Expression and characteristics of a Ca²⁺-dependent endoglucanase from Cytophaga hutchinsonii

Cytophaga hutchinsonii is a Gram-negative bacterium that can degrade crystalline cellulose efficiently with an unknown strategy. Genomic analysis suggested it lacks exoglucanases which are found in many cellulolytic organisms and most of the cellulases in C. hutchinsonii lack recognizable carbohydrate-binding modules (CBMs). CHU_1280, speculated to be an endoglucanase belonging to glycoside hydrolase family 9 (GH9) in C. hutchinsonii, was functionally expressed in Escherichia coli, and evidence was presented suggesting that it may be a processive endoglucanase. In the absence of Ca(2+), CHU_1280 was inactive. But in the presence of Ca(2+), it had a specific activity of 600 U/μmol with carboxymethyl cellulose (CMC) as the substrate. With Ca(2+), CHU_1280 hydrolyzed regenerated amorphous cellulose (RAC) with nearly 80 % of the reducing ends appearing in the soluble fraction, suggesting it degraded cellulose in a processive way. CHU_1280 could bind to cellulose without recognizable CBMs and its binding ability was also Ca(2+)-dependent. Ca(2+) could stabilize the catalytic domain at high temperature, but the denaturation temperature of the whole protein was decreased. C. hutchinsonii might have an exoglucanase-independent cellulases system which included endoglucanases, processive endoglucanases, and β-glucosidases.