Hydrolytic resolution of (R,S)-2-hydroxycarboxylic acid esters in biphasic media: implication for rate-limiting formation or breakdown of tetrahedral intermediates in acylation step

A thermally stable esterase (SNSM‐87) from Klebsiella oxytoca is explored as an enantioselective biocatalyst for the hydrolytic resolution of (R,S)‐2‐hydroxycarboxylic acid esters in biphasic media, where the best methyl esters possessing the highest enantioselectivity and reactivity are selected and elucidated in terms of the structure–enantioselectivity correlations and substrate partitioning in the aqueous phase. With (R,S)‐2‐chloromandelates as the model substrates, an expanded Michaelis–Menten mechanism for the rate‐limiting acylation step is adopted for the kinetic analysis. The Brønsted slope of 25.7 for the fast‐reacting (S)‐2‐chloromandelates containing a difficult leaving alcohol moiety, as well as that of 4.13 for the slow‐reacting (R)‐2‐chloromandelates in the whole range of leaving alcohol moieties, indicates that the breakdown of tetrahedral intermediates to acyl‐enzyme intermediates is rate‐limiting. However, the rate‐limiting step shifts to the formation of tetrahedral intermediates for the (S)‐2‐chloromandelates containing an easy leaving alcohol moiety, and leads to an optimal enantioselectivity for the methyl ester substrate. Biotechnol. Bioeng. 2007; 98: 30–38. © 2007 Wiley Periodicals, Inc.