TY - JOUR TI - Approaching boiling point stability of an alcohol dehydrogenase through computationally-guided enzyme engineering AU - Aalbers, Friso S AU - Fürst, Maximilian JLJ AU - Rovida, Stefano AU - Trajkovic, Milos AU - Gómez Castellanos, J Rubén AU - Bartsch, Sebastian AU - Vogel, Andreas AU - Mattevi, Andrea AU - Fraaije, Marco W A2 - Cole, Philip A VL - 9 PY - 2020 DA - 2020/03/31 SP - e54639 C1 - eLife 2020;9:e54639 DO - 10.7554/eLife.54639 UR - https://doi.org/10.7554/eLife.54639 AB - 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. KW - biocatalysis KW - enzyme engineering KW - oxidations KW - biotechnology KW - alcohol dehydrogenase KW - cofactor JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -