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

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 (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,

SUBMITTER: Aalbers FS 

PROVIDER: S-EPMC7164962 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature