High-throughput screening method for the identification of active and enantioselective ω-transaminases

A new spectrophotometric determination method using CuSO4/MeOH was developed to determine the activity of ω-transaminase. Staining solution of CuSO4/MeOH forms a blue complex with the α-amino acid produced during ω-transaminase reaction. This complex can be easily quantified using UV-Vis spectrophotometer at 595 nm. The analytical data determined using spectrophotometric assay agree well with HPLC analysis data, and the differences between the two results are below 10%. Amino donor and amino acceptor specificities and enantioselectivities of ω-transaminase were simultaneously investigated in 96-well microplates using the CuSO4/MeOH, leading to quite simple, but agreeable rapid determination of the substrate specificities and apparent enantioselectivities of the model ω-transaminase. This method was applied to directed evolution of the ω-transaminase for improving reactivity towards 3-amino-3-phenylpropionic acid, resulting a mutant with threefold higher activity than the wild type.