Robert Longin

Purification and properties of an endo-β-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.

Purification and properties of the endoglucanase C of Clostridium thermocellum produced in Escherichia coli

The celC gene, which codes for a new endoglucanase of Clostridium thermocellum, termed endoglucanase C, was found to be expressed when cloned in Escherichia coli. The enzyme was purified to electrophoretic homogeneneity from E. coli and its biochemical properties were studied. It differs from the previously studied endoglucanases A and B. In particular, endoglucanase C displays features common to endo- and exoglucanases, since it had a high activity on carboxymethylcellulose and on p-nitrophenyl-beta-D-cellobioside where only the agluconic bond was split. In addition, the enzyme was able to release cellobiose units from G3, G4 and G5 cellodextrins. Endoglucanase C was characterized by Western blot in a culture supernatant from C. thermocellum grown on cellulose, using an antiserum raised against the enzyme produced by E. coli.

Development of an improved bottle-system for cultivation of strict anaerobes (methanogens)

A simple and efficient bottle-system developed for growth of obligate anaerobes, particularly methanogens, in liquid culture is described. The system consists of a modified plasma bottle fitted with a T-tube permitting direct optical density measurements and cultivation of cells in large quantities under complete anaerobiosis and elevated pressures.