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  Methodology Development in Directed Evolution: Exploring Options when Applying Triple-Code Saturation Mutagenesis

Qu, G., Lonsdale, R., Yao, P., Li, G., Liu, B., Reetz, M. T., et al. (2018). Methodology Development in Directed Evolution: Exploring Options when Applying Triple-Code Saturation Mutagenesis. Chembiochem, 19(3), 239-246. doi:10.1002/cbic.201700562.

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 Creators:
Qu, Ge1, Author
Lonsdale, Richard2, 3, Author              
Yao, Peiyuan1, Author
Li, Guangyue2, 3, Author              
Liu, Beibei1, Author
Reetz, Manfred T.1, 2, 3, Author              
Sun, Zhoutong1, Author
Affiliations:
1Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin, 300308, China, ou_persistent22              
2Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society, ou_1445588              
3Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse, 35032, Marburg, Germany, ou_persistent22              

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Free keywords: alcohol dehydrogenase; biocatalysis; directed evolution; saturation mutagenesis; stereoselectivity
 Abstract: Directed evolution of stereo- or regioselective enzymes as catalysts in asymmetric transformations is of particular interest in organic synthesis. Upon evolving these biocatalysts, screening is the bottleneck. To beat the numbers problem most effectively, methods and strategies for building "small but smart" mutant libraries have been developed. Herein, we compared two different strategies regarding the application of triple-code saturation mutagenesis (TCSM) at multiresidue sites of the Thermoanaerobacter brockii alcohol dehydrogenase by using distinct reduced amino-acid alphabets. By using the synthetically difficult-to-reduce prochiral ketone tetrahydrofuran-3-one as a substrate, highly R- and S-selective variants were obtained (92-99 % ee) with minimal screening. The origin of stereoselectivity was provided by molecular dynamics analyses, which is discussed in terms of the Bürgi-Dunitz trajectory.

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Language(s): eng - English
 Dates: 2017-10-182018-01-042018-02-02
 Publication Status: Published online
 Pages: 8
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1002/cbic.201700562
 Degree: -

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Title: Chembiochem
  Other : Chembiochem
Source Genre: Journal
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Publ. Info: Weinheim, Germany : Wiley-VCH
Pages: - Volume / Issue: 19 (3) Sequence Number: - Start / End Page: 239 - 246 Identifier: ISSN: 1439-4227
CoNE: https://pure.mpg.de/cone/journals/resource/110978984568897