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

Zhang, Cong, Wang, Ying, Li, Zhe, Zhou, Xiangru, Zhang, Weican, Zhao, Yue, Lu, Xuemei
Applied microbiology and biotechnology 2014 v.98 no.15 pp. 6679-6687
Cytophaga hutchinsonii, reducing sugars, glucose, genome, polymerization, viscosity, cellobiose, mutation, carboxymethylcellulose, amino acids, active sites, carbohydrate binding, genomics, endo-1,4-beta-glucanase, bacteria, Escherichia coli
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.