Bifidobacterium longum endogalactanase liberates galactotriose from type I galactans

S.W.A. Hinz, M.I. Pastink, L.A.M. van den Broek, J.P. Vincken, A.G.J. Voragen

Research output: Contribution to journalArticleAcademicpeer-review

35 Citations (Scopus)

Abstract

A putative endogalactanase gene classified into glycoside hydrolase family 53 was revealed from the genome sequence of Bifidobacterium longum strain NCC2705 (Schell et al., Proc. Natl. Acad. Sci. USA 99:14422-14427, 2002). Since only a few endo-acting enzymes from bifidobacteria have been described, we have cloned this gene and characterized the enzyme in detail. The deduced amino acid sequence suggested that this enzyme was located extracellularly and anchored to the cell membrane. galA was cloned without the transmembrane domain into the pBluescript SK(-) vector and expressed in Escherichia coll. The enzyme was purified from the cell extract by anion-exchange and size exclusion chromatography. The purified enzyme had a native molecular mass of 329 kDa, and the subunits had a molecular mass of 94 kDa, which indicated that the enzyme occurred as a tetramer. The optimal pH of endogalactanase activity was 5.0, and the optimal temperature was 37°C, using azurine-cross-linked galactan (AZCL-galactan) as a substrate. The Km and Kmax for AZCL-galactan were 1.62 mM and 99 U/mg, respectively. The enzyme was able to liberate galactotrisaccharides from (ß1¿4)galactans and (ß1¿4)galactooligosaccharides, probably by a processive mechanism, moving toward the reducing end of the galactan chain after an initial midchain cleavage. GalA's mode of action was found to be different from that of an endogalactanase from Aspergillus aculeatus. The enzyme seemed to be able to cleave (ß1¿3) linkages. Arabinosyl side chains in, for example, potato galactan hindered GalA.
Original languageEnglish
Pages (from-to)5501-5510
JournalApplied and Environmental Microbiology
Volume71
Issue number9
DOIs
Publication statusPublished - 2005

Fingerprint

Galactans
Bifidobacterium longum
galactans
enzyme
Enzymes
enzymes
Aspergillus aculeatus
molecular weight
galactooligosaccharides
Escherichia
Bifidobacterium
gene
Glycoside Hydrolases
anion exchange
Aspergillus
Solanum tuberosum
hydrolases
Cell Extracts
potato
cleavage

Keywords

  • aspergillus-niger
  • beta-galactosidase
  • molecular-cloning
  • swiss-model
  • purification
  • oligosaccharides
  • expression
  • prediction
  • aculeatus
  • bacteria

Cite this

@article{2b3c2c8c34c844a88b79f32f2c466d25,
title = "Bifidobacterium longum endogalactanase liberates galactotriose from type I galactans",
abstract = "A putative endogalactanase gene classified into glycoside hydrolase family 53 was revealed from the genome sequence of Bifidobacterium longum strain NCC2705 (Schell et al., Proc. Natl. Acad. Sci. USA 99:14422-14427, 2002). Since only a few endo-acting enzymes from bifidobacteria have been described, we have cloned this gene and characterized the enzyme in detail. The deduced amino acid sequence suggested that this enzyme was located extracellularly and anchored to the cell membrane. galA was cloned without the transmembrane domain into the pBluescript SK(-) vector and expressed in Escherichia coll. The enzyme was purified from the cell extract by anion-exchange and size exclusion chromatography. The purified enzyme had a native molecular mass of 329 kDa, and the subunits had a molecular mass of 94 kDa, which indicated that the enzyme occurred as a tetramer. The optimal pH of endogalactanase activity was 5.0, and the optimal temperature was 37°C, using azurine-cross-linked galactan (AZCL-galactan) as a substrate. The Km and Kmax for AZCL-galactan were 1.62 mM and 99 U/mg, respectively. The enzyme was able to liberate galactotrisaccharides from ({\ss}1¿4)galactans and ({\ss}1¿4)galactooligosaccharides, probably by a processive mechanism, moving toward the reducing end of the galactan chain after an initial midchain cleavage. GalA's mode of action was found to be different from that of an endogalactanase from Aspergillus aculeatus. The enzyme seemed to be able to cleave ({\ss}1¿3) linkages. Arabinosyl side chains in, for example, potato galactan hindered GalA.",
keywords = "aspergillus-niger, beta-galactosidase, molecular-cloning, swiss-model, purification, oligosaccharides, expression, prediction, aculeatus, bacteria",
author = "S.W.A. Hinz and M.I. Pastink and {van den Broek}, L.A.M. and J.P. Vincken and A.G.J. Voragen",
year = "2005",
doi = "10.1128/AEM.71.9.5501-5510.2005",
language = "English",
volume = "71",
pages = "5501--5510",
journal = "Applied and Environmental Microbiology",
issn = "0099-2240",
publisher = "American Society for Microbiology",
number = "9",

}

Bifidobacterium longum endogalactanase liberates galactotriose from type I galactans. / Hinz, S.W.A.; Pastink, M.I.; van den Broek, L.A.M.; Vincken, J.P.; Voragen, A.G.J.

