Bifidobacterium bifidum Lacto-N-Biosidase, a Critical Enzyme for the Degradation of Human Milk Oligosaccharides with a Type 1 Structure

ABSTRACT Breast-fed infants often have intestinal microbiota dominated by bifidobacteria in contrast to formula-fed infants. We found that several bifidobacterial strains produce a lacto-N-biosidase that liberates lacto-N-biose I (Galβ1,3GlcNAc; type 1 chain) from lacto-N-tetraose (Galβ1,3GlcNAcβ1,3Galβ1,4Glc), which is a major component of human milk oligosaccharides, and subsequently isolated the gene from Bifidobacterium bifidum JCM1254. The gene, designated lnbB, was predicted to encode a protein of 1,112 amino acid residues containing a signal peptide and a membrane anchor at the N and C termini, respectively, and to possess the domain of glycoside hydrolase family 20, carbohydrate binding module 32, and bacterial immunoglobulin-like domain 2, in that order, from the N terminus. The recombinant enzyme showed substrate preference for the unmodified β-linked lacto-N-biose I structure. Lacto-N-biosidase activity was found in several bifidobacterial strains, but not in the other enteric bacteria, such as clostridia, bacteroides, and lactobacilli, under the tested conditions. These results, together with our recent finding of a novel metabolic pathway specific for lacto-N-biose I in bifidobacterial cells, suggest that some of the bifidobacterial strains are highly adapted for utilizing human milk oligosaccharides with a type 1 chain.

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Bifidobacterium bifidum Lacto-N-Biosidase, a Critical Enzyme for the Degradation of Human Milk Oligosaccharides with a Type 1 Structure

Applied and Environmental Microbiology ◽

10.1128/aem.01847-09 ◽

2009 ◽

Vol 75 (19) ◽

pp. 6414-6414

Author(s):

Jun Wada ◽

Takuro Ando ◽

Masashi Kiyohara ◽

Hisashi Ashida ◽

Motomitsu Kitaoka ◽

...

Keyword(s):

Human Milk ◽

Bifidobacterium Bifidum ◽

Human Milk Oligosaccharides ◽

Milk Oligosaccharides

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Sharing of human milk oligosaccharides degradants within bifidobacterial communities in faecal cultures supplemented with Bifidobacterium bifidum

Scientific Reports ◽

10.1038/s41598-018-32080-3 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 38

Author(s):

Aina Gotoh ◽

Toshihiko Katoh ◽

Mikiyasu Sakanaka ◽

Yiwei Ling ◽

Chihaya Yamada ◽

...

Keyword(s):

Human Milk ◽

Bifidobacterium Bifidum ◽

Human Milk Oligosaccharides ◽

Milk Oligosaccharides

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Bifidobacterium longum subsp. infantis uses two different β-galactosidases for selectively degrading type-1 and type-2 human milk oligosaccharides

Glycobiology ◽

10.1093/glycob/cwr116 ◽

2011 ◽

Vol 22 (3) ◽

pp. 361-368 ◽

Cited By ~ 75

Author(s):

Erina Yoshida ◽

Haruko Sakurama ◽

Masashi Kiyohara ◽

Masahiro Nakajima ◽

Motomitsu Kitaoka ◽

...

Keyword(s):

Human Milk ◽

Bifidobacterium Longum ◽

Human Milk Oligosaccharides ◽

Milk Oligosaccharides

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A novel class of human milk oligosaccharides based on 6-galactosyllactose and containing N-acetylglucosamine branches extended by oligogalactoses

10.1101/2021.05.27.445961 ◽

2021 ◽

Author(s):

Franz-Georg Hanisch ◽

Clemens Kunz

Keyword(s):

Human Milk ◽

Infant Nutrition ◽

Maldi Mass Spectrometry ◽

The Novel ◽

Human Milk Oligosaccharides ◽

Milk Oligosaccharides ◽

New Class ◽

Terminal Galactose ◽

Branched Chains

Human milk oligosaccharides (HMOs) have attracted much attention in recent years not only as a prebiotic factor, but in particular as an essential component in infant nutrition related to their impact in innate immunity. The backbone structures of complex HMOs generally contain single or repetitive lacto-N-biose (type 1) or lactosamine (type 2) units in either linear or branched chains extending from a lactose core. While all known branched structures originate from 3,6-substitution of the lactosyl core galactose, we here describe a new class of HMOs that tentatively branch at terminal galactose of 6-galactosyllactose. Another novel feature of this class of HMOs was found in linear oligo-galactosyl chains linked to one of the N-acetylglucosamine (GlcNAc) branches. The novel structures exhibit general formulas with hexose vs. hexosamine contents of 5/2 to 8/2 and can be designated as high-galactose (HG)-HMOs. In addition, up to three fucosyl residues are linked to the octa- to dodecasaccharides, which were detected in two human milk samples from Lewis blood group defined donors. Structural analyses of methylated glycans and their alditols comprised MALDI mass spectrometry, ESI-(CID)MS and linkage analyses by GC-MS of the derived partially methylated alditol acetates. Enzymatic degradation by application of β1-3,4-specific galactosidase supported the presence of terminal galactose linked [beta]1-6 to one of the two GlcNAc branches.

