Enhancing the Thermal Robustness of an Enzyme by Directed Evolution: Least Favorable Starting Points and Inferior Mutants Can Map Superior Evolutionary Pathways

In a previous directed evolution study, the B‐FIT approach to increasing the thermal robustness of proteins was introduced and applied to the lipase from Bacillus subtilis . It is based on the general concept of iterative saturation mutagenesis (ISM), according to which sites in an enzyme are subjected to saturation mutagenesis, the best hit of a given library is then used as a template for randomization at other sites, and the process is continued until the desired catalyst improvement has been achieved. The appropriate choice of the ISM sites is crucial; in the B‐FIT method the criterion is residues characterized by highest B factors available from X‐ray crystallography data. In the present study, B‐FIT was employed in order to increase the thermal robustness of the epoxide hydrolase from Aspergillus niger . Several rounds of ISM resulted in the best variant showing a 21 °C increase in the ${T{{\,60\hfill \atop 50\hfill}}}$ value, an 80‐fold improvement in half‐life at 60 °C, and a 44 kcal mol −1 improvement in inactivation energy. Seven other variants were also evolved with moderate yet significant improvements; these were characterized by 10–14 °C increases in ${T{{\,60\hfill \atop 50\hfill}}}$ , 20–30‐fold improvement in half‐lives at 60 °C and 15–20 kcal mol −1 elevations in activation energy. Unexpectedly, in the ISM process the best variants were obtained from essentially neutral or even inferior mutant parents, that is, when a given library contains no improved mutants. This constitutes a practical way to escape from what appear to be local minima (“dead ends”) in the fitness landscape—a finding of notable significance in directed evolution.