A cellulase assay coupled to cellobiose dehydrogenase.
An assay for cellulase activity based on the oxidation of cellobiose, formed during the cellulase reaction, with ferricyanide and a cellobiose dehydrogenase derived from the cellulolytic fungus Sporotrichum (Chrysosporium) thermophile is presented. Due to the restricted specificity of this enzyme for cellobiose and cellodextrins, glucose, which may be formed by the action of some cellulolytic components or by beta-glucosidase, does not contribute to the result. The negative interference of beta-glucosidase may be eliminated by glucono-delta-lactone inhibition. The assay, which is not influenced by cellobiose back-inhibition of the cellulase reaction, like the usual cellulase tests based on the increase in reducing power, is basically unspecific with respect to endo- or exo-acting enzymes giving rise to a total cellulase activity. With the use of an amorphous cellulose substrate (reprecipitated cellulose after dissolving in concentrated phosphoric acid), unpredictable effects due to cooperativity between endo- and exo-enzyme components were eliminated. An analytical procedure giving a linear response between activity and enzyme concentration and between activity and time of incubation has been worked out.
D002237:Carbohydrate Dehydrogenases / Q000032:analysis; D002475:Cellobiose / Q000378:metabolism*; D002480:Cellulase / Q000032:analysis*; D004187:Disaccharides / Q000378:metabolism*; D005292:Ferricyanides; D010084:Oxidation-Reduction; D013175:Sporothrix / Q000201:enzymology; D001617:beta-Glucosidase / Q000032:analysis
D004187:Disaccharides; D005292:Ferricyanides; C007931:hexacyanoferrate III; D002475:Cellobiose; D002237:Carbohydrate Dehydrogenases; C019859:cellobiose-quinone oxidoreductase; D001617:beta-Glucosidase; D002480:Cellulase