Thermobifida fusca Cel6A is an endoglucanase with relatively high activity on crystalline cellulose. Trp 231 is a substrate-binding residue in the −2 sub-site that is within the active-site cleft. Mutating Trp 231 to Ala, Asp, or His causes significant decreases in activity on both oligosaccharides and cellulose. The largest decreases in activity are seen on short oligosaccharides, which is clearly due to altered oligosaccharide binding, as shown by docking studies. The mutation causes a significant drop in the production of soluble products from crystalline cellulose, with little change in the production of insoluble reducing ends. This result is due to weaker rebinding of the mutant enzyme to cellulose after hydrolysis before it completely dissociates, while the initial binding of the mutant catalytic domain is only slightly reduced. The Asp mutant enzyme had a much smaller increase in activity with increased temperature than the wild type, but it is not clear why this occurs. However, this mut...