title: Combinatorial library based engineering of Candida antarctica lipase A for enantioselective transacylation of sec-alcohols in organic solvent.
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 (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.
authors: Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, 10691 Stockholm (Sweden).
authors: Ylva
authors: Y
authors: Wikmark
authors: Maria
authors: M
authors: Svedendahl Humble
authors: Jan-E
authors: JE
authors: Bäckvall
mesh_terms: D000215:Acylation; D000438:Alcohols / Q000737:chemistry*; D002175:Candida / Q000201:enzymology*; D020650:Combinatorial Chemistry Techniques; D008049:Lipase / Q000737:chemistry*; D008958:Models, Molecular; D009930:Organic Chemicals / Q000737:chemistry*; D015202:Protein Engineering; D012997:Solvents / Q000737:chemistry*; D013237:Stereoisomerism
chemical_list: D000438:Alcohols; D009930:Organic Chemicals; D012997:Solvents; D008049:Lipase
keywords: biocatalysis; kinetic resolution; lipase A; protein engineering; secondary alcohols