Giver L, Gershenson A, Freskgard P O, Arnold F H
Division of Chemistry and Chemical Engineering, Mail Code 210-41, California Institute of Technology, Pasadena, CA 91125, USA.
Proc Natl Acad Sci U S A. 1998 Oct 27;95(22):12809-13. doi: 10.1073/pnas.95.22.12809.
We have used in vitro evolution to probe the relationship between stability and activity in a mesophilic esterase. Previous studies of these properties in homologous enzymes evolved for function at different temperatures have suggested that stability at high temperatures is incompatible with high catalytic activity at low temperatures through mutually exclusive demands on enzyme flexibility. Six generations of random mutagenesis, recombination, and screening stabilized Bacillus subtilis p-nitrobenzyl esterase significantly (>14 degreesC increase in Tm) without compromising its catalytic activity at lower temperatures. Furthermore, analysis of the stabilities and activities of large numbers of random mutants indicates that these properties are not inversely correlated. 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 low-temperature activity are very rare. Unless both properties are constrained (by natural selection or screening) the evolution of one by the accumulation of single amino acid substitutions typically comes at the cost of the other, regardless of whether the two properties are inversely correlated or not correlated at all.
我们利用体外进化来探究嗜温酯酶稳定性与活性之间的关系。先前对在不同温度下进化以实现特定功能的同源酶的这些特性的研究表明，由于对酶柔韧性的相互排斥的要求，高温下的稳定性与低温下的高催化活性不相容。经过六代随机诱变、重组和筛选，枯草芽孢杆菌对硝基苄酯酶的稳定性显著提高（熔解温度升高超过14℃），且不影响其在较低温度下的催化活性。此外，对大量随机突变体的稳定性和活性分析表明，这些特性并非呈负相关。虽然增强的热稳定性不一定以活性为代价，但分子适应的过程很重要。在保持低温活性的同时提高热稳定性的突变非常罕见。除非这两种特性都受到限制（通过自然选择或筛选），否则通过单个氨基酸取代的积累来进化其中一种特性通常会以牺牲另一种特性为代价，无论这两种特性是呈负相关还是根本不相关。
