Fan_2026_J.Org.Chem__

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.