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

Elife. 2020 Mar 31;9:e54639. doi: 10.7554/eLife.54639.


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.

Keywords: E. coli; alcohol dehydrogenase; biocatalysis; biotechnology; cofactor; computational biology; enzyme engineering; molecular biophysics; oxidations; structural biology; systems biology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohol Dehydrogenase / chemistry*
  • Alcohol Dehydrogenase / genetics
  • Computers, Molecular
  • Crystallization
  • Enzyme Stability
  • Escherichia coli / genetics*
  • Gene Library
  • Kinetics
  • Mutation*
  • Protein Conformation
  • Protein Engineering / methods*
  • Saccharomycetales / enzymology
  • Transition Temperature*


  • Alcohol Dehydrogenase

Supplementary concepts

  • Starmerella magnoliae

Associated data

  • PDB/6TQ3
  • PDB/6TQ5
  • PDB/6TQ8