Enzymatic Synthesis,Molecular Dynamics Simulation For Novel Human Milk-like 1,3-di-oleic-2-medium Chain Triacylglycerol And It’s Effects On Hepatocyte Lipid Metabolism

Medium and long chain triglycerides（MLCTs）are structural triglycerides thatcontain both long-chain fatty acids and medium-chain fatty acids on the glycerol skeleton.Because of their unique metabolic pathway in human body and their positive role in the immune system,MLCTs have become a research hotspot in the field of lipids.Up to now,there are four kinds of MLCTs:MLL,MML,LML and MLM.Most of them are different in chain length and position.Studies have shown that MLCTs play an important role in reducing body weight and preventing obesity;At the same time,MLCTs play an important role in providing essential fatty acids.Therefore,MLCTs have high potential nutritional value or can be used as a substitute for vegetable oil.1,3-dioleic acid-2-medium chain fatty acid（OMO）triglycerides are new type of triglycerides with similar structure to human milk ester 1,3-dioleic acid-2-palmitic acid（OPO）.They are LML type MLCTs composing of two oleic acids distributed in sn-1,3 position of glycerol and one medium chain fatty acid distributed in sn-2 position of glycerol.Theoretically,this kind of triglycerides may not only have the function of MLCTs,but also have part of the function of breast milk ester.However,there is no report on the structure and function of OMO triglycerides.In order to further understand the properties and functions of enzymatic synthesis of OMO triglycerides and promote the development and application of OMO triglycerides in functional food industry,MLCTs with high content of OMO triglycerides were synthesized by enzymatic synthesis of Cinnamomum camphora seed oil（CCSO）and oleic acid,coconut oil（CO）and high oleic acid rapeseed oil（HORO）,GC/HPLC-APCI-MS was used to analyze the content and distribution of fatty acids;Four kinds of lipases（Lipozyme RM IM,Lipozyme TL IM,Novozym435 and AO IM）were used to catalyze the synthesis of OMO triglycerides in a trace water-in-oil system;Molecular dynamics and quantum chemistry were used to investigate the effects of external factors on the formation of transition tetrahedron and reaction energy barrier in the synthesis of lipase and OMO triglycerides;Finally,the effect and mechanism of OMO triglycerides on oleic acid-induced lipid deposition in human liver LO2 cells were studied.The main results are as follows:1.MLCTs with high content of OMO triacylglycerols were synthesized by the acidolysis reaction between Cinnamomum camphora seed oil（CCSO）and oleic acid,catalyzed by lipozyme RM IM.In addition,the simultaneous rate of acyl migration（RAM）was investigated during the esterification process.The maximal yield of OMO-structured triacylglycerols（YST）was up to 64.45%under the optimum condition:the molar ratio of CCSO to oleic acid,1:4;enzyme dosage,10 wt%;temperature,60 ^oC;and reaction time,24 h.RAM was 28.66%at same time.It is shown that YST positively related to RAM at single experiments of the molar ratio（r²=0.999,P<0.01）,enzyme dosage（r²=0.988,P<0.01）,below 60 ^oC of temperature（r²=0.923,P<0.05）and within 24 h of reaction time（r²=0.940,P<0.05）.However,YST was negatively correlated with RAM at a temperature higher than 60^oC（r²=-0.682,P>0.05）or a reaction time longer than 24 h（r²=-0.594,P>0.05）.The effect of temperature on the reaction could be verified by molecular dynamics simulation,opened enzyme lids were increased with temperature elevated,and helped substrates invade into catalytic triad.When temperature rose to higher than 333 K（60^oC）,rearrangement of the catalytic triad was promoted,which resulted in both decreasing enzyme catalytic activity and irritating acyl migration.This study could help understand the trigger cause of acyl migration at high temperature,and conclusively reaction temperature was a vital factor to balance YST and RAM in a solvent-free system.OMO structured triacylglycerols might possess a potential application as a new type of nutritional lipids for daily diet.2.New structured lipids with OMO triacylglycerols were synthesized by promoting acyl migration in Lipozyme RM IM catalyzed interesterification between coconut oil（CO）and high oleic rapeseed oil（HORO）.Results from an orthogonal design L₂₅（5⁵）showed that the maximal yield of OMO-structured triacylglycerols was 45.65%under the following conditions:the molar ratio of CO to HORO,50:50;enzyme dosage,12wt%;reaction temperature,60 ^oC;reaction time,2 h;water activity,0.07.Low water activity showed a high rate of acyl migration（10.86%vs 5.07%no water system）,which promoted OMO