The processive endocellulase CelF, a major component of the Clostridium cellulolyticum cellulosome: purification and characterization of the recombinant form.
The recombinant form of the cellulase CelF of Clostridium cellulolyticum, tagged by a C-terminal histine tail, was overproduced in Escherichia coli. The fusion protein was purified by affinity chromatography on a Ni-nitrilotriacetic acid column. The intact form of CelF (Mr, 79,000) was rapidly degraded at the C terminus, giving a shorter stable form, called truncated CelF (Mr, 71,000). Both the entire and the truncated purified forms degraded amorphous cellulose (kcat = 42 and 30 min(-1), respectively) and microcrystalline cellulose (kcat = 13 and 10 min(-1), respectively). The high ratio of soluble reducing ends to insoluble reducing ends released by truncated CelF from amorphous cellulose showed that CelF is a processive enzyme. Nevertheless, the diversity of the cellodextrins released by truncated CelF from phosphoric acid-swollen cellulose at the beginning of the reaction indicated that the enzyme might randomly hydrolyze beta-1,4 bonds. This hypothesis was supported by viscosimetric measurements and by the finding that CelF and the endoglucanase CelA are able to degrade some of the same cellulose sites. CelF was therefore called a processive endocellulase. The results of immunoblotting analysis showed that CelF was associated with the cellulosome of C. cellulolyticum. It was identified as one of the three major components of cellulosomes. The ability of the entire form of CelF to interact with CipC, the cellulosome integrating protein, or mini-CipC1, a recombinant truncated form of CipC, was monitored by interaction Western blotting (immunoblotting) and by binding assays using a BIAcore biosensor-based analytical system.
Reverbel-Leroy
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Bioénergétique et Ingéniérie des Protéines, Centre National de la Recherche Scientifique, IBSM-IFR1, Marseille, France.
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Belaich
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Belaich
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D001426:Bacterial Proteins / Q000378:metabolism; D002241:Carbohydrates / Q000032:analysis; D002352:Carrier Proteins / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D003013:Clostridium / Q000201:enzymology*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002241:Carbohydrates; D002352:Carrier Proteins; C098847:CipC protein, Clostridium cellulolyticum; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D002480:Cellulase