A High-Throughput Screening Method for the Directed Evolution of Hydroxynitrile Lyase towards Cyanohydrin Synthesis |
| |
| Authors: | Yu-Cong Zheng Liang-Yi Ding Qiao Jia Zuming Lin Prof. Dr. Ran Hong Prof. Dr. Hui-Lei Yu Prof. Dr. Jian-He Xu |
| |
| Affiliation: | 1. 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;2. 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 |
| |
| Abstract: | 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. |
| |
| Keywords: | amino-benzamidoxime cyanohydrins directed evolution high-throughput screening hydrocyanation |
|
|
