Molecular Enzymology Group, University of Groningen, Groningen, Netherlands; Department of Biology and Biotechnology “L. Spallanzani”, University of Pavia, Pavia, Italy
Molecular Enzymology Group, University of Groningen, Groningen, Netherlands; MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, United Kingdom
Stefano Rovida
Department of Biology and Biotechnology “L. Spallanzani”, University of Pavia, Pavia, Italy
Milos Trajkovic
Molecular Enzymology Group, University of Groningen, Groningen, Netherlands
J Rubén Gómez Castellanos
Department of Biology and Biotechnology “L. Spallanzani”, University of Pavia, Pavia, Italy
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 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 Tm of 88 °C (+45 °C relative to wild type). This work demonstrates the value of enzyme stabilization through computational library design.
