Combining a chitosan-immobilized phospholipase D with a mixed micelle system for highly efficient synthesis of phosphatidylserine

This study developed a highly efficient immobilized phospholipase D (PLD) system using a magnetic chitosan-based carrier with the natural crosslinking agent genipin (Fe 3 O 4 @CS-Gnp-PLD). Systematic characterization confirmed successful PLD immobilization, with chitosan's abundant amino and hydroxyl groups providing stable binding sites that enhanced both enzyme loading and stability. In the sodium deoxycholate-based mixed micelle system, the Fe 3 O 4 @CS-Gnp-PLD achieved an 8.05-fold higher phosphatidylserine (PS) conversion rate than free PLD, owing to enhanced substrate solubility and improved enzyme accessibility. The immobilized enzyme demonstrated exceptional operational stability, retaining 80% activity after 6 reuse cycles and 72% activity after 12 days of storage at 4 °C. This integrated platform, combining chitosan-based enzyme immobilization with a mixed micelle system, offers an efficient, stable, and recyclable approach for sustainable PS biomanufacturing.