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Purification and partial characterization of an acid β-fructosidase from sweet-pepper (Capsicum annuum L.) fruit

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Abstract

The present study describes the biochemical characteristics of an acid β-fructosidase (EC 3.2.1.26) purified from the fruit of sweet pepper (Capsicum annuum L.). The soluble form, which constitutes more than 95% of the total activity at pH 4.5, hydrolyzes sucrose, raffinose, and stachyose. Its pH and temperature optima are 4.5 and 55 °C, respectively. Metal cations such as Ag+ and Hg2+ strongly inhibit its activity, suggesting the presence of at least one sulfhydryl group at the catalytic site. After purification of the enzyme by means of ammonium sulfate fractionation, gel chromatography (diethyl-aminoethyl-Sephacel, hydroxylapatite, concanavalin A-Sepharose), and preparative gel electrophoresis, the purified enzyme was shown to be a 42 kDa glycoprotein interacting specifically with concanavalin A. After complete chemical deglycosylation with trifluoromethanesulfonic acid, the molecular weight of the constitutive polypeptide was estimated to be 39 kDa. The enzyme glycans were characterized using both affino- and immunodetection. The enzyme has at least two N-linked oligosaccharide sidechains, one of the high-mannose type, and the other of the complex type. The high-mannose glycan has a low molecular weight (1 kDa), and is responsible for the interaction between the enzyme and concanavalin A. The complex-type glycan has an estimated molecular weight of 2 kDa. It contains one β1 → 2-linked xylose residue, probably one fucose residue α 1 → 3-linked to the chitobiose unit, and no terminal galactose residue. The two glycans, associated to the 39 kDa polypeptide, constitute the acid β-fructosidase of the sweet-pepper fruit.

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Abbreviations

βF:

β-fructosidase

ConA:

concanavalin A

DEAE:

diethylaminoethyl

DTNB:

dithionitrobenzoic acid

endo F:

endo-β-N-acetylglucosamidase F

endo H:

endo-β-N-acetylglucosamidase H

NEM:

N-ethylmaleimide

PCMB:

parachloromercurobenzoate

PNGase:

glycopeptide-N-glycosidase

TFMS:

trifluoromethane sulfonic acid

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Authors and Affiliations

  1. Département de phytologie, FSAA, Université Laval, G1K 7P4, Québec, Canada

    D. Michaud & S. Yelle

  2. Laboratoire des Transports Intracellulaires, UFR des Sciences, Université de Rouen, CNRS URA 203, B.P. 118, F-76821, Mont Saint-Aignan, France

    A. Seye, A. Driouich & L. Faye

Authors
  1. D. Michaud
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  2. A. Seye
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  3. A. Driouich
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  4. S. Yelle
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  5. L. Faye
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Additional information

This work was partly supported by a grant from the Commission Permanente de Coopération Franco-Québécoise to L. Faye, and S. Yelle. D. Michaud was a recipient of a graduate scholarship from the Natural Science and Engineering Research Council of Canada.

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Michaud, D., Seye, A., Driouich, A. et al. Purification and partial characterization of an acid β-fructosidase from sweet-pepper (Capsicum annuum L.) fruit. Planta 191, 308–315 (1993). https://doi.org/10.1007/BF00195687

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  • DOI: https://doi.org/10.1007/BF00195687

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