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Molecular cloning and characterization of a novel β-1,3-xylanase possessing two putative carbohydrate-binding modules from a marine bacterium Vibrio sp. strain AX-4

Biochemical Journal ◽

10.1042/bj20050190 ◽

2005 ◽

Vol 388 (3) ◽

pp. 949-957 ◽

Cited By ~ 23

Author(s):

Masashi KIYOHARA ◽

Keishi SAKAGUCHI ◽

Kuniko YAMAGUCHI ◽

Toshiyoshi ARAKI ◽

Takashi NAKAMURA ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Marine Bacterium ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

Amino Acid Residues ◽

Reading Frame ◽

Carbohydrate Binding Modules ◽

Hydrolysis Of ◽

Hydrolase Family

We cloned a novel β-1,3-xylanase gene, consisting of a 1728-bp open reading frame encoding 576 amino acid residues, from a marine bacterium, Vibrio sp. strain AX-4. Sequence analysis revealed that the β-1,3-xylanase is a modular enzyme composed of a putative catalytic module belonging to glycoside hydrolase family 26 and two putative carbohydrate-binding modules belonging to family 31. The recombinant enzyme hydrolysed β-1,3-xylan to yield xylo-oligosaccharides with different numbers of xylose units, mainly xylobiose, xylotriose and xylotetraose. However, the enzyme did not hydrolyse β-1,4-xylan, β-1,4-mannan, β-1,4-glucan, β-1,3-xylobiose or p-nitrophenyl-β-xyloside. When β-1,3-xylo-oligosaccharides were used as the substrate, the kcat value of the enzyme for xylopentaose was found to be 40 times higher than that for xylotetraose, and xylotriose was extremely resistant to hydrolysis by the enzyme. A PSI-BLAST search revealed two possible catalytic Glu residues (Glu-138 as an acid/base catalyst and Glu-234 as a nucleophile), both of which are generally conserved in glycoside hydrolase superfamily A. Replacement of these two conserved Glu residues with Asp and Gln resulted in a significant decrease and complete loss of enzyme activity respectively, without a change in their CD spectra, suggesting that these Glu residues are the catalytic residues of β-1,3-xylanase. The present study also clearly shows that the non-catalytic putative carbohydrate-binding modules play an important role in the hydrolysis of insoluble β-1,3-xylan, but not that of soluble glycol-β-1,3-xylan. Furthermore, repeating a putative carbohydrate-binding module strongly enhanced the hydrolysis of the insoluble substrate.

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Bifidobacterium bifidum Extracellular Sialidase Enhances Adhesion to the Mucosal Surface and Supports Carbohydrate Assimilation

mBio ◽

10.1128/mbio.00928-17 ◽

2017 ◽

Vol 8 (5) ◽

Cited By ~ 23

Author(s):

Keita Nishiyama ◽

Yuji Yamamoto ◽

Makoto Sugiyama ◽

Takashi Takaki ◽

Tadasu Urashima ◽

...

Keyword(s):

Human Milk ◽

Type A ◽

Bifidobacterium Bifidum ◽

Mucosal Surface ◽

Blood Type ◽

Human Milk Oligosaccharides ◽

Milk Oligosaccharides ◽

Adhesion Properties ◽

Mechanistic Basis

ABSTRACT Bifidobacterium is a natural inhabitant of the human gastrointestinal (GI) tract. We studied the role of the extracellular sialidase (SiaBb2, 835 amino acids [aa]) from Bifidobacterium bifidum ATCC 15696 in mucosal surface adhesion and carbohydrate catabolism. Human milk oligosaccharides (HMOs) or porcine mucin oligosaccharides as the sole carbon source enhanced B. bifidum growth. This was impaired in a B. bifidum ATCC 15696 strain harboring a mutation in the siabb2 gene. Mutant cells in early to late exponential growth phase also showed decreased adhesion to human epithelial cells and porcine mucin relative to the wild-type strain. These results indicate that SiaBb2 removes sialic acid from HMOs and mucin for metabolic purposes and may promote bifidobacterial adhesion to the mucosal surface. To further characterize SiaBb2-mediated bacterial adhesion, we examined the binding of His-tagged recombinant SiaBb2 peptide to colonic mucins and found that His-SiaBb2 as well as a conserved sialidase domain peptide (aa 187 to 553, His-Sia) bound to porcine mucin and murine colonic sections. A glycoarray assay revealed that His-Sia bound to the α2,6-linked but not to the α2,3-linked sialic acid on sialyloligosaccharide and blood type A antigen [GalNAcα1-3(Fucα1-2)Galβ] at the nonreducing termini of sugar chains. These results suggest that the sialidase domain of SiaBb2 is responsible for this interaction and that the protein recognizes two distinct carbohydrate structures. Thus, SiaBb2 may be involved in Bifidobacterium-mucosal surface interactions as well as in the assimilation of a variety of sialylated carbohydrates. IMPORTANCE Adhesion to the host mucosal surface and carbohydrate assimilation are important for bifidobacterium colonization and survival in the host gastrointestinal tract. In this study, we investigated the mechanistic basis for B. bifidum extracellular sialidase (SiaBb2)-mediated adhesion. SiaBb2 cleaved sialyl-human milk oligosaccharides and mucin glycans to produce oligosaccharides that supported B. bifidum growth. Moreover, SiaBb2 enhanced B. bifidum adhesion to mucosal surfaces via specific interactions with the α2,6 linkage of sialyloligosaccharide and blood type A antigen on mucin carbohydrates. These findings provide insight into the bifunctional role of SiaBb2 and the adhesion properties of B. bifidum strains. IMPORTANCE Adhesion to the host mucosal surface and carbohydrate assimilation are important for bifidobacterium colonization and survival in the host gastrointestinal tract. In this study, we investigated the mechanistic basis for B. bifidum extracellular sialidase (SiaBb2)-mediated adhesion. SiaBb2 cleaved sialyl-human milk oligosaccharides and mucin glycans to produce oligosaccharides that supported B. bifidum growth. Moreover, SiaBb2 enhanced B. bifidum adhesion to mucosal surfaces via specific interactions with the α2,6 linkage of sialyloligosaccharide and blood type A antigen on mucin carbohydrates. These findings provide insight into the bifunctional role of SiaBb2 and the adhesion properties of B. bifidum strains.

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