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 hide
3.2.1.140
-
RECOMMENDED NAME
GeneOntology No.
lacto-N-biosidase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
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
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-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
condensation
hydrolysis
hydrolysis of O-glycosyl bond
-
-
-
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transglycosylation
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
CAS REGISTRY NUMBER
COMMENTARY hide
146359-52-6
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183511-11-7
lacto-N-biosidase (Streptomyces strain 142)
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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
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-
Manually annotated by BRENDA team
subsp. longum, gene LnbX and LnbY
UniProt
Manually annotated by BRENDA team
strain L-101
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-
Manually annotated by BRENDA team
strain L-101
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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
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-
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
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-
Manually annotated by BRENDA team
strain 142
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Manually annotated by BRENDA team
strain 142
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2'-fucosyllactose + H2O
lactose + fucose
show the reaction diagram
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Fuc-alpha-(1->2)-Gal-beta-(1->4)-Glc
-
-
?
4-nitrophenyl-GalNAc-beta-(1->3)-GlcNAc + H2O
4-nitrophenol + GalNAc-beta-(1->3)-GlcNAc
show the reaction diagram
-
-
-
?
4-nitrophenyl-lacto-N-bioside + H2O
4-nitrophenol + lacto-N-biose
show the reaction diagram
-
-
-
?
D-Gal-(1,3)-beta-GlcNAc-(1,3)-beta-D-Gal-(1,4)-beta-D-Glc-pyridylamine + H2O
?
show the reaction diagram
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
Galbeta1-3GlcNAcbeta-p-nitrophenol + H2O
Galbeta1-3GlcNAc + p-nitrophenol
show the reaction diagram
Galbeta1-3GlcNAcbeta-p-nitrophenol + lactose
Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc + ?
show the reaction diagram
lacto-N-difucohexaose + H2O
lactose + Fuc-alpha-(1->2)-Gal-beta-(1->3)[Fuc-alpha-(1->4)]GlcNAc
show the reaction diagram
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Fuc-alpha-(1->2)Gal-beta-(1->3)[Fuc-alpha-(1->4)]GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc
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-
?
lacto-N-fucopentaose I + H2O
lactose + Fuc-alpha-(1->2)-Gal-beta-(1->3)-GlcNAc
show the reaction diagram
lacto-N-hexaose + H2O
lactose + lacto-N-tetraose
show the reaction diagram
Gal-beta-(1->3)-GlcNAc-beta-(1->3)-[Gal-beta-(1->4)-GlcNAc-beta-(1->6)]-beta-(1->4)-Glc
-
-
?
lacto-N-tetraose + H2O
lacto-N-biose
show the reaction diagram
lacto-N-tetraose + H2O
lactose + lacto-N-biose
show the reaction diagram
p-nitrophenyl-Gal-(1,3)-beta-GalNAc + H2O
?
show the reaction diagram
p-nitrophenyl-Gal-(1,3)-beta-GlcNAc + H2O
?
show the reaction diagram
p-nitrophenyl-lacto-N-bioside + H2O
p-nitrophenol + lacto-N-biose
show the reaction diagram
pyridylamine-oligosaccharides which have type 1 chains at the nonreducing terminus + H2O
lacto-N-biose + ?
show the reaction diagram
sialyllacto-N-tetraose a + H2O
lactose + Fuc-alpha-(1->2)-Gal-beta-(1->3)[Fuc-alpha-(1->4)]GlcNAc
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
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-fucopentaose I + H2O
lactose + Fuc-alpha-(1->2)-Gal-beta-(1->3)-GlcNAc
show the reaction diagram
lacto-N-tetraose + H2O
lactose + lacto-N-biose
show the reaction diagram
sialyllacto-N-tetraose a + H2O
lactose + Fuc-alpha-(1->2)-Gal-beta-(1->3)[Fuc-alpha-(1->4)]GlcNAc
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
required for proper folding of the enzyme LnbX
Mg2+
required for proper folding of the enzyme LnbX
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Gal-beta-(1->3)-GlcNAc-thiazoline
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-
GalNAc-thiazoline
specific inhibition
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GlcNAc-thiazoline
specific inhibition
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guanidine hydrochloride
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lacto-N-biose
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N-acetylglucosamine-thiazoline
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-
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
protein LnbY
protein LnbY is absolutely required for proper folding of the enzyme LnbX and its activity. EDTA has no effect on the specific activity of the purified enzyme
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.186
4-nitrophenyl-GalNAc-beta-(1->3)-GlcNAc
recombinant enzyme, pH 5.4, 25C
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0.119
4-nitrophenyl-lacto-N-bioside
recombinant enzyme, pH 5.4, 25C
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0.0068
Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc-pyridylamine
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14.6
lacto-N-fucopentaose I
recombinant enzyme, pH 5.4, 25C
0.401
lacto-N-tetraose
recombinant enzyme, pH 5.4, 25C
0.0068
p-nitrophenyl-Gal-(1,3)-beta-GlcNAc
pH 4.5, 25°C
0.068
p-Nitrophenyl-lacto-N-bioside
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pH 4.5, 25C
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additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
39.4
4-nitrophenyl-GalNAc-beta-(1->3)-GlcNAc
Bifidobacterium longum
A0A024QYS6
recombinant enzyme, pH 5.4, 25C
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96.1
4-nitrophenyl-lacto-N-bioside
Bifidobacterium longum
A0A024QYS6
recombinant enzyme, pH 5.4, 25C
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13.5
lacto-N-fucopentaose I
Bifidobacterium longum
A0A024QYS6
recombinant enzyme, pH 5.4, 25C
113 - 282
lacto-N-tetraose
89
p-nitrophenyl-Gal-(1,3)-beta-GlcNAc
