Enzymatic synthesis of phosphatidylserine using bile salt mixed micelles

Phosphatidylserine (PS) rich in polyunsaturated fatty acids of the n-3 series was obtained by enzymatic synthesis with phospholipase D (PLD) and a marine lipid extract as substrate. Synthesis was performed using mixed micelles composed of either sodium deoxycholate (SDC) or sodium cholate (SC). To limit the use of surfactant and to monitor the performance of PLD, the mixed micelles were characterized both in terms of bile salt/lipid molar ratio in the aggregates and of mean diameter. A fractional factorial experiment was selected to study the effect of pH, temperature, enzyme, L-serine concentrations, bile salt/lipid molar ratio and Ca(2+) content (in the case of SC only) on PS synthesis. The amount of L-serine was the main factor governing the equilibrium between transphosphatidylation and hydrolysis reaction. Increasing the bile salt/lipid molar ratio decreased PS synthesis yield. In contrast, pH (6.5-8) and temperature (35-45°C) did not affect PLD activity in the tested conditions. This statistical approach allowed determining a combination of parameters (pH, temperature, bile salt/lipid molar ratio, enzyme and alcohol acceptor concentrations) for PS synthesis. After 24 h, the transphosphatidylation reaction led to 57±2% and 56±3% of PS in the phospholipid mixtures with SDC and SC, respectively. In both cases, about 10% of phosphatidic acid was present as a side-product. On the whole, this work provided fundamental basis for a possible development of enzymatic PLD technology using food-grade emulsifiers to produce PS complying with industrial constraints for nutritional applications.