一种用于定向进化羟腈酶用于氰醇合成的高通量筛选方法。
A High-Throughput Screening Method for the Directed Evolution of Hydroxynitrile Lyase towards Cyanohydrin Synthesis.
机构信息
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai Collaborative Innovation Centre for Biomanufacturing, Frontiers Science Center for Materiobiology and Dynamic Chemistry, Shanghai, 200237, P. R. China.
CAS Key Laboratory of Synthetic Chemistry of Natural Substances, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry (CAS), Shanghai, 200032, P. R. China.
出版信息
Chembiochem. 2021 Mar 16;22(6):996-1000. doi: 10.1002/cbic.202000658. Epub 2021 Jan 12.
Chiral cyanohydrins are useful intermediates in the pharmaceutical and agricultural industries. In nature, hydroxynitrile lyases (HNLs) are a kind of elegant tool for enantioselective hydrocyanation of carbonyl compounds. However, currently available methods for demonstrating hydrocyanation are still stalled at precise, but low-throughput, GC or HPLC analyses. Herein, we report a chromogenic high-throughput screening (HTS) method that is feasible for the cyanohydrin synthesis reaction. This method was highly anti-interference and sensitive, and could be used to directly profile the substrate scope of HNLs either in cell-free extract or fermentation clear broth. This HTS method was also validated by generating new variants of PcHNL5 that presented higher catalytic efficiency and stronger acidic tolerance in variant libraries.
手性氰醇是医药和农业行业中有用的中间体。在自然界中，羟腈酶（HNLs）是一种用于对羰基化合物进行对映选择性氰化氢加成的优雅工具。然而，目前用于展示氰化氢加成的方法仍然停留在精确但高通量低的 GC 或 HPLC 分析上。在此，我们报告了一种用于氰醇合成反应的显色高通量筛选（HTS）方法。该方法具有高度抗干扰性和灵敏度，可用于直接分析无细胞提取物或发酵澄清液中 HNL 的底物范围。该 HTS 方法还通过生成 PcHNL5 的新变体进行了验证，这些变体在变体文库中表现出更高的催化效率和更强的耐酸性。
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