Bioanalysis for Biocatalysis: Multiplexed Capillary Electrophoresis–Mass Spectrometry Assay for Aminotransferase Substrate Discovery and Specificity Profiling

In this work, we introduce an entirely automated enzyme assay based on capillary electrophoresis coupled to electrospray ionization mass spectrometry termed MINISEP-MS for multiple interfluent nanoinjections–incubation–separation–enzyme profiling using mass spectrometry. MINISEP-MS requires only nanoliters of reagent solutions and uses the separation capillary as a microreactor, allowing multiple substrates to be assayed simultaneously. The method can be used to rapidly profile the substrate specificity of any enzyme and to measure steady-state kinetics in an automated fashion. We used the MINISEP-MS assay to profile the substrate specificity of three aminotransferases (E. coli aspartate aminotransferase, E. coli branched-chain amino acid aminotransferase, and Bacillus sp. YM-1 d-amino acid aminotransferase) for 33 potential amino acid substrates and to measure steady-state kinetics. Using MINISEP-MS, we were able to recapitulate the known substrate specificities and to discover new amino acid substrates for these industrially relevant enzymes. Additionally, we were able to measure the apparent K M and k cat parameters for amino acid donor substrates of these aminotransferases. Because of its many advantages, the MINISEP-MS assay has the potential of becoming a useful tool for researchers aiming to identify or create novel enzymes for specific biocatalytic applications.