Cloning and characterization of a novel α-galactosidase fromBifidobacterium breve203 capable of synthesizing Gal-α-1,4 linkage

Abstract

A novel alpha-galactosidase gene (aga2) was cloned from Bifidobacterium breve 203. It contained an ORF of 2226-bp nucleotides encoding 741 amino acids with a calculated molecular mass of 81.5 kDa. The recombinant enzyme Aga2 was heterogeneously expressed, purified and characterized. Regarding substrate specificity for hydrolysis, Aga2 was highly active towards p-nitrophenyl-alpha-d-galactopyranoside (pNPG). The Km value for pNPG was estimated to be 0.27 mM and for melibiose it was estimated to be 4.3 mM. Aga2 was capable of catalyzing transglycosylation as well as hydrolysis. The enzyme synthesized a trisaccharide (Gal-alpha-1, 4-Gal-alpha-1, 6-Glc) using melibiose as a substrate. It was a new oligosaccharide produced by glycosidase and contained Gal-alpha-1,4 linkage, a novel galactosidic link formed by microbial alpha-galactosidase. In the presence of pNPG as a donor, Aga2 was able to catalyze glycosyl transfer to various acceptors including monosaccharides, disaccharides and sugar alcohols.

Performances of new isolates of Bifidobacterium on fermentation of soymilk

“…Bifi dobacterium adolescentis DSM 20083 strain secreted α-galactosidase extracellularly; that was suggested to be a tetrametric structure of the protein [32]. Later, two groups -Goulas et al [33] as well as Zhao et al [34] -reported cloning, expression and characterisation of α-galactosidase from B. bifi dum NCBIM B41171 and B. breve 203, respectively, and it had been found a monomeric nature of this galactosidase with molecular mass about 80 kDa. Tochikura et al [35] however, found that bifi dobacteria exhibit higher hydrolyzing activity toward various p-nitrophenyl glycosides than other intestinal bacteria.…”

Carbohydrate metabolism in Bifidobacteria

“…In spite of this, a-galactosidases, capable of catalyzing hydrolysis as well as transglycosylation of a-galacto-oligosaccharides, have only been studied in four bifidobacterial species, i.e. B. adolescentis [62,146], B. bifidum [38], B. breve [167] and B. longum subsp. longum [31].…”

Metabolism of Four α-Glycosidic Linkage-Containing Oligosaccharides by Bifidobacterium breve UCC2003

“…Stachyose and raffinose (sugars of the so-called raffinose family, which also includes verbascose) are present in a wide variety of plants (20), while the related sugar melibiose (though not a member of the raffinose family) is also found in many plants and is particularly abundant in soybean roots and stems (21). To metabolize such ␣-galacto-oligosaccharides, bifidobacteria require ␣-galactosidase enzyme activity, which has been identified and characterized in five bifidobacterial species or strains: Bifidobacterium bifidum JCM 1254 (22), Bifidobacterium adolescentis (23,24), Bifidobacterium bifidum NCIMB 41171 (25), Bifidobacterium breve 203 (26), and Bifidobacterium longum subsp. longum (27,28).…”