Qualitative assays for cellulases and xylanases

Qualitative assays have been developed either to select microbial strains producing high levels of cellulases and xylanases, or to identify and/or characterize these enzymes in a given sample. Insoluble xylan and RBB-xylan are ideal substrates to select microorganisms producing xylanase on solid agar medium (Sprey and Lambert, 1983; Farkas et al., 1985). Similarly, methods using either RBB-CM-cellulose or CM-cellulose stained with Congo Red can be used to select microorganisms producing endoglucanase activity (Kluepfel, 1988).

The capacity of Congo Red to complex with either polymeric xylan or CM-cellulose, but not with small oligosaccharide products, is conveniently used to detect xylanase and endoglucanase activities in zymograms after the fractionation of protein mixtures by SDS/IEF-PAGE (Coughlan, 1988). RBB-xylan and RBB-CM-cellulose can be used for similar purposes (Biely et al., 1985). The advantages of the latter method are: (i) that the dyed fragments released from RBB-xylan/CM-cellulose diffuse from the detection gel into the separating gel and further help to identify the position of a xylanase or endoglucanase, which can subsequently be eluted; and (ii) the hydrolysis of the dyed substrate can be visually followed and the reaction terminated when necessary. The use ofthese substrates facilitates the identification of multiple forms of xylanase or endoglucanase produced by different microorganisms. Likewise, chromogenic and fluorogenic cello- and xylo-oligosaccharides have been successfully used to identify multiple forms of cellulases and xylanases after the separation of crude enzyme mixtures by SDS/IEF-PAGE (Biely et al., 1992; Kalogiannis et al., 1996).

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