Abstract
Ionic liquids (ILs) are organic salts molten at room temperature that can be used for a wide variety of applications. Many ILs, such as 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]), have been shown to remove a significant fraction of the complex biopolymer lignin from biomass during pretreatment. Valorizing lignin via biological pathways (e.g., enzymes) holds promise but is limited by the low biocompatibility of many ILs used for pretreatment. The discovery of thermostable enzymes and the application of enzyme engineering techniques have yielded biocatalysts capable of withstanding high concentrations of ILs. Converting lignin from a waste product to value-added chemicals is vital to the success of future cellulosic biorefineries. To that end, we screened the activity of the lignolytic enzyme laccase from a hyperthermophilic bacterium (Thermus thermophilus) in aqueous [C2C1Im][OAc]. Despite the thermophilicity (Topt > 90°C) of this laccase, significant activity loss (> 50%) was observed in only 2% (w/v) [C2C1Im][OAc]. Kinetics studies show that the IL can bind to the free enzyme and the enzyme-substrate complex. Docking simulations suggest that the cation favors binding to a region close to the active site. We then used a rational design strategy to improve the activity of the laccase in [C2C1Im][OAc]. A total of 8 single amino acid mutations were made; however, there were no significant improvements in the activity of the mutants in [C2C1Im][OAc] compared to the wild type. The results of this study shed light on the complex nature of enzyme-IL interactions and the challenges faced when designing a biological lignin valorization strategy.
Document Type
Article
Publication Date
7-24-2020
Digital Object Identifier (DOI)
https://doi.org/10.3389/fenrg.2020.00158
Funding Information
The authors acknowledge the support from the National Science Foundation under Cooperative Agreement Nos. 1355438 and 1632854 and the National Institute of Food and Agriculture, United States Department of Agriculture, Hatch-Multistate project under accession number 1018315. This material is based upon research supported by the Chateaubriand Fellowship of the Office for Science & Technology of the Embassy of France in the United States.
Related Content
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Repository Citation
Stevens, Joseph Craig; Rodgers, David W.; Dumon, Claire; and Shi, Jian, "Characterization and Enzyme Engineering of a Hyperthermophilic Laccase toward Improving Its Activity in Ionic Liquid" (2020). Biosystems and Agricultural Engineering Faculty Publications. 234.
https://uknowledge.uky.edu/bae_facpub/234
Supplementary Material
Included in
Biochemistry, Biophysics, and Structural Biology Commons, Bioresource and Agricultural Engineering Commons
Notes/Citation Information
Published in Frontiers in Energy Research, v. 8, article 158.
© 2020 Stevens, Rodgers, Dumon and Shi.
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