Production of Diacylglycerol‐Mixture of Regioisomers with High Purity by Two‐Step Enzymatic Reactions Combined with Molecular Distillation

Abstract Pure diacylglycerol (DAG) is of vital importance for the biomedical and dietetic properties research of DAG. In this study, we aimed to develop an effective process to produce DAG‐mixture of regioisomers with high purity. Firstly, DAGs and monoacylglycerols (MAGs) were synthesized by enzymatic esterification of glycerol and free fatty acids (FFAs) from camellia oil with catalysis of Penicillium camembertii lipase, and the obtained reaction mixture was composed of 49.9 % DAG [33.4 % for 1,3‐DAG and 16.5 % for 1,2 (2,3)‐DAG], 31.6 % MAG and 18.5 % FFA. Secondly, a monoacylglycerol lipase (lipase CBD‐MGLP), which was produced by recombinant Escherichia coli in our laboratory, was employed to hydrolyze MAG in the above reaction mixture, and the MAG content decreased to 1.9 % under the optimal conditions with 375 U/g (U/w, with respect to the mass of MAG in the mixture) of CBD‐MGLP loading, temperature of 45 °C, mass ratio of esterification mixture to Tris–HCl buffer (w/w) 10:10, and pH of Tris–HCl buffer 9.0. Then, the hydrolytic products were further purified by molecular distillation at low temperature of 130 °C under a pressure of 10 Pa [equivalent to 377 °C at 101.325 kPa (1 atm)], and the DAG purity was up to 98.0 % (66.6 % for 1,3‐DAG and 31.4 % for 1,2‐DAG) in the final products. This indicated that two‐step enzymatic reactions combined with molecular distillation at low temperature could be a feasible and prospective process to produce DAG‐mixture of regioisomers with high purity.