A specific chromophoric substrate for activity assays of 1,3-1,4-β-D-glucan 4-glucanohydrolases

The synthesis of 4-methylumbelliferyl 3-β-O-cellobiosyl-β-D-glucopyranoside (3a) and its use as specific substrate to monitor enzyme activity of 1,3-1,4-β-D-glucan 4-glucanohydrolases are described. The chromophoric substrate 3a is prepared by a chemoenzymatic approach starting from barley grain, whose β-D-glucan polysaccharide is degraded down to a tri- (1) and tetrasaccharide (2) by an extracellular extract of recombinant E. coli expressing and secreting Bacillus licheniformis 1,3-1,4-β-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.