Bakers yeast (Saccharomyces cerevisiae) whole-cell bioconversions of naringenin 7-O-beta- glucoside revealed considerable beta-glucosidase activity which impairs any strategy to generate or modify flavonoid glucosides in yeast transformants. Up to ten 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 alpha- or beta-glucosides identified three efficient beta-glucosidases (EXG1, SPR1 and YIR007W) which were further assayed with natural flavonoid beta-glucoside substrates and product verification by TLC or 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 or SPR1 were completely abolished by Val168Tyr mutation, which confirmed the relevance of this residue as reported for other glucosidases. Most importantly, biotransformation experiments with knock-out yeast strains revealed that solely EXG1 knock-out strains lost the capability of hydrolyzing flavonoid glucosides.
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