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Computational Library Design for Increasing Haloalkane Dehalogenase Stability

Authors

  • Robert J. Floor,

    1. Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen (The Netherlands)
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    • These authors contributed equally to this work.

  • Dr. Hein J. Wijma,

    1. Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen (The Netherlands)
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    • These authors contributed equally to this work.

  • Dana I. Colpa,

    1. Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen (The Netherlands)
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  • Dr. Aline Ramos-Silva,

    1. Department of Biotechnology, Institute of Biomedical Sciences, University of Sao Paulo, Av. Prof. Lineu Prestes, 2415. Sao Paulo (Brazil)
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  • Peter A. Jekel,

    1. Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen (The Netherlands)
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  • Dr. Wiktor Szymański,

    1. Center for Systems Chemistry, Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen (The Netherlands)
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  • Prof. Dr. Ben L. Feringa,

    1. Center for Systems Chemistry, Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen (The Netherlands)
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  • Prof. Dr. Siewert J. Marrink,

    1. Department of Biophysical Chemistry, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 7, 9747 AG Groningen (The Netherlands)
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  • Prof. Dr. Dick B. Janssen

    Corresponding author
    1. Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen (The Netherlands)
    • Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen (The Netherlands)===

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Abstract

We explored the use of a computational design framework for the stabilization of the haloalkane dehalogenase LinB. Energy calculations, disulfide bond design, molecular dynamics simulations, and rational inspection of mutant structures predicted many stabilizing mutations. Screening of these in small mutant libraries led to the discovery of seventeen point mutations and one disulfide bond that enhanced thermostability. Mutations located in or contacting flexible regions of the protein had a larger stabilizing effect than mutations outside such regions. The combined introduction of twelve stabilizing mutations resulted in a LinB mutant with a 23 °C increase in apparent melting temperature (Tm,app, 72.5 °C) and an over 200-fold longer half-life at 60 °C. The most stable LinB variants also displayed increased compatibility with co-solvents, thus allowing substrate conversion and kinetic resolution at much higher concentrations than with the wild-type enzyme.

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