A 1-step microplate method for assessing the substrate range of l-α-amino acid aminotransferase

Aminotransferase enzymes catalyse the reversible substitution of a keto group for an amino group. While this reaction is highly stereoselective with respect to the amino group, each enzyme can usually catalyse the turnover of a number of different substrates. As the substrate range cannot be inferred from the sequence, it remains an early bottleneck when selecting an enzyme for a biocatalysis application. We have developed a simple first round characterisation method applicable to the broad range of aminotransferases that accept l-glutamate, the central junction of cellular transamination, as one of the amino donors. The assay is based on l-glutamate detection by its highly specific dehydrogenase enzyme in a coupled assay, ending in the reduction of the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-tetrazolium-5-carboxanilide (XTT). While products of most tetrazolium salts are water-insoluble, XTT is reduced to a water soluble colored formazan, allowing direct spectrophotometric detection. The reaction is carried out in microplate format using a single endpoint measurement and is thus suitable for automation.