Chemoenzymatic synthesis of a bifunctionalized cellohexaoside as a specific substrate for the sensitive assay of cellulase by fluorescence quenching.
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.
Boyer
Viviane
V
Centre de Recherches sur les Macromolécules Végétales, CERMAV-CNRS, Université Joseph Fourier B.P. 53, 38041 Grenoble cedex 9, France.
Fort
Sébastien
S
Frandsen
Torben P
TP
Schülein
Martin
M
Cottaz
Sylvain
S
Driguez
Hugues
H
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
D007202:Indicators and Reagents; D009282:Naphthalenesulfonates; D009844:Oligosaccharides; C099423:cellohexaose; C063126:5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid; D002480:Cellulase