Study On The Regulation Of Polyphenol Oxidase Activity In Apples Based On Surfactants

Currently,researchers usually focus on studying the properties of purified enzyme,and the study about how to modulate the properties of enzyme in plants and other complex organisms is still rare.In this dissertation,catechol was used as substrate to study the activity and thermal stability of polyphenol oxidase(PPO)from Fuji apple,and the regulatory effects of conventional single chain surfactants(cetyl trimethyl ammonium bromide(CTAB)and dodecyl trimethyl ammonium bromide(DTAB)),genini surfactants(pentane-1,5-bis(dimethyldodecyl ammonium bromide)(C12-C5-C12),octamethylene-1,5-bis(dimethyldodecyl ammonium bromide)(C12-Cs-C12),dodecane-1,12-bis(dimethyldodecylammonium bromide)(C12-C12-C12),pentane-1,5-bis(dimethylcetyl ammonium bromide)(C16-C5-C16)and octamethylene-1,8-bis(dimethylcetyl ammonium bromide)(C16-C8-C16))and the complex systems formed by single chain surfactants and gemini surfactants on the activity and stability of PPO were explored.The results showed that the optimum experimental conditions for PPO enzyme were pH 7.0,the reaction temperature 30℃ and the ionic strength 1.5 M.In the presence of CTAB or DTAB,PPO activity could be increased by 70%or 45%at 30℃ once the concentration of CTAB reached 0.3 mM or that of DTAB reached 15 mM.Compared with single chain surfactants,20-30 μM of gemini surfactant C16-C8-C16,C16-C5-C16,C12-C5-C12,C12-C8-C12 and C12-C12-C12 could increase PPO activity by 45%,45%,35%,30%and 35%respectively.Moreover,the complex systems of single chain surfactant CTABwith gemini surfactants C16-C8-C16,C16-C5-C16 and C12-C5-C12 were found to increase PPO activity by 90%,100%and 80%,respectively,and the complex systems of DTAB with gemini surfactants C16-C8-C16,C16-C5-C16 and C12-C5-C12 were found to increase PPO activity by 80%,140%and 60%,respectively.Besides,the addition of surfactants could increase the thermal stability of PPO enzyme obviously,making the enzyme retain high activity at elevated temperature.By using conductivity meter,surface tension meter and fluorescence spectrophotometer,it was found that the PPO enzyme reduced the cmc value of the surfactant,and the surfactant could cause the conformation of PPO changed.In addition,the surfactants had little effect on the michaelis-menten constant(Km)of PPO enzyme,but increased the maximum initial reaction rate(vmax),indicating that(1)the addition of the surfactant did not enhance the affinity between the enzyme and the substrate and that the active region of the enzyme was not destroyed,and(2)the enzyme and the surfactant could form a more active intermediate,which might combine with the substrate much easier and make PPO enzyme activity increased.