Determination of the lipase stereoselectivities using circular dichroism (CD); lipases produce chiral di- O-acylglycerols from achiral tri- O-acylglycerols

Abstract

A general method was described to determine the optical purity of 1,2 (or 2,3)-di- O-acylglycerols via a key compound, 3 (or dibenzoyl-sn- glycerol ( 3 or 3'). The chiral di- O-acylglycerols were first silylated and the acyl groups were removed by the Grignard degradation to 3 (or 1) O-tert-butyldimethylsilyl-sn-glycerol and subsequent benzoylation lead to the key compound 3 or 3' without racemization. The optical purity was determined from the strong exciton Cotton effect of 3 (+) or 3' (−) at 238 nm in the concentration of ca. 1 mM. The method was successfully applied to determine the stereoselectivities of lipases (EC 3.1.1.3) from three origins, bacteria, mammal and fungus such as Pseudomonas (AP, 89% optical purity, sn-1 preference), porcine pancreatin (PPL, 9.3% optical purity, sn-3 preference) and Candida (CC, sn-2 preference) using tripalmitin. The similar studies were extended to tri- O-benzoylglycerol (6) and tri- O-(cyclohexanecarbonyl)glycerol (5). All the enzymes showed high stereoselectivities with tri- O-benzoylglycerol. PPL and AP showed high and low stereoselectivities with tri- O-(cyclohexanecarbonyl) glycerol, while low and high stereoselectivities with tri- O-palmitoylglycerol, respectively. The results show that the stereoselectivities are ruled by the origins of lipases and acyl groups. The structures of the recognition site might be associated with enantioselectivities of the enzymes.