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Characterization of hyperbranched glycopolymers produced in vitro using enzymes

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Abstract

Asymmetrical flow field flow fractionation (AF4) has proven to be a very powerful and quantitative method for the determination of the macromolecular structure of high molar mass branched biopolymers, when coupled with multi-angle laser light scattering (MALLS). This work describes a detailed investigation of the macromolecular structure of native glycogens and hyperbranched α-glucans (HBPs), with average molar mass ranging from 2 × 106 to 4.3 × 107 g mol−1, which are not well fractionated by means of classical size-exclusion chromatography. HBPs were enzymatically produced from sucrose by the tandem action of an amylosucrase and a branching enzyme mimicking in vitro the elongation and branching steps involved in glycogen biosynthesis. Size and molar mass distributions were studied by AF4, coupled with online quasi-elastic light scattering (QELS) and transmission electron microscopy. AF4-MALLS-QELS has shown a remarkable agreement between hydrodynamic radii obtained by online QELS and by AF4 theory in normal mode with constant cross flow. Molar mass, size, and dispersity were shown to significantly increase with initial sucrose concentration, and to decrease when the branching enzyme activity increases. Several populations with different size range were observed: the amount of small size molecules decreasing with increasing sucrose concentration. The spherical and dense global conformation thus highlighted was partly similar to native glycogens. A more detailed study of HBPs synthesized from low and high initial sucrose concentrations was performed using complementary enzymatic hydrolysis of external chains and chromatography. It emphasized a more homogeneous branching pattern than native glycogens with a denser core and shorter external chains.

Characterization of hyperbranched glycopolymers. TEM Transmission electron microscopy. AF4-MALLS-QELS Asymmetrical flow field flow fractionation coupled with multi-angle laser-light scattering and quasi-elastic light scattering

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Acknowledgments

The authors thank the Agence Nationale de la Recherche for financial support (grant number ANR-09-CP2D-07-01), as well as Marion De Carvahlo, Bruno Pontoire and Nelly Monties for technical assistance.

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Authors and Affiliations

  1. UR1268 Biopolymères Interactions Assemblages, INRA, F-44300, Nantes, France

    Agnès Rolland-Sabaté, Sophie Guilois & Alain Buléon

  2. Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, 31077, Toulouse, France

    Florent Grimaud, Sandrine Laguerre & Gabrielle Potocki-Véronèse

  3. INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, 31400, Toulouse, France

    Florent Grimaud, Sandrine Laguerre & Gabrielle Potocki-Véronèse

  4. CNRS, UMR5504, 31400, Toulouse, France

    Florent Grimaud, Sandrine Laguerre & Gabrielle Potocki-Véronèse

  5. CERMAV-CNRS (affiliated with Université Joseph Fourier, member of Institut de Chimie Moléculaire de Grenoble and Institut Carnot PolyNat), BP 53, 38041, Grenoble cedex 9, France

    Christine Lancelon-Pin & Jean-Luc Putaux

  6. UGSF, UMR 8576 CNRS—Université Lille 1, sciences et technologies, Bât. C9, 59655, Villeneuve d’Ascq, France

    Xavier Roussel & Christophe D’Hulst

  7. Novozymes A/S, Krogshoejvej, 36, 2880, Bagsvaerd, Denmark

    Anders Viksø-Nielsen

Authors
  1. Agnès Rolland-Sabaté
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  2. Sophie Guilois
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  3. Florent Grimaud
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  4. Christine Lancelon-Pin
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  5. Xavier Roussel
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  6. Sandrine Laguerre
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  7. Anders Viksø-Nielsen
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  8. Jean-Luc Putaux
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  9. Christophe D’Hulst
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  10. Gabrielle Potocki-Véronèse
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  11. Alain Buléon
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Corresponding author

Correspondence to Agnès Rolland-Sabaté.

Additional information

Published in the topical collection Field-Flow Fractionation with guest editors S. Kim R. Williams and Karin D. Caldwell.

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Rolland-Sabaté, A., Guilois, S., Grimaud, F. et al. Characterization of hyperbranched glycopolymers produced in vitro using enzymes. Anal Bioanal Chem 406, 1607–1618 (2014). https://doi.org/10.1007/s00216-013-7403-2

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  • DOI: https://doi.org/10.1007/s00216-013-7403-2

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