A pH-Based High-Throughput Screening of Sucrose-Utilizing Transglucosidases for the Development of Enzymatic Glucosylation Tools

Authors

  • Elise Champion Dr.,

    1. Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse (France), CNRS, UMR5504, F-31400 Toulouse (France), INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse (France), Fax: (+33) 5 61 55 94 00
    Search for more papers by this author
    • These authors contributed equally to this work.

  • Claire Moulis Dr.,

    1. Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse (France), CNRS, UMR5504, F-31400 Toulouse (France), INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse (France), Fax: (+33) 5 61 55 94 00
    Search for more papers by this author
    • These authors contributed equally to this work.

  • Sandrine Morel Dr.,

    1. Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse (France), CNRS, UMR5504, F-31400 Toulouse (France), INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse (France), Fax: (+33) 5 61 55 94 00
    Search for more papers by this author
  • Laurence A. Mulard Dr.,

    1. Institut Pasteur, Unité de Chimie des Biomolécules, CNRS URA 2128, 28 rue du Dr. Roux, F-75015 Paris (France)
    Search for more papers by this author
  • Pierre Monsan Prof.,

    1. Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse (France), CNRS, UMR5504, F-31400 Toulouse (France), INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse (France), Fax: (+33) 5 61 55 94 00
    Search for more papers by this author
  • Magali Remaud-Siméon Prof.,

    1. Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse (France), CNRS, UMR5504, F-31400 Toulouse (France), INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse (France), Fax: (+33) 5 61 55 94 00
    Search for more papers by this author
  • Isabelle André Dr.

    1. Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse (France), CNRS, UMR5504, F-31400 Toulouse (France), INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse (France), Fax: (+33) 5 61 55 94 00
    Search for more papers by this author

Abstract

Sucrose-utilizing transglucosidases are valuable enzymatic tools for the diversification of carbohydrate-based molecules. Among them, recombinant amylosucrase from Neisseria polysaccharea is a glucansucrase that naturally catalyzes the synthesis of an amylose-like polymer as well as the transglucosylation of exogenous hydroxylated acceptors. A semirational engineering approach was recently undertaken to redesign the enzyme active site and adapt it to the glucosylation of a nonnatural acceptor, allyl 2-N-acetyl-2-deoxy-α-D-glucopyranoside (α-D-GlcpNAc[BOND]OAll), to produce a key building block in the chemoenzymatic synthesis of Shigella flexneri 1b serotype O-antigen repeating unit. This prior work shows the beneficial effect of single amino acid mutations at two positions (228 and 290) on the recognition of the acceptor by amylosucrase. On the basis of these first results, a library of about 8000 amylosucrase variants combining mutations at these two positions is constructed by saturation mutagenesis. The library is prescreened using a novel pH-sensitive colorimetric screening method for the detection of sucrose-utilizing amylosucrase variants, thereby reducing by about 95 % the size of the library to be subsequently screened for acceptor glucosylation. Active clones (5 % of the initial library) are then screened for acceptor recognition, leading to the isolation of 20 variants of potential interest for the production of the target disaccharide α-D-Glcp-(1→4)-α-D-GlcpNAc.

Ancillary