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PreprintCited by 3Enzyme Production and CharacterizationPolysaccharides and Plant Cell WallsGut microbiota and health

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

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Lindstad, Lars Jordhøy, Mrozek, Pascal Michael, Boehlich, Gordon Jacob, Leivers, Shaun, Pope, Phillip B., La Rosa, Sabina Leanti, Westereng, Bjørge·2025·Biochemistry
DOI 10.64898/2025.12.12.693890PMC PMC13276840

Preprint — published as 10.1021/acs.biochem.6c00194

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Abstract

β-Mannans are widespread in the human diet as components of plant-derived foods and as food additives. Several classes of β-mannans are decorated with acetylations, which are key for their physicochemical properties and protection against enzymatic degradation. While the process for depolymerization of acetylated β-mannans has been described in depth for members of the phylum Bacillota, there is limited mechanistic knowledge on how Bacteroidota utilize 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 Bc CExxx enzyme, representing a novel carbohydrate esterase (CE) family, exhibits high specificity by selectively removing axially oriented 2- O -acetyl groups from mannose residues. In contrast, Bc CE7 functions as a broad-spectrum esterase, capable of deacetylating oligosaccharides derived from structurally diverse substrates, including β-mannans, xylans, and acetylated cellulose. In transesterification reactions, Bc CExxx showed activity on both mannooligosaccharides and polymeric glucomannan. Overall, our findings provide new insight into the strategies that beneficial Bacteroides have evolved to deacetylate complex β-mannans in the human gut. Significance Statement: β-Mannans, commonly found in various plant-based foods and used as food additives, can be metabolized by gut microbes, potentially impacting host health. Acetylation of β-mannans enhances their resistance to enzymatic breakdown, making carbohydrate esterases crucial for gut bacteria to utilize these carbohydrates. This study explores the roles of two carbohydrate esterases (CEs) from a human commensal Bacteroides species in deacetylating complex β-mannans. The two esterases together remove acetylations at different positions on mannose units. Bc CE7 is a versatile esterase that deacetylates a variety of oligosaccharides, while the newly discovered Bc CExxx is highly active towards 2- O -acetylations on mannose units in both oligosaccharides and polymeric β-mannan. This research enhances our understanding of how Bacteroidota species deacetylate β-mannans.

