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 K(m)(app) of 11 microM and a K(cat)(app) of 3.5s(-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.
Miller
Antonia G
AG
School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand.
Hegge
Stephan
S
Uhlmann
Andrea
A
Gerrard
Juliet A
JA
D000594:Amino Acid Oxidoreductases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001232:Aspergillus fumigatus / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D052160:Benzothiazoles; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005632:Fructose / Q000031:analogs & derivatives*; D066298:In Vitro Techniques; D007700:Kinetics; D015416:Maillard Reaction; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011437:Propylamines; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013379:Substrate Specificity; D013451:Sulfonic Acids; D013696:Temperature
D052160:Benzothiazoles; D017931:DNA Primers; D011437:Propylamines; D011994:Recombinant Proteins; D013451:Sulfonic Acids; C453492:fructosylpropylamine; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D005632:Fructose; D000594:Amino Acid Oxidoreductases; C103012:amadoriase