Determination of the lipase stereoselectivities using circular dichroism (CD); lipases produce chiral di-O-acylglycerols from achiral tri-O-acylglycerols.
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.
Uzawa
H
H
Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, Sendai, Japan.
Noguchi
T
T
Nishida
Y
Y
Ohrui
H
H
Meguro
H
H
D000818:Animals; D002175:Candida; D002942:Circular Dichroism; D004075:Diglycerides / Q000737:chemistry*; D008049:Lipase / Q000737:chemistry*; D011549:Pseudomonas; D013237:Stereoisomerism; D013379:Substrate Specificity; D013552:Swine; D014280:Triglycerides / Q000737:chemistry*
D004075:Diglycerides; D014280:Triglycerides; C005859:tripalmitin; D008049:Lipase