Determining the regioselectivity of immobilized lipases in triacylglycerol acidolysis reactions

Lipase regioselectivity is the ability to distinguish between primary (i.e., sn-1,3) and secondary (sn-2) ester functionalities in a triacylglycerol molecule, which is of importance in the manufacture of structured lipids. Unlike existing methods of assessment, which utilize hydrolysis reactions, an alternative technique to assess the regioselectivity of lipases in triacylglycerol transesterification reactions has been developed. An acidolysis reaction is performed between triolein and decanoic, lauric, or stearic acids under conditions that minimize acyl migration, and products are analyzed by silver-ion complexation liquid chromatography, enabling detection of specific triacylglycerol positional isomers. From the rate of formation of these isomers the relative selectivity of the lipase for sn-2 and sn-1,3 ester bonds is determined. With lipases known to lack regioselectivity, the rate of reaction at sn-2 was similar to that at sn-1,3 from the start of the reaction. With sn-1,3 selective lipases, the formation of triacylglycerol isomers with decanoic acid in the secondary position was not detected at any point in the reaction. Regioselectivity as a function of reaction progress was monitored. Two lipases from the genus Pseudomonas exhibited activity toward all positions, but the rate at sn-2 was much reduced, and no incorporation of decanoic acid into this position was detectable until a high degree of conversion had been achieved.