In a directed evolution aimed at improving enzymatic activity, a situation occurs where highly active variants can no longer be obtained from a template protein because the template is already located at a peak (local maximum) in the fitness landscape of activity for the sequence space. To overcome this situation, the template needs to descend the mountain (lose activity) once and climb another higher mountain. However, there is no solid guideline of how the template should go down. Here, we propose a stability index. Previous studies have shown that protein evolution is potentially governed by stability, and that proteins with low activity but high stability are more favorable templates for producing highly active variants. In our earlier works on conventional directed evolution by random mutagenesis of an esterase from Sulfolobus tokodaii, we identified variants with 3-fold higher activity than the wild-type as the highest activity variants. In this work, as a first step, stability-keeping variants were selected by five rounds of random mutagenesis and screening based on halo formation assay using the substrate tributyrin at 70 °C after heat treatment for 30 min at 90 °C. These variants are likely to be scattered at the feet of various mountains in the fitness landscape. Next, these variants were pooled and used as parental proteins for a conventional experiment with activity-based selection, where the activity of variants was assayed using their cell-free extracts on the substrate p-nitrophenyl butyrate at 75 °C. After two rounds of random mutagenesis, we successfully obtained a variant with 9-fold higher activity than the wild-type. These results indicate that the two-step selection by stability and activity enables us more easily to produce markedly activity-improving variants.

中文翻译：

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通过基于稳定性选择的定向进化产生的高活性酶

在旨在提高酶活性的定向进化中，由于模板已经位于序列空间活性适应度景观中的峰值（局部最大值），因此无法再从模板蛋白中获得高活性变体的情况会发生。为了克服这种情况，模板需要下山（失去活动）一次并爬上另一座更高的山。但是，没有关于模板应该如何下降的可靠指南。在这里，我们提出了一个稳定性指数。先前的研究表明，蛋白质进化可能受稳定性控制，低活性但高稳定性的蛋白质是更有利的模板，可用于产生高活性变体。在我们早期通过随机诱变来自 Sulfolobus tokodaii 的酯酶进行常规定向进化的工作中，我们将活性比野生型高 3 倍的变体鉴定为最高活性变体。在这项工作中，作为第一步，在 90°C 下热处理 30 分钟后，使用底物三丁酸甘油酯在 70°C 下通过五轮随机诱变和筛选选择保持稳定性的变体。这些变种很可能散落在健身景观中的各个山脚下。接下来，将这些变体合并并用作基于活性的选择的常规实验的亲本蛋白，其中在 75°C 下使用它们在底物对硝基苯丁酸盐上的无细胞提取物测定变体的活性。经过两轮随机诱变，我们成功获得了活性比野生型高 9 倍的变体。