Functional expression of human liver cytosolic β-glucosidase in Pichia pastoris

Insights into its role in the metabolism of dietary glucosides

Authors

  • Jean-Guy Berrin,

    1. Nutrition, Health and Consumer Sciences Division, Institute of Food Research, Norwich, UK;
    2. Institut Méditerranéen de Recherche en Nutrition, Faculté des Sciences et Techniques de Saint-Jérôme, Marseilles, France
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  • W. Russell McLauchlan,

    1. Nutrition, Health and Consumer Sciences Division, Institute of Food Research, Norwich, UK;
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  • Paul Needs,

    1. Nutrition, Health and Consumer Sciences Division, Institute of Food Research, Norwich, UK;
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  • Gary Williamson,

    1. Nutrition, Health and Consumer Sciences Division, Institute of Food Research, Norwich, UK;
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  • Antoine Puigserver,

    1. Institut Méditerranéen de Recherche en Nutrition, Faculté des Sciences et Techniques de Saint-Jérôme, Marseilles, France
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  • Paul A. Kroon,

    1. Nutrition, Health and Consumer Sciences Division, Institute of Food Research, Norwich, UK;
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  • Nathalie Juge

    1. Nutrition, Health and Consumer Sciences Division, Institute of Food Research, Norwich, UK;
    2. Institut Méditerranéen de Recherche en Nutrition, Faculté des Sciences et Techniques de Saint-Jérôme, Marseilles, France
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P. A. Kroon, Nutrition, Health & Consumer Sciences Division, Institute of Food Research, Colney Lane, Norwich, NR4 7UA, UK. Fax: + 44 1603 255038, Tel.: + 44 1603 255236, E-mail: paul.kroon@bbsrc.ac.uk

Abstract

Human tissues such as liver, small intestine, spleen and kidney contain a cytosolic β-glucosidase (CBG) that hydrolyses various β-d-glycosides, but whose physiological function is not known. Here, we describe the first heterologous expression of human CBG, a system that facilitated␣a detailed assessment of the enzyme specificity towards dietary glycosides. A full-length CBG cDNA (cbg-1) was cloned from a human liver cDNA library and expressed in the methylotrophic yeast Pichia pastoris at a secretion yield of ≈ 10 mg·L−1. The recombinant CBG (reCBG) was purified from the supernatant using a single chromatography step and was shown to be similar to the native enzyme isolated from human liver in terms of physical properties and specific activity towards 4-nitrophenyl-β-d-glucoside. Furthermore, the reCBG displayed a broad specificity with respect to the glycone moiety of various aryl-glycosides (β-d-fucosides, α-l-arabinosides, β-d-glucosides, β-d-galactosides, β-l-xylosides, β-d-arabinosides), similar to the native enzyme. For the first time, we show that the human enzyme has significant activity towards many common dietary xenobiotics including glycosides of phytoestrogens, flavonoids, simple phenolics and cyanogens with higher apparent affinities (Km) and specificities (kcat/Km) for dietary xenobiotics than for other aryl-glycosides. These data indicate that human CBG hydrolyses a broad range of dietary glucosides and may play a critical role in xenobiotic metabolism.

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