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 W-transaminase. Staining solution Of CuSO4/MeOH forms a blue complex with the alpha-amino acid produced during omega-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 omega-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 omega-transaminase. This method was applied to directed evolution of the w-transaminase for improving reactivity towards 3-amino-3-phenylpropionic acid, resulting a mutant with threefold higher activity than the wild type. (C) 2004 Elsevier Inc. All rights reserved.