Salt-induced increase in the yield of enzymatically synthesized phosphatidylinositol and the underlying mechanism.

Abstract

The purpose of this study was to improve the efficiency of enzymatic synthesis of phosphatidylinositol (PI) from phosphatidylcholine (PC) and myo-inositol in a phospholipase D (PLD)-mediated transphosphatidylation. A conventional biphasic reaction system consisting of ethyl acetate and an aqueous buffer afforded PI with a yield of 14 mol%. In contrast, the reaction performed in the presence of high concentration (0.8-4.3 M) of NaCl in the aqueous phase showed improved PI yield in a NaCl concentration-dependent manner. At 4.3 M NaCl, PI yield of as much as 35 mol% was achieved. The increase in the PI yield offered by other tested salts varied; however, we observed that some salts caused inactivation of the enzyme when used at high concentrations. Although NaCl at high concentration increased the apparent hydrolytic activity on aggregated PC, it decreased the activity towards monomeric PC, indicating that high concentration of salt intrinsically inhibits the enzyme. Binding assays revealed that PLD re-localized from the aqueous phase to the solvent-buffer interface, where the enzymatic reaction takes place, in the presence of both, the salt and PC. Hence, we concluded that improvement of the PI synthesis in the presence of salt occurs mainly due to the accumulation of the enzyme at the interface by strengthening the hydrophobic interactions, by which the apparent activation outweighs the salt-induced inhibitory effect. Using this improved system, several PI with defined structures, namely sn-1, 2-dioleoyl-PI, sn-1-palmitoyl-2-oleoyl-PI, and sn-1-stearoyl-2-arachidonoyl-PI, were successfully synthesized with overall yields of 25-37%, and PI isomeric purities of 91-96%.

DOI: 10.1016/j.jbiosc.2016.02.011

Cite this paper

@article{Muraki2016SaltinducedII, title={Salt-induced increase in the yield of enzymatically synthesized phosphatidylinositol and the underlying mechanism.}, author={Michiko Muraki and Jasmina Damnjanovi{\'c} and Hideo Nakano and Yugo Iwasaki}, journal={Journal of bioscience and bioengineering}, year={2016}, volume={122 3}, pages={276-82} }