Skip to main content
ARS Home » Northeast Area » Ithaca, New York » Robert W. Holley Center for Agriculture & Health » Plant, Soil and Nutrition Research » Research » Publications at this Location » Publication #207381
Title: Characterization of a family 45 glycosyl hydrolase from Fibrobacter succinogenes S85

Author
item PARK, JAE SEON - CORNELL UNIVERSITY
item Russell, James
item WILSON, DAVID - CORNELL UNIVERSITY

Submitted to: Anaerobe
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/28/2007
Publication Date: 3/19/2007
Citation: Park, J.S., J.B. Russell, and D.B. Wilson. 2007. Characterization of a family 45 glycosyl hydrolase from Fibrobacter succinogenes S85. Anaerobe 13:83-88.

Interpretive Summary: Rumen bacteria give ruminant animals the ability to metabolize cellulosic materials that would otherwise be indigestible. In the 1960's researchers, attempted to extract "true" cellulases from the three most active cellulose-digesting ruminal bacteria, but extracts from F. succinogenes had virtually no activity. Only growing cultures of F. succinogenes digest cellulose. A variety of enzymes from F. succinogenes have been cloned and characterized, but none of these enzymes had very little activity against insoluble native cellulose. The genome sequence of F. succinogenes is now available and has a "cellulase" gene from a group designated as "family 45". This gene does not encode a cellulose binding motif, but some of the enzymes from this family have good activity against crystalline cellulose. Based on this information, primers were designed to clone this gene and express it in E. coli. The purified protein digested cellulose, but the rate of this hydrolysis was much too low to explain the rate of cellulose digestion by growing cultures. Because the F. succinogenes S85 endoglucanase genes do not encode carbohydrate binding modules, it appears that F. succinogenes has a novel mechanism of cellulose degradation. Research on ruminal cellulose digestion has the potential to enhance the nutrition of cattle.

Technical Abstract: Fibrobacter succinogenes is one of the most active cellulolytic bacteria ever isolated from the rumen, but enzymes from F. succinogenes capable of hydrolyzing native (insoluble) cellulose at a rapid rate have not been identified. However, the genome sequence of F. succinogenes is now available, and it was hoped that this information would yield new insights into the mechanism of cellulose digestion. The genome has a "cellulase" gene from a group designated as "family 45" and some of the enzymes in this family have good activity against native cellulose. The gene encoding the family 45 glycosyl hydrolase from F. succinogenes S85 was cloned into Escherichia coli JM109(DE3) using pMAL-c2 as a vector. Recombinant E. coli cells produced a soluble fusion protein (MAL-F45) that was purified on a maltose affinity column and characterized. MAL-F45 was most active on carboxymethylcellulose between pH 6 and 7 and it hydrolyzed cellopentaose and cellohexaose but not cellotetraose. It also cleaved p-nitrophenyl-cellopentose into cellotriose and p-nitrophenyl-cellobiose. MAL-F45 produced cellobiose, cellotriose and cellotetraose from acid swollen cellulose and bacterial cellulose, but the rate of this hydrolysis was much too low to explain the rate of cellulose digestion by growing cultures. Because the F. succinogenes S85 genome lacks dockerin and cohesin sequences, does not encode any known processive cellulases, and most of its endoglucanase genes do not encode carbohydrate binding modules, it appears that F. succinogenes has a novel mechanism of cellulose degradation.