Characterization of CenC, an enzyme from Cellulomonas fimi with both endo- and exoglucanase activities

Abstract

The cenC gene, encoding ␤-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؇C. The enzyme was extremely active on soluble, fluorophoric, and chromophoric glycosides (4-methylumbelliferyl ␤-glycosides, 2-chloro-4-nitrophenyl-␤-D-cellobioside, and 2-chloro-4-nitrophenyl-lactoside) and efficiently hydrolyzed carboxymethyl cellulose, barley ␤-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 cellulase 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 a semiprocessive enzyme with both endo-and exoglucanase activities.

Molecular Cloning and Expression of Cellulase and Polygalacturonase Genes in E. coli as a Promising Application for Biofuel Production

“…On the other hand, the activity of the celB enzyme on CMC substrate, reported by [86,87], suggests that this enzyme has both endo-and exo-cellulase activities [85]. This suggestion has been supported by other reports of cellulases with both endo-and exo-activities [88,89]. Analysis of β-glucan hydrolysis products of a GH-12 member β-(1,4)-glucanase from Aspergillus japonicas by gel-permeation chromatography revealed the formation of glucose in the initial part of the reaction [90].…”

Characterization of a Cellulase Containing a Family 30 Carbohydrate-Binding Module (CBM) Derived from Clostridium thermocellum CelJ: Importance of the CBM to Cellulose Hydrolysis

“…4). These data closely resemble those obtained for CenC (35), suggesting that CBM30-Ig-CM9 hydrolyzes CMC by a semiproessive mechanism; i.e., the enzyme is a semiprocessive enzyme with both endo-and exoglucanase activities. Synergistic action between CBM30-Ig-CM9 and the endoglucanse CelC suggests that these two cellulases hydrolyze the insoluble substrate in different ways.…”

“…However, the classification of ␤-1,4-glucanases into two mutually exclusive groups, endoglucanases and cellobiohydrolases, may be inappropriate. For example, Cellulomonas fimi CenC, a cellulase of family 9, first attacks an internal ␤-1,4-glucosidic bond and removes only a few successive cellobiosyl residues from the new reducing end before it jumps to a new site (35). Clostridium cellulolyticum cellulase Cel9E (formerly CelE) and Cel48F (formerly CelF) also have a similar hydrolytic activity (12,27).…”

CelE, a Multidomain Cellulase from Clostridium cellulolyticum : a Key Enzyme in the Cellulosome?

“…2), seems to have the closest activity pattern (18), but it does not decrease the viscosity of CMC at all (19). CenC seems to be a better CMCase (equally or more active on this substrate than either CenA or CenB from the same organism [33]), but as was observed for CelE this activity is not correlated with an important decrease in viscosity, and the main product of PASC and BMCC hydrolysis is cellobiose (98%). These data are in agreement with the existence of a class of cellulases (associating CBM4, Ig, and GH9 domains) which acts first by random mode on cellulose and mainly releases cellobiose.…”

“…To our knowledge, enzymatic properties of only two enzymes presenting associations of CBM4, Ig, and GH9 domains have been described so far: CenC from Cellulomonas fimi, which is considered by Tomme et al (33) to be a semiprocessive enzyme, and CelK from C. thermocellum (18,19). The latter, which presents the greatest structural similarity to CelE (Fig.…”