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

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....

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Detalles Bibliográficos
Autores principales: Aalbers, Friso S, Fürst, Maximilian JLJ, Rovida, Stefano, Trajkovic, Milos, Gómez Castellanos, J Rubén, Bartsch, Sebastian, Vogel, Andreas, Mattevi, Andrea, Fraaije, Marco W
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7164962/
https://www.ncbi.nlm.nih.gov/pubmed/32228861
http://dx.doi.org/10.7554/eLife.54639
Descripción
Sumario: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 (ΔT(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.