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Engineering Cel7A carbohydrate binding module and linker for reduced lignin inhibition
Author(s) -
Strobel Kathryn L.,
Pfeiffer Katherine A.,
Blanch Harvey W.,
Clark Douglas S.
Publication year - 2016
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.25889
Subject(s) - cellulase , lignin , chemistry , cellulose , carbohydrate binding module , trichoderma reesei , biochemistry , hydrolysis , linker , directed evolution , protein engineering , glycoside hydrolase , thermostability , enzyme , mutant , site directed mutagenesis , organic chemistry , computer science , gene , operating system
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.