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

• Published in: Microbiology (Society for General Microbiology) 2002-01, Vol.148 (1), p.247-255
• Main Authors: Zverlov, Vladimir V, Velikodvorskaya, Galina A, Schwarz, Wolfgang H
• Format: Article
• Language: English
• Subjects: cellobiohydrolase; Cellobiose - metabolism; Cellulase - chemistry; Cellulase - genetics; Cellulase - metabolism; Cellulose - metabolism; Cellulose 1,4-beta-Cellobiosidase; celO gene; Clostridium - enzymology; Clostridium - genetics; Clostridium thermocellum; Crystallization; Hydrolysis; Molecular Sequence Data; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Analysis, DNA; Substrate Specificity
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/00221287-148-1-247

Institute of Molecular Genetics, Russian Academy of Science, Kurchatov Sq., 123182 Moscow, Russia 1 Research Group Microbial Biotechnology, Technische Universität München, Am Hochanger 4, D-85350 Freising-Weihenstephan, Germany 2 Author for correspondence: Wolfgang H. Schwarz. Tel: [phone]. Fax: [phone]. 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 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. 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; p NP, p -nitrophenyl The GenBank accession number for the sequence determined in this work is AJ275975 .