Study On The Correlation Between The Conformational Change And Catalytic Behavior Of Lipase Induced By High Pressure

High pressure?HP?technology,an emerging food biotechnology,could be used in the study of structural changes of the protein.It helps to reveal the structure-function relationship of proteins,particularly that of enzymes,which would lay foundations for the directional modification of enzymes.Lipase is one of the most important enzymes in food industry.This study selected lipase as the model enzyme to probe the changes in lipase's catalytic behavior and conformation as well as thermal stability after HP treatment.Additionally,the in situ detecting system and molecular simulation were employed to probe the kinetics of catalysis and conformational states under HP.The structure-function relationship of lipase was further discussed based on the above information.The main research contents are as following:?1?The modification of the activities of Rhizopus chinensis lipase?RCL?and Candida rugosa lipase?CRL?by HP were studied.At 40?,the activities of the lipases increased and then decreased with pressure.The highest hydrolysis activities of the lipases were observed after HP treatment under 200 MPa for 10 min.The esterification activity of RCL reached the maximal activity under 200 MPa,while that of CRL achieved the maximal value under 300MPa.The synthesis of hexyl octanoate catalyzed by CRL followed the Ping-Pong Bi-Bi mechanism with inhibition by both substrates,which was not modified by HP treatment.?2?The conformational changes of pure RCL after HP treatment were probed.The secondary and tertiary structure of RCL changed little at pressures below 400 MPa and that the enzyme was in native-like state.The contents of?-helix and?-sheet kept at 45%and 25%,respectively.Meanwhile,the parameter(K_sv)for quenching intrinsic fluorescence of RCL maintained at 10.40.As pressure increased to 500 MPa,RCL began to unfold and the content of?-helix decreased 11%with the conformational rearrangements,the slight increase in K_sv and the remarkable changes in tertiary structure.A part of secondary and tertiary structure of RCL disappeared after HP treatment at pressure over 500 MPa,which caused the unfolding and the loosely compacted structure of RCL.Meanwhile,K_sv increased to 12.52 and the intrinsic fluorescence intensity of RCL increased markedly with red shift of the maximal wavelength from 331 to 348 nm.Furthermore,the extrinsic fluorescence intensity increased three folds that at 0.1 MPa and water molecules penetrated into RCL molecules with exposure of some hydrophobic regions.These changes resulted in the irreversible denaturation of RCL.?3?The behaviors of catalytic region of RCL under HP were studied.Under pressures below 200 MPa,the lid of RCL locating above the active center tended to open by increasing the distance between them to 8.3?without influencing the lipase's interfacial activation.In addition,the interaction between substrate and catalytic center was slightly enhanced with the interaction energy increasing form 14.32 to 15.07 kJ/mol when the pressure increased from0.1 to 200 MPa.All of these together caused the increase of RCL activity under this pressure range.Contrary to this,the lid got more closed with the distance below 7.7?under pressure range of 400-600 MPa.Meanwhile,HP influenced the interaction between catalytic residues,for instance,the hydrogen bond interaction between His257 and Asp204 was weakened and the binding affinity between substrates and the catalytic center was reduced under pressure of400-600 MPa,for example,the interaction energy was decreased to 11.33 kJ/mol at 600 MPa.Besides,the microenvironment of Trp224 in the cavity near the catalytic region may be modified by HP of 600 MPa.Together with the loss of ordered structure of RCL,these changes caused the noteworthy decrease in RCL activity.?4?In the p-nitrophenol laurate hydrolysis system catalyzed by RCL,the tendency of catalytic ability under HP was the same as that measured after depressurization.After the HP treatment under 200 MPa,the optimal temperature was 40?.Under these conditions the Michaelis constant and the activation volume were the lowest,and the affinity,the catalytic efficiency were the highest.The interfacial activation was not changed after HP treatment under 0.1-400 MPa and 40?,while it was markedly weakened under 500 MPa.The thermal stability of RCL under different pressure-temperature combinations showed that the antagonistic effect occurred at the range of 0.1-350 MPa at 50-60?but the synergistic effect was observed and RCL was destabilized by treatment under pressure over 350 MPa.The effect of monovalent cations on the activity of RCL approximately followed the Hofmeister serise?K⁺>Na>⁺>Li⁺?.At 0.1 mol/L,K⁺and Li⁺improved the thermal stability of RCL,it may be due to that they changed the conformation of RCL.At 1.0 mol/L,the polyhydroxy alcohol with 3 or more hydroxyl groups could well protect RCL by maintaining the enzyme's conformation.?5?The conformation under HP was observed by in situ detecting system.Under pressure of 50-200 MPa and 20-40?,the fluorescence intensity was approximately 10%higher than that at 0.1 MPa.It was still higher than that at 0.1 MPa even after depressurization,so the changes in Trp surroundings was partially irreversible.At 60?,HP inhibited the thermal treatment-induced increase of fluorescence intensity and protected the tertiary structure of RCL.In addition,the secondary structure and the intactness of lid was protected by HP,which were responsible for the high catalytic ability and stability of RCL under 200 MPa at 60?.