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Combined Mass Mapping and Biochemical Characterization of Grape β-Glycosidase-enriched Extract

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Abstract

A β-glucosidase enzyme activity was enriched from skins of ripe grape berry by cell wall fractionation, hydrophobic interaction and cation-exchange chromatographies. This enriched enzyme extract contained several β-glycosidase activities hydrolyzing a wide range of synthetic and natural monoglycosides and diglycosides, as well as a β-fructosidase activity. The enzyme extract was further characterized by two-dimensional gel electrophoresis coupled to peptide mass fingerprinting of eight spots using MALDI-TOF mass spectrometry. No β-glucosidase but a β-fructosidase associated to the relevant spot at 66 kDa/pI 5.1 was identified. Taken together all results issued from the biochemical characterization, the substrate specificity and the mass spectrometry-based identification of this enriched enzyme extract, we propose that this protein could be a specific β-fructosidase isoform associated with a broad spectrum of β-glycosidase activities in grape berry skin and involved in cell wall modifications which occur during the ripening-induced thickness of the grape.

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Abbreviations

2-D:

Two-dimensional

DTT:

Dithiothreitol

EDTA:

Ethylenediaminetetraacetic acid

FPLC:

Fast protein liquid chromatography

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

IEF:

Isoelectrofocusing

IPG:

Immobilized pH gradient

HPLC:

High performance liquid chromatography

MALDI-TOF:

Matrix assisted laser desorption/ionization-time of flight

MS:

Mass spectrometry

PEG:

Polyethylene glycol

PMF:

Peptide mass fingerprinting

PMSF:

Phenylmethylsulphonyl fluoride

p-NP:

p-Nitrophenol

PSD:

Post-source decay

PVP:

Polyvinylpyrrolidone

TLC:

Thin layer chromatography

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Acknowledgements

This work was supported by the “Région Languedoc-Roussillon” and by “Institut National de Recherche Agronomique”. The authors thank Prof. Watanabe (University of Shizuoka, Japan) and Bernard Astruc (Listel-Jarras vineyard, Aigues-Mortes, France) for grateful gift of primeveroside substrates and grape berries of Muscat, respectively. We wish also to thank Dr Gil Morrot (INRA, Montpellier) for his support, Dr Bernard Henrissat (CNRS, Marseille) for ‘fruitful’ discussions and Drs. Simon Knight and David Schooley for critical reading of the manuscript.

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Authors and Affiliations

  1. UMR UMI/ENSAM/INRA Science Pour l’Oenologie, INRA, 2 Place Pierre Viala, F-34060, Montpellier Cedex 1, France

    Jean-Emmanuel Sarry, Jérôme Grimplet, Marie-José Vallier, Martine Pradal & Charles Romieu

  2. University of Pennsylvania School of Medicine, Philadelphia, PA, 19104, USA

    Jean-Emmanuel Sarry

  3. Laboratoire de Protéomique, INRA, 2 Place Pierre Viala, F-34060, Montpellier Cedex 1, France

    Nicolas Sommerer

  4. UPR 9056 UMI/CNRS Laboratoire de Botanique, Mycologie & Phytochimie, Faculté de Pharmacie, Université Montpellier I, Avenue Charles Flahault, F-34060, Montpellier Cedex 2, France

    Laurence Mondolot & Claude Andary

  5. UMR UMII/ENSAM/INRA Ingénierie des Réactions Biologiques-Bioproductions, Université Montpellier II, Place Eugène Bataillon, F-34095, Montpellier Cedex 5, France

    Ziya Günata

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  1. Jean-Emmanuel Sarry
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Correspondence to Jean-Emmanuel Sarry.

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Sarry, JE., Grimplet, J., Sommerer, N. et al. Combined Mass Mapping and Biochemical Characterization of Grape β-Glycosidase-enriched Extract. Protein J 27, 258–266 (2008). https://doi.org/10.1007/s10930-008-9134-3

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