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

摘要

A new spectrophotometric determination method using CuSO_4/MeOH was developed to determine the activity of omega-transaminase.Staining solution of CuSO_4/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 CuSO_4/MeOH,leading to quite simple,but agreeable rapid determination of the substrate specificities and apparent enantioselectivities of the mode omega-transaminase.This method was applied to directed evolution of the omega-transaminase for improving reactivity towards 3-amino-3-phenylpropionic acid,resulting a mutant with threefold higher activity than the wild type.