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Thermobifida fusca cellulases exhibit limited surface diffusion on bacterial micro-crystalline cellulose

Authors

  • Jose M. Moran-Mirabal,

    Corresponding author
    1. Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York 14850; telephone: 607-255-5544; fax: 607-255-4080,
    2. Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario, Canada L8S 4M1; telephone: 905-525-9140 ext. 24507; fax: 905-522-2509
    • Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York 14850; telephone: 607-255-5544; fax: 607-255-4080,.
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  • Jacob C. Bolewski,

    1. Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York 14850; telephone: 607-255-5544; fax: 607-255-4080,
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  • Larry P. Walker

    Corresponding author
    1. Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York 14850; telephone: 607-255-5544; fax: 607-255-4080,
    • Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York 14850; telephone: 607-255-5544; fax: 607-255-4080,.
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Abstract

Elucidation of cellulase–cellulose interactions is key to modeling biomass deconstruction and in understanding the processes that lead to cellulase inactivation. Here, fluorescence recovery after photobleaching and single molecule tracking (SMT) experiments are used to assess the surface diffusion of Thermobifida fusca cellulases on bacterial micro-crystalline cellulose. Our results show that cellulases exhibit limited surface diffusion when bound to crystalline cellulose and that a large fraction of the cellulases remain immobile even at temperatures optimal for catalysis. A comparison of our experimental results to Monte Carlo (MC) simulations, which use published diffusion coefficients to model cellulase displacements, shows that even those enzymes that are mobile on the cellulose surface exhibit significantly slower diffusive motions than previously reported. In addition, it is observed that the enzymes that show significant displacements exhibit complex, non-steady surface motions, which suggest that cellulose–bound cellulases exist in molecular states with different diffusive characteristics. These results challenge the notion that cellulases can freely diffuse over cellulose surfaces without catalyzing bond cleavage. Biotechnol. Bioeng. 2013; 110: 47–56. © 2012 Wiley Periodicals, Inc.

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