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Literature summary for 3.2.1.140 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
Search for more literature for 3.2.1.140

Cloned(Commentary)

Cloned (Comment) Organism
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
cloned in Escherichia coli Rosetta(DE3) Bifidobacterium bifidum JCM 1254

Protein Variants

Protein Variants Comment Organism
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]

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

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
110000
-
 apparent molecular mass, SDS-PAGE Bifidobacterium bifidum JCM 1254
112000
-
 truncated enzyme without signal peptide and membrane anchor, amino acids 35-1064, SDS-PAGE, and calculated from amino acids Bifidobacterium bifidum
112000
-
 calculated mass Bifidobacterium bifidum JCM 1254
120000
-
 full enzyme, calculated from 350 bp PCR product of 1,112 amino acids Bifidobacterium bifidum
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)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
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 ?
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

Organism UniProt Comment Textmining
Bifidobacterium bifidum B3TLD6 strain JCM1254 with highest activity, taken for gene sequencing and further characterization, also activity in strains JCM1255, and JCM7004
-
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
-
Bifidobacterium longum
-
 enzyme activity in strains JCM1217, JCM1222, JCM7054, found in infant intestines
-
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
-
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
-
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)

Purification (Comment) Organism
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
recombinant protein, as the C-terminally His6-tagged protein Bifidobacterium bifidum JCM 1254

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
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)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
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 ?
-
 ?
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 ?
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 ?
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
-
 ?
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
-
 ?
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 ?
-
 ?
additional information the recombinant enzyme shows substrate preference for the unmodified beta-linked lacto-biose I structure Bifidobacterium bifidum JCM 1254 ?
-
 ?
p-nitrophenyl-Gal-(1,3)-beta-GalNAc + H2O 30% enzyme activity compared to p-nitrophenyl-Gal-(1,3)-beta-GlcNAc Bifidobacterium bifidum ?
-
 ?
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 ?
-
 ?
p-nitrophenyl-Gal-(1,3)-beta-GlcNAc + H2O 30% relative activity compared to p-nitrophenyl-lacto-N-bioside Bifidobacterium bifidum JCM 1254 ?
-
 ?
p-nitrophenyl-lacto-N-bioside + H2O most active substrate Bifidobacterium bifidum JCM 1254 p-nitrophenol + lacto-N-biose
-
 ?

Subunits

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

Synonyms

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

Temperature Optimum [°C]

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

Temperature Stability [°C]

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

Turnover Number [1/s]

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

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
4.5
-
-
 Bifidobacterium longum
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
4.5
-
 p-nitrophenyl-lacto-N-bioside as substrate Bifidobacterium bifidum JCM 1254
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
6
-
 using pyridylamino oligosaccharides as substrates Bifidobacterium bifidum JCM 1254

pH Stability

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