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Applied and Environmental Microbiology, September 2005, p. 5501-5510, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5501-5510.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Bifidobacterium longum Endogalactanase Liberates Galactotriose from Type I Galactans

Sandra W. A. Hinz, Marieke I. Pastink, Lambertus A. M. van den Broek, Jean-Paul Vincken, and Alphons G. J. Voragen*

Laboratory of Food Chemistry, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands

Received 19 January 2005/ Accepted 9 April 2005

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 coli. 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 Vmax 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.

* Corresponding author. Mailing address: Laboratory of Food Chemistry, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands. Phone: 31 317 483209. Fax: 31 317 484893. E-mail: Fons.Voragen{at}WUR.NL.

Applied and Environmental Microbiology, September 2005, p. 5501-5510, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5501-5510.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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