Characterization of four β-glucosidases acting on isoflavone-glycosides from Bifidobacterium pseudocatenulatum IPLA 36007
2017
Guadamuro, Lucía | Flórez, Ana Belén | Alegría, Ángel | Vázquez, Lucía | Mayo, Baltasar
Bifidobacterium pseudocatenulatum IPLA 36007 acts on isoflavone glycosides, releasing their corresponding aglycones. This strain-specific activity might be a key step in making isoflavones bioavailable and harnessing their oestrogenic activity. To investigate the molecular mechanisms involved in this activity, four glycosyl hydrolase-encoding genes in the IPLA 36007 genome (AW18_01575, AW18_09810, AW18_08145, and AW18_08090) were selected, synthesized with heterologous promoter and terminator signals (r-β-gluA, r-β-gluB, r-β-gluD and r-β-gluE, respectively), cloned into Escherichia coli, overexpressed as His-tagged proteins, and the enzymes purified and characterized. All four enzymes – GluAHis, GluBHis, GluDHis and GluEHis – proved to have β-glucosidase activity and deglycosylated (although at different rates) the isoflavone glycosides daidzin and genistin, releasing the aglycone moieties daidzein and genistein, respectively. GluDHis and GluEHis were also shown to hydrolyse β-glucosyl disaccharides such as cellobiose and gentiobiose, while GluAHis and GluBHis did not. Differences in activity were recorded for all four β-glucosidases at different pHs and temperatures under otherwise similar assay conditions, suggesting they have complementary activities under different environmental conditions. Two of the recombinant genes, r-β-gluA, and r-β-gluD, were cloned and expressed in the model lactic acid bacterium Lactococcus lactis, suggesting starter and probiotic organisms could be endowed with β-glucosidase activity. B. pseudocatenulatum IPLA 36007 contains additional β-glucosidases to those studied in this work, indicating a high level of redundancy for this enzymatic activity. Knowledge of glycoside-degrading enzymes should facilitate the development of novel, more effective or more selective prebiotics or functional foods for the promotion of bifidobacterial numbers in the human gut. It might also be of interest in the development of novel probiotics with specific health-promoting activities.
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