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Literature summary extracted from

  • Wada, J.; Ando, T.; Kiyohara, M.; Ashida, H.; Kitaoka, M.; Yamaguchi, M.; Kumagai, H.; Katayama, T.; Yamamoto, K.
    Bifidobacterium bifidum lacto-N-biosidase, a critical enzyme for the degradation of human milk oligosaccharides with a type 1 structure (2008), Appl. Environ. Microbiol., 74, 3996-4004.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.2.1.140 Bifidobacterium bifidum grown anaerobically in GAM medium, enzyme gene inserted into plasmid pMW118, expression of truncated form missing signal peptide and membrane anchor (era 35-1064) in Escherichia coli rosetta(DE3) pLacI with plasmid pET23b-lnbB, LB medium, 37°C Bifidobacterium bifidum
3.2.1.140 cloned in Escherichia coli Rosetta(DE3) Bifidobacterium bifidum JCM 1254

Protein Variants

EC Number Protein Variants Comment Organism
3.2.1.140 additional information truncated enzyme without N-terminal signal peptide of 34 amino acids and C-terminal membrane anchor from 1065-1108 Bifidobacterium bifidum

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.2.1.140 0.0068
-
 p-nitrophenyl-Gal-(1,3)-beta-GlcNAc pH 4.5, 25°C Bifidobacterium bifidum
3.2.1.140 0.068
-
 p-Nitrophenyl-lacto-N-bioside pH 4.5, 25°C Bifidobacterium bifidum JCM 1254

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
3.2.1.140 110000
-
 apparent molecular mass, SDS-PAGE Bifidobacterium bifidum JCM 1254
3.2.1.140 112000
-
 truncated enzyme without signal peptide and membrane anchor, amino acids 35-1064, SDS-PAGE, and calculated from amino acids Bifidobacterium bifidum
3.2.1.140 112000
-
 calculated mass Bifidobacterium bifidum JCM 1254
3.2.1.140 120000
-
 full enzyme, calculated from 350 bp PCR product of 1,112 amino acids Bifidobacterium bifidum
3.2.1.140 120000
-
 predicted mass, 1112 amino acid residues. The deduced amino acid sequence contains a signal peptide and a membrane anchor at the N-terminal and C-terminal ends. This result strongly suggests that LnbB is a membrane-tethered protein with a large extracellular region Bifidobacterium bifidum JCM 1254

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.2.1.140 D-Gal-(1,3)-beta-GlcNAc-(1,3)-beta-D-Gal-(1,4)-D-Glc + H2O Bifidobacterium longum lacto-N-tetraose, major component of human milk oligosaccharides lactose + D-galactosyl-beta-1,3-N-acetyl-D-glucosamine lacto-N-biose I ?
3.2.1.140 D-Gal-(1,3)-beta-GlcNAc-(1,3)-beta-D-Gal-(1,4)-D-Glc + H2O Bifidobacterium bifidum lacto-N-tetraose, major component of human milk oligosaccharides lactose + D-galactosyl-beta-1,3-N-acetyl-D-glucosamine lacto-N-biose I ?

