Oligosaccharide dehydrogenase-catalyzed assay for the determination of polysaccharides

Oligosaccharide dehydrogenase (ODH), an enzyme known to have a broad selectivity for reducing sugars of low molecular weight, was investigated to determine its catalytic properties with larger polysaccharides. Six substrates were studied: pullulan standards with molecular weights of between 5,400 and 90,900, debranched starch, and dextran. In addition, maltotriose, isomaltotriose, maltose, and glucose were used as substrates for comparison. ODH catalyzed the oxidation of the large pullulans with a degree of polymerization of at least 560. Isomaltotriose and dextran were not oxidized. ODH activity for the pullulans, expressed as the rate constant Kps, was only three times lower than that for maltose. When the oxidation of sugars with ODH was coupled to a color-forming reaction, quantitative spectrophotometric determination of sugars was possible using either Meldola's blue or N-methylphenazinium as electron acceptors in combination with nitrotetrazolium blue. Linear calibration curves for maltose, maltotriose, and debranched starch were obtained using this ODH method and compared with curves from the conventional spectrophotometric copper sulfate method. This work demonstrates that ODH can be advantageously used for the determination of polysaccharides.