Deacetylation of β-Mannans by Two Complementary Carbohydrate Esterases from the Human Gut Microbe Bacteroides cellulosilyticus

β-Mannans are widespread in the human diet as components of plant-derived foods and as food additives. Several β-mannans are decorated with acetylations, which are key to their physicochemical properties and protection against enzymatic degradation. Depolymerization of acetylated β-mannans has been described in depth for members of the phylum Bacillota, while there is limited mechanistic knowledge on how Bacteroidota utilizes these glycans. Here, we combined proteomics and biochemical analyses to functionally characterize a pair of carbohydrate esterases (CEs) from Bacteroides cellulosilyticus that, together, deacetylate complex β-mannans. We demonstrate that the newly identified BcCE25 enzyme, representing a new carbohydrate esterase (CE) family, exhibits high specificity toward axially oriented 2-O-acetyl groups on mannose residues. In contrast, BcCE7 functions as a broad-spectrum esterase, capable of deacetylating oligosaccharides from structurally diverse substrates. Overall, our findings provide new insight into the strategies that beneficial Bacteroides have evolved to deacetylate complex β-mannans in the human gut.