Affiliation:
1. Institut für Pharmazeutische Biologie, Philipps-Universität Marburg, Deutschhausstr. 17A, 35037 Marburg/Lahn, Germany
2. AZTI-Tecnalia/Food Research Division, Parque Tecnológico de Bizkaia, Astondo Bidea 609, 48160 Derio, Spain
Abstract
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.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
55 articles.
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