Enzymatic hydrolysis is a crucial step in the conversion of cellulosic biomass to bioethanol. Glycosyl hydrolases (GH) like cellulases are enzymes that catalyze the hydrolysis of polysaccharides such as cellulose to monosaccharides. Lignocellulosic biomass, such as corn stover, is readily available and can be converted into biofuels. While lignocellulosic biomass is composed of both polysaccharides and lignin, only binding to polysaccharides results in useful products. Thus, it is desired that the Carbohydrate Binding Module (CBM) protein domain of glycosyl hydrolases have maximal productive binding to cellulose and minimal non-productive binding to lignin. Our group has recently shown that negatively supercharging enzymes surfaces is effective in reducing enzyme binding to lignin (Whitehead 2017). However, such supercharging also results in a slight reduction of reducing sugar yields due to reduced cellulose binding, which is not desired for the purpose of obtaining glucose. To address this gap in knowledge of how supercharged enzymes work, a library of mutants for CBM family 2 was designed and these CBM mutants were cloned simultaneously into both an endo-cellulase (Cel5A from T. fusca) and an exo-cellulase fusion protein vector (Cel6B from T. fusca). The synergistic activities of the CBM2a fused Cel5A and Cel6B enzymes reveals information regarding the potential for an effective cellulase enzyme cocktail that can efficiently convert lignocellulosic biomass to bioethanol while reducing inhibition due to non-productive lignin binding.

T.A. Whitehead, C. K. Bandi, M. Berger, J. Park and S. P. S. Chundawat, ACS Sustain. Chem. Eng., 2017, 5, 6247–6252.