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Literature summary 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.
    View publication on PubMedView publication on EuropePMC

Activating Compound

EC Number Activating Compound Comment Organism Structure
3.2.1.140 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)

EC Number Cloned (Comment) Organism
3.2.1.140 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

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

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.2.1.140 0.119
-
 4-nitrophenyl-lacto-N-bioside recombinant enzyme, pH 5.4, 25°C Bifidobacterium longum
3.2.1.140 0.186
-
 4-nitrophenyl-GalNAc-beta-(1->3)-GlcNAc recombinant enzyme, pH 5.4, 25°C Bifidobacterium longum
3.2.1.140 0.401
-
 lacto-N-tetraose recombinant enzyme, pH 5.4, 25°C Bifidobacterium longum
3.2.1.140 14.6
-
 lacto-N-fucopentaose I recombinant enzyme, pH 5.4, 25°C Bifidobacterium longum

Metals/Ions

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

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
3.2.1.140 161000
-
 2 * 161000, recombinant nontagged enzyme, SDS-PAGE Bifidobacterium longum
3.2.1.140 356000
-
 recombinant nontagged enzyme, gel filtration Bifidobacterium longum

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.2.1.140 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
-
 ?
3.2.1.140 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
-
 ?
3.2.1.140 lacto-N-tetraose + H2O Bifidobacterium longum Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc lactose + lacto-N-biose
-
 ?
3.2.1.140 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
-
 ?
3.2.1.140 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 ?
-
 ?
3.2.1.140 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 ?
-
 ?
3.2.1.140 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
-
 ?
3.2.1.140 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

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

Purification (Commentary)

EC Number Purification (Comment) Organism
3.2.1.140 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)

EC Number Renatured (Comment) Organism
3.2.1.140 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]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
3.2.1.140 37.2
-
 purified recombinant non-tagged enzyme, pH 5.4, 30°C Bifidobacterium longum

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.2.1.140 4-nitrophenyl-GalNAc-beta-(1->3)-GlcNAc + H2O
-
 Bifidobacterium longum 4-nitrophenol + GalNAc-beta-(1->3)-GlcNAc
-
 ?
3.2.1.140 4-nitrophenyl-lacto-N-bioside + H2O
-
 Bifidobacterium longum 4-nitrophenol + lacto-N-biose
-
 ?
3.2.1.140 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
-
 ?
3.2.1.140 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
-
 ?
3.2.1.140 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
-
 ?
3.2.1.140 lacto-N-tetraose + H2O Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc Bifidobacterium longum lactose + lacto-N-biose
-
 ?
3.2.1.140 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
-
 ?
3.2.1.140 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 ?
-
 ?
3.2.1.140 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 ?
-
 ?
3.2.1.140 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 ?
-
 ?
3.2.1.140 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 ?
-
 ?
3.2.1.140 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
-
 ?
3.2.1.140 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

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

Synonyms

EC Number Synonyms Comment Organism
3.2.1.140 BLLJ_1505
-
 Bifidobacterium longum
3.2.1.140 BLLJ_1506
-
 Bifidobacterium longum

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.2.1.140 60
-
-
 Bifidobacterium longum

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.2.1.140 13.5
-
 lacto-N-fucopentaose I recombinant enzyme, pH 5.4, 25°C Bifidobacterium longum
3.2.1.140 39.4
-
 4-nitrophenyl-GalNAc-beta-(1->3)-GlcNAc recombinant enzyme, pH 5.4, 25°C Bifidobacterium longum
3.2.1.140 96.1
-
 4-nitrophenyl-lacto-N-bioside recombinant enzyme, pH 5.4, 25°C Bifidobacterium longum
3.2.1.140 113
-
 lacto-N-tetraose recombinant enzyme, pH 5.4, 25°C Bifidobacterium longum
3.2.1.140 282
-
 lacto-N-tetraose recombinant enzyme, pH 5.4, 25°C Bifidobacterium longum

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.2.1.140 5.4
-
-
 Bifidobacterium longum

General Information

EC Number General Information Comment Organism
3.2.1.140 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]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.2.1.140 0.932
-
 lacto-N-fucopentaose I recombinant enzyme, pH 5.4, 25°C Bifidobacterium longum
3.2.1.140 211
-
 4-nitrophenyl-GalNAc-beta-(1->3)-GlcNAc recombinant enzyme, pH 5.4, 25°C Bifidobacterium longum
3.2.1.140 282
-
 lacto-N-tetraose recombinant enzyme, pH 5.4, 25°C Bifidobacterium longum
3.2.1.140 806
-
 4-nitrophenyl-lacto-N-bioside recombinant enzyme, pH 5.4, 25°C Bifidobacterium longum