Purification and properties of an endo-beta-1,4-glucanase from Clostridium thermocellum.

The extracellular cellulolytic enzymes of the thermophilic anaerobe Clostridium thermocellum occur as a protein complex or aggregate which, until now, has not been resolved into individual enzyme components. By using QAE-Sephadex A50 chromatography in the presence of 6 M urea, it was possible to split the complex into distinct protein fractions. One of these fractions contained an endo-beta-1,4-glucanase which was isolated at a high degree of purity and was identified by its ability to hydrolyze trinitrophenylated carboxymethylcellulose. The enzyme is of monomeric nature, with a molecular weight of 56,000. It has an isoelectric pH of 6.2 and an optimum pH of 6.0. It hydrolyzed carboxymethylcellulose and, at a slower rate, cellulose powder. The major end products of cellulose degradation are glucose, cellobiose and cellotriose; cellotetrose is formed as an intermediate product. No specific small molecular weight activator or inhibitor was found except cellobiose and, to a lesser extent, glucose, which at high concentrations partially inhibit the activity of the enzyme. The temperature dependence of the enzyme is related to the thermophilic character of the producing microorganism.