A newly described cellulosomal cellobiohydrolase, CelO, from Clostridium thermocellum: investigation of the exo-mode of hydrolysis, and binding capacity to crystalline cellulose.
The sequence of the celO gene from Clostridium thermocellum F7 was determined. The gene product, cellulase CelO (Ct-Cel5F), had a modular structure consisting of a carbohydrate-binding module of the CBM3 family and a catalytic domain of the glycosyl hydrolase family 5. The presence of the dockerin module indicated that the enzyme was a component of the cellulosome complex. The thermostable recombinant gene product was active on cellodextrins, barley beta-glucan, carboxymethylcellulose and insoluble cellulose. Cellobiose was the only product released from amorphic and crystalline cellulose, cellotetraose and higher cello-oligosaccharides, identifying CelO as a cellobiohydrolase. The cleavage pattern of p-nitrophenyl beta-D-cellotetraoside, blockage of the hydrolysis of NaBH(4)-reduced cellopentaose and the reduction in substrate viscosity suggested activity from the reducing end in a processive mode after making random cuts. Binding to insoluble, i.e. amorphous, and crystalline cellulose was mediated by the carbohydrate-binding module CBM3b, with a preference for the crystalline substrate.
Zverlov
Vladimir V
VV
Institute of Molecular Genetics, Russian Academy of Science, Kurchatov Sq., 123182 Moscow, Russia1.
Velikodvorskaya
Galina A
GA
Institute of Molecular Genetics, Russian Academy of Science, Kurchatov Sq., 123182 Moscow, Russia1.
Schwarz
Wolfgang H
WH
Research Group Microbial Biotechnology, Technische Universität München, Am Hochanger 4, D-85350 Freising-Weihenstephan, Germany2.
D002475:Cellobiose / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D003460:Crystallization; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D011994:Recombinant Proteins; D002475:Cellobiose; D002482:Cellulose; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase