The Effect Of Lactic Acid-heat On Activity And Conformation Of Polyphenol Oxidase

Polyphenol oxidase (PPO) is a kind of copper-protein enzyme, which widelydistributes plants, animal, and fungi. The different origins of polyphenol oxidasemake it have different biological characteristic. Polyphenol oxidase can causedenzymatic Browning, which caused the loss of sensory quality and nutrition of fruitsand vegetables. So, the research about biological characteristic and passivation ofpolyphenol oxidase is necessary. In the study, we carried out some research: thebiological characteristic and passivation of polyphenol oxidase (CPPO) fromChoerospondias axillaris; the effect on activity and conformation of polyphenoloxidase induced by lactic acid, heat or lactic acid-heat treatment. The mainconclusions are as follows:(1) The biological characteristic and passivation of CPPO: The optimumreaction temperature of CPPO is50℃. CPPO has low activity in low temperature.The optimal pH is7.0, and controlling pH is a good way to inhibit the enzymeactivity. The main substrates of CPPO are two phenolic substances such as catechol,TBC,4-methyl catechol; three phenolic substances such as gallic acid and focalgallic acid have low catalytic activity. Six kinds of inhibitors for CPPO havedifferent result and the strength of inhibition is ascorbic acid> Na2SO3> citricacid> tartaric acid> l-cysteine CaCl2(2)The effect of lactic acid on activity and conformation of polyphenol oxidase:The relative activity of PPO decreased gradually with the increasing of lactic acidconcentration, the relative activity of PPO dropped to1.20%of its original activityat40mmol/L. The activity of PPO was inhibited significantly by lactic acid at40mmol/L. By the detection of sulfhydryl content (SH), the SH content decreasedgradually with increasing lactic acid concentration and it showed a liner relationshipthe activity of PPO (R2=0.9861). When PPO is induced by different concentrationsof lactic acid, the conformation of PPO was gradually unfolded which induced thedecreasing of relative activity.(3) The effect of heat treatment on activity and conformation of polyphenol oxidase: Heat treatment can effectively inhibit PPO activity, and the strength ofpassivation increased gradually with the extension of time and the enzyme activitywas only28.79%incubated55℃with30min. The inactivation of PPO induced byheat treatment fit the first-order model, and it’s enzymatic parameters are K/min-1=0.0425and D/min=54.19. PPO fit Michaelis-Menten equation model, and Vmaxdecreases with increasing treatment time. UV spectrum and fluorescence spectrumshowed that PPO conformation is destroyed,(4)The effect of lactic acid-heat treatment on activity and conformation of PPO:The means of lactic acid-heat treatment is effectively and quickly. Lactic acid andheat treatment or combined treatment have inhibitory effect on PPO, the strength ofinhibition of combined treatment is much stronger than lactic acid treatment or heattreatment. The relative activity of PPO incubated at55℃with90min or10mmol/L lactic acid with90min is34.03%and98.08%, respectively. And therelative activity of PPO incubated at55℃,10mmol/L with45min is9.89%. UVspectrum and fluorescence spectrum showed that heat treatment or combinedtreatment can change the molecular conformation and the breaking strength andspeed of PPO molecular conformation caused by combined treatment is higher thanlactic acid treatment and heat treatment lonely.