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A single-molecule analysis reveals morphological targets for cellulase synergy

Nature Chemical Biology volume 9, pages 356–361 (2013)Cite this article

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Abstract

The mechanisms of enzyme activity on solid substrates are not well understood. Unlike enzyme catalysis in aqueous solutions, enzyme activity on surfaces is complicated by adsorption steps and structural heterogeneities that make enzyme-substrate interactions difficult to characterize. Cellulase enzymes, which catalyze the depolymerization of cellulose, show binding specificities for different cellulose surface morphologies, but the influence of these specificities on the activity of multienzyme mixtures has remained unclear. We developed a metric to quantify binding-target arrangements determined by photoactivated localization microscopy, and we used that metric to show that combinations of cellulases designed to bind within similar but nonidentical morphologies can have synergistic activity. This phenomenon cannot be explained with the binary crystalline or amorphous classifications commonly used to characterize cellulase-binding targets. Our results reveal a strategy for improving the activity of cellulolytic mixtures and demonstrate a versatile method for investigating protein organization on heterogeneous surfaces.

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Figure 1: Equilibrium binding patterns of CBMs bound to cellulose.
Figure 2: The CBM order parameter.
Figure 3: Order parameters and synergy values.

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Acknowledgements

We thank S. Bauer for his assistance in conducting the compositional analysis on cotton and miscanthus. We thank A.L. McEvoy for providing image processing software and J.W. Chu, A.S. Gross, K. Haas and A.L. McEvoy for helpful discussions. J.M.F. is the recipient of a US National Science Foundation predoctoral fellowship and J.L., H.W.B and D.S.C. acknowledge support from the Energy Biosciences Institute (grant no. 50000029463).

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Authors and Affiliations

  1. Energy Biosciences Institute, University of California, Berkeley, California, USA

    Jerome M Fox, Phillip Jess, Jan Liphardt, Douglas S Clark & Harvey W Blanch

  2. Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, USA

    Jerome M Fox, Douglas S Clark & Harvey W Blanch

  3. California Institute for Quantitative Biosciences (QB3), University of California–Berkeley, Berkeley, California, USA

    Phillip Jess & Jan Liphardt

  4. Department of Plant and Microbial Biology, University of California, Berkeley, California, USA

    Rakesh B Jambusaria

  5. Department of Chemistry, University of California, Berkeley, California, USA

    Genny M Moo

  6. Department of Physics, University of California, Berkeley, California, USA

    Jan Liphardt

  7. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Jan Liphardt

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Contributions

J.M.F. designed and built fusion constructs, carried out imaging and hydrolysis experiments, developed the mathematical analysis and wrote the paper. P.J. constructed the PALM microscope and assisted with imaging. R.B.J. and G.M.M. built fusion constructs. J.L., D.S.C. and H.W.B. analyzed data and wrote the paper.

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Correspondence to Jan Liphardt, Douglas S Clark or Harvey W Blanch.

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Fox, J., Jess, P., Jambusaria, R. et al. A single-molecule analysis reveals morphological targets for cellulase synergy. Nat Chem Biol 9, 356–361 (2013). https://doi.org/10.1038/nchembio.1227

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