Modern
enzyme development relies to an increasing extent on strategies based on diversity generation followed by screening for variants with optimised properties. In principle, these directed evolution strategies might be used for optimising any
enzyme property, which can be screened for in an economically feasible way, even if the molecular basis of that property is not known. Stability is an interesting property of
enzymes because (1) it is of great industrial importance, (2) it is relatively easy to screen for, and (3) the molecular basis of stability relates closely to contemporary issues in
protein science such as the protein folding problem and
protein folding diseases. Thus, engineering enzyme stability is of both commercial and scientific interest. Here, we review how directed evolution has contributed to the development of stable
enzymes and to new insight into the principles of protein stability. Several recent examples are described. These examples show that directed evolution is an effective strategy to obtain stable
enzymes, especially when used in combination with rational or semi-rational engineering strategies. With respect to the principles of protein stability, some important lessons to learn from recent efforts in directed evolution are (1) that there are many structural ways to stabilize a
protein, which are not always easy to rationalize, (2) that
proteins may very well be stabilized by optimizing their surfaces, and (3) that high thermal stability may be obtained without forfeiture of catalytic performance at low temperatures.