- Rapid and Quantitative Profiling of Substrate Specificity of ω-Transaminases for Ketones
-
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.
- Han, Sang-Woo,Shin, Jong-Shik
-
p. 3287 - 3295
(2019/06/21)
-
- The hydrolysis of epoxides catalyzed by inorganic ammonium salts in water: Kinetic evidence for hydrogen bond catalysis
-
Naturally-occurring inorganic ammonium ions have been recently reported as efficient catalysts for some organic reactions in water, which contributes to the understanding of the chemistry in some natural environments (soils, seawater, atmospheric aerosols, .) and biological systems, and is also potentially interesting for green chemistry as many of their salts are cheap and non-toxic. In this work, the effect of NH4+ ions on the hydrolysis of small epoxides in water was studied kinetically. The presence of NH4+ increased the hydrolysis rate by a factor of 6 to 25 compared to pure water and these catalytic effects were shown not to result from other ions, counter-ions or from acid or base catalysis, general or specific. The small amounts of amino alcohols produced in the reactions were identified as the actual catalysts by obtaining a strong acceleration of the reactions when adding these compounds directly to the epoxides in water. Replacing the amino alcohols by other strong hydrogen-bond donors, such as trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP) gave the same results, demonstrating that the kinetics of these reactions was driven by hydrogen-bond catalysis. Because of the presence of many hydrogen-bond donors in natural environments (for instance amines and hydroxy-containing compounds), hydrogen-bond catalysis is likely to contribute to many reaction rates in these environments.
- Nozière,Fache,Maxut,Fenet,Baudouin,Fine,Ferronato
-
p. 1583 - 1590
(2018/02/06)
-