Engineering a Carboxylesterase for Highly Efficient Enantioselective Acyl Transfer Reaction

Amino acid steric engineering as an enzyme engineering strategy is generally indispensable for the creation of an ideally configured catalytic cavity with maximized catalytic effect. The conventional approach in the typical form of hypo-steric engineering relies on the intentional decrease of steric size of an amino acid as a control handle for the purposeful increase of catalytic cavity size and accordingly, elevation of catalytic adaptability. Herein, we have developed a hyper-steric engineering method, with the intentional increase of steric size of an amino acid exploited as a superior alternative handle for the targeted increase of catalytic cavity size and correspondingly, elevation in catalytic activity and/or enantioselectivity. In particular, a carboxylesterase has been engineered for the achievement of highly efficient acyl transfer to alcohols. With the R386V,R390V variant (VIII) of a metagenomically identified family VIII carboxylesterase, EstFF1, a high 105:1 alcohol over VIII molar ratio and a low 2:1 vinyl acetate over alcohol molar ratio are sufficient for effecting highly productive acyl transfer to a broad scope of primary alcohols over hydrolysis. The hyper-steric engineering of G359 to L359 (IX) on the R386V variant (II) enables acyl transfer to a broad scope of both aromatic/alkyl and alkyl/alkyl secondary alcohols, at the quantitative or near-quantitative S-enantioselectivity level. The high catalytic activity and/or enantioselectivity demonstrated herein promise hyper-steric engineering as a powerful conceptual framework for expanding the toolbox of enzymatic catalysis.