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  Multiparameter Optimization in Directed Evolution: Engineering Thermostability, Enantioselectivity, and Activity of an Epoxide Hydrolase

Li, G., Zhang, H., Sun, Z., Liu, X., & Reetz, M. T. (2016). Multiparameter Optimization in Directed Evolution: Engineering Thermostability, Enantioselectivity, and Activity of an Epoxide Hydrolase. ACS Catalysis, 6(6), 3679-3687. doi:10.1021/acscatal.6b01113.

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 Creators:
Li, Guangyue1, 2, Author              
Zhang, Hui3, Author
Sun, Zhoutong1, 2, Author              
Liu, Xinqi3, Author
Reetz, Manfred T.1, 2, Author              
Affiliations:
1Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society, ou_1445588              
2Philipps-Universität Marburg, Fachbereich Chemie, ou_persistent22              
3State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, 300071 Tianjin, People’s Republic of China, ou_persistent22              

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Free keywords: directed evolution; enantioselectivity; thermostability; activity; saturation mutagenesis
 Abstract: The challenge of optimizing several parameters in the directed evolution of enzymes remains a central issue. In this study we address the thermostability, enantioselectivity, and activity of limonene epoxide hydrolase (LEH) as the catalyst in the hydrolytic desymmetrization of cyclohexene oxide with formation of (R,R)- and (S,S)-cyclohexane-1,2-diol. Wild type LEH shows a thermostability of T-50(30) = 41 C and an enanioselectivity of 2% ee (S,S). Two approaches are described herein. In one strategy, the mutations generated previously by Janssen, Baker, and co-workers for notably increased thermostability are combined with mutations evolved earlier for enhanced enantioselectivity. Although highly enantioselective R,R and S,S variants (92-93% ee) with increases in T-50(30) by 10-11 C were obtained, relative to wild type LEH the tradeoff in activity was significant. The second strategy based on the simultaneous optimization of both parameters using iterative saturation mutagenesis (ISM) with minimized tradeoff in activity proved to be superior. Several notably improved variants were observed, a reasonable "compromise" being R,R- and S,S-selective LEH variants (80-94% ee) showing enhanced thermostability by 5-10 degrees C and still reasonable levels of activity. Analysis of the X-ray structure of the S,S variant (94% ee) with and without diol product sheds light on the origin of altered stereoselectivity.

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Language(s): eng - English
 Dates: 2016-05-022016-06-03
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1021/acscatal.6b01113
 Degree: -

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Title: ACS Catalysis
  Abbreviation : ACS Catal.
Source Genre: Journal
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Publ. Info: Washington, DC : ACS
Pages: - Volume / Issue: 6 (6) Sequence Number: - Start / End Page: 3679 - 3687 Identifier: ISSN: 2155-5435
CoNE: https://pure.mpg.de/cone/journals/resource/2155-5435