Screening is the bottleneck of directed evolution. In order to address this problem, a series of novel semi-rational designed strategies have been developed based on combinatorial active-site saturation test and iterative saturation mutagenesis, including single code saturation mutagenesis, double code saturation mutagenesis and triple code saturation mutagenesis. By creation of "small and smart" high qualified mutant libraries and combinatorial mutagenesis of specific sites, these new strategies have been successfully applied in multiparameter optimization, e.g. stereo/regioselectivity and activity. This review summarized recent advances in directed evolution and its applications in biocatalysis field.
筛选是制约酶定向进化改造的瓶颈。为解决这一难题，近年来一系列基于组合活性中心饱和突变(Combinatorial active-site saturation test，CAST) 及迭代饱和突变 (Iterative saturation mutagenesis，ISM) 的半理性设计新方法被开发出来，包括单密码子饱和突变 (Single code saturation mutagenesis，SCSM)、双密码子饱和突变 (Double code saturation mutagenesis，DCSM) 和三密码子饱和突变 (Triple code saturation mutagenesis，TCSM)。通过构建“小而精”的高质量突变体文库，对特定靶点进行组合突变，并成功应用于多种生物催化剂的立体/区域选择性及催化活力等多参数的改造。文中综述了近年来定向进化技术的最新进展及其在生物催化剂定向改造中的应用。.
Keywords: biocatalysis; directed evolution; saturation mutagenesis; stereoselectivity.