Rhizopus oryzae lipase-catalyzed stereospecific esterification of 2-monoradylglycerols — a comparison to corresponding triradylglycerol hydrolysis

Abstract

In a model elaborated earlier to understand and predict the stereopreference ofRhizopus oryzae lipase (ROL) catalyzed hydrolysis of triradylglycerols, we identified the degree of flexibility of the C1′-X′ bond (X = O for ether, N for amide, C for alkyl, methylene, and a phenylring, respectively) adjacent to C2 of glycerol being responsible for the discrimination of the enantiomers (Kovac et al., Eur. J. Lipid Sci. Technol.2000, 61—72). During catalysis of forward and back reaction — hydrolysis and esterification — in either case the carbonyl carbon of the sn-1 or sn-3 fatty acid binds to the active site serine of ROL leading to a covalently bound intermediate, which was simulated in the model. Thus, we assumed that stereoselectivity of ROL in esterification of corresponding 2-monoradylglycerols with oleic acid in cyclohexane should follow the same model. As predicted by this model 2-monoradylglycerols with “rigid” phenyl and amide substituents were esterified at thesn-3 position, and those with “ flexible” ether substituents at thesn-1 position. However, enantiomeric excess of wild type ROL in esterifying 2-monaradylglycerols with flexible benzylether and methylene substituents differed by around 50% as compared to hydrolysis experiments with corresponding triradylglyc-erols. In addition esterification of 2-monoradylglycerol with flexible ether substituent by ROL L258F/L254F double mutant was essentially non-selective compared to corresponding triradylglycerol where enantiomeric excess was 58%sn-1. Whether water activity was a factor determining these discrepancies was investigated for ROL- and double mutant enzyme-catalyzed esterification of the ether and methylene substrates under controlled water activities from 0.02—0.85. In all cases stereoselectivities ob-served were independent from water activities. In conclusion, the model describing the stereoselective course of aqueous hydrolysis of triradylglycerols catalyzed by ROL in most cases applies to the esterification reaction in organic solvent. Differences in stereoselectivity observed are attributed to reduced possibilities for interaction of 2-monoradylglycerol substrates with the binding sites of ROL as compared to those of triradylglycerol substrates.