Porter_2015_Protein.J_34_82

Directed evolution is a common tool employed to generate enzymes suitable for industrial use. High thermal stability is often advantageous or even a requirement for biocatalysts, as such the evolution of protein stability is of practical as well as academic interest. Even when evolving enzymes for new or improved catalytic functions, stability is an important factor since it can limit the accumulation rate and number of desired active site mutations. Dienelactone hydrolase, a small monomeric protein, has been previously evolved via a three-stage process to possess enhanced activity and specificity toward non-physiological substrates. In addition to seven active site mutations there were three surface mutations that were thought to increase the stability of the enzyme and compensate for the destabilizing active site mutations. Here, the individual influence of the three surface mutations--Q110L, Y137C and N154D--on the thermal and chemical stability of DLH has been assessed. While the Q110L and N154D mutations improved the thermal stability, the influence of the Y137C mutation was more complex. Individually it was destabilizing both thermally and chemically, but when in the presence of the Q110L and N154D mutations its effect was neutralized in relation to thermal but not chemical stability. In the context of a directed evolution experiment, these compensatory surface mutations play important roles. However, our results show that detrimental mutations can arise, thus the simultaneous monitoring of stability changes while evolving enzymes for enhanced catalytic properties can be beneficial.