Immobilization of Bioimprinted Phospholipase D and Its Catalytic Behavior for Transphosphatidylation in the Biphasic System

Phosphatidylserine (PS) holds considerable importance in both the food and medical sectors; however, its biosynthesis is critically dependent on phospholipase D (PLD). The practical application of PLD is constrained by pronounced side reactions in its free form and by reduced selectivity when immobilized. To address these challenges, this study employed a sequential strategy involving bioimprinting to hyperactivate PLD, followed by microencapsulation via ionotropic gelation within an alginate–chitosan matrix. This approach induced conformational rigidification, enabling PLD to maintain its hyperactivated state in aqueous environments. Under optimal conditions, the encapsulation efficiency reached 78.56%, and the enzyme activity recovery achieved 105.27%. The immobilized bioimprinted PLD demonstrated exceptional catalytic performance, achieving a 94.68% PS yield within 20 min, which significantly surpassed that of free PLD (85.82% in 150 min) and non-imprinted immobilized PLD (90.34% in 60 min). This represents 7.27-fold and 2.14-fold efficiency improvements, respectively. Furthermore, the biocatalyst exhibited outstanding storage stability, thermal stability, and reusability (77.53% yield after 8 cycles). To our knowledge, this is the first report combining bioimprinting with alginate-chitosan microencapsulation via ionotropic gelation, which yielded remarkably enhanced PLD activity. These findings highlight the strong potential of this method for efficient PS production.