CelG from Clostridium cellulolyticum: a multidomain endoglucanase acting efficiently on crystalline cellulose.
The gene coding for CelG, a family 9 cellulase from Clostridium cellulolyticum, was cloned and overexpressed in Escherichia coli. Four different forms of the protein were genetically engineered, purified, and studied: CelGL (the entire form of CelG), CelGcat1 (the catalytic domain of CelG alone), CelGcat2 (CelGcat1 plus 91 amino acids at the beginning of the cellulose binding domain [CBD]), and GST-CBD(CelG) (the CBD of CelG fused to glutathione S-transferase). The biochemical properties of CelG were compared with those of CelA, an endoglucanase from C. cellulolyticum which was previously studied. CelG, like CelA, was found to have an endo cutting mode of activity on carboxymethyl cellulose (CMC) but exhibited greater activity on crystalline substrates (bacterial microcrystalline cellulose and Avicel) than CelA. As observed with CelA, the presence of the nonhydrolytic miniscaffolding protein (miniCipC1) enhanced the activity of CelG on phosphoric acid swollen cellulose (PASC), but to a lesser extent. The absence of the CBD led to the complete inactivation of the enzyme. The abilities of CelG and GST-CBD(CelG) to bind various substrates were also studied. Although the entire enzyme is able to bind to crystalline cellulose at a limited number of sites, the chimeric protein GST-CBD(CelG) does not bind to either of the tested substrates (Avicel and PASC). The lack of independence between the two domains and the weak binding to cellulose suggest that this CBD-like domain may play a special role and be either directly or indirectly involved in the catalytic reaction.
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Laboratoire de Bioénergétique et Ingénierie des Protéines, IBSM, Centre National de la Recherche Scientifique, Marseille, France.
Gaudin
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Belaich
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Tardif
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Belaich
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D001426:Bacterial Proteins; D001665:Binding Sites; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002482:Cellulose; C081472:celG protein, Bacteria; D002480:Cellulase