Characterization of CenC, an enzyme from Cellulomonas fimi with both endo- and exoglucanase activities.
The cenC gene, encoding beta-1,4-glucanase C (CenC) from Cellulomonas fimi, was overexpressed in Escherichia coli with a tac-based expression vector. The resulting polypeptide, with an apparent molecular mass of 130 kDa, was purified from the cell extracts by affinity chromatography on cellulose followed by anion-exchange chromatography. N-terminal sequence analysis showed the enzyme to be properly processed. Mature CenC was optimally active at pH 5.0 and 45 degrees C. The enzyme was extremely active on soluble, fluorophoric, and chromophoric glycosides (4-methylumbelliferyl beta-glycosides, 2'-chloro-4'-nitrophenyl-beta-D-cellobioside, and 2'-chloro-4'-nitrophenyl-lactoside) and efficiently hydrolyzed carboxymethyl cellulose, barley beta-glucan, lichenan, and, to a lesser extent, glucomannan. CenC also hydrolyzed acid-swollen cellulose, Avicel, and bacterial microcrystalline cellulose. However, degradation of the latter was slow compared with its degradation by CenB, another C. fimi cellulose belonging to the same enzyme family. CenC acted with inversion of configuration at the anomeric carbon, in accordance with its classification as a family 9 member. The enzyme released mainly cellobiose from soluble cellodextrins and insoluble cellulose. Attack appeared to be from the reducing chain ends. Analysis of carboxymethyl cellulose hydrolysis suggests that CenC is semiprocessive enzyme with both endo- and exoglucanase activities.
Tomme
P
P
Department of Microbiology, University of British Columbia, Vancouver, Canada.
Kwan
E
E
Gilkes
N R
NR
Kilburn
D G
DG
Warren
R A
RA
D001483:Base Sequence; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism*; D003912:Dextrins / Q000378:metabolism; D044643:Glucan 1,4-beta-Glucosidase; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000235:genetics / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism; D006093:Gram-Positive Asporogenous Rods / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D011108:Polymers / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D013237:Stereoisomerism; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism*
D003912:Dextrins; D006027:Glycosides; D009844:Oligosaccharides; D011108:Polymers; D011994:Recombinant Proteins; D002482:Cellulose; C068994:cellodextrin; D001617:beta-Glucosidase; D004693:Glucan Endo-1,3-beta-D-Glucosidase; D044643:Glucan 1,4-beta-Glucosidase