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

• Published in: Angewandte Chemie International Edition
• Main Authors: Wikmark, Ylva, Svedendahl Humble, Maria, Bäckvall, Jan‐E.
• Format: Article in Journal/Newspaper
• Language: English
• Published: Wiley 2015
• Subjects: Antarc*; Antarctica
• Online Access: http://dx.doi.org/10.1002/anie.201410675; https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1002%2Fanie.201410675; https://onlinelibrary.wiley.com/doi/pdf/10.1002/anie.201410675

Abstract 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 (His 6 ‐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.