Synthesis of 2-monoglycerides by alcoholysis of palm oil and tuna oil using immobilized lipasesCommercial immobilized lipases were used for the synthesis of 2-monoglycerides (2-MG) by alcoholysis of palm and tuna oils with ethanol in organic solvents. Several parameters were studied, i.e., the type of immobilized lipases, water activity, type of solvents and temperatures. The optimum conditions for alcoholysis of tuna oil were at a water activity of 0.43 and a temperature of 60 °C in methyl-tert-butyl ether for ~12 h. Although immobilized lipase preparations from Pseudomonas sp. and Candida antarctica fraction B are not 1,3-regiospecific enzymes, they were considered to be more suitable for the production of 2-MG by the alcoholysis of tuna oil than the 1,3-regiospecific lipases (Lipozyme RM IM from Rhizomucor miehei and lipase D from Rhizopus delemar). With Pseudomonas sp. lipase a yield of up to 81% 2-MG containing 80% PUFA (poly-unsaturated fatty acids) from tuna oil was achieved. The optimum conditions for alcoholysis of palm oil were similar as these of tuna oil alcoholysis. However, lipase D immobilized on Accurel EP100 was used as catalyst at 40 °C with shorter reaction times (12 h). This lead to a yield of ~ 60% 2-MG containing 55.0-55.7% oleic acid and 18.7-21.0% linoleic acid.

A new method for the lipase-catalyzed synthesis of structured TAG (ST) is described. First, sn1,3-dilaurin or -dicaprylin were enzymatically synthesized using different published methods. Next, these were esterified at the sn2-position with oleic acid or its vinyl ester using different lipases. Key to successful enzymatic synthesis of ST was the choice of a lipase with appropriate FA specificity, i.e., one that does not act on the FA already present in the sn1,3-DAG, but that at the same time exhibits high selectivity and activity toward the FA to be introduced. Reactions were performed in the presence of organic solvents or in solvent-free systems under reduced pressure. With this strategy, mixed ST containing the desired compounds 1,3-dicapryloyl-2-oleyl-glycerol or 1,3-dilauroyl-2-oleyl-glycerol (CyOCy or LaOLa) were obtained at 87 and 78 mol% yield, respectively, using immobilized lipases from Burkholderia cepacia (Amano PS-D) in n-hexane at 60°C. However, regiospecific analysis with porcine pancreatic lipase indicated that in CyOCy, 25.7% caprylic acid and in LaOLa 11.1% lauric acid were located at the sn2-position. Oleic acid vinyl ester was a better acyl donor than oleic acid. Esterification of sn1,3-DAG and free oleic acid gave very low yield (<20%) of ST in a solvent system and moderate yield (>50%) in a solvent-free system under reduced pressure.Paper no. J10669 in JAOCS 81, 151-155 (February 2004). KEY WORDS:Burkholderia cepacia lipase, Candida antarctica lipase B, 1,3-DAG, immobilized lipases, structured TAG.The nutritional value of TAG and their physicochemical properties not only are determined by the FA composition but also depend on the positional distribution of the acyl groups bonded to the glycerol. Structured TAG (ST) of the ABA-type containing medium-chain FA (MCFA; e.g., C 8 ) in sn1,3-positions and a long-chain unsaturated FA (e.g., C 16 -C 22 ) in the sn2-position are an effective energy source for patients with malabsorption, e.g., pancreatic insufficiency. A range of methods for the enzymatic synthesis of these compounds have been already described (1,2). This includes acidolysis of a TAG (3,4) or interesterification between two TAG (5,6). Unfortunately, yields of ABA-type TAG are low, and a variety of by-products are formed. These are difficult to separate from the desired product. Significantly higher yields and purities can be achieved by a two-step process developed in our laboratories (7-9). In the first step, highly pure 2-MAG are obtained by alcoholysis of a TAG with ethanol using a 1,3-regiospecific lipase followed by crystallization. The 2-MAG thus obtained are subsequently esterified with a suitable FA in the second step to obtain the desired ST. More recently, we also reported the synthesis of ST containing PUFA from tuna oil in the sn2-position (10). However, crystallization of 2-MAG containing unsaturated FA is rather difficult due to the very low m.p. (i.e., -56°C for 2-docosahexaenoic acid glycerol ester). An alternative would be the use of sn1,3-diacylglycerols (1,3-DAG) ...

Lipase-catalyzed acidolysis and phospholipase D-catalyzed transphosphatidylation of phosphocholineTwo approaches on enzymatic phospholipid modification were studied: (1) transphosphatidylation of the 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and ethanolamine in biphasic and anhydrous organic solvent systems by phospholipase D (PLD) and (2) incorporation of oleic acid into the sn1-position of DLPC in organic solvents with different immobilized lipases at controlled water activity. First, DLPC was chemically synthesized from glycerophosphocholine and lauric acid. Next, PLD-catalyzed head group exchange of DLPC with ethanolamine was studied using an enzyme from Streptomyces antibioticus expressed recombinantly in E. coli. A comparison of the free PLD with the biocatalyst activated by a salt-activation technique using KCl showed that the salt-activated enzyme (PLD-KCl) was 10-12 folds more active based on the amount of protein used. Thus, DLPC was quantitatively converted to 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine in an anhydrous solvent system within 12 h at 60 7C. For the acidolysis of DLPC with oleic acid, among the four lipases studied (CAL-B, Lipozyme TL IM, Lipozyme RM IM and lipase D immobilized on Accurel EP-100), Lipozyme TL IM showed the highest activity and incorporation of oleic acid. A quantitative incorporation was achieved at 40 7C using a 8-fold molar excess of oleic acid in n-hexane at a water activity of 0.11.