ANALYTICAL BIOCHEMISTRY 164,72-77 (1987) Activity Staining of Cellulases in Polyacrylamide Gels Containing Mixed Linkage P-Glucans WOLFGANG H. SCHWARZ, KARIN BRONNENMEIER,FOLKEGRABNITZ, AND WALTER L. STAUDENBAUER Institute for Microbiology, Technical University Munich, Arcisstrasse 21 I D-8000 Munich 2. Federal Republic of Germany Received December 9, 1986 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 &glucan (barley &glucan, lichenan) in the separation gel. Zymograms are pre- pared after a renaturation treatment and incubation by staining the gel with Congo red. This method is suitable for the detection of @-glucanases with different substrate specificities cleaving fl- 1,4-, j3- 1,4- 1,3-, or p- 1,3-glucans. Cellobiohydrolase activities can be detected by adding 4-methylumbelliferyl-O-D-cellobioside to the incubation buffer. The gels are subsequently stained with Coomassie blue to establish identical molecular weights of&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. 0 1987 Academic Press. Inc. KEY WORDS: cellulase; P-glucanase; cellobiohydrolase; gel electrophoresis; Clostridium ther- mocellum; Clostridium stercorarium. Enzymes which hydrolyze &lucans are of great biotechnological potential for the deg- radation of cellulose and plant P-glucans containing both p-1,4 and p-1,3 linkages. Three types of enzymatic activities can be distinguished: 1,4-&D-glucan glucanohydro- lase (CMCase),’ 1,3-P-D-glucan glucanohy- drolase, and 1,3- 1,C/3+glucan glucanohy- drolase. There is a strong correlation be- tween the ability of bacterial isolates to degrade soluble p- 1,3- and /3-1,3- 1,4glucans and their ability to degrade cellulose (1). Activity staining after SDS-PAGE appears to be the most suitable analytical procedure for the characterization of these enzymatic activities. This technique combines the ad- ’ Abbreviations used: CMC, carboxymethylcellulose; CMCase, carboxymethylcellulasese; DTT, dithiothreitol; MUC, 4-methylumbelliferyl-8-D-cellobioside; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dode- cyl sulfate. vantage of enzyme identification with the resolution and molecular weight dependence of gel electrophoresis in the presence of SDS. Beguin (2) visualized bands with endo- 1,4-p- glucanase activity after electrophoresis in the presence of SDS by blotting the slab gels on CMC agar. Upon incubation and staining of the agar replicas with Congo red, the loca- tions corresponding to the endoglucanases were revealed by their lack of color. How- ever, diffusion of renatured enzymes out of polyacrylamide gels is slow and inefficient (3). Therefore, detection methods employing agar replicas suffer from low sensitivity and a loss of resolution. In the course of our work on cellulases of thermophilic bacteria we developed a simpler and more sensitive detection proce- dure by incorporating mixed linkage P-glu- cans, such as barley /3-glucan or lichenan, into the gels before polymerization. This in situ method has several advantages. First, the 0003-2697187 $3.00 Copyright 0 1987 by Academic Press. Inc. All rights of reproduction in any form reserved. 72