synthesis due to medium-chain fatty acid migration to the sn-2position.In a low water content（5%）system of the molecular dynamics simulation,water molecules stabilized the whole structure of RM IM through hydrogen bonding,which helped fix lipase-catalyzed active sites,making substrates more easily inserted into active sites,resulting in increased enzyme activity.3.In the present study,a kind of structured lipids,namely OMO triacylglycerols,were synthesized through lipase-catalyzed reactions using coconut oil and rapeseed acid as materials in a trace water-in-oil system.Experimental analysis and computational simulations were undertaken to compare the stability of four lipases including Lipozyme RM IM,Lipozyme TL IM,Novozym 435,and Aspergillus oryzae immobilized lipase（AO IM）,and illustrate catalytic mechanism of Novozym435 during the synthesis of OMO.Fourier transform infrared and molecular dynamics simulation results demonstrated that a decrease in ordered structure（α-helix andβ-sheet）led to a reduction in enzyme activity.Compared with Lipozyme RM IM and Novozym 435,Lipozyme TL IM and AO IM exhibited better stability due to a short chain lid in TL IM,which covers activity sites,and hydrogen bonds formed between activity center of AO IM and water.Among four lipases,AO IM exhibited best catalytic performance:a OMO yield of 30.7%at 3 h and a good stability of long-term（48 h）.Density functional theory results demonstrated that specifically,during the synthesis of OMO triacylglycerol,the addition of Novozym 435（derived from Candida antarctica lipase B,CALB）substantially lowered reaction barriers（64.4KJ/mol with CALB vs.332.7 KJ/mol with no lipase）,aiding in the generation of OMO because of the formations of transitional tetrahedral intermediates.A trace water-in-oil system was a green and efficient alternative for lipase-catalyzed production of OMO,and this study provided crucial insights into the stability/instability and catalytic mechanisms of lipase in the synthesis of structured lipids.4.Reaction environments play a vital role on the structural stability and catalytic activity of enzyme.In this study,molecular dynamic（MD）simulation and Fourier transform infrared（FTIR）spectra were used to investigate the effects of aqueous and five non-aqueous systems including hexane,methanol,supercritical CO₂（sc CO₂）,ionic liquids of 1-butyl-3-methylimidazolium tetrafluoroborate（[BMIM][BF₄]）,and triolein（treated as a solvent-free system）,on the structural properties of lipase Candida antarctica lipase B（CALB）.The analysis of root mean square deviation（RMSD）showed that the structural variations of CALB in hexane（0.239 nm）and in triolein（0.188 nm）were lower than that in an aqueous system（0.275 nm）,implying a desirable stability on CALB structure when dissolved in hexane and triolein.Moreover,analysis of the radius of gyration（Rg）presented that the compactness of the CALB structure was significantly reduced（P<0.05）in methanol,resulting in a decrease of lipase stability.The secondary structure analysis revealed that in non-aqueous systems,the amounts ofβ-sheet andγ-turn of the lipase were reduced and the amounts ofα-helix and random coil were increased.More primary structure of lipase was preserved in non-aqueous systems than in aqueous system based on tertiary structure analysis in terms of residue contact maps.FTIR result verified that hexane and triolein are suitable reaction environments,which favor the stability of CALB.This study contributes to a better understanding of the correlations between reaction environments and CALB structure,identifying suitable reaction environments for CALB stability.5.This research investigated the effects of different triglycerides on lipid deposition and the underlying mechanism in oleic acid-induced human hepatic LO2 cells.Cells were treated with vehicle,oleic acid（OA）,OA+coconut oil（CO）,OA+rapeseed oil（RO）,OA+physical mixture oil of CO and RO（CROM）,OA+interesterified oil of CO and RO（CROI,rich in medium-and long-chain triglycerides（MLCTs））,OA+OMO（a kind of MLCTs）and OA+1,3-di-oleic-2-palmitic triglyceride（OPO）for24 h.Compared with the OA group,OA+CROI and OA+OMO treatment significantly reduced（P<0.05）the contents of total triglyceride and total cholesterol.Moreover,CROI and OMO down-regulated the protein expression of diacylglycerol acyltransferase（DGAT1）,and up-regulated the protein expression of peroxisome proliferators activated receptorα（PPARα）.This study showed that CROI and OMO triglycerides,abundant in MLCTs,attenuated lipid accumulation and regulated the expression of lipid metabolism-related proteins in LO2 cells induced by oleic acid.