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1 (a) Various structures of β-mannans found in plants. Structural differences and degree of acetylation vary between sources, as well as the processing of material for β-mannan isolation. NR end, nonreducing end; R end, reducing end. (b) Growth of B. cellulosilyticus in minimal media supplemented with 5 mg/mL glucose, AcGGM, or KGM as the sole carbon source. (c) The heat map presents the more abundant proteins from B. cellulosilyticus when grown on AcGGM and KGM compared to glucose. Each substrate was tested in triplicate, and the color intensity shows the protein abundance as Log2 of the label-free quantification (LFQ) values (13 to 25, in blue and red, respectively). (d) Genomic representation of the MUL (BACCELL_02371–02383) with three additional genes (BACCELL_02125–02126 and BACCELL_04534) involved in β-mannan processing (based on proteomics data).
1 (a) Various structures of β-mannans found in plants. Structural differences and degree of acetylation vary between sources, as well as the processing of material for β-mannan isolation. NR end, nonreducing end; R end, reducing end. (b) Growth of B. cellulosilyticus in minimal media supplemented with 5 mg/mL glucose, AcGGM, or KGM as the sole carbon source. (c) The heat map presents the more abundant proteins from B. cellulosilyticus when grown on AcGGM and KGM compared to glucose. Each substrate was tested in triplicate, and the color intensity shows the protein abundance as Log2 of the label-free quantification (LFQ) values (13 to 25, in blue and red, respectively). (d) Genomic representation of the MUL (BACCELL_02371–02383) with three additional genes (BACCELL_02125–02126 and BACCELL_04534) involved in β-mannan processing (based on proteomics data).CC BY · PubMed Central
2 MALDI-ToF spectra of activity on β-mannan for BcCE7 and BcCE25. (a) AcGGM from Norway spruce was tested with the two esterases, both separately and in combination. The top row shows untreated AcGGM, while the second and third rows show treatment with BcCE7 and BcCE25, respectively. Both esterases were active on the substrate, reducing peak intensities of masses corresponding to acetylated oligos. Treatments with both esterases combined (bottom row) completely removed acetylations. BcCE7 and BcCE25 were tested for activity on transacetylated mannotetraose with one (m/z 731) and two (m/z 773) acetylations in specific positions at O-3 (panel b) and O-2 (panel c), produced with the previously characterized RiCE2 and RiCE17, respectively. (b) Both esterases showed some activity toward mannotetraose with 3-O-acetylations during the reaction time. (c) BcCE25 and BcCE7 act on 2-O-acetylated mannotetraose, with BcCE25 completely removing all acetyl groups, while BcCE7 shows only a minor effect. The reactions were performed with 1 μM enzyme concentration and 0.1 mg/mL substrate in 10 mM sodium phosphate pH 5.9 buffer run at 25 °C with stirring. Reactions with AcGGM and specific substrate were performed for 24 and 1 h, respectively.
2 MALDI-ToF spectra of activity on β-mannan for BcCE7 and BcCE25. (a) AcGGM from Norway spruce was tested with the two esterases, both separately and in combination. The top row shows untreated AcGGM, while the second and third rows show treatment with BcCE7 and BcCE25, respectively. Both esterases were active on the substrate, reducing peak intensities of masses corresponding to acetylated oligos. Treatments with both esterases combined (bottom row) completely removed acetylations. BcCE7 and BcCE25 were tested for activity on transacetylated mannotetraose with one (m/z 731) and two (m/z 773) acetylations in specific positions at O-3 (panel b) and O-2 (panel c), produced with the previously characterized RiCE2 and RiCE17, respectively. (b) Both esterases showed some activity toward mannotetraose with 3-O-acetylations during the reaction time. (c) BcCE25 and BcCE7 act on 2-O-acetylated mannotetraose, with BcCE25 completely removing all acetyl groups, while BcCE7 shows only a minor effect. The reactions were performed with 1 μM enzyme concentration and 0.1 mg/mL substrate in 10 mM sodium phosphate pH 5.9 buffer run at 25 °C with stirring. Reactions with AcGGM and specific substrate were performed for 24 and 1 h, respectively.CC BY · PubMed Central
3 Transesterification reactions with a range of different substrates. Transesterification reactions of mannotetraose with (a) BcCE7 and (b) BcCE25 with vinyl acetate, -propionate, and -butyrate. For CE7, acetyl and propionyl (to a low extent) groups were attached to the substrate, while no transbutyrylation was observed, whereas BcCE25 transesterified mannotetraose with all donors tested, although to a lesser extent for the larger donors. (c) Transacetylation of GalMan3, (d) Gal2Man5, and (e) chemically deacetylated Norway spruce with BcCE7 and BcCE25. (f) Comparison of degradation with the endomannanase (RiGH26) of KGM without (black) or with (blue) a pretreatment with BcCE25 transacetylation in vinyl acetate. Transesterification reactions were conducted with 1 mg/mL substrate in 10 mM sodium phosphate (pH 5.9) and 200 nM enzyme with vinyl acetate/propionate/butyrate donors added to 50% of the sample volume and run overnight with stirring at 25 °C. Abbreviations: Ac, acetyl; Prop, propyl; But, butyryl; Man, mannose; Gal, Galactose; m/z, mass/charge; DP, degree of polymerization.
3 Transesterification reactions with a range of different substrates. Transesterification reactions of mannotetraose with (a) BcCE7 and (b) BcCE25 with vinyl acetate, -propionate, and -butyrate. For CE7, acetyl and propionyl (to a low extent) groups were attached to the substrate, while no transbutyrylation was observed, whereas BcCE25 transesterified mannotetraose with all donors tested, although to a lesser extent for the larger donors. (c) Transacetylation of GalMan3, (d) Gal2Man5, and (e) chemically deacetylated Norway spruce with BcCE7 and BcCE25. (f) Comparison of degradation with the endomannanase (RiGH26) of KGM without (black) or with (blue) a pretreatment with BcCE25 transacetylation in vinyl acetate. Transesterification reactions were conducted with 1 mg/mL substrate in 10 mM sodium phosphate (pH 5.9) and 200 nM enzyme with vinyl acetate/propionate/butyrate donors added to 50% of the sample volume and run overnight with stirring at 25 °C. Abbreviations: Ac, acetyl; Prop, propyl; But, butyryl; Man, mannose; Gal, Galactose; m/z, mass/charge; DP, degree of polymerization.CC BY · PubMed Central
4 1H-Spectrum (400 MHz, 298 K) of mannotetraose after (top) and before (bottom) enzymatic transacetylation by BcCE25. The signal at 2.19 indicated the presence of unbound acetate. The introduction of the acetyl group at O-2 shifts the resonance frequencies of nearby protons downfield by ∼0.2 ppm for H-1 and ∼1.4 ppm for H-2. Abbreviation: HDO, hydrogen–deuterium oxide.
4 1H-Spectrum (400 MHz, 298 K) of mannotetraose after (top) and before (bottom) enzymatic transacetylation by BcCE25. The signal at 2.19 indicated the presence of unbound acetate. The introduction of the acetyl group at O-2 shifts the resonance frequencies of nearby protons downfield by ∼0.2 ppm for H-1 and ∼1.4 ppm for H-2. Abbreviation: HDO, hydrogen–deuterium oxide.CC BY · PubMed Central
5 HSQC-TOCSY spectrum (D2O, 400 MHz, 298 K) showing acetylation of the nonreducing-end mannose. Horizontal axis: 1H NMR (full spectrum in Figure S4g) Vertical axis: DEPTQ (Full Spectrum in Figure S4h). The nonreducing-end mannose can be easily differentiated from the in-chain mannoses by the lower chemical shift of C-4 (69.9 ppm vs 79.5 ppm, represented by red and green circles/arrows, respectively). Furthermore, anomerically linked mannoses can be differentiated from the reducing-end mannose by the chemical shift of their respective anomeric carbons (C-1). C-1 of nonacetylated mannose resonated at 103.1 ppm, C-1 of O-2 acetylated mannose resonated at 102.1 ppm (based on the correlations represented by black and blue circles/arrows, respectively), and the reducing end C-1 resonates at a much lower chemical shift of 96.8 ppm. The full HSQC-TOCSY spectrum is given in Figure S4i.
5 HSQC-TOCSY spectrum (D2O, 400 MHz, 298 K) showing acetylation of the nonreducing-end mannose. Horizontal axis: 1H NMR (full spectrum in Figure S4g) Vertical axis: DEPTQ (Full Spectrum in Figure S4h). The nonreducing-end mannose can be easily differentiated from the in-chain mannoses by the lower chemical shift of C-4 (69.9 ppm vs 79.5 ppm, represented by red and green circles/arrows, respectively). Furthermore, anomerically linked mannoses can be differentiated from the reducing-end mannose by the chemical shift of their respective anomeric carbons (C-1). C-1 of nonacetylated mannose resonated at 103.1 ppm, C-1 of O-2 acetylated mannose resonated at 102.1 ppm (based on the correlations represented by black and blue circles/arrows, respectively), and the reducing end C-1 resonates at a much lower chemical shift of 96.8 ppm. The full HSQC-TOCSY spectrum is given in Figure S4i.CC BY · PubMed Central
6 Proposed β-mannan degradation pathway in B. cellulosilyticus. Polymeric β-mannans are depolymerized on the surface of B. cellulosilyticus by the endomannanase BcGH26B, and the products are imported into the cell by the SusC-SusD-like transporter proteins. In the periplasm, the galactosyl and acetyl decorations are removed by the α-galactosidase BcGH36 and the acetyl esterases BcCE7 and BcCE25. The mannanase BcGH26A generates mannobiose or short mannooligosaccharides, and the exoacting glucosidase BcGH3 removes glucose residues. Mono- and disaccharides are further transported into the cytosol by a sugar transporter. Mannobiose is epimerized to mannosyl-glucose by the epimerase. The phosphorylase BcGH130_1 phosphorolyses mannosyl-glucose to mannose-1-phosphate and glucose. The monosaccharides then enter glycolysis. The specific roles of GH5_2 and GH5_7 are not displayed due to the lack of functional information.
6 Proposed β-mannan degradation pathway in B. cellulosilyticus. Polymeric β-mannans are depolymerized on the surface of B. cellulosilyticus by the endomannanase BcGH26B, and the products are imported into the cell by the SusC-SusD-like transporter proteins. In the periplasm, the galactosyl and acetyl decorations are removed by the α-galactosidase BcGH36 and the acetyl esterases BcCE7 and BcCE25. The mannanase BcGH26A generates mannobiose or short mannooligosaccharides, and the exoacting glucosidase BcGH3 removes glucose residues. Mono- and disaccharides are further transported into the cytosol by a sugar transporter. Mannobiose is epimerized to mannosyl-glucose by the epimerase. The phosphorylase BcGH130_1 phosphorolyses mannosyl-glucose to mannose-1-phosphate and glucose. The monosaccharides then enter glycolysis. The specific roles of GH5_2 and GH5_7 are not displayed due to the lack of functional information.CC BY · PubMed Central

