Study On Selective Synthesis Of Diacylglycerol By Lipase In Natural Deep Eutectic Solvents And Its Molecular Mechanism

As a generally recognized safe health functional oil,diacylglycerol(DAG)has been widely used in food and medicine fields.It is an important means for the selectively enrichment of single products to introduce the organic solvents or ionic liquids to regulate the balance of enzymatic reactions and enzyme substrate specificity during the enzymatic synthesis of DAG.Completely composed of small molecule metabolites in living organisms,natural deep eutectic solvent(NADES)is a new type of green solvent system,which is reported not only a good protector of lipase,but also suitable for a variety of enzyme catalysis.At present,NADESs are mainly used in the preparation of biodiesel,glycolipids and epoxy lipids in the field of enzymatic lipid modification.There are few studies on the preparation of DAG,and the molecular mechanism of how the solvent system regulates the selective synthesis of structural esters is not clear.Based on this,choline chloride based NADESs and betaine based NADESs were prepared in this study,and their formation mechanism and physicochemical properties were characterized and analyzed.Then,a green and efficient catalytic system for enzymatic esterification of DAG was established with the introduction of them,and the reasons for the promotion of DAG accumulation in NADESs were discussed from three aspects:the change of water activity and reaction interface during esterification,as along as the Thermokinetics of enzymatic hydrolysis.Finally,the molecular mechanism of enzymatic selective synthesis of 1,3-DAG in NADESs was studied by combining experimental studies and molecular dynamics simulations.The research results lay a theoretical foundation for rational regulation of the composition of reaction products in the process of enzyme catalysis.The specific research content and results are as follows:(1)Ten different classes of choline chloride based NADESs and betaine based NADESs were prepared in this study,and the structure of solvents were analyzed by FT-IR and NMR,then the physical and chemical properties of NADESs and the effect of water content and temperature on the properties of some solvents were studied in detail.It was shown that the NADESs were formed mainly through the hydrogen bond interactions between components,and the strength of hydrogen bond between molecules was related to the type of hydrogen bond donor.The polarity,acid-base and density of NADESs are related to the kind of their hydrogen bond donors.The water activity of NADESs ranges from 0.081 to 0.232,all of them are in the low water activity state,and when the water content increases to 50%,the hydrogen bond network of NADESs will be destroyed.(2)In this study,three immobilized lipases were used to synthesize DAG by enzymatic esterification reaction in the ten NADESs prepared in(1),and the solvent-free system was used as control.After screening and analysis,it was determined that the optimal immobilized lipase was Novozym 435,and the optimal reaction solvent was the ChCl:Gly.The study found that compared to the solvent free system,the yield of enzymatic esterification synthesis of DAG and 1,3-DAG in the ChCl:Gly significantly increased,while the yield of by-product monoacylglycerol significantly decreased.A green,efficient and effective catalytic system for the enzymatic esterification of DAG was established.Subsequently,the process of DAG synthesis catalyzed by lipase Novozym 435 in ChCl:Gly was investigated by single factor and response surface experiments.The optimum esterification conditions were determined as follows:substrate molar ratio of 3.79,reaction temperature of 50.84℃,enzyme addition of 4.87%,reaction time of 7.17 h.The actual yield of DAG reached 68.18%under the optimized conditions.Finally,the reason why the content of DAG can be significantly increased in the ChCl:Gly was preliminarily explored from the changes in water activity and reaction interface during esterification,as well as the thermodynamics of enzymatic hydrolysis.(3)In this study,it was found that compared to solvent-free systems,sugar alcohol NADESs can effectively improve the selectivity of 1,3-DAG during enzymatic esterification.The molecular mechanism was explored from the effects of NADESs on the acyl transfer of 1,3-DAG and the site specificity of lipase,respectively.The results showed that the solvent properties of NADESs were not the main factor affecting the acyl transfer of 1,3-DAG,but the main factor affecting the synthesis of 1,3-DAG by regulating the water activity during the esterification reaction to regulate the reaction balance.The influence of NADESs on the enzyme structure can’t directly reflect the influence on its position selectivity.However,combining the results of circular dichroism,fluorescence spectroscopy,and molecular dynamics simulation,it can be seen that NADESs can maintain the overall conformation and secondary structure stability of the lipase CALB,and the stability of the tertiary structure is maintained by the hydrogen bond interaction between NADES and CALB.Finally,from the free energy landscape obtained by molecular dynamics simulation for CALB in solvent-free and NADESs systems simulating the binding of catalytic substrates,glycerol and oleic acid,the probability of reacting hydroxyl group at sn-1,3 position of glycerol with oleic acid was higher than that at sn-2 position in NADESs systems,and NADESs effectively improved the sn-1,3 position specificity of CALB.In conclusion,this study suggests that NADESs enhances the selectivity of 1,3-DAG mainly by synchronously regulating the water activity of the esterification reaction system and the sn-1,3 position specificity of lipases.