The Effect Of Ultra High Pressure On The Lipase Activity And Preliminary Studies On The Mechanism

Recently, non-thermal processing has become a hot topic of the food processing. As oneof the non-thermal processings, ultra high pressure (UHP), which could both sterilize andkeep quality, was the first technology admitted by the United States Department ofAgriculture-Food Safety and Inspection Service (USDA-FSIS). At present, although the effectof pressure on the stability and rates of reaction for many enzymes has been researched, thereis still a lack of experimental data for the majority of known enzymes. Also, the effect of UHPon enzyme structure is scare. Selecting lipase as the research object, this paper studied theeffect of UHP on the activity, stability and conformation of the lipase, besides the effect ofUHP on the hydrolysis reaction system of lipase was also studied.Firstly, the effect of high pressure on the lipase activity was studied. The results showedthat: between40and60℃, the effect of UHP on the lipase activity was not obvious, while athigh temperature range from70℃to80℃, the effect turned obvious. At70℃, the activityof lipase treated with high pressure was obviously higher than that under0.1MPa, at200MPa the maximum increased by32%compared with0.1MPa. At70℃, the activitydecreased with the extension of experiment time at0.1MPa, while the rate of decreasedactivity was slowed down under200MPa. The optimum pH was turned to alkaline underUHP treatment. The lipase activity was increased by5%-10%under high pressure after48h.Secondly, changes of lipase conformation after high pressure were analysed byNative-PAGE, Circular Dichroism (CD), fluorescence spectrum and Differential ScanningCalorimetry (DSC). The results investigated that: The electrophoretic band wasn’t changed,and it declared there was no dissociation and association after UHP. CD of lipase showed thatpressure could make the secondary structure change, the α-helix contents raised and β-sheetcontents decreased as the pressure increased. The fluorescent intensity of lipase wasstrengthened at339nm after UHP treatment, which indicated that after UHP treatment thetryptophan residues were more exposed. After UHP treatment, the denatured temperature wasslightly increased while ΔH was changed greatly. Under200MPa, ΔH reached to maximum,which indicated the thermostability of the enzyme was improved by UHP.Thirdly, the effect of UHP on the stability of lipase was studied. The results indicatedthat: between40and60℃, the effect of200MPa on the stability of lipase was not obvious,while it was obviously stable at70℃and80℃. The effect of sorbitol on lipase stabilizationwas the strongest in the low molecular alcohols. It could decrease the negative effect of heatand high pressure, but the effect of UHP was dominated, the low molecular alcohols justplayed a supplementary role. The effect of sorbitol on the secondary structure of lipase wasdetected by CD measurement, which showed that the α-helix contents raised and β-sheet contents decreased compared with the control.Lastly, the effect of UHP on the reaction system of lipase was studied. The resultsshowed that: The enzyme-catalyzed reaction time of equilibrium just needed30min at70℃,200MPa. At70℃, the yield was lower under0.1MPa, but the yield increased under UHPtreatment. The optimum pH was turned to alkalinity under200MPa, which declared that thereaction might be more appropriate for alkalinity under UHP.