Engineering a Carboxylesterase for Enantioselective Acyl Transfer Reaction

Amino acid steric engineering is generally indispensable for the creation of an ideally configured enzyme catalytic cavity with maximized catalytic effect. The conventional typical strategy 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 opposite handle for the targeted increase of catalytic cavity size and correspondingly elevation in catalytic activity and/or enantioselectivity. In particular, a carboxylesterase, EstFF1, has been engineered for the achievement of highly efficient acyl transfer to alcohols. With the R386V, R390V variant ( VIII ), a high 10 5:1 alcohol over VIII molar ratio and a low 2:1 vinyl acetate over alcohol molar ratio are sufficient for effecting 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 ) tilts away W358 and leads to the increase of catalytic cavity size, thus enabling acyl transfer to a broad scope of both aromatic/alkyl and alkyl/alkyl secondary alcohols with quantitative or near-quantitative S -enantioselectivity.