Negatively Supercharging Cellulases Render Them Lignin-Resistant

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 Ruminoclostridium thermocellum family 5 CeIE and a CeIE-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.