Conversion of phosphatidylcholine to phosphatidylserine by various phospholipases D in the presence of l- or d-serine

Abstract

The conversion of phosphatidylcholine (PC) to phosphatidylserine (PS) by four phospholipase D preparations was studied in the presence of l-serine or d-serine. The biphasic reaction was carried out at 30 ± 0.5°C using aqueous phase containing phospholipase D with serine and ethyl acetate containing PC. As a solvent candidate for PS formation, ethyl acetate and diethyl ether demonstrated efficient transphosphatidylation. Among the phospholipase D preparations examined, three from Streptomyces and one from cabbage origin, a Streptomyces phospholipase D displayed the highest catalytic activity for PS formation and the apparent rate constant ratio of transphosphatidylation and hydrolysis, (k′3/k3)app of the phospholipase D was the highest. At a concentration of 3.4 M serine, which is the solubility of serine in buffer of pH 5.6 at 30 ± 0.5°C, the phospholipase D yielded 98.5 ± 0.5 and 100% of PS using 17.8 mM PC with l-serine and d-serine, respectively. The increase in serine concentration exhibited no inhibition. PC was almost completely converted to PS (98 ± 0.8% yield) below 53.4 mM PC, keeping other reaction conditions constant. At all the concentrations of both the stereoisomers of serine, the initial rate of transphosphatidylation was higher with d-serine than with l-serine using all the Streptomyces phospholipase D preparations. On the other hand, cabbage phospholipase D barely showed any transphosphatidylation in the presence of d-serine and displayed only hydrolytic activity, while partial transphosphatidylation was observed with l-serine, yielding a maximum 41.3 ± 1.9% of PS. The PS products formed with l- and d-isomers of serine by the phospholipase D retained their original stereoisomeric forms of serine. Repeated batch operation conducted with the phospholipase D immobilized in amphiphilic gels showed some encouraging results and 96 ± 1.45% of PC to PS conversions were achieved in the first four batch cycles.