The possible structural and catalytic functions of the nine tryptophan amino acid residues, including Trp(54), Trp(105), Trp(112), Trp(141), Trp(148), Trp(165), Trp(186), Trp(198), and Trp(203) in Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (Fs beta-glucanase), were characterized using site-directed mutagenesis, initial rate kinetics, fluorescence spectrometry, and structural modeling analysis. Kinetic studies showed that a 5-7-fold increase in K(m) value for lichenan was observed for W141F, W141H, and W203R mutant Fs beta-glucanases, and approximately 72-, 56-, 30-, 29.5-, 4.9-, and 4.3-fold decreases in k(cat) relative to that for the wild-type enzyme were observed for the W54F, W54Y, W141H, W203R, W141F, and W148F mutants, respectively. In contrast, W186F and W203F, unlike the other 12 mutants, exhibited a 1.4- and 4.2-fold increase in k(cat), respectively. W165F and W203R were the only two mutants that exhibited a 4-7-fold higher activity relative to the wild-type enzyme after they were incubated at pH 3.0 for 1 h. Fluorescence spectrometry indicated that all of the mutations on the nine tryptophan amino acid residues retained a folding similar to that of the wild-type enzyme. Structural modeling and kinetic studies suggest that Trp(54), Trp(141), Trp(148), and Trp(203) play important roles in maintaining structural integrity in the substrate-binding cleft and the catalytic efficiency of the enzyme.