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Phospholipase D (PLD) plays a pivotal role in the biosynthesis of phosphatidylserine (PS), but its practical application is constrained by limitations in stability and reusability. In this study, we successfully fabricated the Fe3O4@SiO2–graphene oxide (GO) nanocomposite by chemical binding of Fe3O4@SiO2 and GO. Subsequently, PLD was immobilized onto the nanocomposite via physical adsorption, with the aim of enhancing catalytic stability, reducing mass transfer resistance, and improving reusability. Under optimal conditions, the immobilization efficiency reached 84.4%, with a PLD loading capacity of 111.4 mg/gsupport. The optimal pH for PS production by immobilized PLD shifted from 6.0 to 6.5, while the optimal temperature increased from 45 °C to 50 °C. Notably, the immobilized PLD demonstrated a shorter reaction time and a higher PS yield, achieving a 95.4% yield within 90 min, compared to the free PLD (78.1% yield within 150 min), representing a 1.04-fold improvement in production efficiency. Furthermore, the immobilized PLD exhibited outstanding storage stability and thermal stability, along with remarkable reusability. Even after being reused for 10 cycles, the PS yield still stays as high as 78.3%. These findings strongly suggest that the Fe3O4@SiO2–GO immobilized PLD has the potential for the efficient production of PS. [ABSTRACT FROM AUTHOR]

Phosphatidylserine (PS) has significant applications in various sectors, such as the medical and food industries. However, its production relies heavily on phospholipase D (PLD), a crucial tool that is hindered by issues like poor stability and irrecoverability. Immobilization presents itself as an effective solution to overcome these limitations. In this study, magnetic graphene oxide (MGO) modified with an amino (NH2) group was synthesized and utilized for PLD immobilization. The activity of the immobilized PLD (MGO-PLD) reached 3062 U/gMGO, with a specific activity of 33.9 U/mgPLD, virtually identical to that of the free PLD. MGO-PLD was utilized to synthesize PS efficiently in a biphasic system. Under optimal conditions, the PS yield reached 18.66 g/L, with a conversion rate of 92.8% and a productivity of 3.11 g/L/h. Notably, MGO-PLD retained an impressive PS conversion rate of 77.4% even after seven repetitive usages. Moreover, MGO-PLD displayed enhanced thermal and pH resistance properties compared to free PLD, alongside augmented storage stability. After an 8-week preservation at 4 °C, its residual activity was maintained at 76.3%. This study provides a sustainable and highly efficient pathway for the biocatalytic synthesis of PS. [ABSTRACT FROM AUTHOR]