CHARACTERIZATION OF CENC, AN ENZYME FROM CELLULOMONAS-FIMI WITH BOTH ENDOGLUCANASE 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 expressio n vector. The resulting polypeptide, with an apparent molecular mass o f 130 kDa, was purified from the cell extracts by affinity chromatogra phy 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 e xtremely 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 c ellulose, Avicel, and bacterial microcrystalline cellulose. However, d egradation of the latter was slow compared with its degradation by Cen B, another C. fimi cellulase belonging to the same enzyme family. CenC acted with inversion of configuration at the anomeric carbon, in acco rdance with its classification as a family 9 member. The enzyme releas ed mainly cellobiose from soluble cellodextrins and insoluble cellulos e. Attack appeared to be from the reducing chain ends. Analysis of car boxymethyl cellulose hydrolysis suggests that CenC is a semiprocessive enzyme with both endo- and exoglucanase activities.