Study On The Enzymatic Properties Of Polyphenol Oxidase And Peroxidase In The Lonicera Confusa

A polyphenol oxidase (PPO), located in the bud of Lonicera Confusa, was extracted, purified, and characterized. Ammonium sulfate precipitation and DEAE-cellulose were used to purify the PPO25.48-fold. The molecular mass of PPO was28.8kDa. as determined by size exclusion chromatography combined with multiangle laser light scattering (MALLS) and refractive index (RI). The optimum pH value and temperature for PPO activity in the presence of L-3,4-dihydroxyphenylalanine (L-dopa) as a substrate were8.5and15℃. respectively. In the thermal stability test, the enzyme was stable after60min at10and20℃, and the PPO activity remained above90%. As the increase of temperature, the thermal stability was reduced. The PPO exhibited both diphenolase and triphenolase activities. The Km for1-dopa was determined to be2.26mmol/L. Using Vmax/Km as a specificity constant, pyrocatechol was the better substrate for PPO. Moreover, ascorbic acid was a strong inhibitor that inhibited more than78%of PPO activity at1mmol/L.A Peroxidase (POD), located in the bud of Lonicera Confusa. was extracted and characterized. The POD of Lonicera Confusa by using guaiacol as substrate, the optimum pH was6as weak acid and the optimum temperature was low of only15℃. The POD was high thermal stability, enzyme activity changed very littleafter pretreatment60min below50℃. When the temperature reached high temperature (80℃), the enzyme activity was inactivated. The Km for guaiacol and H2O2was determined to be14.33mmol/L and0.2mmol/L, respectively. Common chemical compounds had a certain impact on POD. Ascorbic acid, which has the strongest inhibitory effect on POD, inhibited more than90%of POD activity just at the concentration of lmmol/L. The common metal ions also had certain effects on enzyme activity.To choose the best drying method and improve the quality of Lonicerae Confusae. the effects of different drying methods on the enzyme activities and the contents of active compounds from the bud of Lonicerae Confusae was studied. Dehydrating methods of vacuum microwave drying, air blast drying and shade drying were chosen to deal with buds of Lonicerae Confusae. The enzyme activity and the content of total phenoltotal, total flavonoids were determined by spectrophotometer. The contents of chlorogenic acid and luteoloside from different drying methods were determined by HPLC. Using the vacuum microwave drying method, the activities of polyphenol oxidase and peroxidase showed a minimum value, the residual activity remained only30%and13%resulting in a slight browning. Total phenols, total flavonoids, chlorogenic acid and luteoloside were all the highest compared with other two drying methods. The residual enzyme activity still remained above80%dried by shade drying method, the browning happened seriously, the content of active compounds was the least. The different drying methods had greater impact on the active compounds and enzyme activities, the vacuum microwave drying was the best method.