Activity staining of cellulases in polyacrylamide gels containing mixed linkage beta-glucans

Abstract

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.