Information on EC 3.2.1.140 - lacto-N-biosidase

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The expected taxonomic range for this enzyme is: Actinobacteridae

EC NUMBER
COMMENTARY
3.2.1.140
-
RECOMMENDED NAME
GeneOntology No.
lacto-N-biosidase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
beta-D-Gal-(1->3)-beta-D-GlcNAc-(1->3)-beta-D-Gal-(1->4)-D-Glc + H2O = beta-D-Gal-(1->3)-D-GlcNAc + beta-D-Gal-(1->4)-D-Glc
show the reaction diagram
-
-
-
-
lacto-N-tetraose + H2O = lacto-N-biose + lactose
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
condensation
B3TLD6, -
lacto-N-biose + lactose + enzyme
condensation
Bifidobacterium bifidum JCM1254
-
lacto-N-biose + lactose + enzyme
-
hydrolysis
B3TLD6, -
-
hydrolysis
Bifidobacterium bifidum JCM1254
-
-
-
hydrolysis of O-glycosyl bond
-
-
-
-
transglycosylation
B3TLD6, -
-
transglycosylation
Bifidobacterium bifidum JCM1254
-
-
-
SYSTEMATIC NAME
IUBMB Comments
oligosaccharide lacto-N-biosylhydrolase
The enzyme from Streptomyces specifically hydrolyses the terminal lacto-N-biosyl residue (beta-D-Gal-(1->3)-D-GlcNAc) from the non-reducing end of oligosaccharides with the structure beta-D-Gal-(1->3)-beta-D-GlcNAc-(1->3)-beta-D-Gal-(1->R). Lacto-N-hexaose (beta-D-Gal-(1->3)-beta-D-GlcNAc-(1->3)-beta-D-Gal-(1->3)-beta-D-GlcNAc-(1->3)-beta-D-Gal-(1->4)-D-Glc) is hydrolysed to form first lacto-N-tetraose plus lacto-N-biose, with the subsequent formation of lactose. Oligosaccharides in which the non-reducing terminal Gal or the penultimate GlcNAc are replaced by fucose or sialic acid are not substrates. Asialo GM1 tetraose (beta-D-Gal-(1->3)-beta-D-GalNAc-(1->3)-beta-D-Gal-(1->4)-D-Glc) is hydrolysed very slowly, but lacto-N-neotetraose (beta-D-Gal-(1->4)-beta-D-GalNAc-(1->3)-beta-D-Gal-(1->4)-D-Glc) is not a substrate
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
lacto-N-biosidase
-
-
-
-
lacto-N-biosidase
B3TLD6
-
lacto-N-biosidase
Bifidobacterium bifidum JCM1254
B3TLD6
-
-
lacto-N-biosidase
-
-
lacto-N-biosidase (Streptomyces strain 142)
-
-
-
-
LnbB
Bifidobacterium bifidum JCM1254
B3TLD6
-
-
CAS REGISTRY NUMBER
COMMENTARY
146359-52-6
-
183511-11-7
lacto-N-biosidase (Streptomyces strain 142)
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
strain JCM1254 with highest activity, taken for gene sequencing and further characterization, also activity in strains JCM1255, and JCM7004
UniProt
Manually annotated by BRENDA team
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
-
-
Manually annotated by BRENDA team
Bifidobacterium bifidum JCM1254
strain JCM1254 with highest activity, taken for gene sequencing and further characterization, also activity in strains JCM1255, and JCM7004
UniProt
Manually annotated by BRENDA team
enzyme activity in strains JCM1217, JCM1222, JCM7054, found in infant intestines; 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
-
-
Manually annotated by BRENDA team
strain L-101
-
-
Manually annotated by BRENDA team
Microbacterium sp. L-101
strain L-101
-
-
Manually annotated by BRENDA team
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
-
-
Manually annotated by BRENDA team
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
-
-
Manually annotated by BRENDA team
strain 142
-
-
Manually annotated by BRENDA team
Streptomyces sp. 142
strain 142
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
D-Gal-(1,3)-beta-GlcNAc-(1,3)-beta-D-Gal-(1,4)-beta-D-Glc-pyridylamine + H2O
?
show the reaction diagram
B3TLD6, -
lacto-N-tetraose-pyridylamine, no activity on alpha-linked disaccharides, beta-linked p-nitrophenyl monosaccarides such as p-nitrophenyl-beta-BlcNAc and p-nitrophenyl-beta-GalNAc, 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
-
-
?
