Literature summary for 3.2.1.140 extracted from

Sakurama, H.; Kiyohara, M.; Wada, J.; Honda, Y.; Yamaguchi, M.; Fukiya, S.; Yokota, A.; Ashida, H.; Kumagai, H.; Kitaoka, M.; Yamamoto, K.; Katayama, T.
Lacto-N-biosidase encoded by a novel gene of Bifidobacterium longum subspecies longum shows unique substrate specificity and requires a designated chaperone for its active expression (2013), J. Biol. Chem., 288, 25194-25206.

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Activating Compound

Activating Compound	Comment	Organism	Structure
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	Bifidobacterium longum

Cloned(Commentary)

Cloned (Comment)	Organism
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	Bifidobacterium longum

Cloned (Comment)

Organism

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

Bifidobacterium longum

Inhibitors

Inhibitors	Comment	Organism	Structure
additional information	EDTA does not affect the specific activity of the purified enzyme	Bifidobacterium longum

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.119	-	4-nitrophenyl-lacto-N-bioside	recombinant enzyme, pH 5.4, 25°C	Bifidobacterium longum
0.186	-	4-nitrophenyl-GalNAc-beta-(1->3)-GlcNAc	recombinant enzyme, pH 5.4, 25°C	Bifidobacterium longum
0.401	-	lacto-N-tetraose	recombinant enzyme, pH 5.4, 25°C	Bifidobacterium longum
14.6	-	lacto-N-fucopentaose I	recombinant enzyme, pH 5.4, 25°C	Bifidobacterium longum

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Ca2+	required for proper folding of the enzyme LnbX	Bifidobacterium longum
Mg2+	required for proper folding of the enzyme LnbX	Bifidobacterium longum

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
161000	-	2 * 161000, recombinant nontagged enzyme, SDS-PAGE	Bifidobacterium longum
356000	-	recombinant nontagged enzyme, gel filtration	Bifidobacterium longum

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
lacto-N-fucopentaose I + H2O	Bifidobacterium longum	Fuc-alpha-(1->2)-Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc	lactose + Fuc-alpha-(1->2)-Gal-beta-(1->3)-GlcNAc	-	?
lacto-N-fucopentaose I + H2O	Bifidobacterium longum JCM 1217	Fuc-alpha-(1->2)-Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc	lactose + Fuc-alpha-(1->2)-Gal-beta-(1->3)-GlcNAc	-	?
lacto-N-tetraose + H2O	Bifidobacterium longum	Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc	lactose + lacto-N-biose	-	?
lacto-N-tetraose + H2O	Bifidobacterium longum JCM 1217	Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc	lactose + lacto-N-biose	-	?
additional information	Bifidobacterium longum	the native substrate are human milk oligosaccharides. Among the different structures, four molecular species, namely 2'-fucosyllactose, lacto-N-tetraose, lacto-N-fucopentaose I, and lacto-N-difucohexaose I, are most abundantly present and comprise more than 50% of the total oligosaccharides, unless the milk is derived from non-secretor or Lewis-negative subjects	?	-	?
additional information	Bifidobacterium longum JCM 1217	the native substrate are human milk oligosaccharides. Among the different structures, four molecular species, namely 2'-fucosyllactose, lacto-N-tetraose, lacto-N-fucopentaose I, and lacto-N-difucohexaose I, are most abundantly present and comprise more than 50% of the total oligosaccharides, unless the milk is derived from non-secretor or Lewis-negative subjects	?	-	?
sialyllacto-N-tetraose a + H2O	Bifidobacterium longum	Neu5Ac-alpha-(2->3)-Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Gal	lactose + Fuc-alpha-(1->2)-Gal-beta-(1->3)[Fuc-alpha-(1->4)]GlcNAc	-	?
sialyllacto-N-tetraose a + H2O	Bifidobacterium longum JCM 1217	Neu5Ac-alpha-(2->3)-Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Gal	lactose + Fuc-alpha-(1->2)-Gal-beta-(1->3)[Fuc-alpha-(1->4)]GlcNAc	-	?

Organism

Organism	UniProt	Comment	Textmining
Bifidobacterium longum	A0A024QYS6	subsp. longum, gene LnbX and LnbY	-
Bifidobacterium longum JCM 1217	A0A024QYS6	subsp. longum, gene LnbX and LnbY	-

Purification (Commentary)

Purification (Comment)	Organism
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	Bifidobacterium longum

Renatured (Commentary)

Renatured (Comment)	Organism
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	Bifidobacterium longum

Renatured (Comment)

Organism

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

Bifidobacterium longum

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
37.2	-	purified recombinant non-tagged enzyme, pH 5.4, 30°C	Bifidobacterium longum

