Combinatorial Library Based Engineering of Candida antarctica Lipase A for Enantioselective Transacylation of sec‐Alcohols in Organic Solvent

A method for determining lipase enantioselectivity in the transacylation of sec‐alcohols in organic solvent was developed. The method was applied to a model library of Candida antarctica lipase A (CalA) variants for improved enantioselectivity (E values) in the kinetic resolution of 1‐phenylethanol in isooctane. A focused combinatorial gene library simultaneously targeting seven positions in the enzyme active site was designed. Enzyme variants were immobilized on nickel‐coated 96‐well microtiter plates through a histidine tag (His₆‐tag), screened for transacylation of 1‐phenylethanol in isooctane, and analyzed by GC. The highest enantioselectivity was shown by the double mutant Y93L/L367I. This enzyme variant gave an E value of 100 (R), which is a dramatic improvement on the wild‐type CalA (E=3). This variant also showed high to excellent enantioselectivity for other secondary alcohols tested.