A continuous enzyme assay and characterisation of fructosyl amine oxidase enzymes (EC 1.5.3)

Enzymatic reversal of the Maillard reaction is a growing area of research. Fructosyl amine oxidase enzymes (EC 1.5.3) have attracted recent attention through demonstration of their ability to deglycate Amadori products, low molecular weight intermediates formed during the early stage of the Maillard reaction. Although stopped assays have been described, a bottleneck in current studies is the lack of continuous kinetic assays. Here, we describe the development of a continuous, coupled enzyme assay and its successful application to determining optimal storage conditions and the steady-state kinetic parameters of an enzyme from this group, amadoriase I. A Kmapp of 11 μM and a Kcatapp of 3.5 s−1 were determined using this assay using fructosyl propylamine as a substrate, which differ from previous reports. This method was also used to test the activity of two site-directed mutants of amadoriase I, H357N and S370A, which were found to be catalytically inactive.