Study On Effects Of Solvent And Water Activity On Lipase Selectivity And Acyl Migration In Enzymatic Synthesis Of 2-AG

2-Arachidonoylglycerol（2-AG）is an endogenous cannabinoid that has the functions of relieving stress,regulating energy metabolism and protecting retinal nerves.Enzymatic ethanolysis is a common method for the preparation of 2-AG.However,2-AG is extremely unstable and easily acyl-transferred to 1-AG,resulting in the loss of physiological activity.In addition,the enzymatic synthesis of 2-AG depends heavily on the 1,3-position selectivity of lipase.Previous studies have shown that conditions such as solvent and water activity may affect the acyl migration of 2-AG or the position selectivity of lipase.However,in the enzymatic ethanolysis reaction system,the mechanism of the effect of reaction conditions on its acyl migration and position selectivity are still not clear and need to be further explored.Based on the above problems,in this paper,2-monoacylglycerols（2-MAG）rich in 2-AG were obtained by enzymatic ethanolysis of microbial oil.The ethanolysis reaction was taken as the research object to explore the effects of solvent and water activity on the 1,3-position selectivity of lipase and the acyl migration of 2-AG.The main contents are as follows:Firstly,2-MAG was prepared by enzymatic ethanolysis of Mortierella alpina oil rich in arachidonic acid（ARA）as the raw material.The ethanolysis process was optimized.The results showed that the immobilized lipase Lipozyme TL IM from Thermomyces lanuginosus showed good ethanolysis efficiency and economic applicability.The optimum reaction condition was as follows:enzyme dosage 15%（w/w,oil weight）,molar ratio of ethanol to oil 40:1,reaction solvent t-butanol 1.5 m L/g oil,optimal water activity 0.53,reaction temperature 30°C,and reaction time 6 h.The content of 2-MAG in the crude product reached 35.05%with the yield of 91.11%.After two-step solvent extraction,the purified 2-MAG product was obtained with2-MAG content of 94.79%,ARA content of 51.83%and purification yield of 80.57%.Secondly,a simple reaction system for the acyl migration of 2-MAG was constructed.The purified 2-MAG was used as the substrate,and inactivated Lipozyme TL IM was added to investigate the effects of solvent and water activity on the acyl migration of 2-MAG.The results showed that after 20 days of storage,the[2-MAG]/[MAG]₀ ratio had a very significant negative correlation with the log P value of solvent at water activities of 0.11 and 0.97;When the water activity was 0.53 and 0.74,there was no significant correlation between[2-MAG]/[MAG]₀ ratio and log P value of solvents,indicating that the interaction of water and solvent caused“shielding”for the solvent at medium water activity.When the solvent was t-butanol,the[2-MAG]/[MAG]₀ ratios in the systems with water activities of 0.11,0.53,0.74,and 0.97 were66.44%,79.98%,69.74%,and 85.98%,respectively.No significant correlation was found with acyl migration,suggesting that the effect of water activity on the acyl migration of 2-MAG was more complex than that of the solvent.Thirdly,the 2-MAG enzymatic ethanolysis reaction model was established and the kinetic parameters were solved to explore the effects of solvent and water activity on the lipase position selectivity and 2-MAG acyl migration,respectively.The purified 2-MAG was used as the substrate for the ethanolysis reaction,and Lipozyme TL IM was used as the catalyst for the enzymatic ethanolysis reaction.The rate constants of each reaction in the enzymatic reaction system were solved by the molar concentration-time curve.The rate constants of k₁to k₆ with the mean square error（MSE）less than 0.03 were obtained using particle swarm optimization（PSO）algorithm,and the accuracy of the estimated value was verified by comparing the theoretical curve with the actual curve.The results showed that the curve fitting effect performed well,indicating that the method could accurately estimate the rate constants of ethanolysis reaction.Finally,the rate constant k₁ and the value of k₅/k₃ were used as indicators to characterize the position selectivity of acyl migration and lipase,respectively,and the effects of solvent and water activity on the position selectivity and acyl migration of the lipase were analyzed,respectively.The results showed that with the increase of log P value of solvent k₅/k₃ was 119.99,101.69,102.29,67.68 and 787.40,respectively,and there was a significant correlation between log P and k₅/k₃.The value of k₅/k₃ increased at first and then decreased with the increase of water activity,indicating that Lipozyme TL IM had the strongest sn-1,3 selectivity when the water activity was 0.53.In the enzymatic reaction system,the changing law of acyl migration with the activity of solvent and water was consistent with that in the non-catalytic system.According to the obtained inner law,the reasons for the effect of solvent and water activities on the yield of 2-MAG in the enzymatic reaction were explained.In summary,2-MAG products rich in ARA were prepared from microbial oil by enzymatic ethanolysis.Studies with two reaction models showed that highly polar solvents exhibited stronger inhibition of acyl migration（except at the water activity of 0.74）but were not conducive to enhancing lipase selectivity.The effect of water activity on acyl migration was relatively complex,while lipase had a specific optimal water activity.Lipozyme TL IM had strong sn-1,3 selectivity at water activity of 0.53.The results provide a theoretical support for the realization of selective and targeted synthesis of structural lipids and the enzymatic reaction to produce a variety of structural lipid products.