title: Chemoenzymatic synthesis of a bifunctionalized cellohexaoside as a specific substrate for the sensitive assay of cellulase by fluorescence quenching.
abstract: A new bifunctionalized cellohexaose derivative was synthesized as a specific substrate for continuous assay of cellulases by resonance energy transfer. This cellohexaoside has a naphthalene moiety (EDANS) as a fluorescent energy donor at the reducing end and a 4-(4'-dimethylaminobenzeneazo)-benzene derivative as an acceptor chromophore at the non-reducing end. The key steps for the preparation of the target molecule involved transglycosylation reactions of cellobiosyl and cellotetraosyl fluoride donors onto cellobiosyl acceptors catalysed by the E197A mutant of cellulase Cel7B from Humicola insolens. Upon digestion with various cellulases, the energy transfer was disrupted and an increase of fluorescence was observed.
authors.affiliation: Centre de Recherches sur les Macromolécules Végétales, CERMAV-CNRS, Université Joseph Fourier B.P. 53, 38041 Grenoble cedex 9, France.
authors.forename: Viviane
authors.initials: V
authors.lastname: Boyer
authors.forename: Sébastien
authors.initials: S
authors.lastname: Fort
authors.forename: Torben P
authors.initials: TP
authors.lastname: Frandsen
authors.forename: Martin
authors.initials: M
authors.lastname: Schülein
authors.forename: Sylvain
authors.initials: S
authors.lastname: Cottaz
authors.forename: Hugues
authors.initials: H
authors.lastname: Driguez
mesh_terms: D002480:Cellulase / Q000032:analysis*; D004735:Energy Transfer; D005470:Fluorometry; D007202:Indicators and Reagents; D009282:Naphthalenesulfonates / Q000737:chemistry; D009844:Oligosaccharides / Q000138:chemical synthesis*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
chemical_list: D007202:Indicators and Reagents; D009282:Naphthalenesulfonates; D009844:Oligosaccharides; C099423:cellohexaose; C063126:5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid; D002480:Cellulase