Rational design of phospholipase D to enhance thermostability and catalytic activity for efficient biocatalytic...

Phospholipase D (PLD) is an essential enzyme for the enzymatic synthesis of phosphatidylserine (PS), but its widespread application is limited by its low stability and activity. Herein, a highly active and thermostable multi-mutant PLD_M4 was obtained through a comprehensive strategy based on various computer-aided mutant design and systematic clustering analysis, extending its half-life from 3.7 to 89.8 min and enhancing its activity from 42.7 to 413.1 U/mL. The PLD_M4 exhibited a higher PS synthesis efficiency in water-organic solvent system, reaching 119.1 g/L. The synthesis of PS within various edible oils was also achieved, producing PS-enriched edible oils (approximately 19.8 g/L) and creating opportunities to construct food grade PLD-mediated reaction system. These results highlight the proposed method providing an efficient tool and a promising mutant with enhanced thermal stability and activity, and expand the practical application of PLD in the food industry.