Directed evolution has emerged as the most productive enzyme engineering method, with stereoselectivity playing a crucial role when evolving mutants for application in synthetic organic chemistry and biotechnology. In order to reduce the screening effort (bottleneck of directed evolution), improved methods for the creation of small and smart mutant libraries have been developed, including the combinatorial active-site saturation test (CAST) which involves saturation mutagenesis at appropriate residues surrounding the binding pocket, and iterative saturation mutagenesis (ISM). Nevertheless, even CAST/ISM mutant libraries require a formidable screening effort. Thus far, rational design as the alternative protein engineering technique has had only limited success when aiming for stereoselectivity. Here, we highlight a recent methodology dubbed focused rational iterative site-specific mutagenesis (FRISM), in which mutant libraries are not involved. It makes use of the tools that were previously employed in traditional rational enzyme design, but, inspired by CAST/ISM, the process is performed in an iterative manner. Only a few predicted mutants need to be screened, a fast process which leads to the identification of highly enantioselective and sufficiently active mutants.

中文翻译：

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聚焦理性迭代位点特异性诱变 (FRISM)。

定向进化已成为最有效的酶工程方法，立体选择性在进化突变体以应用于合成有机化学和生物技术时起着至关重要的作用。为了减少筛选工作（定向进化的瓶颈），已经开发了用于创建小型智能突变文库的改进方法，包括组合活性位点饱和测试 (CAST)，它涉及在结合周围的适当残基处进行饱和诱变口袋和迭代饱和诱变（ISM）。然而，即使是 CAST/ISM 突变体文库也需要艰巨的筛选工作。迄今为止，作为替代蛋白质工程技术的合理设计在以立体选择性为目标时仅取得了有限的成功。这里，我们强调了最近的一种称为集中合理迭代位点特异性诱变（FRISM）的方法，其中不涉及突变文库。它利用了以前在传统理性酶设计中使用的工具，但受 CAST/ISM 的启发，该过程以迭代方式进行。只需要筛选少数预测的突变体，这是一个快速的过程，可以识别出高度对映选择性和足够活跃的突变体。