A fluorescence-quenched chitopentaose for the study of endo-chitinases andchitobiosidases

A new fluorogenic substrate displaying intramolecular fluorescence energy t ransfer (FRET) has been synthetized from N-I,N-II,N-III,N-IV-tetra-acetyl-c hitopentaose. Two molecules, a fluorophore (5-(2-aminoethyl) amino-1-naphta lene-sulfonic acid; EDANS) and a quenching group (dimethylaminophenylazophe nyl; DAB) were chemically introduced on to the chitopentaose, one at each e nd. Among eight enzymes tested, only endo-chitinase and chitobiosidase acti vities could be specifically assayed by monitoring the variation of fluores cence after enzymatic hydrolysis of this substrate. Chitobiases and N-acety l-beta-glucosaminidases are not active on the compound, the presence of a b ulky chromogenic group at the 2 position of the nonreducing end of the subt rate preventing the binding and thus hydrolysis by these two exo-enzymes. T he observation that chitobiosidases are able to hydrolyse a chitooligosacch aride functionalized on both extremities demonstrates the possibility of an endo-action for this class of chitinases, which are generally classified a s exo-enzymes. This fluorogenic chitooligosaccharide should prove to be ver y useful for the detection and the convenient assay of chitinolytic activit ies at nanomolar concentrations.