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Intra- and extracellular beta-galactosidases from Bifidobacterium bifidum and B. infantis: molecular cloning, heterologous expression, and comparative characterization.

ABSTRACT: Three beta-galactosidase genes from Bifidobacterium bifidum DSM20215 and one beta-galactosidase gene from Bifidobacterium infantis DSM20088 were isolated and characterized. The three B. bifidum beta-galactosidases exhibited a low degree of amino acid sequence similarity to each other and to previously published beta-galactosidases classified as family 2 glycosyl hydrolases. Likewise, the B. infantis beta-galactosidase was distantly related to enzymes classified as family 42 glycosyl hydrolases. One of the enzymes from B. bifidum, termed BIF3, is most probably an extracellular enzyme, since it contained a signal sequence which was cleaved off during heterologous expression of the enzyme in Escherichia coli. Other exceptional features of the BIF3 beta-galactosidase were (i) the monomeric structure of the active enzyme, comprising 1,752 amino acid residues (188 kDa) and (ii) the molecular organization into an N-terminal beta-galactosidase domain and a C-terminal galactose binding domain. The other two B. bifidum beta-galactosidases and the enzyme from B. infantis were multimeric, intracellular enzymes with molecular masses similar to typical family 2 and family 42 glycosyl hydrolases, respectively. Despite the differences in size, molecular composition, and amino acid sequence, all four beta-galactosidases were highly specific for hydrolysis of beta-D-galactosidic linkages, and all four enzymes were able to transgalactosylate with lactose as a substrate.

SUBMITTER: Moller PL

PROVIDER: S-EPMC92867 | biostudies-literature | 2001 May

REPOSITORIES: biostudies-literature

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Intra- and extracellular beta-galactosidases from Bifidobacterium bifidum and B. infantis: molecular cloning, heterologous expression, and comparative characterization.

Møller P L PL Jørgensen F F Hansen O C OC Madsen S M SM Stougaard P P

Applied and environmental microbiology 20010501 5

Three beta-galactosidase genes from Bifidobacterium bifidum DSM20215 and one beta-galactosidase gene from Bifidobacterium infantis DSM20088 were isolated and characterized. The three B. bifidum beta-galactosidases exhibited a low degree of amino acid sequence similarity to each other and to previously published beta-galactosidases classified as family 2 glycosyl hydrolases. Likewise, the B. infantis beta-galactosidase was distantly related to enzymes classified as family 42 glycosyl hydrolases. ...[more]

PMID: 11319112

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Project description:Two genes encoding beta-galactosidase isoenzymes, beta-galI and beta-galIII, from Bifidobacterium infantis HL96 were revealed on 3.6- and 2.4-kb DNA fragments, respectively, by nucleotide sequence analysis of the two fragments. beta-galI (3,069 bp) encodes a 1,022-amino-acid (aa) polypeptide with a predicted molecular mass of 113 kDa. A putative ribosome binding site and a promoter sequence were recognized at the 5' flanking region of beta-galI. Further upstream a partial sequence of an open reading frame revealed a putative lactose permease gene transcribing divergently from beta-galI. The beta-galIII gene (2,076 bp) encodes a 691-aa polypeptide with a calculated molecular mass of 76 kDa. A rho-independent transcription terminator-like sequence was found 25 bp downstream of the termination codon. The amino acid sequences of beta-GalI and beta-GalIII are homologous to those found in the LacZ and the LacG families, respectively. The acid-base, nucleophilic, and substrate recognition sites conserved in the LacZ family were found in beta-GalI, and a possible acid-base site proposed for the LacG family was located in beta-GalIII, which featured a glutamate at residue 160. The coding regions of the beta-galI and beta-galIII genes were each cloned downstream of a T7 promoter for overexpression in Escherichia coli. The molecular masses of the overexpressed proteins, as estimated by polyacrylamide gel electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, agree with their predicted molecular weights. beta-GalI and beta-GalIII were specific for beta-D-anomer-linked galactoside substrates. Both are more active in response to ONPG (o-nitrophenyl-beta-D-galactopyranoside) than in response to lactose, particularly beta-GalIII. The galacto-oligosaccharide yield in the reaction catalyzed by beta-GalI at 37 degrees C in 20% (wt/vol) lactose solution was 130 mg/ml, which is more than six times higher than the maximum yield obtained with beta-GalIII. The structure of the major trisaccharide produced by beta-GalI catalysis was characterized as O-beta-D-galactopyranosyl-(1-3)-O-beta-D-galactopyranosyl-(1-4)-D-glucopyranose (3'-galactosyl-lactose).

Project description: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.

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Dataset Information

Intra- and extracellular beta-galactosidases from Bifidobacterium bifidum and B. infantis: molecular cloning, heterologous expression, and comparative characterization.

Publications

Intra- and extracellular beta-galactosidases from Bifidobacterium bifidum and B. infantis: molecular cloning, heterologous expression, and comparative characterization.

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