Abstract
Phosphatidylserine (PS) has been widely used in the fields of food and medicine, among others, owing to its unique chemical structure and health benefits. However, the phospholipase D (PLD)-mediated enzymatic production of PS remains a challenge due to the low transphosphatidylation activity of PLD. Therefore, in the present study, we designed a maltose-binding protein (MBP) tag and a PLD co-expression method to achieve the expression of soluble PLD in Escherichia coli. A "reconstruct substrate pocket" strategy was then proposed based on the catalytic mechanism and molecular dynamics simulation, expanding the substrate pocket and manipulating the coordination of l-Ser within the active site. The best mutant (SrMBPPLDMu6) exhibited a 2.04-fold higher transphosphatidylation/hydrolysis ratio than the wild-type Furthermore, under optimal conditions, Mu6 produced 58.6 g/L PS with 77.2% conversion, within 12 h on a 3 L scale, which demonstrates the potential of the proposed method for industrial application.
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