DIRECTED EVOLUTION OF A THERMOSTABLE ESTERASE

We have used in vitro evolution to probe the relationship between stab ility and activity in a mesophilic esterase, Previous studies of these properties in homologous enzymes evolved for function at different te mperatures have suggested that stability at high temperatures is incom patible with high catalytic activity at low temperatures through mutua lly exclusive demands on enzyme flexibility. Six generations of random mutagenesis, recombination, and screening stabilized Bacillus subtili s p-nitrobenzyl esterase significantly (>14 degrees C increase in T-m) without compromising its catalytic activity at lower temperatures. Fu rthermore, analysis of the stabilities and activities of large numbers of random mutants indicates that these properties are not inversely c orrelated. Although enhanced thermostability does not necessarily come at the cost of activity, the process by which the molecule adapts is important. Mutations that increase thermostability while maintaining l ow-temperature activity are very rare. Unless both properties are cons trained (by natural selection or screening) the evolution of one by th e accumulation of single amino acid substitutions typically comes at t he cost of the other, regardless of whether the two properties are inv ersely correlated or not correlated at all.