Approaching boiling point stability of an alcohol dehydrogenase through computationally-guided enzyme engineering.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 32228861.
- Also identified by DOI 10.7554/eLife.54639 and PMC identifier 7164962.
- Licence recorded as CC BY.
- The licence permits redistribution, so the abstract is shown in full and the full text is available from the publisher.
Abstract
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<i>T</i><sub>m</sub> ≥ +3 °C). By combining mutations, a 10-fold mutant was generated with a <i>T</i><sub>m</sub> 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 <i>T</i><sub>m</sub> of 88 °C (+45 °C relative to wild type). This work demonstrates the value of enzyme stabilization through computational library design.
Medical subject headings
- Alcohol Dehydrogenase
- Escherichia coli
- Mutation
- Protein Engineering
- Transition Temperature