Studying the Effect of CBM Supercharging on Endo-Exo Cellulase Synergism

Lignocellulosic biomass, composed of cellulose, hemicellulose and lignin, is broken down to simple sugars in nature by enzyme cocktails secreted by microbes. Glucose, produced by the breakdown of cellulose mediated by cellulase enzymes, can be used for fermentation to ethanol, which can be used as transportation fuel. However, the cost of enzymes is currently the bottleneck limiting wider commercial adoption of biofuels. Non-productive binding of enzymes to lignin and cellulose, is proposed to be a key deterrent to increased cellulose hydrolysis. Recently, collaborative work from our lab (Whitehead et al., 2017) has shown that supercharging cellulase enzymes through modification of surface residues to incorporate charged residues, is an effective strategy to reduce inhibition of cellulase activity by lignin. However, supercharged enzymes in this study showed a reduction in reducing sugar yields despite desirable performance in terms of lignin inhibition. To address this gap in our knowledge of how supercharged enzymes work, we designed a library of mutants for CBM family 2 using RosettaDesign and these CBM mutants were cloned simultaneously into an endo-cellulase (Cel5A from T.fusca) and an exo-cellulase vector (Cel6B from T.fusca). We report our work on the design, cloning, purification and preliminary work on characterization of the synergistic activities of wild-type enzymes followed by the mutants. This work has implications for designing efficient cellulase enzyme cocktails that have reduced lignin inhibition and hence lead to cost effective conversion of lignocellulosic biomass to biofuels.