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...

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Bibliographic Details
Published in:Angewandte Chemie International Edition
Main Authors: Wikmark, Ylva, Svedendahl Humble, Maria, Bäckvall, Jan-E
Format: Text
Language:English
Published: WILEY-VCH Verlag 2015
Subjects:
Communications
Antarc*
Antarctica
Online Access:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4471580/
http://www.ncbi.nlm.nih.gov/pubmed/25676632
https://doi.org/10.1002/anie.201410675
Description
Summary: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.

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