title: Engineering Cel7A carbohydrate binding module and linker for reduced lignin inhibition.
abstract: Non-productive binding of cellulases to lignin inhibits enzymatic hydrolysis of biomass, increasing enzyme requirements and the cost of biofuels. This study used site-directed mutagenesis of the Trichoderma 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. CBM mutations that added hydrophobic or positively charged residues decreased the specificity for cellulose, while mutations that added negatively charged residues increased the specificity. Linker mutations that altered predicted glycosylation patterns selectively impacted lignin affinity. Beneficial mutations were combined to generate a mutant with 2.5-fold less lignin affinity while fully retaining cellulose affinity. 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. Biotechnol. Bioeng. 2016;113: 1369-1374. © 2015 Wiley Periodicals, Inc.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, 94720.
authors.forename: Kathryn L
authors.initials: KL
authors.lastname: Strobel
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, 94720.
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Pfeiffer
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, 94720.
authors.forename: Harvey W
authors.initials: HW
authors.lastname: Blanch
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, 94720. clark@berkeley.edu.
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Clark
mesh_terms: D000327:Adsorption; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics*; D002482:Cellulose / Q000737:chemistry* / Q000235:genetics; D003432:Cross-Linking Reagents / Q000737:chemistry; D004789:Enzyme Activation; D006868:Hydrolysis; D008031:Lignin / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
chemical_list: D003432:Cross-Linking Reagents; D002482:Cellulose; D008031:Lignin; D002480:Cellulase
keywords: biomass conversion; cellulase; lignin; protein engineering