Experimental and kinetic insights into enzymatic synthesis of phosphatidylglycerol in an oscillatory baffled reactor

• A new aqueous synthesis route of PG was developed and optimized. • 63.5% PG conversion was achieved in 20 mins at a 250 ml scale without any PA. • A kinetic model was established, reproducing PG and PC profiles at R 2 > 0.95. • The kinetic and experimental insights assist process design, scaleup and operation. Phosphatidylglycerol (PG) is a valuable product across pharmaceuticals, cosmetics and food industries, the conventional phospholipase D (PLD) syntheses however require organic solvents and very long reaction times to reach 50-74% yield at millilitre scale. The novelty of this study is that we have developed a solvent-free, fully aqueous synthesis route of PG using PLD-catalysed transphosphatidylation of phosphatidylcholine (PC) with glycerol in a 250 mL oscillatory baffled reactor (OBR). By optimising temperature, PLD concentration, glycerol-to-PC ratio and mixing, we achieved 63.5% PG conversion within 20 minutes with no detectable byproduct. Time-resolved kinetic analysis has revealed a three-phase mechanism in this reaction: an initial Michaelis–Menten behaviour, followed by product inhibition and eventual enzyme deactivation. We have then developed a multi-parameter kinetic model integrating intrinsic enzyme kinetics with operational variables, enabling quantitative predictions of reaction concentration, conversion and selectivity at high confidence level (R2>0.95). Coupling the green, solvent-free process with reactor intensification and mechanistic modelling establishes a scalable framework for PG manufacture and offers regulatory and sustainability advantages by avoiding volatile organic solvents and simplifying downstream processing.