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Journal of Bacteriology, March 2008, p. 1976-1984, Vol. 190, No. 6
0021-9193/08/$08.00+0     doi:10.1128/JB.01667-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Cel9D, an Atypical 1,4-β-D-Glucan Glucohydrolase from Fibrobacter succinogenes: Characteristics, Catalytic Residues, and Synergistic Interactions with Other Cellulases{triangledown} ,{dagger}

Meng Qi, Hyun-Sik Jun,{ddagger} and Cecil W. Forsberg*

Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada

Received 15 October 2007/ Accepted 3 January 2008

The increasing demands of renewable energy have led to the critical emphasis on novel enzymes to enhance cellulose biodegradation for biomass conversion. To identify new cellulases in the ruminal bacterium Fibrobacter succinogenes, a cell extract of cellulose-grown cells was separated by ion-exchange chromatography and cellulases were located by zymogram analysis and identified by peptide mass fingerprinting. An atypical family 9 glycoside hydrolase (GH9), Cel9D, with less than 20% identity to typical GH9 cellulases, was identified. Purified recombinant Cel9D enhanced the production of reducing sugar from acid swollen cellulose (ASC) and Avicel by 1.5- to 4-fold when mixed separately with each of four other glucanases, although it had low activity on these substrates. Cel9D degraded ASC and cellodextrins with a degree of polymerization higher than 2 to glucose with no apparent endoglucanase activity, and its activity was restricted to β-1->4-linked glucose residues. It catalyzed the hydrolysis of cellulose by an inverting mode of reaction, releasing glucose from the nonreducing end. Unlike many GH9 cellulases, calcium ions were not required for its function. Cel9D had increased kcat/Km values for cello-oligosaccharides with higher degrees of polymerization. The kcat/Km value for cellohexaose was 2,300 times higher than that on cellobiose. This result indicates that Cel9D is a 1,4-β-D-glucan glucohydrolase (EC 3.2.1.74) in the GH9 family. Site-directed mutagenesis of Cel9D identified Asp166 and Glu612 as the candidate catalytic residues, while Ser168, which is not present in typical GH9 cellulases, has a crucial structural role. This enzyme has an important role in crystalline cellulose digestion by releasing glucose from accessible cello-oligosaccharides.

* Corresponding author. Mailing address: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada. Phone: (519) 824-4120, ext. 53433. Fax: (519) 837-1802. E-mail: cforsber{at}uoguelph.ca

{triangledown} Published ahead of print on 18 January 2008.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

{ddagger} Present address: Section on Cellular Differentiation, NICHD, National Institute of Health, Bldg. 10, Room 10N325, 9000 Rockville Pike, Bethesda, MD 20892.

Journal of Bacteriology, March 2008, p. 1976-1984, Vol. 190, No. 6
0021-9193/08/$08.00+0     doi:10.1128/JB.01667-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Watson, B. J., Zhang, H., Longmire, A. G., Moon, Y. H., Hutcheson, S. W. (2009). Processive Endoglucanases Mediate Degradation of Cellulose by Saccharophagus degradans. J. Bacteriol. 191: 5697-5705 [Abstract] [Full Text]  


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