ß-Glactosidase from Bifidobacterium adolescentis DSM20083 prefers ß(1,4)-galactosides over lactose

S.W.A. Hinz, L.A.M. van den Broek, G. Beldman, J.P. Vincken

Research output: Contribution to journalArticleAcademicpeer-review

51 Citations (Scopus)

Abstract

A -galactosidase gene (-Gal II) from Bifidobacterium adolescentis DSM 20083 was cloned into a pbluescript SK (–) vector and expressed in Escherichia coli. The recombinant enzyme was purified from the cell extract by anion-exchange and size-exclusion chromatography. -Gal II had a native molecular mass of 235 kDa and the subunits had a molecular mass of 81 kDa, indicating that -Gal II occurs as a trimer. The enzyme was classified as belonging to glycosyl hydrolase family 42. The optimal pH was 6.0 and the optimal temperature was 50°C, using p-nitrophenyl--d-galactopyranoside as a substrate. The Km and Vmax for Gal(1–4)Gal were 60 mM and 1,129 U/mg, respectively. The recombinant -Gal II was highly active towards Gal(1–4)Gal and Gal(1–4)Gal-containing oligosaccharides; only low activity was observed towards Gal(1–3)Gal, lactose, and Gal(1–3)GalOMe. No activity was found towards Gal(1–6)Gal, Gal(1–4)Man, Gal(1–4)Gal, Gal(1–3)Gal(1–4)Gal, cellobiose, maltose and sucrose. -Gal II was inhibited at high substrate concentrations (100 mg/ml) and no transglycosylation activity was found. At lower substrate concentrations (10 mg/ml) only low transglycosylation activity was found; the Gal/[Gal(1–4)]2Gal peak area ratio was 9:1.
LanguageEnglish
Pages276-284
JournalApplied Microbiology and Biotechnology
Volume66
Issue number3
DOIs
Publication statusPublished - 2004

Fingerprint

Galactosides
Lactose
Galactosidases
Cellobiose
Maltose
Hydrolases
Enzymes
4-O-alpha-D-galactopyranosyl-D-galactose
Bifidobacterium adolescentis
Cell Extracts
Oligosaccharides
Galactose
Gel Chromatography
Anions
Sucrose
Escherichia coli
Temperature
Genes

Keywords

  • fermentation properties
  • oligosaccharides
  • purification
  • sequence
  • infantis
  • bifidum
  • system
  • classification
  • prediction
  • expression

Cite this

@article{ccdc4ffec9704434ac6553623fcaebb6,
title = "{\ss}-Glactosidase from Bifidobacterium adolescentis DSM20083 prefers {\ss}(1,4)-galactosides over lactose",
abstract = "A -galactosidase gene (-Gal II) from Bifidobacterium adolescentis DSM 20083 was cloned into a pbluescript SK (–) vector and expressed in Escherichia coli. The recombinant enzyme was purified from the cell extract by anion-exchange and size-exclusion chromatography. -Gal II had a native molecular mass of 235 kDa and the subunits had a molecular mass of 81 kDa, indicating that -Gal II occurs as a trimer. The enzyme was classified as belonging to glycosyl hydrolase family 42. The optimal pH was 6.0 and the optimal temperature was 50°C, using p-nitrophenyl--d-galactopyranoside as a substrate. The Km and Vmax for Gal(1–4)Gal were 60 mM and 1,129 U/mg, respectively. The recombinant -Gal II was highly active towards Gal(1–4)Gal and Gal(1–4)Gal-containing oligosaccharides; only low activity was observed towards Gal(1–3)Gal, lactose, and Gal(1–3)GalOMe. No activity was found towards Gal(1–6)Gal, Gal(1–4)Man, Gal(1–4)Gal, Gal(1–3)Gal(1–4)Gal, cellobiose, maltose and sucrose. -Gal II was inhibited at high substrate concentrations (100 mg/ml) and no transglycosylation activity was found. At lower substrate concentrations (10 mg/ml) only low transglycosylation activity was found; the Gal/[Gal(1–4)]2Gal peak area ratio was 9:1.",
keywords = "fermentation properties, oligosaccharides, purification, sequence, infantis, bifidum, system, classification, prediction, expression",
author = "S.W.A. Hinz and {van den Broek}, L.A.M. and G. Beldman and J.P. Vincken",
year = "2004",
doi = "10.1007/s00253-004-1745-9",
language = "English",
volume = "66",
pages = "276--284",
journal = "Applied Microbiology and Biotechnology",
issn = "0175-7598",
publisher = "Springer Verlag",
number = "3",

}

ß-Glactosidase from Bifidobacterium adolescentis DSM20083 prefers ß(1,4)-galactosides over lactose. / Hinz, S.W.A.; van den Broek, L.A.M.; Beldman, G.; Vincken, J.P.