Organism

EC Number Organism UniProt Comment Textmining
3.2.1.140 Bifidobacterium bifidum B3TLD6 strain JCM1254 with highest activity, taken for gene sequencing and further characterization, also activity in strains JCM1255, and JCM7004
-
3.2.1.140 Bifidobacterium bifidum JCM 1254
-
 lacto-N-biosidase activity is found in several bifidobacterial strains: strains Bifidobacterium bifidum JCM 1255 and JCM 7004, Bifidobacterium longum JCM 1217, JCM 1222, JCM 7045, but not in the other enteric bacteria, such as clostridia, bacteroides, and lactobacilli
-
3.2.1.140 Bifidobacterium longum
-
 enzyme activity in strains JCM1217, JCM1222, JCM7054, found in infant intestines
-
3.2.1.140 Bifidobacterium longum
-
 lacto-N-biosidase activity is found in several bifidobacterial strains: strains Bifidobacterium bifidum JCM 1254, JCM 1255, and JCM 7004, Bifidobacterium longum JCM 1217, JCM 1222, and JCM 7045, but not in the other enteric bacteria, such as clostridia, bacteroides, and lactobacilli
-
3.2.1.140 no activity in Bifidobacterium breve
-
 strains found in infant intestines, bacteria grown anaerobically in basal lactose medium, 37 degrees Celsius, enzyme assay 3 h - overnight, in 100 mM potassium phosphate buffer pH 7.0 and 4 mM substrate lacto-N-tetraose, HPLC with absorbance measurement of N-acetyl group at 214 nm
-
3.2.1.140 no activity in Bifidobacterium catenulatum
-
 strains found in infant intestines, bacteria grown anaerobically in basal lactose medium, 37 degrees Celsius, enzyme assay 3 h - overnight, in 100 mM potassium phosphate buffer pH 7.0 and 4 mM substrate lacto-N-tetraose, HPLC with absorbance measurement of N-acetyl group at 214 nm
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.2.1.140 Ni2+-charged HiTrap chelating column chromatography, followed by superdex 200 10/300 GL column chromatography, purity determined by SDS-PAGE with Coomassie brilliant blue R250 staining Bifidobacterium bifidum
3.2.1.140 recombinant protein, as the C-terminally His6-tagged protein Bifidobacterium bifidum JCM 1254

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
3.2.1.140 additional information
-
 enzyme activity in strains JCM1217, JCM1222, JCM7054, bacteria grown anaerobically in basal lactose medium, 37°C, enzyme assay 3 h - overnight, in 100 mM potassium phosphate buffer pH 7.0 and 4 mM substrate lacto-N-tetraose, HPLC with absorbance measurement of N-acetyl group at 214 nm Bifidobacterium longum

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.2.1.140 D-Gal-(1,3)-beta-GlcNAc-(1,3)-beta-D-Gal-(1,4)-beta-D-Glc-pyridylamine + H2O lacto-N-tetraose-pyridylamine, no activity on alpha-linked disaccharides, beta-linked p-nitrophenyl monosaccharides such as 4-nitrophenyl beta-D-N-acetylglucosamine and 4-nitrophenyl beta-D-N-acetyl-D-galactosamine, no activity on ganglioside GA1 structure with beta-linked galacto-N-biose, no activity on fucosylated forms of lacto-N-tetraose or lacto-N-neotetraose Bifidobacterium bifidum ?
-
 ?
3.2.1.140 D-Gal-(1,3)-beta-GlcNAc-(1,3)-beta-D-Gal-(1,4)-D-Glc + H2O lacto-N-tetraose, major component of human milk oligosaccharides Bifidobacterium longum lactose + D-galactosyl-beta-1,3-N-acetyl-D-glucosamine lacto-N-biose I ?
3.2.1.140 D-Gal-(1,3)-beta-GlcNAc-(1,3)-beta-D-Gal-(1,4)-D-Glc + H2O lacto-N-tetraose, major component of human milk oligosaccharides Bifidobacterium bifidum lactose + D-galactosyl-beta-1,3-N-acetyl-D-glucosamine lacto-N-biose I ?
3.2.1.140 lacto-N-tetraose + H2O lacto-N-tetraose is a major component of human milk oligosaccharides, substrate used to examine the LnbB activities in various bacterial strains Bifidobacterium longum lactose + lacto-N-biose
-
 ?
3.2.1.140 lacto-N-tetraose + H2O lacto-N-tetraose is a major component of human milk oligosaccharides, substrate used to examine the LnbB activities in various bacterial strains Bifidobacterium bifidum JCM 1254 lactose + lacto-N-biose
-
 ?
3.2.1.140 additional information no activity on beta-linked p-nitrophenyl-monosaccharides, ganglioside GA1 structure with a beta-linked galacto-N-biose, fucosylated forms of lacto-N-tetraose or lacto-N-neoteraose Bifidobacterium bifidum JCM 1254 ?
-
 ?
3.2.1.140 additional information the recombinant enzyme shows substrate preference for the unmodified beta-linked lacto-biose I structure Bifidobacterium bifidum JCM 1254 ?
-
 ?
3.2.1.140 p-nitrophenyl-Gal-(1,3)-beta-GalNAc + H2O 30% enzyme activity compared to p-nitrophenyl-Gal-(1,3)-beta-GlcNAc Bifidobacterium bifidum ?
-
 ?
3.2.1.140 p-nitrophenyl-Gal-(1,3)-beta-GlcNAc + H2O lacto-N-biosyl-beta-p-nitrophenyl, more genereal lacto-N-tetraose with 1-alcanols (methanol, ethanol, 1-propanol, 1-butanol) or p-nitrophenyl-beta-lacto-N-biose + lactose with enzyme Bifidobacterium bifidum ?
-
 ?
3.2.1.140 p-nitrophenyl-Gal-(1,3)-beta-GlcNAc + H2O 30% relative activity compared to p-nitrophenyl-lacto-N-bioside Bifidobacterium bifidum JCM 1254 ?
-
 ?
3.2.1.140 p-nitrophenyl-lacto-N-bioside + H2O most active substrate Bifidobacterium bifidum JCM 1254 p-nitrophenol + lacto-N-biose
-
 ?

