The galacto-oligosaccharide (GOS) OLIGOMATE 55N (Yakult) is a mixture of oligosaccharides, the main component of which is 4′-galactosyllactose (4′-GL). Numerous reports have shown that GOSs are non-digestible, reach the colon and selectively stimulate the growth of bifidobacteria. The product has been used as a food ingredient and its applications have expanded rapidly. However, the bifidobacterial glycoside hydrolases and transporters responsible for utilizing GOSs have not been characterized sufficiently. In this study, we aimed to identify and characterize genes responsible for metabolizing 4′-GL in strain Yakult. We attempted to identify Yakult genes induced by 4′-GL using transcriptional profiling during growth in basal medium containing 4′-GL with a custom microarray. We found that BbrY_0420, which encodes solute-binding protein (SBP), and BbrY_0422, which encodes β-galactosidase, were markedly upregulated relative to that during growth in basal medium containing lactose. Investigation of the substrate specificity of recombinant BbrY_0420 protein using surface plasmon resonance showed that BbrY_0420 protein bound to 4′-GL, but not to 3′-GL and 6′-GL, structural isomers of 4′-GL. Additionally, BbrY_0420 had a strong affinity for 4-galactobiose (4-GB), suggesting that this SBP recognized the non-reducing terminal structure of 4′-GL. Incubation of purified recombinant BbrY_0422 protein with 4′-GL, 3′-GL, 6′-GL and 4-GB revealed that the protein efficiently hydrolysed 4′-GL and 4-GB, but did not digest 3′-GL, 6′-GL or lactose, suggesting that BbrY_0422 digested the bond within Gal1,4-β-Gal. Thus, BbrY_0420 (SBP) and BbrY_0422 (β-galactosidase) had identical, strict substrate specificity, suggesting that they were coupled by co-induction to facilitate the transportation and hydrolysis of 4′-GL.


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