ω-Transaminases (ω-TAs) have gained growing attention owing to their capability for asymmetric synthesis of chiral amines from ketones. Reliable high-throughput activity assay of ω-TAs is essential in carrying out extensive substrate profiling and establishing a robust screening platform. Here we report spectrophotometric and colorimetric methods enabling rapid quantitation of ω-TA activities toward ketones in a 96-well microplate format. The assay methods employ benzylamine, a reactive amino donor for ω-TAs, as a cosubstrate and exploit aldehyde dehydrogenase (ALDH) as a reporter enzyme, leading to formation of benzaldehyde detectable by ALDH owing to concomitant NADH generation. Spectrophotometric substrate profiling of two wild-type ω-TAs of opposite stereoselectivity was carried out at 340 nm with 22 ketones, revealing subtle differences in substrate specificities that were consistent with docking simulation results obtained with cognate amines. Colorimetric readout for naked eye detection of the ω-TA activity was also demonstrated by supplementing the assay mixture with color-developing reagents whose color reaction could be quantified at 580 nm. The colorimetric assay was applied to substrate profiling of an engineered ω-TA for 24 ketones, leading to rapid identification of reactive ketones. The ALDH-based assay is expected to be promising for high-throughput screening of enzyme collections and mutant libraries to fish out the best ω-TA candidate as well as to tailor enzyme properties for efficient amination of a target ketone.
All Science Journal Classification (ASJC) codes
- Physical and Theoretical Chemistry
- Organic Chemistry
- Inorganic Chemistry