CAC0915, an endoglucanase whose genetic coding exists in Clostridium acetobutylicum but which is not normally expressed by it, was expressed by Escherichia coli after is gene was synthesized. It belongs to glycoside hydrolase family 44 and has been structurally and kinetically characterized. The crystal structure of CAC0915 was solved to 2.2 Å resolution, revealing a TIM barrel structure with a Greek key fold and b-sandwich domain. The catalytic acid, Glu180, and base, Glu352, are located 5.5 Å apart on the fourth and seventh beta strands of the TIM barrel. The catalytic acid is 4.5 Å from the glycosidic oxygen suggesting a rearrangement occurs upon ligand binding. This rearrangement maybe facilitated by a calcium atom, located behind the catalytic acid, which forms a kink in the peptide backbone. A catalytic triad composed of Glu180-His277-Ser351 raises the pK_a of the catalytic acid.

Substrates bind in a cleft composed of nine subsites, –4 through +5. Subsites –4 through   –1 contain three aromatic residues (Trp58, Tyr65, and Trp385) which form stacking interactions with the substrate. Four hydrogen bonds are also present in the minus subsites, two of which help distort the pyranose ring in subsite –1. Subsites +1 to +5 contain one hydrogen bond, in subsite +2, and two aromatic residues (Trp320 and Trp324) in subsites +4 and +5. The topography of the binding cleft suggests the substrate will have higher affinity for subsites –1 to –4 than for subsites +1 to +5. This is consistent with the results of reaction product analysis on cellopentaose and cellohexaose substrates using thin layer chromatography. Cellopentaose produces cellotetraose and glucose and cellohexaose produces cellotriose and cellobiose, respectively, as primary products. However, both substrates also produce secondary reaction products, showing the substrate can bind in subsites –3 to +2 or –3 to +3.

CAC0915 hydrolyzes carboxylmethyl cellulose, birchwood xylan, larchwood xylan, cellopentaose, and cellohexaose. It is not active on mannan, laminarin, Avicel, pullulan, or lichenan. Michaelis–Menten kinetic parameters were determined on carboxymethyl cellulose (k_cat 18.9 U/mg, K_M 0.263 g/L), birchwood xylan (k_cat 31.6 U/mg, K_M 0.412 g/L) and larchwood xylan (k_cat 29.5 U/mg, K_M 0.278 g/L). CAC0915 is optimally active at pH 5.0 and has an activation energy for activity of 31.3 ± 2.3 kJ/mol-K and an activation energy for inactivation of 227 ± 20.5 kJ/mol-K.