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Metadata

Authors
Lindstad, L. J., Mrozek, P. M., Boehlich, G. J., Leivers, S., Pope, P. B., La Rosa, S. L., & Westereng, B.
Affiliations
  • Norwegian University of Life Sciences (NO)
  • Nofima (NO)
  • Translational Research Institute (AU)
Corresponding author
Westereng, Bjørge
ORCID
Venue
Biochemistry
Publisher
openRxiv
Published
2025-12-12
Volume
65
Issue
12
Pages
1966-1977
ISSN
0006-2960 (ISSN-L)
DOI
10.64898/2025.12.12.693890
PMC
PMC13276840
OpenAlex
W7162395345
Cited by
3
References
2
Citations by year
2026: 3
FWCI
8.64
Type
journal article
Language
en
Open access
Licence
Locations
  • Biochemistry (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
Funders
Norges Forskningsråd (244259, 295 910, 309558)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Polysaccharides and Plant Cell Walls
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
Keywords
β-mannan, carbohydrate esterases, Bacteroides cellulosilyticus, deacetylation, human gut microbiome, Bacteroidetes, glycan degradation, enzyme specificity, oligosaccharides
MeSH terms
Acetylation, Bacterial Proteins, Bacteroides, Esterases, Humans, Mannans, Substrate Specificity

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