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.
D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002480:Cellulase / Q000302:isolation & purification*; D002482:Cellulose / Q000378:metabolism; D002850:Chromatography, Gel; D003013:Clostridium / Q000201:enzymology*; D007526:Isoelectric Point; D008970:Molecular Weight; D014301:Trinitrobenzenes; D014508:Urea
D014301:Trinitrobenzenes; D014508:Urea; D002482:Cellulose; D002480:Cellulase; D002266:Carboxymethylcellulose Sodium