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A newly described cellulosomal cellobiohydrolase, CelO, from Clostridium thermocellum: investigation of the exo-mode of hydrolysis, and binding capacity to crystalline cellulose

Institute of Molecular Genetics, Russian Academy of Science, Kurchatov Sq., 123182 Moscow, Russia¹
Research Group Microbial Biotechnology, Technische Universität München, Am Hochanger 4, D-85350 Freising-Weihenstephan, Germany²

Author for correspondence: Wolfgang H. Schwarz. Tel: +49 8161 71 5445. Fax: +49 8161 71 5475. e-mail: schwarz{at}mikro.biologie.tu-muenchen.de

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 ß-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 ß-D-cellotetraoside, blockage of the hydrolysis of NaBH₄-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.

Keywords: cellulase CelO, cellulosome, carbohydrate-binding module CBM3, reducing end

Abbreviations: CBM, carbohydrate-binding module; CMC, carboxymethylcellulose; GHF, glycosyl hydrolase family; PASC, phosphoric acid swollen cellulose; pNP, p-nitrophenyl

The GenBank accession number for the sequence determined in this work is AJ275975.

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