Shigehisa Akira, Sotoya Hidetsugu, Sato Takashi, Hara Taeko, Matsumoto Hoshitaka, Matsuki Takahiro
Yakult Central Institute, 5-11 Izumi, Kunitachi, Tokyo 186-8650, Japan.
Microbiology (Reading). 2015 Jul;161(7):1463-70. doi: 10.1099/mic.0.000100. Epub 2015 Apr 22.
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 Bifidobacterium breve strain Yakult. We attempted to identify B. breve 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.
低聚半乳糖（GOS）OLIGOMATE 55N（养乐多）是一种低聚糖混合物，其主要成分是4'-半乳糖基乳糖（4'-GL）。大量报告表明，低聚半乳糖不可消化，可到达结肠并选择性刺激双歧杆菌的生长。该产品已被用作食品成分，其应用迅速扩大。然而，负责利用低聚半乳糖的双歧杆菌糖苷水解酶和转运蛋白尚未得到充分表征。在本研究中，我们旨在鉴定和表征短双歧杆菌养乐多菌株中负责代谢4'-GL的基因。我们试图在含有4'-GL的基础培养基中生长期间，使用定制微阵列通过转录谱分析来鉴定由4'-GL诱导的短双歧杆菌养乐多基因。我们发现，编码溶质结合蛋白（SBP）的BbrY_0420和编码β-半乳糖苷酶的BbrY_0422相对于在含有乳糖的基础培养基中生长时显著上调。使用表面等离子体共振对重组BbrY_0420蛋白的底物特异性进行研究表明，BbrY_0420蛋白与4'-GL结合，但不与4'-GL的结构异构体3'-GL和6'-GL结合。此外，BbrY_0420对4-半乳糖二糖（4-GB）具有很强的亲和力，表明该SBP识别4'-GL的非还原末端结构。将纯化的重组BbrY_0422蛋白与4'-GL、3'-GL、6'-GL和4-GB一起孵育表明，该蛋白有效地水解了4'-GL和4-GB，但不消化3'-GL、6'-GL或乳糖，这表明BbrY_0422水解了Gal1,4-β-Gal内的键。因此，BbrY_0420（SBP）和BbrY_0422（β-半乳糖苷酶）具有相同、严格的底物特异性，表明它们通过共诱导偶联以促进4'-GL的转运和水解。
