Abstract：
A fluorescence-quenching-based assay system to determine the hydrolytic activity of endo-β-N-acetylglucosaminidases (ENGases), which act on the innermost N-acetylglucosamine (GlcNAc) residue of the chitobiose segment of core-fucosylated N-glycans, was constructed using a dual-labeled fluorescent probe with a hexasaccharide structure. The fluorogenic probe was evaluated using a variety of ENGases, including Endo-M W251N mutant, Endo-F3, and Endo-S, which recognize core fucosylated N-glycans. The occurrence of a hydrolysis reaction was detected by observing an increased fluorescence intensity, ultimately allowing the ENGase activities to be easily and quantitatively evaluated, with the exception of Endo-S. The obtained results clearly indicated the substrate specificities of the examined ENGases.
摘要：
一种基于荧光猝灭的测定系统，用于确定内切β-N-乙酰氨基葡萄糖苷酶（ENGases）的水解活性，作用于核心岩藻糖基化N-聚糖的壳二糖片段的最内层N-乙酰葡糖胺（GlcNAc）残基，使用具有六糖结构的双标记荧光探针构建。使用各种ENGases评估荧光探针，包括Endo-MW251N突变体，Endo-F3和Endo-S,其识别核心岩藻糖基化N-聚糖。通过观察增加的荧光强度来检测水解反应的发生。最终允许容易和定量地评估ENGase活性，除了Endo-S获得的结果清楚地表明了所检查的ENGase的底物特异性。