D-Gal-(1,3)-beta-GlcNAc-(1,3)-beta-D-Gal-(1,4)-beta-D-Glc-pyridylamine + H2O
?
show the reaction diagram
Bifidobacterium bifidum JCM1254
B3TLD6
lacto-N-tetraose-pyridylamine, no activity on alpha-linked disaccharides, beta-linked p-nitrophenyl monosaccarides such as p-nitrophenyl-beta-BlcNAc and p-nitrophenyl-beta-GalNAc, 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
-
-
?
D-Gal-(1,3)-beta-GlcNAc-(1,3)-beta-D-Gal-(1,4)-D-Glc + H2O
lactose + D-galactosyl-beta-1,3-N-acetyl-D-glucosamine
show the reaction diagram
-
lacto-N-tetraose, major component of human milk oligosaccharides
lacto-N-biose I
-
?
D-Gal-(1,3)-beta-GlcNAc-(1,3)-beta-D-Gal-(1,4)-D-Glc + H2O
lactose + D-galactosyl-beta-1,3-N-acetyl-D-glucosamine
show the reaction diagram
B3TLD6, -
lacto-N-tetraose, major component of human milk oligosaccharides
lacto-N-biose I
-
?
D-Gal-(1,3)-beta-GlcNAc-(1,3)-beta-D-Gal-(1,4)-D-Glc + H2O
lactose + D-galactosyl-beta-1,3-N-acetyl-D-glucosamine
show the reaction diagram
Bifidobacterium bifidum JCM1254
B3TLD6
lacto-N-tetraose, major component of human milk oligosaccharides
lacto-N-biose I
-
?
Galbeta1-3GlcNAcbeta-p-nitrophenol + H2O
Galbeta1-3GlcNAc + p-nitrophenol
show the reaction diagram
Microbacterium sp., Microbacterium sp. L-101
-
-
-
-
?
Galbeta1-3GlcNAcbeta-p-nitrophenol + lactose
Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc + ?
show the reaction diagram
Microbacterium sp., Microbacterium sp. L-101
-
-
Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc, i.e. lacto-neotetraose
-
?
lacto-N-tetraose + H2O
lacto-N-biose
show the reaction diagram
-
-
-
-
-
lacto-N-tetraose + H2O
lacto-N-biose
show the reaction diagram
-
lacto-N-tetraose is a major component of human milk oligosaccharides, substrate used to examine the LnbB activities in various bacterial strains
-
-
?
p-nitrophenyl-Gal-(1,3)-beta-GalNAc + H2O
?
show the reaction diagram
B3TLD6, -
30% enzyme activity compared to p-nitrophenyl-Gal-(1,3)-beta-GlcNAc
-
-
?
p-nitrophenyl-Gal-(1,3)-beta-GalNAc + H2O
?
show the reaction diagram
Bifidobacterium bifidum JCM1254
B3TLD6
30% enzyme activity compared to p-nitrophenyl-Gal-(1,3)-beta-GlcNAc
-
-
?
p-nitrophenyl-Gal-(1,3)-beta-GlcNAc + H2O
?
show the reaction diagram
B3TLD6, -
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
-
-
?
p-nitrophenyl-Gal-(1,3)-beta-GlcNAc + H2O
?
show the reaction diagram
-
30% relative activity compared to p-nitrophenyl-lacto-N-bioside
-
-
?
p-nitrophenyl-Gal-(1,3)-beta-GlcNAc + H2O
?
