Characterization of a multi-function processive endoglucanase CHU_2103 from Cytophaga hutchinsonii

Abstract

Cytophaga hutchinsonii is a Gram-negative gliding bacterium which can efficiently degrade crystalline cellulose by an unknown strategy. Genomic analysis suggests the C. hutchinsonii genome lacks homologs to an obvious exoglucanase that previously seemed essential for cellulose degradation. One of the putative endoglucanases, CHU_2103, was successfully expressed in Escherichia coli JM109 and identified as a processive endoglucanase with transglycosylation activity. It could hydrolyze carboxymethyl cellulose (CMC) into cellodextrins and rapidly decrease the viscosity of CMC. When regenerated amorphous cellulose (RAC) was degraded by CHU_2103, the ratio of the soluble to insoluble reducing sugars was 3.72 after 3 h with cellobiose and cellotriose as the main products, indicating that CHU_2103 was a processive endoglucanase. CHU_2103 could degrade cellodextrins of degree of polymerization ≥3. It hydrolyzed p-nitrophenyl β-D-cellodextrins by cutting glucose or cellobiose from the non-reducing end. Meanwhile, some larger-molecular-weight cellodextrins could be detected, indicating it also had transglycosylation activity. Without carbohydrate-binding module (CBM), CHU_2103 could bind to crystalline cellulose and acted processively on it. Site-directed mutation of CHU_2103 demonstrated that the conserved aromatic amino acid W197 in the catalytic domain was essential not only for its processive activity, but also its cellulose binding ability.

Periplasmic Cytophaga hutchinsonii Endoglucanases Are Required for Use of Crystalline Cellulose as the Sole Source of Carbon and Energy

“…The crystal structure of Cel5B supports this suggestion, since the predicted open active site is consistent with a nonprocessive mode of action. Our results indicating the lack of processivity of Cel5B and Cel9C contradict earlier reports (23,38). The earlier studies lacked controls of known processive and nonprocessive enzymes and used amorphous cellulose (RAC) as the substrate in the processivity assays.…”

“…Three of the nine predicted endoglucanase-encoding genes, cel5A (CHU_1107), cel5B (CHU_2103), and cel9B (CHU_1335), have been disrupted by insertion mutagenesis, but these single-gene mutations did not result in obvious defects in cellulose utilization (22,23).…”

Expression and biochemical characterization of a multifunctional glycosidase from the thermophilic <i>Bacillus licheniformis</i> SR01

“…Mn 2+ and K + had no obvious effect on the enzyme. These results mostly agree with previous reports with the exception of glucanase activity inhibited by Mn 2+ in the studies (Liu et al, 2010;Takehara et al, 1981;Yan, 2008;Zhang et al, 2014). The difference may be due to the structure of gkdA, which has a multifunctional binding region different from the previously mentioned glucanase.…”

Novel Outer Membrane Protein Involved in Cellulose and Cellooligosaccharide Degradation by Cytophaga hutchinsonii

“…Zhu et al reported that an outer membrane endoglucanase, ChCel5A, from C. hutchinsonii could hydrolyze microcrystalline cellulose to cellobiose and cellotriose (15). Another cell surface endoglucanase, CHU_2103, was shown to be a processive endoglucanase (26). However, whether cell surface degradation was the main mechanism of cellulose utilization by C. hutchinsonii was still unknown.…”