Enzymatic Synthesis Of Eicosapentaenoyl And Docosahexaenoyl Ethanolamides

Eicosapentaenoyl and docosahexaenoyl ethanolamides(EPEA and DHEA)are endogenous bioactive lipid bioactive lipid mediators,with having physiological functions,such as immunomodulation,brain development,and anti-tumor,and they are expected to become important therapeutic drugs.Because of the low concentration and unstable structure of EPEA and DHEA,extraction of EPEA and DHEA from natural resource would be not easy.But their efficient production is still unresolved.In this study,use natural deep eutectic solvents(NADES)as a reaction medium to enzymatically synthesize EPEA and DHEA.The optimization of conditions in this process would be studied,which shows potential for functional research.The main results in detail as follows.(1)The effect of NADES used as reaction media on the product yields was investigated.Results showed that using choline chloride-based natural deep eutectic solvents(NADES)as media to improve the production of EPEA and DHEA.Especially in choline chloride-glucose(5:2,molar ratio,CG)solvent system,the highest yields of EPEA and DHEA reached to 91.51% and 74.22% under the conditons(time of 3 h,temperature of 60 °C,enzyme loading of 2195 U),respectively.In particular,Novozym 435 had a stronger preference for eicosapentaenoic acid ethyl ester(EPA-EE)than docosahexaenoic acid ethyl ester(DHA-EE)in other choline chloride-based natural deep eutectic solvents.Especially,Novozym 435 had the strongest preference with an EPEA/DHEA ratio of 2.09 in the presence of choline chloride-malonic(1:1,molar ratio,CM)after 5 h.(2)Evaluated the effect of choline chloride-glucose(5:2,molar ratio,CG)with different water contents on aminolysis.The yields of EPEA and DHEA were remarkably improved in the initial stage of the reaction by adding water.Adding 8.5% water to CG led to a significant increase(14.42% for EPEA and 32.08% for DHEA)in the yields under the contions(time of 1 h,temperature of 60 °C,enzyme loading of 2195 U).(3)The optimized conditions of preparing EPEA and DHEA in CG-8.5%water solvent system were studied.The high yields of EPEA(96.84%)and DHEA(90.06%)were obtained under the optimized conditions(fish oil ethyl esters/ethanolamine molar ratio of 1:2,temperature of 60 °C,reaction time of 1 h,enzyme loading of 2195 U).(4)Explored an separation and purification process of polyunsaturated N-acylethanolamines.The mixture after the aminolysis reaction was subjected to centrifugation,resulting two layers,suggesting that the centrifugation separated the products(EPEA and DHEA)from the NADES and ethanolamine.So the separation was simple and effective.The next purification of EPEA and DHEA was by column chromatography.Then EPEA and DHEA in the mixture were identified by high performance liquid chromatograph and liquid chromatography-mass spectrometry.The results showed that 87.57% of EPEA and DHEA without fish oil ethyl esters(EPA-EE and DHA-EE)was obtained.The approach proposed is an efficient and green technology and the separation of products is simple and effective,which show potential reference for functional studies of EPEA and DHEA.