show the reaction diagram
Bifidobacterium bifidum JCM1254
B3TLD6
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
-
-
?
p-nitrophenyl-lacto-N-bioside + H2O
p-nitrophenol + lacto-N-biose
show the reaction diagram
-
-
-
-
p-nitrophenyl-lacto-N-bioside + H2O
p-nitrophenol + lacto-N-biose
show the reaction diagram
-
-
-
-
-
p-nitrophenyl-lacto-N-bioside + H2O
p-nitrophenol + lacto-N-biose
show the reaction diagram
-
most active substrate
-
-
?
p-nitrophenyl-lacto-N-bioside + H2O
p-nitrophenol + lacto-N-biose
show the reaction diagram
Streptomyces sp. 142
-
-
-
-
p-nitrophenyl-lacto-N-bioside + H2O
p-nitrophenol + lacto-N-biose
show the reaction diagram
Streptomyces sp. 142
-
-
-
-
-
pyridylamine-oligosaccharides which have type 1 chains at the nonreducing terminus + H2O
lacto-N-biose + ?
show the reaction diagram
-
-
from N-acetyllactosamine type of triantennary sugar chain with type 1 structure and N-avcetyllactosamine type of biantennary sugar chain is produced
-
pyridylamine-oligosaccharides which have type 1 chains at the nonreducing terminus + H2O
lacto-N-biose + ?
show the reaction diagram
Streptomyces sp., Streptomyces sp. 142
-
no reaction with pyridylamine-oligosaccharides which contain only type 2 chains. No reaction with oligosaccharides in which the non-reducing terminal Gal or the penultimate GlcNAc are replaced by fucose or sialic acid are
-
-
-
pyridylamine-oligosaccharides which have type 1 chains at the nonreducing terminus + H2O
lacto-N-biose + ?
show the reaction diagram
Streptomyces sp. 142
-
-
from N-acetyllactosamine type of triantennary sugar chain with type 1 structure and N-avcetyllactosamine type of biantennary sugar chain is produced
-
lacto-N-tetraose + H2O
lacto-N-biose
show the reaction diagram
Streptomyces sp. 142
-
-
-
-
-
additional information
?
-
-
no reaction with lacto-N-neotetraose, lacto-N-triose, sialyl lacto-N-tetraose, lacto-N-fucopentaose I, II, or III, asialo-GM1 tetrasaccharide, poly-N-acetyllactosamine
-
-
-
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, the recombinant enzyme shows substrate preference for the unmodified beta-linked lacto-biose I structure
-
-
-
additional information
?
-
Streptomyces sp. 142
-
no reaction with lacto-N-neotetraose, lacto-N-triose, sialyl lacto-N-tetraose, lacto-N-fucopentaose I, II, or III, asialo-GM1 tetrasaccharide, poly-N-acetyllactosamine
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
D-Gal-(1,3)-beta-GlcNAc-(1,3)-beta-D-Gal-(1,4)-D-Glc + H2O
lactose + D-galactosyl-beta-1,3-N-acetyl-D-glucosamine
show the reaction diagram
-
lacto-N-tetraose, major component of human milk oligosaccharides
lacto-N-biose I
-
?
D-Gal-(1,3)-beta-GlcNAc-(1,3)-beta-D-Gal-(1,4)-D-Glc + H2O
lactose + D-galactosyl-beta-1,3-N-acetyl-D-glucosamine
show the reaction diagram
B3TLD6, -
lacto-N-tetraose, major component of human milk oligosaccharides
lacto-N-biose I
-
?
D-Gal-(1,3)-beta-GlcNAc-(1,3)-beta-D-Gal-(1,4)-D-Glc + H2O
lactose + D-galactosyl-beta-1,3-N-acetyl-D-glucosamine
show the reaction diagram
Bifidobacterium bifidum JCM1254
B3TLD6
lacto-N-tetraose, major component of human milk oligosaccharides
lacto-N-biose I
-
?
