Characterization of glycosyl-hydrolases acting on isoflavones from Bifidobacterium pseudocatenulatum IPLA 36007

Trabajo presentado en el 1er Congreso Iberoamericano de Biotecnología: "Bio.Iberoamérica 2016. Biotecnología Integrando Continentes”, celebrado en Salamanca (España) entre el 5 y el 8 de junio de 2016.Glycosidases (glycosyl hydrolases) are a wide and varied group of enzymes able to hydrolyse the glycosidic linkage between two or more sugars or one sugar and a chemical residue of other nature. Bifidobacteria species are currently being used as probiotics for the maintenance of a healthy status. Bifidobacteria have their natural niche in the human and animal gastrointestinal tracts. Glycolytic activities of probiotic bifidobacteria are thought to have a prominent role for these bacteria to colonize and survive in the intestinal ecosystem. Additionally, these enzymatic activities might contribute to their probiotic functionality. Objectives Bifidobacterium pseudocatenulatum IPLA 36007 is a strain from human origin showing deglycosylation activity on isoflavone-glycosides, an essential step for isoflavones to become bioavailable and the subsequent formation of more active compounds such as equol. The genome of IPLA 36007 has recently been sequenced (Alegría et al., 2014) allowing to investigate its genetic potential. Genome analysis showed a vast array of genes encoding glycosyl hydrolases, of which some were tentatively classified as ß-glucosidases. This study aimed to identify and characterize ß-glucosidases from IPLA 36007 acting on isoflavone-glycosides. Results and Discussion Five genes encoding putative ß-glucosidases (Glu-1 through Glu-5) were synthetized under the control of a heterologous promoter working on bifidobacteria and cloned in a pUC19-derived vector. Expression in Escherichia coli proved that four of the genes encode glycosidases, of which three were considered ß-glucosidases (Glu-1, Glu-2 and Glu-4) and one an ¿-glucosidase (Glu-3). The three ß-glucosidases showed activity towards the isoflavone glycoside daidzin producing daidzein. Activity of Glu-4 was shown to be 6- to 10-fold higher than that of Glu-1 and Glu-2, respectively. The genetic determinants were subsequently transferred to pET28a(+), a His-Tag vector designed for convenient cloning and expression of genes, and purification of the corresponding proteins. Recombinant enzymes were purified by immobilized metal affinity chromatography and characterized. The main biochemical properties of the two ß-glucosidases will be presented in the poster.Peer reviewe