title: Identification of a Saccharomyces cerevisiae glucosidase that hydrolyzes flavonoid glucosides.
abstract: Baker's yeast (Saccharomyces cerevisiae) whole-cell bioconversions of naringenin 7-O-β-glucoside revealed considerable β-glucosidase activity, which impairs any strategy to generate or modify flavonoid glucosides in yeast transformants. Up to 10 putative glycoside hydrolases annotated in the S. cerevisiae genome database were overexpressed with His tags in yeast cells. Examination of these recombinant, partially purified polypeptides for hydrolytic activity with synthetic chromogenic α- or β-glucosides identified three efficient β-glucosidases (EXG1, SPR1, and YIR007W), which were further assayed with natural flavonoid β-glucoside substrates and product verification by thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC). Preferential hydrolysis of 7- or 4'-O-glucosides of isoflavones, flavonols, flavones, and flavanones was observed in vitro with all three glucosidases, while anthocyanins were also accepted as substrates. The glucosidase activities of EXG1 and SPR1 were completely abolished by Val168Tyr mutation, which confirmed the relevance of this residue, as reported for other glucosidases. Most importantly, biotransformation experiments with knockout yeast strains revealed that only EXG1 knockout strains lost the capability to hydrolyze flavonoid glucosides.
authors.affiliation: Fondazione Edmund Mach, Istituto Agrario San Michele all'Adige-IASMA, Centro Ricerca e Innovazione, Area Alimentazione, Via E. Mach 1, 38010 San Michele all'Adige (TN), Italy.
authors.forename: Sabine
authors.initials: S
authors.lastname: Schmidt
authors.forename: Sandra
authors.initials: S
authors.lastname: Rainieri
authors.forename: Simone
authors.initials: S
authors.lastname: Witte
authors.forename: Ulrich
authors.initials: U
authors.lastname: Matern
authors.forename: Stefan
authors.initials: S
authors.lastname: Martens
mesh_terms: D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D002863:Chromogenic Compounds / Q000378:metabolism; D005419:Flavonoids / Q000378:metabolism*; D017353:Gene Deletion; D005959:Glucosidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism*; D020125:Mutation, Missense; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
chemical_list: D002863:Chromogenic Compounds; D005419:Flavonoids; D005960:Glucosides; D005959:Glucosidases