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Bifidobacterium longum Endogalactanase Liberates Galactotriose from Type I Galactans

Author:
Hinz, Sandra W. A., Pastink, Marieke I., van den Broek, Lambertus A. M., Vincken, Jean-Paul, Voragen, Alphons G. J.
Source:
Applied and environmental microbiology 2005 v.71 no.9 pp. 5501-5510
ISSN:
0099-2240
Subject:
Bifidobacterium longum, O-glycoside hydrolases, galactans, enzymatic hydrolysis, trisaccharides, enzyme activity, pH, molecular weight, genes, nucleotide sequences
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 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 K[subscript m] and V[subscript max] for AZCL-galactan were 1.62 mM and 99 U/mg, respectively. The enzyme was able to liberate galactotrisaccharides from ({szligbeta}1[rightwards arrow]4)galactans and ({szligbeta}1[rightwards arrow]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 ({szligbeta}1[rightwards arrow]3) linkages. Arabinosyl side chains in, for example, potato galactan hindered GalA.
Agid:
1410982