Abstract: To address the issues of low efficiency and poor safety associated with traditional methods for producing phosphatidylethanolamine (PE), we isolated and identified a strain of Stenotrophomonas maltophilia capable of producing phospholipase D (PLD), and cloned and heterologously expressed its PLD gene smapld. Structural analysis revealed that the N-terminus of SmaPLD contained a non-conserved flexible region (residues 1–52) in the PLD superfamily. Molecular dynamics simulation indicated that this fragment led to increased structural dynamics and decreased stability of the enzyme. By rational design, a N-terminal deletion mutant ΔN-SmaPLD was constructed, which showed significantly enhanced structural rigidity, decreased substrate binding free energy, and greatly improved catalytic activity. The yield of PE obtained with it was 47.66%, compared with 26.37% for the wild-type enzyme. Under optimized conditions of a molar ratio of phosphatidylcholine to ethanolamine 1:3, a volume ratio of aqueous phase to organic phase of 1:2, and a reaction time of 8 hours, the yield of PE catalyzed by ΔN-SmaPLD reached 93.25%. This enzymatic process is mild, green and highly efficient, overcoming the limitations of traditional production methods and providing a new approach for the large-scale preparation of food-grade PE.

Key words: phosphatidylethanolamine; phospholipase D; rational design; enzymatic synthesis; molecular dynamics simulation; food additive