Enhanced Activity and Stability of <i>Acidothermus cellulolyticus</i> Endoglucanase 1 in Ionic Liquids via Engineering Active Site Residues and Non-Native Disulfide Bridges
Samantha R. Summers, Casey J Kraft, Sarah Alamdari, Jim Pfaendtner, Joel L. Kaar
Abstract
We report the rational mutagenesis and engineering of endoglucanase 1 (E1) from Acidothermus cellulolyticus, an industrially relevant cellulase, for improved biomass conversion in ionic liquids. The utility of E1 was specifically improved by mutating active site residues, including 245 and 213, which led to up to a 2-fold increase in activity in 10% w/v 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]). Improvements in activity resulted from altering the balance of affinity between [BMIM][Cl] and the substrate for E1. Furthermore, stability studies revealed that the presence of a tyrosine at position 245 not only prevents competitive inhibition by [BMIM][Cl] but also stabilizes E1 (2.5-fold in 10% w/v [BMIM][Cl]) against [BMIM][Cl]-induced denaturation. These studies uncovered the previously unknown role of aggregation in the inactivation of E1, which was linked to structural fluctuations in the region of residues 120–150 and 160–180. By engineering a non-native disulfide within this region, the stability of E1 toward [BMIM][Cl] was improved even further, which correlated with lower aggregation kinetics. While leading to novel variants of E1 with improved activity and stability in the presence of [BMIM][Cl], our findings suggest a new strategy for reducing the inactivation of cellulases and potentially other enzymes by ionic liquids.