| Polyphenoloxidase（PPO）exists in many kinds of organisms,such as plants,animals,fungi,bacteria and so on.It is a major cause of enzymatic browning which catalyzes two sequential reactions in the presence of oxygen:the hydroxylation of phenols to form o-diphenols and the oxidation of o-diphenols to o-quinones.PPO seriously affects the quality of the food during food processing,storage and transportation,especially fruits and vegetables.The inhibition of the enzyme activity is directly related to food quality and its economic value.Therefore,it is necessary to choose effective and security inhibitors.In this thesis,the inhibitory activity,inhibition type,inhibition kinetics,thermodynamics,conformational changes and variation in microstructure of three polyphenols（gentisic acid,apigenin and cyanidin3-glucoside chloride）on PPO were evaluated by UV-vis spectrophotometry,fluorescence emission spectra,circular dichroism and atomic force microscopy,respectively.In addition,the binding interactions between polyphenols and PPO were analyzed by computational docking simulation from the molecular level.These would provide a theoretical reference on inhibition mechanism of polyphenols on PPO.The main contents of this thesis are summarized as follows:（1）This chapter briefly introduced the sources,structure browning mechanism of PPO,and summarized the research status of PPO,including the methods of PPO inhibition,inhibition kinetics,conformational changes,variation of microstructure and the application of computational docking simulation on the research of PPO.（2）The effects of gentisic acid（GA）,apigenin and cyanidin 3-glucoside chloride（C3G）on the enzymatic characterization of PPO were studied in this chapter.It showed that the relative activity of PPO gradually decreased with increasing concentrations of GA,apigenin and C3G.The half-maximal inhibitory concentration of GA,apigenin and C3G were 2.10?10-3,3.92?10-5 and 3.05?10-55 mol L-1,respectively.Inhibition capability:C3G>apigenin>GA.It showed synergistic effect when GA or cinnamic acid（CA）was mixed with apigenin to inhibit PPO activity,CA and apigenin showed a stronger synergistic effect on the inhibition of PPO than other mixtures.While C3G and apigenin showed additive and subadditive effects on the inhibition of PPO for all concentrations observed.Inhibition of PPO induced by GA and apigenin was reversible,while C3G inhibited PPO activity irreversibly.The Ki andαof GA were measured as 3.10?10-4 mol L-1 and 15.36,respectively.The Ki andαvalues of apigenin were 2.23?10-5 mol L-1 and 1.33,respectively.The thermostability of PPO decreased gradually when GA,apigenin and C3G bound with PPO at different temperature.（3）The interaction mechanisms between GA,apigenin and C3G with PPO were studied in this chapter.GA,apigenin and C3G had quenching effect on the intrinsic fluorescence of PPO,and directly changed the tertiary structure of PPO.The fluorescence intensity of PPO decreased with a continuous increase inconcentration of GA with distinct red shift at maximum peak wavelength.According to binding constant analysis,GA and apigenin both had a binding site on PPO,respectively.The secondary structure of PPO had changed partially with the decrease ofα-helix content and the increase ofβ-sheet content after binding with GA（or apigenin）.After binding with GA and apigenin,the dimension of roughness,height and width of PPO molecules became larger,and it almost had no change when C3G bound with PPO.GA and apigenin inserted into with hydrophobic cavity of PPO,and interacted well with the primary amino acid residues.The docking conformation became more stabilized by hydrogen bond,pi-pi interaction,van der Waals forces and other forces.There might be a covalent bond between C3G and Cys83 around PPO active site.It was also possible that a covalent bond might be formed between C3G and Met294after the broken of glucosidic bond. |
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