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
211
4-nitrophenyl-GalNAc-beta-(1->3)-GlcNAc
Bifidobacterium longum
A0A024QYS6
recombinant enzyme, pH 5.4, 25C
202176
806
4-nitrophenyl-lacto-N-bioside
Bifidobacterium longum
A0A024QYS6
recombinant enzyme, pH 5.4, 25C
202175
0.932
lacto-N-fucopentaose I
Bifidobacterium longum
A0A024QYS6
recombinant enzyme, pH 5.4, 25C
8127
282
lacto-N-tetraose
Bifidobacterium longum
A0A024QYS6
recombinant enzyme, pH 5.4, 25C
5569
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000125
Gal-beta-(1->3)-GlcNAc-thiazoline
pH 4.5, 30C
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37.2
purified recombinant non-tagged enzyme, pH 5.4, 30C
additional information
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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
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
46000
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gel filtration
110000
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apparent molecular mass, SDS-PAGE
112000
120000
356000
recombinant nontagged enzyme, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified wild-type and selenomethionine-labeled enzymes in complex with Gal-beta-(1->3)-GlcNAc and Gal-beta-(1->3)-GlcNAc-thiazoline, sitting drop vapor diffusion method, mixing of 0.0005 ml of 7 mg/ml protein and 10 mM Gal-beta-(1->3)-GlcNAc with an equal volume of reservoir solution containing 0.2 M potassium sodium tartrate tetrahydrate, 0.1 M sodium citrate, pH 5.6, and 2.0 M ammonium sulfate, X-ray diffraction structure determination and analysis at 1.8 A resolution
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 10
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4C, 16 h, stable
26153
4 - 7
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4C, 16 h, stable
26154
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
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30 min, stable
50
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30 min, 20% loss of activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
dilution without Brij58 causes 30-50% inactivation
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stabilized by 0.05% Brij58, 0.05% Nonidet P-40 or 0.01% bovine serum albumin
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STORAGE STABILITY
ORGANISM
UNIPROT
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
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Purification/COMMENTARY
ORGANISM
UNIPROT
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
recombinant His-tagged wild-type and mutant as well as His-tagged selenomethionine-labeled enzymes from Escherichia coli by nickel affinity and anion exchange chromatography and gel filtration to homogeneity
recombinant non-tagged enzyme 3.6fold from Escherichia coli by two different steps of anion exchange chromataography and hydrophobic interaction chromatography, followed by gel filtration. Recombinant His-tagged enzyme from Escherichia coli by nickel affinity and anion exchange chromatography, and gel filtration
recombinant protein, as the C-terminally His6-tagged protein
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Cloned/COMMENTARY
ORGANISM
UNIPROT
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
C-terminally His6-tagged enzyme
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cloned in Escherichia coli Rosetta(DE3)
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gene lnbB, expression of His-tagged wild-type enzyme in Escherichia coli BL21 CodonPlus (DE3)-RIL and of His-tagged selenomethionine-labeled enzyme in Escherichia coli BL21 CodonPlus (DE3)-RIL X
gene lnbX, genomic library construction of Bifidobacterium longum strain JCM1217 in Escherichia coli strain DH5alpha, gene disruption and complementation analysis, recombinant expression of lnbX in non-tagged and C-terminally His-tagged form, coexpression with N-terminall His-tagged LnbY in Escherichia coli. The addition of His-tag to the C-terminus of the protein does not alter the enzymatic properties of LnbX
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D320A
site-directed mutagenesis, the mutant shows significantly reduced kcat, but unaffected Km compared with the wild-type enzyme
D320N
site-directed mutagenesis, the mutant shows significantly reduced kcat, but unaffected Km compared with the wild-type enzyme
H263F
site-directed mutagenesis, the mutant shows significantly reduced kcat compared with the wild-type enzyme
Y419F
site-directed mutagenesis, the mutant shows significantly reduced Km and kcat values compared with the wild-type enzyme
D320A
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site-directed mutagenesis, the mutant shows significantly reduced kcat, but unaffected Km compared with the wild-type enzyme
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D320N
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site-directed mutagenesis, the mutant shows significantly reduced kcat, but unaffected Km compared with the wild-type enzyme
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H263F
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site-directed mutagenesis, the mutant shows significantly reduced kcat compared with the wild-type enzyme
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Y419F
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site-directed mutagenesis, the mutant shows significantly reduced Km and kcat values compared with the wild-type enzyme
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additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
In vitro refolding, overview. Denatured C-terminally His-tagged enzyme LnbX is refolded in the presence and absence of LnbY and metals Ca2and Mg2+. Purified LnbX is denatured in 6 M guanidine HCl. It is diluted 100fold by adding 50 mM HEPES buffer, pH 7.0, containing various concentrations of LnbY and metal ions 0.0-5.0 mM in a total volume of 0.5 ml, followed by immediate dialysis against HEPES buffer containing or not containing metal ions
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
synthesis