Structured Lipid Produced By Lipase Catalyzation And Quantum-Chemistry Studying The Mechanism Of Acyl Migration During The Process Of Intereterification

Partially hydrogenated shortening contains a certain amount of trans fatty acids(TFA),while overtaking TFA may increase the risk of cardiovascular disease.The lipase-catalyzed interesterification are popularly used to modify the properties of synthetic shortening by selecting suitable substrates to meet the physiochemical proportions of shortening.Cinnamomum Camphora Seed Oil(CCSO)is rich in medium-chain fatty acids(MCFAs)(C10:0,59%;C12:0,34.2%).MCFA is not only quickly absorbed by the body,but also lower blood fat and cholesterol.Therefore,it is beneficial to synthesize healthy shortening from CCSO by lipase-catalyzed interesterification.Although region-specific lipase has catalytic selectivity,acyl migration was found during the process of lipase-catalyzed interesterification.Acyl migration not only produces various by-products such as free fatty acids,triglyceride isomers,diglycerides,but also decreases the yield of target structure lipid and substrate utilization rate.The mechanism of acyl migration cannot be studied in detail due to its difficulty of detecting and capture the transition state and intermediate(IM),even though many scholars believe that the acyl migration mechanism is a nucleophilic substitution process by forming a five-membered ring IM.In recent years,computational simulation has been rapidly developed,which can be used to explain and expand the conclusions obtained by experiments.At present,quantum chemical simulation,as a kind of method in computational simulation,has become an important means to understand the catalytic reaction of lipase in the molecular level,furthermore,it can be utilized to find the transition state and also intermediate in the reaction process.Hence,it is meaningful to thoroughly elucidate the mechanism of acyl migration for the industrial production of lipase-catalyzed structural lipids by quantum chemical simulation.Firstly,shortening base with MCFAs,which can be quickly absorbed were synthesized by blending mixtures of CCSO,camellia oleifera(Theaceae)oil(COO)and fully hydrogenated palm oil(FHPO)at different weight ratios by Lipozyme TL IM.The physicochemical properties of the physical blends(PBs)and their interesterified products(IPs)were determined.The results indicated that the melting point was 42.4 °C in IP(CCSO: COO: FHPO=2:2:5),which met the requirements of commercial shortening,the fatty acids were mainly palmitic acid(C16:0,about 40%),stearic acid(C18:0,about 20%)and oleic acid(9cC18:1,about 16%).The solid fat content(SFC)showed that SFC was retained at 25 °C-40 °C,and the ratios of 2:2:4,2:2:5 and 2:2:6 at 35°C were 20.4%;20.5%;25.5% respectively.The X-ray diffraction spectrum indicated that the fat crystal form of PB remained unknown peak,which is more complicated while IP is mainly a structurally stable crystal form of ?'.In addition,morphology of PBs presented very coarse,branched and leaf-like crystals(55 ?m),but small grainy crystals(20 ?m)in IPs due to several new triglyceride structures(CPO;LaOP;LaPP;COS;CPS)resulting from interesterification reaction.A detailed understanding of mechanism of the acyl migration occurred during lipase-catalyzed interesterification plays a vital role in improving the quality and total yield of structured lipids.However,up to now,the mechanism of the acyl migration remains controversial.Here,two pathways(non-catalyzed and lipase-catalyzed)were designed to study the mechanism of the acyl migration.The B3 LYP and 6-31G(d,p)were employed to establish and optimize the intramolecular non-catalyzed pathway model(1,2-diacetate,1,2-DA)in Gaussian 09 software package.The lipase-catalyzed pathway model(1,2-DA-4TGL)containing 123 atoms was firstly established from 1,2-DA and 4TGL(lipase from PDB database)by using molecular docking software(Discovery Studio 2.5).The initial model was then optimized in the Gaussian 09 software by the same conditions.Based on computational results,we concluded that the barrier of the stepwise step lower than the concerted step(41.8 <55.4 kcal/mol)in non-catalyzed pathway,indicating that former step was easier to occur at room temperature.In addition,a stepwise step involved with one water molecule further reduced the energy barrier to 31.7 kcal/mol,denoting a crucial role of water molecule in the reaction.Although,it was hard to occur at the room temperature due to overwhelming energy barrier.On the other hand,the lipase-catalyzed pathway produced a four-step reaction mechanism:(1)His235 deprotonated Ser144,while Ser144 captured the hydroxyl proton on the sn-1 of substrate 1,2-DA,forming an process of double-proton transfer;(2)The oxyanion on the sn-1 of 1,2-DA nucleophilic attack on the sn-2 carbonyl carbon to form a five-membered ring;(3)Ser144 was reprotonated by His235,while Ser144 protonated the newly formed oxyanion of 2-DA to finish double proton return;(4)accompanied by a transfer of proton from the sn-2 carbonyl carbon to the sn-1 ester oxygen,a five-membered ring was opened involved with one water molecule and the 1,2-DA became 1,3-DA,completing the process of acyl migration.The fourth step was a rate-limiting step of the lipase-catalyzed pathway,and the energy barrier was 18.8 kcal/mol,being better agreement with the available experimental value(17.8 kcal/mol).Interestingly,we also found that the catalytic triad(Asp-His-Ser)in lipase can be used as a generalized acid/base and water molecule can be applied as a "proton shuttle" during the process of acyl migration to collaboratively reduce the required energy barrier.Generally,the acyl migration involved lipasecatalyzed pathway is about 2 times lower than the intramolecular non-catalyzed pathway,suggesting that the presence of lipase can accelerate the acyl migration by reducing the energy barrier of the acyl migration reaction.