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

Area of Science:

• Biotechnology
• Enzyme Engineering
• Protein Engineering

Background:

• Phospholipase D (PLD) synthesizes valuable phospholipid derivatives.
• Limited thermal stability of wild-type PLDs restricts industrial use.

Purpose of the Study:

• To enhance the thermal stability of Phospholipase D (PLD) for industrial applications.
• To develop a robust enzyme capable of high-temperature phospholipid catalysis.

Main Methods:

• Computer-aided design integrating ancestral sequence reconstruction (ASR).
• Gibbs folding free energy (ΔΔG) calculations for mutation prediction.
• Iterative accumulation mutagenesis to generate combinatorial mutants (PLD_M5).

Main Results:

• Engineered PLD_M5 exhibited a 13.4°C increase in melting temperature.
• PLD_M5 showed a 415-fold increase in half-life at 50°C.
• Achieved 78.8% phosphatidylserine conversion with 39.4 µg/L/h productivity, retaining catalytic function.

Conclusions:

• ASR and ΔΔG-guided mutations synergistically enhance PLD structural rigidity and thermal stability.
• The engineered PLD enables efficient high-temperature catalysis of phospholipids.
• Expanded industrial application scope for PLD in phospholipid derivative synthesis.