In: Applied and Environmental Microbiology, Vol. 71, No. 9, 2005, p. 5501-5510.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Bifidobacterium longum endogalactanase liberates galactotriose from type I galactans

AU - Hinz, S.W.A.

AU - Pastink, M.I.

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

AU - Vincken, J.P.

AU - Voragen, A.G.J.

PY - 2005

Y1 - 2005

N2 - A putative endogalactanase gene classified into glycoside hydrolase family 53 was revealed from the genome sequence of Bifidobacterium longum strain NCC2705 (Schell et al., Proc. Natl. Acad. Sci. USA 99:14422-14427, 2002). Since only a few endo-acting enzymes from bifidobacteria have been described, we have cloned this gene and characterized the enzyme in detail. The deduced amino acid sequence suggested that this enzyme was located extracellularly and anchored to the cell membrane. galA was cloned without the transmembrane domain into the pBluescript SK(-) vector and expressed in Escherichia coll. The enzyme was purified from the cell extract by anion-exchange and size exclusion chromatography. The purified enzyme had a native molecular mass of 329 kDa, and the subunits had a molecular mass of 94 kDa, which indicated that the enzyme occurred as a tetramer. The optimal pH of endogalactanase activity was 5.0, and the optimal temperature was 37°C, using azurine-cross-linked galactan (AZCL-galactan) as a substrate. The Km and Kmax for AZCL-galactan were 1.62 mM and 99 U/mg, respectively. The enzyme was able to liberate galactotrisaccharides from (ß1¿4)galactans and (ß1¿4)galactooligosaccharides, probably by a processive mechanism, moving toward the reducing end of the galactan chain after an initial midchain cleavage. GalA's mode of action was found to be different from that of an endogalactanase from Aspergillus aculeatus. The enzyme seemed to be able to cleave (ß1¿3) linkages. Arabinosyl side chains in, for example, potato galactan hindered GalA.

AB - A putative endogalactanase gene classified into glycoside hydrolase family 53 was revealed from the genome sequence of Bifidobacterium longum strain NCC2705 (Schell et al., Proc. Natl. Acad. Sci. USA 99:14422-14427, 2002). Since only a few endo-acting enzymes from bifidobacteria have been described, we have cloned this gene and characterized the enzyme in detail. The deduced amino acid sequence suggested that this enzyme was located extracellularly and anchored to the cell membrane. galA was cloned without the transmembrane domain into the pBluescript SK(-) vector and expressed in Escherichia coll. The enzyme was purified from the cell extract by anion-exchange and size exclusion chromatography. The purified enzyme had a native molecular mass of 329 kDa, and the subunits had a molecular mass of 94 kDa, which indicated that the enzyme occurred as a tetramer. The optimal pH of endogalactanase activity was 5.0, and the optimal temperature was 37°C, using azurine-cross-linked galactan (AZCL-galactan) as a substrate. The Km and Kmax for AZCL-galactan were 1.62 mM and 99 U/mg, respectively. The enzyme was able to liberate galactotrisaccharides from (ß1¿4)galactans and (ß1¿4)galactooligosaccharides, probably by a processive mechanism, moving toward the reducing end of the galactan chain after an initial midchain cleavage. GalA's mode of action was found to be different from that of an endogalactanase from Aspergillus aculeatus. The enzyme seemed to be able to cleave (ß1¿3) linkages. Arabinosyl side chains in, for example, potato galactan hindered GalA.

KW - aspergillus-niger

KW - beta-galactosidase

KW - molecular-cloning

KW - swiss-model

KW - purification

KW - oligosaccharides

KW - expression

KW - prediction

KW - aculeatus

KW - bacteria

U2 - 10.1128/AEM.71.9.5501-5510.2005

DO - 10.1128/AEM.71.9.5501-5510.2005

M3 - Article

VL - 71

SP - 5501

EP - 5510

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

SN - 0099-2240

IS - 9

ER -