Engineering of the Conformational Dynamics of Lipase To Increase Enantioselectivity

In order to increase the R-enantioselectivity of Candida antarctica lipase B (CALB) toward (R)-3-t-butyl-dimethyl-silyloxy glutaric acid methyl monoester at 30 °C, we engineered CALB conformational dynamics. Based on structural analysis and molecular dynamics simulations, two key residues (D223 and A281) were identified, and three mutants (D223V, A281S, and D223V/A281S) were designed to decrease the conformational dynamics of the pocket and channel. Computational and experimental evaluations were performed for all mutants, with the D223V/A281S mutant exhibiting high R-enantioselectivity (>99.00%; increased from 8.00%) and high space-time yield (107.54 g L–1 d–1; a 5.70-fold increase).