Approaching boiling point stability of an alcohol dehydrogenase through computationally-guided enzyme engineering.

Autor: Aalbers FS; Molecular Enzymology Group, University of Groningen, Groningen, Netherlands.; Department of Biology and Biotechnology 'L. Spallanzani', University of Pavia, Pavia, Italy., Fürst MJ; Molecular Enzymology Group, University of Groningen, Groningen, Netherlands.; MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, United Kingdom., Rovida S; Department of Biology and Biotechnology 'L. Spallanzani', University of Pavia, Pavia, Italy., Trajkovic M; Molecular Enzymology Group, University of Groningen, Groningen, Netherlands., Gómez Castellanos JR; Department of Biology and Biotechnology 'L. Spallanzani', University of Pavia, Pavia, Italy., Bartsch S; c-LEcta GmbH, Leipzig, Germany., Vogel A; c-LEcta GmbH, Leipzig, Germany., Mattevi A; Department of Biology and Biotechnology 'L. Spallanzani', University of Pavia, Pavia, Italy., Fraaije MW; Molecular Enzymology Group, University of Groningen, Groningen, Netherlands.
Jazyk: angličtina
Zdroj: ELife [Elife] 2020 Mar 31; Vol. 9. Date of Electronic Publication: 2020 Mar 31.
DOI: 10.7554/eLife.54639
Abstrakt: Enzyme instability is an important limitation for the investigation and application of enzymes. Therefore, methods to rapidly and effectively improve enzyme stability are highly appealing. In this study we applied a computational method (FRESCO) to guide the engineering of an alcohol dehydrogenase. Of the 177 selected mutations, 25 mutations brought about a significant increase in apparent melting temperature (Δ T m ≥ +3 °C). By combining mutations, a 10-fold mutant was generated with a T m of 94 °C (+51 °C relative to wild type), almost reaching water's boiling point, and the highest increase with FRESCO to date. The 10-fold mutant's structure was elucidated, which enabled the identification of an activity-impairing mutation. After reverting this mutation, the enzyme showed no loss in activity compared to wild type, while displaying a T m of 88 °C (+45 °C relative to wild type). This work demonstrates the value of enzyme stabilization through computational library design.
Competing Interests: FA, MF, SR, MT, JG, AV, AM, MF No competing interests declared, SB A patent application on the original ADH was filed by c-LEcta (WO 2019/012095)
(© 2020, Aalbers et al.)
Databáze: MEDLINE
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