Engineering the Bridge-like Structure of Esterase Est7 Enhanced the Synthesis of Eicosapentaenoic Acid Monoglyceride by Expanding Substrate Access and Modifying Fatty Acid Selectivity

Although enzyme-driven synthesis of structured glycerides is significant, it still struggles to address the low-efficiency and complex-product challenge, especially for monoglycerides. This study aimed to improve the synthesis of eicosapentaenoic acid monoglyceride (EPAMG) catalyzed by esterase Est7, which exhibited superior catalytic specificity compared to currently characterized enzymes while elucidating the molecular determinants. The bridge-like structure (formed by F145/Q199/F200) of Est7 was proven to govern its catalytic efficiency and specificity. Through Ala-Leu-Phe scanning, the Est7^F145L mutant exhibited the highest esterification activity, generating 56.93% EPAMG in 2 h without the diglyceride identified, which was attributed to the expanded substrate entrance tunnels, the modified fatty acid selectivity, and the catalytic microenvironment. Molecular dynamics simulation showed that the oxyanion hole (S39/G97/N142) and acid-base catalytic residue (H294) of Est7^F145L stabilized its active conformations, accelerating formation and transformations of the intermediate. This study achieved the efficient production of EPAMG, advancing the understanding of catalytic specificity of esterases.