986 Lipase-Catalyzed Transesterification of Phosphatidylcholine at Controlled Water Activity Ingemar Svensson*, Patrick Adlercreutz and Bo Mattiasson Department of Biotechnology, Chemical Center, University of Lund, S-221 00 Lund, Sweden The incorporation of a free fatty acid into the sn-1 posi- tion of phosphatidylcholine by lipase-catalyzed transester- ification was investigated. The thermodynamic water ac- tivity of both the enzyme preparation and the substrate solution was adjusted to the same value prior to the reac- tion. The reaction rate increased with increasing water activity but the yield of modified phosphatidylcholine decreased due to hydrolysis. By using a large excess of the free fatty acid {heptadecanoic acid}, the hydrolysis reaction was slowed down, so a higher yield was obtained at a given degree of incorporation. The best results were obtained with Rhizopus arrhizus lipase immobilized by adsorption on a polypropylene support. With this prepara- tion, a yield of 60% and nearly 50% incorporation of heptadecanoic acid (100% incorporation in the sn-1 posi- tion} was obtained at a water activity of 0.064. The en- zyme preparation had good operational stability and posi- tion specificity. Little incorporation (<1%) was observed in the sn-2 position, when almost all the fatty acid in the sn-1 position was exchanged. KEY WORDS: Lipase, phosphatidylcholine, transesterification, water activity. There are several reasons why lipases and phospholipases are useful for modifying the acyl group composition in natural phospholipids. The mild reaction conditions and the high enzymatic regiospecificity are strong advantages, especially if the lipids are to be used in human food or for medical purposes. By exchanging fatty acids asym- metrically in the phospholipid molecule, new physical properties can be achieved. These modified lipids can be used in lipid]membrane research or as emulsifiers for food, cosmetics or medical substances. A special application of the enzymatic transesterification is the position-specific labelling of phospholipids with radioactive or photoactive acyl groups. Furthermore, biologically active polyunsatu- rated fatty acids that are chemically unstable can be in- corporated under mild conditions. The normal hydrolytic action of lipases and phospho- lipases has been used for preparing phospholipids with different fatty acids in the two carboxyl ester bond posi- tions. After hydrolysis, the lysophospholipid formed can be nonenzymatically esterified with the desired fatty acid. To achieve acylation in a specific position not only must the lipase be position-specific but spontaneous acyl migra- tion must also be considered (1). By reversing the lipase action to esterification or transesterification, it is possible to use the regioselectivity of the lipases in the acylation step and thereby reduce the problem of acyl migration. A report of lipasmcatalyzed regiospecific 1-position (1-pos) transesterification of fatty acid in phosphatidyl- choline (PC) and phosphatidylethanolamine (PE) was *To whom correspondence should be addressed at Department of Biotechnology,ChemicalCenter, University of Lund, P.O. Box 124, S-221 00 Lurid, Sweden. made by Brockerhoff et aL (2). They used Rhizopus delemar lipase in buffer with phosphatidylcholine and oleic acid as substrates. Yoshimoto et al. (3) used poly- ethyleneglycol-modified Candida cylindracea lipase dis- solved in benzene to incorporate polyunsaturated fatty acids into phosphatidylcholine. A two-phase water/oil system was used by Yagi et al. (4) for transesterification of PC and PE with different fatty acids. Yoichiro and Set- suko (5) also used a water/oil system but used sardine oil as source of polyunsaturated fatty acids for incorporation into soy phospholipid. We used a system with immobiliz- ed lipase in toluene for incorporation of heptadeeanoic acid in egg phosphatidylcholine (6). In all systems the predomi- nant problems were the hydrolysis reactions and low yields. Our system is practical because of the easy recov- ery of enzyme and the possibility to keep the water con- tent low to avoid undesired hydrolysis. Transesterification of the fatty acid in the sn-2 position should be possible with phospholipase A 2, but so far no practical method for doing this has been presented. It has been easier to carry out esterification between lysophos- phatidylcholine (LPC) and free fatty acid, but the yield of PC formed was only in the range of 6-7% (7,8). In this work the previous reaction system (6) has been improved. Both the yield of product PC and the amount of new fatty acid incorporated into the sn-1 position of egg PC have been increased. EXPERIMENTAL PROCEDURES Chemicals. I~a-Phosphatidylcholine (PC from egg, 98.5% purity, average molecular weight 762 g/mole) was a gift of Karlshamns AB, Division Lipid Teknik (Stockholm, Sweden). L--a-phosphatidylethanolamine (PE from egg yolk, 98%), L-a-phosphatidylinositol (PI from soybean, sodium salt, 99%), L-a-phosphatidic acid (PA from egg yolk, sodium salt, 98%), heptadecanoic acid (purity 99%) and phospholipase A 2 from porcine pancreas (Type II) were obtained from Sigma Chemicals (St. Louis, MO). Im- mobilized lipases from Rhizomucor miehei (Lipozyme IM 20 and IM 60) were gifts of Novo Industri A/S (Bagsvaerd, Denmark). Lipase from Rhizopus arrhizus (Lipase 80,000) was a gift of Gist-Brocades S.A. (Delft, The Netherlands). Polypropylene support (EP 100, 400-1000 ~m) was do- nated by Akzo (Obernburg, Germany). Sodium methox- ide was obtained from Merck (Darmstadt, Germany). Sol- vents used were of high-performance liquid chromatog- raphy (HPLC)-grade, and all other chemicals were of ana- lytical grade. Preparation of immobilized enzyme. Lipase from Rhizo- pus arrhizus (200 mg unless otherwise stated) was dis- solved in 20.0 mL sodium phosphate buffer (20 mM, pH 6.0). The enzyme solution was mixed with 1.0 g polypro- pylene support (EP 100), which was prewet with ethanol (normally 0.5 mL). After incubation for 24 h in a shaking bath at 25°C, the preparation was filtered and washed two times with distilled water. The preparation was dried over~ night under reduced pressure. JAOCS, Vol. 69, no. 10 (October 1992)