pH and temperature of honeysuckle PPO were9.0and40℃respectively. The enzyme ctivity was stable at the temperature ranging from40-70℃and under alkaline environment．The effects of different metal ions on thehoneysuckle PPO were studied, and the result showed that Fe2+, K+, Mg2+can activatethe activity of PPO; Cu2+，Ba2+，Ca2+could inhibit the activity of PPO; and Zn2+，Mn2+have double function to PPO, which stimulate PPO activity at low levels andinhibit its activity at higher concentration. The inhibitory effects of several commonly used inhibitors as ascorbic acid,4-hexylresorcinol, L-cysteine, citric acid and acetone on PPO activity wereinvestigated. The results showed that ascorbic acid,4-hexylresorcinol, L-cysteine andacetone were reversible inhibition, and IC50values were0.062,0.053,0.140mmol/L,5.55%. Citric acid was irreversible inhibition to PPO, and IC50values were0.048mmol/L. Lineweaver-Burk plotting showed that ascorbic acid,4-hexylresorcinolL-cysteine and acetone were mixed-type, competitive, anti-competitive andnon-competitively inhibitors, with the inhibition constants KIwere also calculated as1.620,4.587,0,1.530mmol/L, and KISwere1.995,0，3.780,1.530mmol/L,respectively.