Progress in methodology of enzyme directed evolution

Directed evolution is an effective way to improve the catalytic properties of enzymes, such as stereoscopic / regional / chemical selectivity, activity, thermal stability, etc Previous studies have shown that the selectivity and activity of the enzyme can be successfully controlled by saturated mutation (SM) of amino acid residues located in the catalytic pocket of the enzyme As a common degenerate codon, NNK can encode 20 kinds of amino acids, but with the increase of mutation sites, the screening amount increases exponentially (20 m, M is the number of sites) More and more attention has been paid to how to reduce the design of codon and build efficient mutant library to overcome the bottleneck of screening Tianjin Industrial Biotechnology, Chinese Academy of Sciences The research team of enzyme molecular engineering and industrial biocatalysis led by sun Zhoutong, a researcher of the Research Institute, focused on this problem, and took limonene cyclohydrolase and P450 monooxygenase as research objects, respectively constructed mutant libraries with NNK, NDT, rationally designed simplified codon and other simplified codon design methods The experimental results show that rationally designed simplified codon under the same screening amount The number of beneficial mutants was significantly increased, which was supported by statistical analysis (Patrick / Firth and Nov calculation) This work proves for the first time that beneficial mutants can be obtained quickly by simplified codon design and high coverage screening This research is supported by the project of Chinese Academy of Sciences Qu Ge, assistant researcher of Tianjin Institute of technology, is the co first author of the paper Sun Zhoutong and Manfred t reetz, Professor of Mapu Coal Institute / Tianjin Institute of technology, Germany, are the co authors.