Combinatorial Library Based Engineering of Candida antarctica Lipase A for Enantioselective Transacylation of sec-Alcohols in Organic Solvent
2015 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 54, no 14, 4284-4288 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2015. Vol. 54, no 14, 4284-4288 p.
biocatalysis, kinetic resolution, lipase A, protein engineering, secondary alcohols
IdentifiersURN: urn:nbn:se:kth:diva-165210DOI: 10.1002/anie.201410675ISI: 000351679600024PubMedID: 25676632ScopusID: 2-s2.0-84925582964OAI: oai:DiVA.org:kth-165210DiVA: diva2:810384
QC 201505072015-05-072015-04-242015-05-07Bibliographically approved