Study On The Catalytic Reaction Kinetics Of Pancreatic Lipase In Macromolecule Crowding Based On Isothermal Titration Calorimetry

Modulation of lipase activity has important implications for both industrial production and health management.Previous studies have focused on the immobilization of industrial lipase,and how to control the catalytic activity of pancreatic lipase during the digestion in vivo remains a question.The common laboratory methods to study enzyme kinetics are spectrophotometric and fluorometric assays,while the isothermal titration calorimetry(ITC)has not been studied in this area.In this thesis,the catalytic kinetics of porcine pancreatic lipase(PPL)in crowding environment was studied based on ITC,and the mechanism of the effects of macromolecule crowding on the lipase activity was explored.Studying on lipase catalytic kinetics with different chain lengths,namely glyceryl trioctanoate and glyceryl trioleate in different varied molecular weight and concentration of polyethylene glycol(PEG),Ficoll and Dextran were also considered.(1)Catalytic reaction kinetics of PPL based on isothermal titration calorimetricThe kinetics of triglycerides catalyzed by PPL was studied based on ITC.PPL concentration,triglyceride concentration and parameters for ITC experiments(reference power,duration and spacing time)were explored.In the single injection assay,the optimal parameters were determined:the PPL concentration was 0.75?mol·L^-1 and the concentration of triglyceride was 12 mmol·L^-1 or 15 mmol·L^-1,the duration was 240 s or 180 s,and spacing time was 1200 s or 1800 s.When the reaction has proceeded to completion,the heat signal returns to the original baseline.And the multiple injection experiment provided a thermal change rate(d Q/dt)at different substrate concentrations.Subsequent 20 injections of 5?L were performed.The PPL concentration of 0.35 nmol·L^-1 and the triglyceride concentration of 20.5 mmol·L^-1,the interval between injections(spacing time:120 s or 150 s)should allow the system to stabilize the thermal power to the new baseline,which must be short enough to avoid the conversion of a significant amounts of substrate.Finally,the kinetic parameters of PPL hydrolysis reaction were fitted through a series of equations.(2)Kinetics of PPL catalytic reaction in Ficoll and DextranOur results showed that the catalytic rate by PPL in Ficoll400 was higher than that in diluting solution,and the catalytic efficiency(k_cat/K_m)was improved.In Dextran20,the catalytic rate by PPL was lower than that in diluting solution,and its catalytic efficiency was decreased.These phenomina indicated that Ficoll400 and Dextran20 had the opposite regulatory effect on the lipase efficiency.However,such regulation did not change when the different chain lengths of triglycerides were used.Further analysis of the spatial structure of PPL and other factors showed that with the Ficoll400 concentration increased,the fluorescence intensity at 350 nm that represent tryptophan residue was increased,the?-helix content in the secondary structure of PPL was increased,and the loop content was decreased.The change of these structures resulted in a more compact PPL conformation.However,in the Dextran20,the fluorescence intensity at 350 nm was decreased,the?-helix content in the secondary structure of PPL was decreased,the loop content was increased.These changes caused the loose of PPL structure,which led to the decreased enzymatic activity.(3)Kinetics of PPL catalysis reaction in PEGOur results showed that when TG-C18:1 was used as substrate for PPL catalytic,the catalytic rate by PPL in PEG200,PEG2000 and PEG20000 was higher than that in diluting solution,the enzyme catalytic efficiency(k_cat/K_m)was improved.When the TG-C8 was used as substrate for PPL catalytic reaction,the catalytic rate by in PEG2000 was higher than that in diluting solution,and the PPL catalytic efficiency increased.In PEG200 and PEG20000,the catalytic rate by PPL was decreased as compared with that in diluting solution,and the enzyme catalytic efficiency(k_cat/K_m)was decreased.Our results also showed that PEG with different varied molecular weight had different regulation effect on the lipase hydrolysis efficiency,which depended on the different chain lengths of triglycerides.The investigation on PPL structure showed that the fluorescence intensity at 350 nm that refered to tryptophan residues increased with increasing PEG concentration.The increase of?-helix content and the decrease of Loop content resulted in a more compact PPL structure and an increaseed enzyme activity.In PEG,?H_appof TG-C18:1and enzyme was much larger than that of TG-C8.However,in PEG200and PEG20000,the?H_appdifference between TG-C18:1 and TG-C8 was not significantly different from that in PEG2000.The above results indicated that the PPL catalytic kinetic parameters in Dextran20 and Ficoll400 had the opposite trend,which may be mainly attributed to the different effects of two polysaccharides on the PPL conformations,and the changes of enzyme activity led to changes in enzymatic efficiency.It is worth mentioning that both medium chain fatty acids and long chain fatty acids in the same macromolecule crowding had the same trends of the PPL catalytic kinetics,that is,no chain-length-dependent.The catalytic rate of both substrates by PPL increased in PEG2000,which mean that the macromolecule crowding was the main factor in affecting the structure of enzyme.In PEG 200 and PEG 20000,the binding affinity of substrate and enzyme may play a key role in enzyme kinetics,which is the substrate-dependent effect.Therefore,our results could provide mature understanding of the mechanism of lipase catalysis in macromolecular crowding.