Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan.
Faculty of Agriculture, Niigata University, Niigata, 950-2181, Japan.
Anal Biochem. 2018 Nov 1;560:1-6. doi: 10.1016/j.ab.2018.08.021. Epub 2018 Aug 25.
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.
一种比色法测定糖的还原端，通常用于定量评价内切型酶对多糖的降解活性。然而，对于β-1,2-葡聚糖酶的酶促测定，还没有建立起合适的比色法，因为β-1,2-葡聚糖酶从β-1,2-葡聚糖中产生β-1,2-葡寡糖。在检测寡糖还原力的各种已知比色法中，Anthon-MBTH 法是最适合开发β-1,2-葡寡糖显色的方法，因为棉子糖和其他β-1,2-葡寡糖在吸光度上的差异相对较小。当用 Anthon-MBTH 法改变加热时间时，β-1,2-葡寡糖的显色几乎相同。二硫苏糖醇的碱的种类和浓度对显色没有明显影响。通常用于酶实验的大多数缓冲成分、盐和螯合剂都不会抑制显色。此外，使用改良的 MBTH 法成功地对一种β-1,2-葡聚糖酶进行了测定。