In: Applied Microbiology and Biotechnology, Vol. 66, No. 3, 2004, p. 276-284.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - ß-Glactosidase from Bifidobacterium adolescentis DSM20083 prefers ß(1,4)-galactosides over lactose

AU - Hinz, S.W.A.

AU - van den Broek, L.A.M.

AU - Beldman, G.

AU - Vincken, J.P.

PY - 2004

Y1 - 2004

N2 - A -galactosidase gene (-Gal II) from Bifidobacterium adolescentis DSM 20083 was cloned into a pbluescript SK (–) vector and expressed in Escherichia coli. The recombinant enzyme was purified from the cell extract by anion-exchange and size-exclusion chromatography. -Gal II had a native molecular mass of 235 kDa and the subunits had a molecular mass of 81 kDa, indicating that -Gal II occurs as a trimer. The enzyme was classified as belonging to glycosyl hydrolase family 42. The optimal pH was 6.0 and the optimal temperature was 50°C, using p-nitrophenyl--d-galactopyranoside as a substrate. The Km and Vmax for Gal(1–4)Gal were 60 mM and 1,129 U/mg, respectively. The recombinant -Gal II was highly active towards Gal(1–4)Gal and Gal(1–4)Gal-containing oligosaccharides; only low activity was observed towards Gal(1–3)Gal, lactose, and Gal(1–3)GalOMe. No activity was found towards Gal(1–6)Gal, Gal(1–4)Man, Gal(1–4)Gal, Gal(1–3)Gal(1–4)Gal, cellobiose, maltose and sucrose. -Gal II was inhibited at high substrate concentrations (100 mg/ml) and no transglycosylation activity was found. At lower substrate concentrations (10 mg/ml) only low transglycosylation activity was found; the Gal/[Gal(1–4)]2Gal peak area ratio was 9:1.

AB - A -galactosidase gene (-Gal II) from Bifidobacterium adolescentis DSM 20083 was cloned into a pbluescript SK (–) vector and expressed in Escherichia coli. The recombinant enzyme was purified from the cell extract by anion-exchange and size-exclusion chromatography. -Gal II had a native molecular mass of 235 kDa and the subunits had a molecular mass of 81 kDa, indicating that -Gal II occurs as a trimer. The enzyme was classified as belonging to glycosyl hydrolase family 42. The optimal pH was 6.0 and the optimal temperature was 50°C, using p-nitrophenyl--d-galactopyranoside as a substrate. The Km and Vmax for Gal(1–4)Gal were 60 mM and 1,129 U/mg, respectively. The recombinant -Gal II was highly active towards Gal(1–4)Gal and Gal(1–4)Gal-containing oligosaccharides; only low activity was observed towards Gal(1–3)Gal, lactose, and Gal(1–3)GalOMe. No activity was found towards Gal(1–6)Gal, Gal(1–4)Man, Gal(1–4)Gal, Gal(1–3)Gal(1–4)Gal, cellobiose, maltose and sucrose. -Gal II was inhibited at high substrate concentrations (100 mg/ml) and no transglycosylation activity was found. At lower substrate concentrations (10 mg/ml) only low transglycosylation activity was found; the Gal/[Gal(1–4)]2Gal peak area ratio was 9:1.

KW - fermentation properties

KW - oligosaccharides

KW - purification

KW - sequence

KW - infantis

KW - bifidum

KW - system

KW - classification

KW - prediction

KW - expression

U2 - 10.1007/s00253-004-1745-9

DO - 10.1007/s00253-004-1745-9

M3 - Article

VL - 66

SP - 276

EP - 284

JO - Applied Microbiology and Biotechnology

T2 - Applied Microbiology and Biotechnology

JF - Applied Microbiology and Biotechnology

SN - 0175-7598

IS - 3

ER -