Substrate-Binding Site Engineering of Thermomyces dupontii Lipase (TDL) to Improve Selectivity for the Synthesis of Functional sn -1,3-Diacylglycerol

Diacylglycerol (DAG) oils have shown unique health functions, including preventing weight gain, lowering serum triacylglycerol (TAG) levels, and improving glucose metabolism. sn -1,3-DAG produced by esterification, hydrolysis, and glycerolysis using regioselective microbial lipases is regarded as the sole compound contributing to DAG’s functions. However, screening sn -1,3- and sn -2-regioselective lipases to selectively produce sn -1,3-DAG is rare. Hence, the present study screened a lipase that selectively synthesizes sn -1,3-DAG using gene mining. A T3/T14 mutant of TDL was obtained, which increased the DAG content to 60.87% and sn -1,3-DAG ratio to 88.76% after alanine scanning, combination, and iterative mutagenesis. Molecular docking and molecular dynamics simulations results showed that the changes of the hydrogen bond at Ser168 increased sn -2 position hydrolysis and sn -1,3 position synthesis selectivity after mutation. Our findings provide a reference for developing lipases with high regioselectivity to produce functional sn -1,3-DAG.