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
4-nitrophenyl-GalNAc-beta-(1->3)-GlcNAc + H2O	-	Bifidobacterium longum	4-nitrophenol + GalNAc-beta-(1->3)-GlcNAc	-	?
4-nitrophenyl-lacto-N-bioside + H2O	-	Bifidobacterium longum	4-nitrophenol + lacto-N-biose	-	?
lacto-N-fucopentaose I + H2O	Fuc-alpha-(1->2)-Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc	Bifidobacterium longum	lactose + Fuc-alpha-(1->2)-Gal-beta-(1->3)-GlcNAc	-	?
lacto-N-fucopentaose I + H2O	Fuc-alpha-(1->2)-Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc	Bifidobacterium longum JCM 1217	lactose + Fuc-alpha-(1->2)-Gal-beta-(1->3)-GlcNAc	-	?
lacto-N-hexaose + H2O	Gal-beta-(1->3)-GlcNAc-beta-(1->3)-[Gal-beta-(1->4)-GlcNAc-beta-(1->6)]-beta-(1->4)-Glc	Bifidobacterium longum	lactose + lacto-N-tetraose	-	?
lacto-N-tetraose + H2O	Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc	Bifidobacterium longum	lactose + lacto-N-biose	-	?
lacto-N-tetraose + H2O	Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc	Bifidobacterium longum JCM 1217	lactose + lacto-N-biose	-	?
additional information	the native substrate are human milk oligosaccharides. Among the different structures, four molecular species, namely 2'-fucosyllactose, lacto-N-tetraose, lacto-N-fucopentaose I, and lacto-N-difucohexaose I, are most abundantly present and comprise more than 50% of the total oligosaccharides, unless the milk is derived from non-secretor or Lewis-negative subjects	Bifidobacterium longum	?	-	?
additional information	the purified enzyme, which consists of LnbX only, hydrolyzes via a retaining mechanism the GlcNAcbeta-(1->3)-Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I, and sialyllacto-N-tetraose a. Inactive against lacto-N-neotetraose, lacto-N-triose II, lacto-N-fucopentaose II, and sialyllacto-N-tetraose b, substrate specificity, overview	Bifidobacterium longum	?	-	?
additional information	the native substrate are human milk oligosaccharides. Among the different structures, four molecular species, namely 2'-fucosyllactose, lacto-N-tetraose, lacto-N-fucopentaose I, and lacto-N-difucohexaose I, are most abundantly present and comprise more than 50% of the total oligosaccharides, unless the milk is derived from non-secretor or Lewis-negative subjects	Bifidobacterium longum JCM 1217	?	-	?
additional information	the purified enzyme, which consists of LnbX only, hydrolyzes via a retaining mechanism the GlcNAcbeta-(1->3)-Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I, and sialyllacto-N-tetraose a. Inactive against lacto-N-neotetraose, lacto-N-triose II, lacto-N-fucopentaose II, and sialyllacto-N-tetraose b, substrate specificity, overview	Bifidobacterium longum JCM 1217	?	-	?
sialyllacto-N-tetraose a + H2O	Neu5Ac-alpha-(2->3)-Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Gal	Bifidobacterium longum	lactose + Fuc-alpha-(1->2)-Gal-beta-(1->3)[Fuc-alpha-(1->4)]GlcNAc	-	?
sialyllacto-N-tetraose a + H2O	Neu5Ac-alpha-(2->3)-Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Gal	Bifidobacterium longum JCM 1217	lactose + Fuc-alpha-(1->2)-Gal-beta-(1->3)[Fuc-alpha-(1->4)]GlcNAc	-	?

Subunits

Subunits	Comment	Organism
dimer	2 * 161000, recombinant nontagged enzyme, SDS-PAGE	Bifidobacterium longum

Synonyms

Synonyms	Comment	Organism
BLLJ_1505	-	Bifidobacterium longum
BLLJ_1506	-	Bifidobacterium longum

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
60	-	-	Bifidobacterium longum

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
13.5	-	lacto-N-fucopentaose I	recombinant enzyme, pH 5.4, 25°C	Bifidobacterium longum
39.4	-	4-nitrophenyl-GalNAc-beta-(1->3)-GlcNAc	recombinant enzyme, pH 5.4, 25°C	Bifidobacterium longum
96.1	-	4-nitrophenyl-lacto-N-bioside	recombinant enzyme, pH 5.4, 25°C	Bifidobacterium longum
113	-	lacto-N-tetraose	recombinant enzyme, pH 5.4, 25°C	Bifidobacterium longum
282	-	lacto-N-tetraose	recombinant enzyme, pH 5.4, 25°C	Bifidobacterium longum

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
5.4	-	-	Bifidobacterium longum

General Information

General Information	Comment	Organism
evolution	the LnbX protein is found only in Bifidobacterium bifidum, Bifidobacterium longum, and a few gut microbes, suggesting that the protein has evolved in specialized niches	Bifidobacterium longum

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.932	-	lacto-N-fucopentaose I	recombinant enzyme, pH 5.4, 25°C	Bifidobacterium longum
211	-	4-nitrophenyl-GalNAc-beta-(1->3)-GlcNAc	recombinant enzyme, pH 5.4, 25°C	Bifidobacterium longum
282	-	lacto-N-tetraose	recombinant enzyme, pH 5.4, 25°C	Bifidobacterium longum
806	-	4-nitrophenyl-lacto-N-bioside	recombinant enzyme, pH 5.4, 25°C	Bifidobacterium longum