Study On The Inhibition Of Coumarin Compounds On Polyphenol Oxidase

In this paper,the inhibition effects of 25 coumarin derivatives on polyphenol oxidase?PPO?were determined,and the results showed that 4-hydroxycoumarin was the most effective inhibitor.Kinetic study,Native-PAGE,circular dichrosim spectra?CD?,fluorescence sprctra and transmission electron microscopy?TEM?were used to analyse the inhibiting mechanisms of 4-hydroxycoumarin on PPO.The effects of different concentrations of 4-hydroxycoumarin on pear juice browning were studied during storage at 20?and 0?.The main results are as follows:The inhibition effects of coumarin derivatives on PPO were compared and the antibrowning effect of 4-hydroxycoumarin was the strongest.With an increase in the concentration of 4-hydroxycoumarin,PPO activity was decreased rapidly.When the concentration of 4-hydroxycoumarin was 3.33 mmol·L^-1,the relative activity of PPO was only 5%.Then the regulating characteristics of 4-hydroxycoumarin on the structure of PPO were revealed.The inhibition type and inhibition constant of 4-hydroxycoumarin on PPO was determined,and 4-hydroxycoumarin showed competitive type reversible inhibition with K_I value of 1.685 mmol·L^-1.The effect of 4-hydroxycoumarin on the structure of PPO was investigated by Native-PAGE,CD,fluorescence sprctra and TEM.The results showed that the stripe number and molecular weight of PPO did not change by 4-hydroxycoumarin,which indicated that 4-hydroxycoumarin had no effect on the primary structure of PPO.CD spectroscopy was used to monitor the secondary structure change of PPO induced by4-hydroxycoumarin,the decrease of?-helix illustrated the increase of surface hydrophobicity on protein,and there were more amino acid residues exposed to hydrophobic environment.With the addition of 4-hydroxycoumarin,the endogenous fluorescence intensity of PPO decreased with a blue shift in the emission peaks,suggesting that the tertiary structure of PPO had changed.After the treatment of 4-hydroxycoumarin,it was found that the diameter of PPO molecules became larger and the molecule number became fewer,which intimated the aggregation of PPO molecules by hydrophobic interaction.In order to test and verify the inhibitory effect of 4-hydroxycoumarin on pear juice browning,we selected 12 pears to observe the color change opear juice with different concentration of 4-hydroxycoumarin treatment.Reasults showed that the color of‘Dangshan'pear,‘Huangguan'pear,d'Anjou,Red Anjou'and Concord pear juice did not change during storage;when the concentration of 4-hydroxycoumarin was 3.33 mmol·L^-1,it had the most effective inhibition on pear juice browing of‘Fengshui'pear and‘Yali'pear during storage;the browning of pear Gem and Goledn Bosc pear juice appeared during storage at 20?,but4-hydroxycoumarin could decreased browning degree;4-hydroxycoumarin(2.00 mmol·L^-1)could effectively inhibit the browning of Comice and Bartlett pear juice during storage.