Remazol Brilliant Blue-xylan: A soluble chromogenic substrate for xylanases

The conjugate of the polysaccharide with the dye, abbreviated as Remazol Brilliant Blue-Xylan (RBB-xylan), represents a convenient substrate for determination of activity and detection of xylanases. This chapter describes assay method that is based on photometric measurements of the enzyme released dyed fragments soluble in the presence of two volumes of ethanol that precipitates the original substrate and its high-molecular-weight fragments. This principle offers to measure xylanase activity in cell-free extracts and media containing larger amount of reducing compounds that would interfere with classical xylanase activity determination. Xylanase activity on the cell surface and on isolated membranes and organelles can be followed in the presence of viable cells consuming xylose and xylooligosaccharides. The detection of xylanase activity in gels employs transparent agar replicas containing RBB-xylan. Diffusion of dyed fragments released in the place of the enzyme into the separation gel or their selective removal from the agar replicas by the solvent system used for the precipitation of unhydrolyzed RBB-xylan in a solution, represents the basis for enzyme detection. A great advantage of the technique is that the substrate present in a 2% agar gel does not precipitate as it does in a solution, so that the agar replicas remain transparent during and after the destaining of the zones of the enzyme-depolymerized substrate.