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
Fe3+
-
-
-
guanidine hydrochloride
-
-
lacto-N-biose
-
-
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.0068
-
p-nitrophenyl-Gal-(1,3)-beta-GlcNAc
B3TLD6, -
pH 4.5, 25°C
0.068
-
p-Nitrophenyl-lacto-N-bioside
-
pH 4.5, 25C
-
0.0068
-
Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc-pyridylamine
-
-
additional information
-
additional information
-
-
-
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
89
-
p-nitrophenyl-Gal-(1,3)-beta-GlcNAc
B3TLD6, -
pH 4.5, 25°C
89
-
p-Nitrophenyl-lacto-N-bioside
-
pH 4.5, 25C
-
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
18.97
-
-
-
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
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
4.5
-
-
p-nitrophenyl-beta-lacto-N-bioside
4.5
-
B3TLD6, -
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
4.5
-
-
p-nitrophenyl-lacto-N-bioside as substrate
5.5
-
-
Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc-pyridylamine
6
-
B3TLD6, -
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
6
-
-
using pyridylamino oligosaccharides as substrates
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
60
-
-
p-nitrophenyl-beta-lacto-N-bioside
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
Microbacterium sp. L-101, Streptomyces sp. 142
-
-
-
Manually annotated by BRENDA team
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
46000
-
-
gel filtration
110000
-
-
apparent molecular mass, SDS-PAGE
112000
-
-
calculated mass
112000
-
B3TLD6, -
truncated enzyme without signal peptide and membrane anchor, amino acids 35-1064, SDS-PAGE, and calculated from amino acids
120000
-
B3TLD6, -
full enzyme, calculated from 350 bp PCR product of 1,112 amino acids
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
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
B3TLD6, -
x * 120000, SDS-PAGE
?
Bifidobacterium bifidum JCM1254
-
x * 120000, SDS-PAGE
-
monomer
-
1 * 60000, SDS-PAGE
monomer
Streptomyces sp. 142
-
1 * 60000, SDS-PAGE
-
pH STABILITY
pH STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
4
10
-
4C, 16 h, stable
4
7
-
4C, 16 h, stable
4
9
-
stable between pH 4 and 9
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
40
-
B3TLD6, -
88% activity after 30 min incubation
40
-
-
retains 88% activity after incubation at 40C for 30 min
45
-
-
30 min, stable
50
-
-
30 min, 20% loss of activity
GENERAL STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
dilution without Brij58 causes 30-50% inactivation
-
stabilized by 0.05% Brij58, 0.05% Nonidet P-40 or 0.01% bovine serum albumin
-
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
4C or -20C, protein concentration 0.01 mg/ml or 0.00005 mg/ml, 0.05% Brij 58, stable for at least 6 months
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
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
B3TLD6, -
recombinant protein, as the C-terminally His6-tagged protein
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
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
B3TLD6, -
cloned in Escherichia coli Rosetta(DE3)
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
B3TLD6, -
truncated enzyme without N-terminal signal peptide of 34 amino acids and C-terminal membrane anchor from 1065-1108
additional information
Bifidobacterium bifidum JCM1254
-
truncated enzyme without N-terminal signal peptide of 34 amino acids and C-terminal membrane anchor from 1065-1108
-
APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
synthesis
-
synthesis of Galbeta(1-3)GlcNAcbeta(1-3)Galbeta(1-4)Glc, i.e. lacto-neotetraose
synthesis
Microbacterium sp. L-101
-
synthesis of Galbeta(1-3)GlcNAcbeta(1-3)Galbeta(1-4)Glc, i.e. lacto-neotetraose
-
analysis
-
the enzyme may be useful in identifying type 1 structure in glycoconjugates and to distinguish type 1 oligosaccharides from type 2 oligosaccharides
analysis
Streptomyces sp. 142
-
the enzyme may be useful in identifying type 1 structure in glycoconjugates and to distinguish type 1 oligosaccharides from type 2 oligosaccharides
-