A High‐Throughput Screening Method for the Directed Evolution of Hydroxynitrile Lyase towards Cyanohydrin Synthesis. (12th January 2021)

• Record Type: Journal Article
• Authors: Zheng, Yu‐Cong; Ding, Liang‐Yi; Jia, Qiao; Lin, Zuming; Hong, Ran; Yu, Hui‐Lei; Xu, Jian‐He
• Is Part Of: Chembiochem. Volume 22:Number 6(2021)
• Page Start: 996
• Page End: 1000
• Publication Date: 2021-01-12
• Subjects: amino-benzamidoxime -- cyanohydrins -- directed evolution -- high-throughput screening -- hydrocyanation; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.202000658
• Languages: English
• ISSNs: 1439-4227

Abstract

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 Pc HNL5 that presented higher catalytic efficiency and stronger acidic tolerance in variant libraries. Abstract : Making biohydrocyanation measurable : Hydroxynitrile lyases (HNLs) catalyze the enantioselective cleavage/formation of cyanohydrins. However, current methods for determining hydrocyanation are not suitable for mass screening of mutants for protein engineering of these biocatalysts. We demonstrate herein a chromogenic high‐throughput screening method for cyanohydrin synthesis that is validated by either substrate profiling or the directed evolution of HNLs.