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Negatively Supercharging Cellulases Render Them Lignin-Resistant

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† Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, Michigan 48824, United States
‡ Department of Biosystems and Agricultural Engineering, Michigan State University, East Lansing, Michigan 48824, United States
§ Department of Chemical & Biochemical Engineering, Rutgers University, Piscataway, New Jersey 08854, United States
∥ Department of Biomedical Engineering, Rutgers University, Piscataway, New Jersey 08854, United States
*Phone: (517) 432-2097. E-mail: [email protected]
*Phone: (848)-445-3678. E-mail: [email protected]
Cite this: ACS Sustainable Chem. Eng. 2017, 5, 7, 6247–6252
Publication Date (Web):May 22, 2017
https://doi.org/10.1021/acssuschemeng.7b01202
Copyright © 2017 American Chemical Society
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    Abstract

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    Nonspecific adsorption of cellulases to lignin hinders enzymatic biomass deconstruction. Here, we tested the hypothesis that negatively supercharging cellulases could reduce lignin inhibition. Computational design was used to negatively supercharge the surfaces of Ruminoclostridiumthermocellum family 5 CelE and a CelE-family 3a carbohydrate binding module fusion. Resulting designs maintained the same expression yield, thermal stability, and nearly identical activity on soluble substrate as the wild-type proteins. Four designs showed complete lack of inhibition by lignin but with lower cellulose conversion compared to original enzymes. Increasing salt concentrations could partially rescue the activity of supercharged enzymes, supporting a mechanism of electrostatic repulsion between designs and cellulose. Results showcase a protein engineering strategy to construct highly active cellulases that are resistant to lignin-mediated inactivation, although further work is needed to understand the relationship between negative protein surface potential and activity on insoluble polysaccharides.

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b01202.

    • DNA and amino acid sequences of all proteins and effect of pH on CelE-CD hydrolysis yield for PASC (PDF)

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