Purification and characterization of a new endoglucanase from Clostridium thermocellum

Abstract

An endoglucanase (1,4-beta-D-glucan glucanohydrolase, EC 3.2.1.4) from the thermophilic anaerobe Clostridium thermocellum was purified to apparent homogeneity without the use of denaturants. No carbohydrate is associated with the endoglucanase. A molecular mass of 76,000 Da was determined by SDS/PAGE. The optimal pH is 7.0 and the enzyme is isoelectric at pH 5.05. The enzyme has a temperature optimum of 70 degrees C and retains approx. 50% of its activity after 48 h at 60 degrees C. Hydrolysis of CM-cellulose takes place with a rapid decrease in viscosity but a slow liberation of reducing sugars, indicating an endoglucanase type of activity. The endoglucanase shows little ability to hydrolyse highly ordered cellulose. Cellobiose inhibits whereas Mg2+ and Ca2+ stimulate the activity. The enzyme is completely inactivated by 1 mM-Hg2+ and is inhibited by a thiol-blocking reagent.

Industrial Applications of Endoglucanase Obtained from Novel and Native Trichoderma atroviride

“…The obtained enzyme can be used for biostoning and denim finishing, without a notable harmful effect on the inner fibers. Similar results have been obtained by Montazer 58 Pseudomonas fluorescens, 59 Clostridium thermocellum, 60 T. harzianum ETS 323, 28 and optimum activity at pH 5.0 and 50 °C. The EG sustained its activity up to 72 h at 50 °C and pH 5.0, it lost only 9.5 % of its initial activity.…”

Cellulase, Clostridia, and Ethanol

“…Thus, our designation of this protein as CelS was erroneous. A second new endonuclease had the following properties: an M r of 76,000, an isoelectric point of 5.05, an optimum pH of 7.0, and an optimum temperature of 70°C (276). Both of these endoglucanases were inhibited by Hg 2ϩ and p-chloromercuribenzoate, suggesting the existence of thiol groups essential for activity or stability.…”

“…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).…”

Hydrolysis of Lignocellulosic Biomass: Current Status of Processes and Technologies and Future Perspectives

“…Several cellulose degrading enzymes from various thermophilic organisms have been investigated. These include cellulases mainly isolated from anaerobic bacteria such as Anaerocellum thermophilum (Zverlov et al, 1998), Clostridium thermocellum (Romaniec et al, 1992), Clostridium stercorarium (Bronnenmeier et al, 1991;Bronnenmeier & Staudenbauer, 1990) and Caldocellum saccharolyticum (Te'o V et l., 1995), Pyrococcus furiosus (Ma & Adams, 1994), Pyrococcus horikoshi (Rahman et al,1998), Rhodothermus strains (Hreggvidsson et al, 1996), Thermotoga sp., (Ruttersmith et al, 1991), Thermotoga marittima (Bronnenmeier et al, 1995), Thermotoga neapolitana (Bok et al, 1998). Xylanase have been detected in Acidothermus cellulolyticus in different Thermus, Bacillus, Geobacillus, Alicyclobacillus and Sulfolobales species (Sakon et al, 1996).…”