title: The GH5 1,4-β-mannanase from Bifidobacterium animalis subsp. lactis Bl-04 possesses a low-affinity mannan-binding module and highlights the diversity of mannanolytic enzymes.
abstract: BACKGROUND
  β-Mannans are abundant and diverse plant structural and storage polysaccharides. Certain human gut microbiota members including health-promoting Bifidobacterium spp. catabolize dietary mannans. Little insight is available on the enzymology of mannan deconstruction in the gut ecological niche. Here, we report the biochemical properties of the first family 5 subfamily 8 glycoside hydrolase (GH5_8) mannanase from the probiotic bacterium Bifidobacterium animalis subsp. lactis Bl-04 (BlMan5_8).
  
  
  RESULTS
  BlMan5_8 possesses a novel low affinity carbohydrate binding module (CBM) specific for soluble mannan and displays the highest catalytic efficiency reported to date for a GH5 mannanase owing to a very high k cat (1828 ± 87 s(-1)) and a low K m (1.58 ± 0.23 g · L(-1)) using locust bean galactomannan as substrate. The novel CBM of BlMan5_8 mediates increased binding to soluble mannan based on affinity electrophoresis. Surface plasmon resonance analysis confirmed the binding of the CBM10 to manno-oligosaccharides, albeit with slightly lower affinity than the catalytic module of the enzyme. This is the first example of a low-affinity mannan-specific CBM, which forms a subfamily of CBM10 together with close homologs present only in mannanases. Members of this new subfamily lack an aromatic residue mediating binding to insoluble cellulose in canonical CBM10 members consistent with the observed low mannan affinity.
  
  
  CONCLUSIONS
  BlMan5_8 is evolved for efficient deconstruction of soluble mannans, which is reflected by an exceptionally low K m and the presence of an atypical low affinity CBM, which increases binding to specifically to soluble mannan while causing minimal decrease in catalytic efficiency as opposed to enzymes with canonical mannan binding modules. These features highlight fine tuning of catalytic and binding properties to support specialization towards a preferred substrate, which is likely to confer an advantage in the adaptation to competitive ecological niches.
authors.affiliation: Department of Biochemistry and Structural Biology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-221 00, Lund, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Morrill
authors.affiliation: Department of Biochemistry and Structural Biology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-221 00, Lund, Sweden.
authors.forename: Evelina
authors.initials: E
authors.lastname: Kulcinskaja
authors.affiliation: Enzyme and Protein Chemistry (EPC), Department of Systems Biology, Technical University of Denmark (DTU), Søltofts Plads, building 224, DK-2800, Kgs Lyngby, Denmark.|Current address: Biochemistry and Bioprocessing, Department of Food Science, University of Copenhagen, Rolighedsvej 30, DK-1958, Fredriksberg C, Denmark.
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Sulewska
authors.affiliation: Active Nutrition, DuPont Nutrition & Health, Sokeritehtaantie 20, 02460, Kantvik, Finland.
authors.forename: Sampo
authors.initials: S
authors.lastname: Lahtinen
authors.affiliation: Department of Biochemistry and Structural Biology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-221 00, Lund, Sweden.
authors.forename: Henrik
authors.initials: H
authors.lastname: Stålbrand
authors.affiliation: Enzyme and Protein Chemistry (EPC), Department of Systems Biology, Technical University of Denmark (DTU), Søltofts Plads, building 224, DK-2800, Kgs Lyngby, Denmark.
authors.forename: Birte
authors.initials: B
authors.lastname: Svensson
authors.affiliation: Enzyme and Protein Chemistry (EPC), Department of Systems Biology, Technical University of Denmark (DTU), Søltofts Plads, building 224, DK-2800, Kgs Lyngby, Denmark. maha@bio.dtu.dk.
authors.forename: Maher
authors.initials: M
authors.lastname: Abou Hachem
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001644:Bifidobacterium / Q000201:enzymology*; D001665:Binding Sites; D020134:Catalytic Domain; D004043:Dietary Fiber / Q000378:metabolism*; D004795:Enzyme Stability; D005690:Galactose / Q000031:analogs & derivatives; D006801:Humans; D008024:Ligands; D008351:Mannans / Q000737:chemistry / Q000378:metabolism*; D008361:Mannosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010802:Phylogeny; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D040681:Structural Homology, Protein; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004043:Dietary Fiber; D008024:Ligands; D008351:Mannans; D011994:Recombinant Proteins; C012990:galactomannan; D008361:Mannosidases; C041867:endo-1,4-beta-D-mannanase; D005690:Galactose