Engineering Cel7A carbohydrate binding module and linker for reduced lignin inhibition

The high cost of hydrolytic enzymes impedes the commercial production of lignocellulosic biofuels. High enzyme loadings are required in part due to their non-productive adsorption to lignin, a major component of biomass. Despite numerous studies documenting cellulase adsorption to lignin, few attempts have been made to engineer enzymes to reduce lignin binding. In this work, we used rational mutagenesis of the Trichoderma reesei Cel7A carbohydrate binding module (CBM) and linker to investigate the mechanisms of adsorption to lignin and engineer a cellulase with increased binding specificity for cellulose. We determined the main residues responsible for lignin adsorption, developed strategies for selectively reducing lignin affinity, and generated a mutant with 2.5-fold greater specificity for cellulose. This mutant was uninhibited by added lignin during hydrolysis of Avicel and generated 40% more glucose than the wild-type enzyme from dilute acid-pretreated Miscanthus.