A BIFUNCTIONALIZED FLUOROGENIC TETRASACCHARIDE AS A SUBSTRATE TO STUDY CELLULASES

Cellulases are usually classified as endoglucanases and cellobiohydrol ases, but the heterogeneity of cellulose, in terms of particle size an d crystallinity, has always represented a problem for the biochemical characterization of the enzymes. The synthesis of a bifunctionalized t etrasaccharide substrate suitable for measuring cellulase activity by resonance energy transfer is described. The substrate, which carries a 5-(2-aminoethylamino)-1-naphthalenesulfonate group on the non-reducin g end and an indolethyl group on the reducing end, was prepared from b eta-lactosyl fluoride and indolethyl beta-cellobioside by a chemoenzym atic approach using the transglycosylating activity of endoglucanase I of Humicola insolens as the key step. The bifunctionalized substrate has been used for the determination of the catalytic constants of H. i nsolens endoglucanase I and cellobiohydrolases I and II; this substrat e could be of general use to measure the kinetic constants of cellulas es able to act on oligomers of degree of polymerization <5. The data a lso provide evidence that cellobiohydrolases I and II are able to degr ade an oligosaccharide substrate carrying non-carbohydrate substituent s at both ends.