Computational-guided semi-rational design to reprogram phospholipase D into industrially robust enzyme

Phospholipase D (PLD) catalyzes the synthesis of rare phospholipid derivatives from phospholipids, and these derivatives possess significant application value and market potential. However, most reported PLDs show poor thermal stability, with melting temperatures below 60 °C, limiting their industrial application. This study employed a computer-aided design strategy integrating ancestral sequence reconstruction (ASR) and Gibbs folding free energy (ΔΔG) calculation to enhance PLD's thermal stability. Through iterative accumulation mutagenesis, a combinatorial mutant PLD_M5 was generated. Its melting temperature and half-life at 50 °C increased by 13.4 °C and 415-fold, respectively. Meanwhile, the mutant retained effective catalytic performance, achieving a phosphatidylserine conversion of 78.8% with a productivity of 39.4 g/L/h. Structural and dynamics simulations revealed that the ASR and ΔΔG mutations synergistically enhanced structural rigidity through distinct mechanisms. This work expands the industrial application scope of PLD and enables high-temperature catalysis of phospholipids.