Studying the Effect of Cellulase Supercharging on Cellulosic Biomass Hydrolysis

Lignocellulosic biomass, composed of cellulose, hemicellulose and lignin, is broken down to simple sugars in nature by enzyme cocktails secreted by various microbes. Glucose, produced by the breakdown of cellulose mediated by cellulase enzymes, can be fermented to ethanol and used as transportation fuel. However, the cost of enzymes is a bottleneck limiting wider commercial adoption of cellulosic biofuels. Non-productive binding of enzymes to lignin and cellulose, is proposed to be a key deterrent to cellulose hydrolysis. Recently, we had shown that supercharging cellulase enzymes through modification of surface residues is an effective strategy to reduce inhibition of cellulase activity by lignin (Whitehead et al., ACS Sustainable Chem. Eng. 2017, 5, 7, 6247–6252). However, supercharged enzymes in this study showed a significant reduction in activity compared to wild-type enzymes despite desirable performance in terms of lignin inhibition. To address this gap in our knowledge of how supercharged enzymes function, we designed a library of mutants for carbohydrate-binding module (CBM) family 2 proteins found naturally associated with an endocellulase Cel5A (from Thermobifida fusca). These mutant CBMs were attached to GFP reporter fuorescent protein to study binding to cellulose and lignin. In addition, the mutants were fused with wild-type Cel5A (T. fusca) catalytic domain to understand the impact of CBM supercharging on enzyme hydrolytic activity on pure cellulose and pretreated lignocellulosic biomass substrates. Here, we report our computational design strategy for generation of the mutant library and binding/activity results which present a comprehensive picture of protein supercharging as a potential strategy to alleviate enzyme non-productive binding to cellulosic biomass.