Enhancement of the enantioselectivity of carboxylesterase A by structure-based mutagenesis

Previously studied Bacillus subtilis carboxylesterases (CesA and CesB) have potential for the kinetic resolution of racemic esters of 1,2-O-isopropylideneglycerol (IPG). CesA exhibits high activity but low enantioselectivity towards IPG-butyrate and IPG-caprylate, while the more enantioselective CesB does not process IPG-butyrate and exhibits several-fold lower activity than CesA towards IPG-caprylate. A sequence and structure comparison allowed us to identify active site residues that may cause the difference in (enantio)selectivities of CesA and CesB towards these IPG esters. This structure-based approach led to the identification of two active site residues in CesA (F166 and F182), as promising candidates for mutagenesis in order to enhance its enantioselectivity. Mutagenesis of positions 166 and 182 in CesA yielded novel variants with enhanced enantioselectivity and without significant loss of catalytic activity. For IPG-butyrate, a CesA double mutant F166V/F182 C (ER = 13) was generated showing a ∼13-fold increased enantioselectivity as compared to wild-type CesA (E = 1). For IPG-caprylate, we designed a CesA double mutant F166 V/F182Y (ER = 9) displaying a ∼5-fold increased enantioselectivity as compared to the wild-type enzyme (ER = 2). These findings, combined with the results of molecular docking experiments, demonstrate the importance of residues at positions 166 and 182 for the enantioselectivity of CesA, and may contribute to the development of efficient biocatalysts.

► Critical amino acids responsible for the large difference in enantioselectivity between two related esterases have been identified.
► Targeted mutagenesis of these enantioselectivity determining residues has resulted in the isolation of carboxylesterase variants with superior enantioselectivity.
► The novel esterases can be used for the synthesis of S-IPG, the major building block for the pharmaceutically important class of β-blockers.