A specific chromophoric substrate for activity assays of 1,3-1,4-beta-D-glucan 4-glucanohydrolases.
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.
Malet
C
C
Department of Organic Chemistry, CETS Institut Químic de Sarrià, Universitat Ramon Llull, Barcelona, Spain.
Vallés
J
J
Bou
J
J
Planas
A
A
D001709:Biotechnology; D002240:Carbohydrate Sequence; D002863:Chromogenic Compounds / Q000138:chemical synthesis / Q000737:chemistry; D005960:Glucosides / Q000138:chemical synthesis / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000032:analysis / Q000378:metabolism*; D006923:Hymecromone / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009844:Oligosaccharides / Q000138:chemical synthesis / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
C105681:4-methylumbelliferyl-3-O-cellobiosyl-glucopyranoside; D002863:Chromogenic Compounds; D005960:Glucosides; D009844:Oligosaccharides; D006923:Hymecromone; D006026:Glycoside Hydrolases