Purification and characterization of endoglucanase Ss from Clostridium thermocellum

Abstract

The extracellular cellulolytic enzymes of the thermophilic anaerobe Clostridium thermocellum occur as a protein complex or aggregate known as the cellulosome. By using a combination of ion-exchange, adsorption and hydrophobic-interaction chromatography, it was possible to isolate from extracellular broth a specific endoglucanase of interest without the use of denaturants. The endoglucanase was identified as the cellulosomal subunit Ss by the use of specific antibodies. The enzyme has an Mr of 83,000, an isoelectric point of 3.55, optimum pH of 6.6 and optimum temperature of 70 degrees C. It hydrolyses CM-cellulose and, at a higher rate, the cellodextrins, cellotetraose and cellopentaose, but does not hydrolyse a crystalline cellulose such as Avicel. Cellobiose and cellotriose are also immune to attack. It differs from endoglucanases previously isolated by others and a 76,000-Mr endoglucanase recently isolated in this laboratory.

Catalytic and Thermodynamic Characterization of Endoglucanase (CMCase) from Aspergillus oryzae cmc-1

“…1a,b). Carboxymethylcellulases are produced by a variety of microbes, and their molecular weights have been reported in the literature (Table 3) [37][38][39][40][41][42][43][44][45][46][47][48].…”

Optimization of Growth Medium and Enzyme Assay Conditions for Crude Cellulases Produced by a Novel Thermophilic and Cellulolytic Bacterium, Anoxybacillus sp. 527

“…Effect of Ca 2+ has also been reported by other researchers. For example: (1) Ca 2+ can enhance carboxymethylcellulase (CMCase) activity by 20% compared with that of the control [43]; (2) in the presence of Ca 2+ (5 mM), the activity of an endoglucanase from Clostridium thermocellum is stimulated [44]; and (3) with 5 mM Ca 2+ , cellulose hydrolysis by enzymes from C. thermocellum proceeds to completion [22]. The cellulase enzymes produced by strain 527 demonstrated good activities between 50 and 70°C with the maximum activity at 70°C (Fig.…”

Cellulase, Clostridia, and Ethanol

“…These included one having a molecular mass of 83 kDa, an isoelectric point of 3.55, an optimum pH of 6.6 and an optimum temperature of 70°C (80). It hydrolyzed CMC and, at a higher rate, cellotetraose and cellopentaose; Avicel, cellotriose, and cellobiose were not attacked.…”

“…We and others devoted considerable efforts to the isolation and purification of a number of proteins of the C. thermocellum system as well as the cloning, expression, and sequencing of their relevant genes in Escherichia coli and other hosts (80,96,148,163,276,277). Of particular interest was our gene sequencing of S L (96,277) and the gene sequencing of S S (341).…”