Identification of a novel phospholipase D with high transphosphatidylation activity and its application in synthesis of phosphatidylserine and DHA-phosphatidylserine

Phosphatidylserine (PS) and docosahexaenoic acid-phosphatidylserine (DHA-PS) have significant nutritional and biological functions, which are extensively used in functional food industries. Phospholipase D (PLD)-mediated transphosphatidylation of phosphatidylcholine (PC) or DHA-PC with L-serine, is an effective method for PS and DHA-PS preparation. However, because of the hydrolysis activity of PLD, PC and DHA-PC would be converted to the undesirable byproduct, phosphatidic acid (PA) and DHA-PA. In this study, a novel phospholipase D (PLD_a2) was firstly cloned from Acinetobacter radioresistens a2 with high transphosphatidylation activity and no hydrolysis activity. In the PLD-catalyzed synthesis process (12 h), both the transphosphatidylation conversion rate and selectivity of PS and DHA-PS were about 100%, which is the only PLD enzyme reported with this superiority up till now. In comparison with the majority of other known PLDs, PLD_a2 exerted the highest activity at neutral pH, and it was stable from pH 4.0 to pH 9.0. In addition, PLD_a2 had excellent thermal stability, with an optimum reaction temperature of 40 °C and keeping more than 80% activity from 20 °C to 60 °C. The high catalytic selectivity mechanism of PLD_a2 was explained by utilizing homology modeling, two-step docking, and binding energy and conformation analysis. PLD_a2 ensured a stable supply of the biocatalyst with its most preponderant transphosphatidylation activity and PS selectivity, and had great potential in phospholipids industrial production.