Phosphatidylserine synthesis via bilayer PLD in pickering emulsion

Phosphatidylserine (PS) is a vital phospholipid with broad applications in food, cosmetic, and pharmaceutical industries. To overcome the limitations of free phospholipase D (PLD), such as high cost, poor stability, and non-reusability, we developed a novel bilayer immobilization strategy using amino-functionalized magnetic nanoparticles. Magnetic Fe 3 O 4 nanoparticles were sequentially modified with tannic acid (TA) and 3-aminopropyltriethoxysilane (APTES), and genipin (Gnp) was employed as a biocompatible crosslinker to immobilize PLD via combined adsorption and covalent binding. Structural and functional characterization via SEM, FTIR, XRD, and TGA confirmed successful carrier modification and stable enzyme attachment. The optimized immobilized enzyme (Fe 3 O 4 @TA-APTES-Gnp-PLD) exhibited significantly enhanced catalytic performance. Under optimized reaction conditions, it achieved a PS conversion rate 11.27-fold higher than that of free PLD in a Pickering emulsion system, and 7.7-fold higher in a conventional two-phase system. The immobilized PLD also demonstrated excellent operational stability, retaining over 77 % of its initial activity after six consecutive reaction cycles, and 73 % after 12 days of storage at 4 °C. This work presents a sustainable, efficient, and reusable biocatalytic platform for PS synthesis, with strong potential for industrial-scale phospholipid production.