Charge engineering of cellulases improves ionic liquid tolerance and reduces lignin inhibition

• Published in: Biotechnology and bioengineering 2014-08, Vol.111 (8), p.1541-1549
• Main Authors: Nordwald, Erik M., Brunecky, Roman, Himmel, Michael E., Beckham, Gregg T., Kaar, Joel L.
• Format: Article
• Language: English
• Subjects: 1-butyl-3-methylimidazolium chloride; biofuels; Biomass; biomass conversion; Cellulase; Cellulases - chemistry; Cellulases - genetics; Cellulases - metabolism; Cellulose; Cellulose - metabolism; Charge; Conversion; Correlation analysis; enzyme engineering; Enzymes; Hydrolysis; Hypocrea jecorina; Imidazoles - metabolism; Inhibition; Ionic liquids; Ionic Liquids - metabolism; Lignin; Lignin - metabolism; Protein Engineering; Static Electricity; Tolerances; Trichoderma - enzymology; Trichoderma - genetics; Trichoderma - metabolism; Trichoderma reesei cellulase
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.25216

ABSTRACT We report a novel approach to concurrently improve the tolerance to ionic liquids (ILs) as well as reduce lignin inhibition of Trichoderma reesei cellulase via engineering enzyme charge. Succinylation of the cellulase enzymes led to a nearly twofold enhancement in cellulose conversion in 15% (v/v) 1‐butyl‐3‐methylimidazolium chloride ([BMIM][Cl]). The improvement in activity upon succinylation correlated with the apparent preferential exclusion of the [Cl] anion in fluorescence quenching assays. Additionally, modeling analysis of progress curves of Avicel hydrolysis in buffer indicated that succinylation had a negligible impact on the apparent KM of cellulase. As evidence of reducing lignin inhibition of T. reesei cellulase, succinylation resulted in a greater than twofold increase in Avicel conversion after 170 h in buffer with 1 wt% lignin. The impact of succinylation on lignin inhibition of cellulase further led to the reduction in apparent KM of the enzyme cocktail for Avicel by 2.7‐fold. These results provide evidence that naturally evolved cellulases with highly negative surface charge densities may similarly repel lignin, resulting in improved cellulase activity. Ultimately, these results underscore the potential of rational charge engineering as a means of enhancing cellulase function and thus conversion of whole biomass in ILs. Biotechnol. Bioeng. 2014;111: 1541–1549. © 2014 Wiley Periodicals, Inc. Modification of the density of negative surface charges on Trichoderma reesei cellulase increases the conversion of cellulose by preventing denaturing interactions with ionic liquids and reducing lignin inhibition.