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‐phe...
| Published in: | Angewandte Chemie International Edition |
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| Main Authors: | , , |
| Format: | Article in Journal/Newspaper |
| Language: | English |
| Published: |
Wiley
2015
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| Subjects: | |
| 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 |
| Summary: | 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. |
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