Colorimetric determination of β-1,2-glucooligosaccharides for an enzymatic assay using 3-methyl-2-benzothiazolinonehydrazone

A colorimetric determination method measuring the reducing ends of sugars is usually used for quantitative evaluation of polysaccharide-degrading activity of endo-type enzymes. However, no appropriate colorimetric method has been established for enzymatic assay of β-1,2-glucanases, which produce β-1,2-glucooligosaccharides from β-1,2-glucans. The Anthon-MBTH method has been potentially the most adaptable for color development of β-1,2-glucooligosaccharides among various known colorimetric methods for detecting the reducing power of oligosaccharides, since the difference between sophorose and other β-1,2-glucooligosaccharides in absorbance is relatively small. Almost the same color development was obtained for β-1,2-glucooligosaccharides when the heating time with the Anthon-MBTH method was changed. The kind of base and concentration of dithiothreitol did not markedly affect the color development. Most buffer components, salts and a chelating reagent used for usual enzymatic experiments did not inhibit color development. Furthermore, assay was performed successfully for a β-1,2-glucanase using the modified MBTH method.