Activity staining of cellulases in polyacrylamide gels containing mixed linkage beta-glucans.
Endoglucanase and cellobiohydrolase components of thermophilic cellulases can be detected in situ after gel electrophoresis in the presence of sodium dodecyl sulfate by incorporating a mixed linkage beta-glucan (barley beta-glucan, lichenan) in the separation gel. Zymograms are prepared after a renaturation treatment and incubation by staining the gel with Congo red. This method is suitable for the detection of beta-glucanases with different substrate specificities cleaving beta-1,4-, beta-1,4-1,3-, or beta-1,3-glucans. Cellobiohydrolase activities can be detected by adding 4-methylumbelliferyl-beta-D-cellobioside to the incubation buffer. The gels are subsequently stained with Coomassie blue to establish identical molecular weights of beta-glucanase and protein bands. Applications of this technique for the comparison of cellulases and for the identification of cellulase components expressed from recombinant clones are presented.
D002236:Carbohydrate Conformation; D002480:Cellulase / Q000032:analysis; D043366:Cellulose 1,4-beta-Cellobiosidase; D003013:Clostridium / Q000201:enzymology / Q000235:genetics; D003224:Congo Red; D004274:DNA, Recombinant; D002523:Edible Grain / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D043326:Glucan 1,3-beta-Glucosidase; D044643:Glucan 1,4-beta-Glucosidase; D005936:Glucans; D005959:Glucosidases / Q000032:analysis*; D006026:Glycoside Hydrolases / Q000032:analysis / Q000235:genetics; D013194:Staining and Labeling; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000032:analysis* / Q000235:genetics
D004274:DNA, Recombinant; D005936:Glucans; D003224:Congo Red; D005959:Glucosidases; D006026:Glycoside Hydrolases; D001617:beta-Glucosidase; D002480:Cellulase; D043326:Glucan 1,3-beta-Glucosidase; C028020:licheninase; D044643:Glucan 1,4-beta-Glucosidase; D043366:Cellulose 1,4-beta-Cellobiosidase