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Combinatorial Library Based Engineering of Candida antarctica Lipase A for Enantioselective Transacylation of sec ‐Alcohols in Organic Solvent
Author(s) -
Wikmark Ylva,
Svedendahl Humble Maria,
Bäckvall JanE.
Publication year - 2015
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201410675
Subject(s) - candida antarctica , kinetic resolution , chemistry , enantioselective synthesis , lipase , active site , solvent , enzyme , organic chemistry , protein engineering , acylation , stereochemistry , catalysis
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.