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

Abstract

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 (PLDa2) was firstly cloned from Acinetobacter radioresistens a2 with high transphosphatidylation activity and no hydrolysis activity. In the PLD-catalyzed synthesis process (12h), 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, PLDa2 exerted the highest activity at neutral pH, and it was stable from pH 4.0 to pH 9.0. In addition, PLDa2 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 PLDa2 was explained by utilizing homology modeling, two-step docking, and binding energy and conformation analysis. PLDa2 ensured a stable supply of the biocatalyst with its most preponderant transphosphatidylation activity and PS selectivity, and had great potential in phospholipids industrial production.