Characterization of a family 45 glycosyl hydrolase from Fibrobacter succinogenes S85.
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 single family 45 beta-glucanase gene, 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.
Seon Park
Jae
J
Department of Biochemistry, Molecular and Cell Biology, Cornell University and Agricultural Research Service, USDA, Ithaca, NY 14853, USA.
Russell
James B
JB
Wilson
David B
DB
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002475:Cellobiose / Q000378:metabolism; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D045848:Fibrobacter / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013780:Tetroses / Q000378:metabolism
D001426:Bacterial Proteins; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D013780:Tetroses; D002475:Cellobiose; C099423:cellohexaose; C019193:maltopentaose; C048468:cellotetraose; D002482:Cellulose; D006867:Hydrolases; D002266:Carboxymethylcellulose Sodium