Efficient secretion expression of phospholipase D in Bacillus subtilis and its application in synthesis of phosphatidylserine by enzyme immobilization.

Transphosphatidylation catalyzed by phospholipase D has gained increasing attention for producing phosphatidylserine (PS), which can be used in functional food and medicine. In this study, we investigated the effects of six signal peptides on the secretion of PLD (PLD) from Streptomyces antibioticus TCCC 21059 in the food-grade GRAS bacterium Bacillus subtilis. It indicated that the optimal signal peptide DacB with an Ala-X-Ala sequence motif at the C-terminus showed the highest secretory expression ability, resulting in increased production of 2.84 U/mL PLD. Then PLD was immobilized on the epoxy-based carriers, and one of these carriers allowed PLD loading of up to 2.7 mg/g. The immobilized PLD was more stable over a wide range of pH value (4.5-7.5) and temperature (16 °C-60 °C) than free PLD. Subsequently, the synthesis of PS from soybean phosphatidylcholine (PC) was carried out in purely aqueous solution using immobilized PLD, leading to a high yield of 65%. The immobilized PLD catalyst maintained a relative PS production of 60% after 5 recycles. Notably, the use of toxic solvent was completely eliminated in the whole process, which would be more profitable for the application of PS.