Subunits

EC Number Subunits Comment Organism
3.2.1.140 ? x * 120000, SDS-PAGE Bifidobacterium bifidum

Synonyms

EC Number Synonyms Comment Organism
3.2.1.140 lacto-N-biosidase
-
 Bifidobacterium longum
3.2.1.140 lacto-N-biosidase
-
 Bifidobacterium bifidum
3.2.1.140 LnbB
-
 Bifidobacterium longum
3.2.1.140 LnbB
-
 Bifidobacterium bifidum
3.2.1.140 LnbB
-
 Bifidobacterium bifidum JCM 1254

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.2.1.140 25
-
 assay at Bifidobacterium longum
3.2.1.140 25
-
 assay at Bifidobacterium bifidum JCM 1254

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
3.2.1.140 40
-
 88% activity after 30 min incubation Bifidobacterium bifidum
3.2.1.140 40
-
 retains 88% activity after incubation at 40°C for 30 min Bifidobacterium bifidum JCM 1254

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.2.1.140 89
-
 p-Nitrophenyl-lacto-N-bioside pH 4.5, 25°C Bifidobacterium bifidum JCM 1254
3.2.1.140 89
-
 p-nitrophenyl-Gal-(1,3)-beta-GlcNAc pH 4.5, 25°C Bifidobacterium bifidum

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.2.1.140 4.5
-
-
 Bifidobacterium longum
3.2.1.140 4.5
-
 for p-nitrophenyl-Gal-(1,3)-beta-GlcNAc, 50 mM buffers pH 2.5-6.0: citrate-phophate buffer, pH 6.0-8.0: sodium phosphate buffer, pH 8.0-9.0: Tris-HCl buffer Bifidobacterium bifidum
3.2.1.140 4.5
-
 p-nitrophenyl-lacto-N-bioside as substrate Bifidobacterium bifidum JCM 1254
3.2.1.140 6
-
 for D-Gal-(1,3)-beta-GlcNAc-(1,3)-beta-D-Gal-(1,4)-beta-D-Glc-pyridylamine, 50 mM buffers pH 2.5-6.0: citrate-phophate buffer, pH 6.0-8.0: sodium phosphate buffer, pH 8.0-9.0: Tris-HCl buffer Bifidobacterium bifidum
3.2.1.140 6
-
 using pyridylamino oligosaccharides as substrates Bifidobacterium bifidum JCM 1254

pH Stability

EC Number pH Stability pH Stability Maximum Comment Organism
3.2.1.140 4 9
-
 Bifidobacterium bifidum
3.2.1.140 4 9 stable between pH 4 and 9 Bifidobacterium bifidum JCM 1254