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Purification and characterization of an endoglucanase (1,4-beta-D-glucan glucanohydrolase) from Clostridium thermocellum.

ABSTRACT: An endoglucanase (1,4-beta-D-glucan glucanohydrolase, EC 3.2.1.4) was purified from Clostridium thermocellum by procedures that included centrifugation, ultrafiltration, selective precipitation, ion-exchange Sephadex chromatography and preparative gel electrophoresis. The 22-fold-purified enzyme behaved as a homogeneous protein under non-denaturing conditions. The enzyme represented a significant component (greater than 25%) of total extracellular endoglucanase activity, but was purified in low yield by the procedures employed. The native molecular weight of the endoglucanase was determined by ultracentrifugational analysis, amino acid composition and polyacrylamide-gel electrophoresis, and varied between 83000 and 94000. The enzyme contained 11.2% carbohydrate and was isoelectric at pH 6.72. The pH and temperature optima of the endoglucanase were 5.2 and 62 degrees C respectively. The enzyme lacked cysteine and was low in sulphur-containing amino acids. The purified endoglucanase displayed: high activity towards carboxymethylcellulose, celloheptaose, cellohexaose and cellopentaose; low activity towards Avicel microcrystalline cellulose and cellotetraose; no detectable activity towards cellotriose or cellobiose; increased activity towards cello-oligosaccharides with increasing degree of polymerization. The internal glycosidic bonds of cello-oligosaccharides were cleaved by the enzyme in preference to external linkages. The apparent Michaelis constant ([S]0.5V) and Vmax. for cellopentaose and cellohexaose hydrolysis were 2.30 mM and 39.3 mumol/min per mg of protein, and 0.56 mM and 58.7 mumol/min per mg of protein, respectively.

SUBMITTER: Ng TK

PROVIDER: S-EPMC1163377 | biostudies-other | 1981 Nov

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:Clostridium thermocellum is a paradigm for efficient cellulose degradation and a promising organism for the production of second generation biofuels. It owes its high degradation rate on cellulosic substrates to the presence of supra-molecular cellulase complexes, cellulosomes, which comprise over 70 different single enzymes assembled on protein-backbone molecules of the scaffold protein CipA. RESULTS:Although all 24 single-cellulosomal cellulases were described previously, we present the first comparative catalogue of all these enzymes together with a comprehensive analysis under identical experimental conditions, including enzyme activity, binding characteristics, substrate specificity, and product analysis. In the course of our study, we encountered four types of distinct enzymatic hydrolysis modes denoted by substrate specificity and hydrolysis product formation: (i) exo-mode cellobiohydrolases (CBH), (ii) endo-mode cellulases with no specific hydrolysis pattern, endoglucanases (EG), (iii) processive endoglucanases with cellotetraose as intermediate product (pEG4), and (iv) processive endoglucanases with cellobiose as the main product (pEG2). These modes are shown on amorphous cellulose and on model cello-oligosaccharides (with degree of polymerization DP 3 to 6). Artificial mini-cellulosomes carrying combinations of cellulases showed their highest activity when all four endoglucanase-groups were incorporated into a single complex. Such a modeled nonavalent complex (n = 9 enzymes bound to the recombinant scaffolding protein CipA) reached half of the activity of the native cellulosome. Comparative analysis of the protein architecture and structure revealed characteristics that play a role in product formation and enzyme processivity. CONCLUSIONS:The identification of a new endoglucanase type expands the list of known cellulase functions present in the cellulosome. Our study shows that the variety of processivities in the enzyme complex is a key enabler of its high cellulolytic efficiency. The observed synergistic effect may pave the way for a better understanding of the enzymatic interactions and the design of more active lignocellulose-degrading cellulase cocktails in the future.

Dataset Information

Purification and characterization of an endoglucanase (1,4-beta-D-glucan glucanohydrolase) from Clostridium thermocellum.

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