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

Friso S. Aalbers, Maximilian Jlj Fürst, Stefano Rovida, Milos Trajkovic, J. Rubén Gómez Castellanos, Sebastian Bartsch, Andreas Vogel, Andrea Mattevi, Marco W. Fraaije*

*Bijbehorende auteur voor dit werk

OnderzoeksoutputAcademicpeer review

23 Citaten (Scopus)
118 Downloads (Pure)

Samenvatting

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 (ΔTm ≥ +3 °C). By combining mutations, a 10-fold mutant was generated with a Tm of 94 °C (+51 °C relative to wildtype), 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 wildtype, while displaying a Tm of 88 °C (+45 °C relative to wildtype). This work demonstrates the value of enzyme stabilization through computational library design.

Originele taal-2English
Artikelnummer e54639
Aantal pagina's18
TijdschrifteLife
Volume9
DOI's
StatusPublished - 31-mrt.-2020

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