ABSTRACT: Enzymatic synthesis of PS by phospholipase D (PLD)-mediated transphosphatidylation in an aqueous media was investigated. The purpose of this study was to establish a novel synthetic method where no toxic organic solvents were used. An attempt to react soybean lecithin (simply dispersed in an aqueous buffer) with an aqueous solution of L-serine and PLD was unsuccessful, giving only 20% of PS. By contrast, a suspension of lecithin adsorbed on fine powders such as silica was effectively converted into PS in an aqueous solution of L- serine and PLD. After screening various powders for use as the lecithin adsorbent, calcium sulfate was found to be the best with respect to lecithin conversion. In addition, calcium sulfate did not require prior adsorption of lecithin (i.e., the reaction pro- ceeded effectively simply by adding the powder to an aqueous mixture of lecithin, L-serine, and PLD). With this “aqueous sus- pension system” of calcium sulfate, up to 180 mg/mL lecithin was completely converted, resulting in more than 80% PS in 24 h. The synthesized PS could easily be recovered from the pow- der by extracting with a mixture of n-hexane, ethanol, and di- luted HCl. Paper no. J10491 in JAOCS 80, 653–657 (July 2003). KEY WORDS: Lecithin, phosphatidylserine, phospholipase D, transphosphatidylation. PS is a natural phospholipid that is especially abundant in an- imal brain tissue (1). Recently, PS has been shown to have therapeutic effects on several memory-related disorders. For example, Delwaide et al. (2) reported that oral administration of bovine brain cortex-derived PS (BC-PS) to patients with senile dementia improved their cognitive disorders. Another example is the report by Crook et al. (3) that patients with age-associated memory impairment treated with BC-PS had improved performance on tests related to learning and mem- ory tasks of daily life. One of the possible sources for preparing PS is animal or- gans such as bovine brain. In fact, many biochemical re- searchers have used BC-PS for clinical tests (2–5). However, these BC-PS preparations might not be suitable for human use as they may transmit infectious diseases such as bovine spongiform encephalopathy. In addition, animal organs them- selves are not likely to be appropriate sources of PS for in- dustrial-scale production because of their low availability. An alternative for supplying PS is transphosphatidylation, in which the polar head group of lecithin (such as soybean lecithin) is exchanged with L-serine by phospholipase D (PLD) (6,7). The pharmacological effects of PS enzymati- cally synthesized from soybean lecithin were confirmed using several experimental animals (8–10). Generally, transphosphatidylation is carried out in a bipha- sic system consisting of a water-immiscible organic solvent phase (e.g., diethyl ether or ethyl acetate) containing lecithin and an aqueous buffer phase containing the enzyme and L- serine (6,7). Regarding PS as food for human use, however, the use of such toxic organic solvents should be avoided in its production. In this aspect, Dittrich and Ulbrich-Hofmann (11) demonstrated synthesis of phosphatidylglycerol in purely aqueous media. In this article, we report a novel reaction system for transphosphatidylation without using toxic organic solvents. The new system (named an “aqueous suspension” system) features use of a suspension of fine powder-adsorbed phos- pholipid in an aqueous buffer. MATERIALS AND METHODS Chemicals and enzyme. Soybean lecithin (SLP-PC70) was a gift from Tsuru Lecithin Industries Co. Ltd. (Mie, Japan). The phospholipid composition of the lecithin was: PC 83%, PE 6%, PA 8%, and others 3%. Silica gel (Wako gel C-300), octadecylated silica gel (Wakogel 50C18) and activated carbon were from Wako Pure Chemical Industries (Osaka, Japan). Calcium sulfate powder (Type SF-CS) was from Mutsumi Chemicals Co. Ltd. (Mie, Japan). Calcium carbonate powder (Softon 3200) was from Shiraishi Calcium Co. Ltd. (Osaka, Japan). Calcium py- rophosphate powder was from Taihei Yogyo Yakuhin Co. Ltd. (Gifu, Japan). Diatomaceous earth (Celite, grade: Hyflo Super-Cel) was from Johns-Manville (Denver, CO). PLD was prepared in this laboratory from a recombinant strain of Escherichia coli bearing the PLD gene of Strepto- myces antibioticus (12,13). PLD activity was measured using Copyright © 2003 by AOCS Press 653 JAOCS, Vol. 80, no. 7 (2003) *To whom correspondence should be addressed at Laboratory of Molecular Biotechnology, Department of Biological Mechanisms and Functions, Grad- uate School of Bio- and Agro-Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. E-mail: yamanetu@agr.nagoya-u.ac.jp An Aqueous Suspension System for Phospholipase D-Mediated Synthesis of PS Without Toxic Organic Solvent Yugo Iwasaki a , Yukiko Mizumoto a , Takahiro Okada b , Takaya Yamamoto b , Kentaro Tsutsumi b , and Tsuneo Yamane a, * a Laboratory of Molecular Biotechnology, Department of Biological Mechanisms and Functions, Graduate School of Bio- and Agro-Science, Nagoya University, Nagoya 464-8601, Japan, and b Rinoru Oil Mills Co., Ltd., Nihon Seimei Edobashi Building 2-13-12, Tokyo 103-0027, Japan