Malet C, Vallés J, Bou J, Planas A
Department of Organic Chemistry, CETS Institut Químic de Sarrià, Universitat Ramon Llull, Barcelona, Spain.
J Biotechnol. 1996 Jul 31;48(3):209-19. doi: 10.1016/0168-1656(96)01511-8.
The synthesis of 4-methylumbelliferyl 3-beta-O-cellobiosyl-beta-D-glucopyranoside (3a) and its use as specific substrate to monitor enzyme activity of 1,3-1,4-beta-D-glucan 4-glucanohydrolases are described. The chromophoric substrate 3a is prepared by a chemoenzymatic approach starting from barley grain, whose beta-D-glucan polysaccharide is degraded down to a tri- and tetrasaccharide by an extracellular extract of recombinant E. coli expressing and secreting Bacillus licheniformis 1,3-1,4-beta-glucanase. The trisaccharide 1 is further chemically transformed into the title compound. Its use as substrate for an enzyme activity assay, the specificity of cleavage, and kinetic parameters are reported. As it undergoes a single glycosidic bond hydrolysis with release of 4-methylumbelliferone, direct UV monitoring of the reaction provides a sensitive kinetic assay of the enzyme action.
描述了4-甲基伞形酮基3-β-O-纤维二糖基-β-D-吡喃葡萄糖苷（3a）的合成及其作为监测1,3-1,4-β-D-葡聚糖4-葡聚糖水解酶活性的特异性底物的用途。发色底物3a通过化学酶法从大麦粒开始制备，其β-D-葡聚糖多糖被表达并分泌地衣芽孢杆菌1,3-1,4-β-葡聚糖酶的重组大肠杆菌的细胞外提取物降解为三糖和四糖。三糖1进一步化学转化为标题化合物。报道了其作为酶活性测定底物的用途、裂解特异性和动力学参数。由于它经历单一糖苷键水解并释放4-甲基伞形酮，反应的直接紫外监测提供了酶作用的灵敏动力学测定。
