Data_Sheet_1_Characterization and Enzyme Engineering of a Hyperthermophilic Laccase Toward Improving Its Activity in Ionic Liquid.DOCX

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.

离子液体（ILs）系指在室温下熔融的有机盐，广泛应用于众多领域。众多离子液体，例如1-乙基-3-甲基咪唑醋酸盐（[C2C1Im][OAc]），在预处理过程中已被证实能够从生物质中去除复杂生物聚合物木质素的大量部分。通过生物途径（例如酶）对木质素进行增值化利用具有广阔前景，但受到许多用于预处理的离子液体生物相容性低这一局限性的制约。热稳定酶的发现以及酶工程技术的应用已产生能够承受高浓度离子液体生物催化剂。将木质素从废弃物转化为增值化学品对于未来纤维素生物精炼厂的成功至关重要。为此，我们对一种嗜热菌（Thermus thermophilus）产生的木质素降解酶laccase在含水[C2C1Im][OAc]中的活性进行了筛选。尽管此laccase具有嗜热性（最适温度Topt > 90°C），但在仅含2%（w/v）[C2C1Im][OAc]的溶液中观察到显著的活性损失（>50%）。动力学研究表明，离子液体可以与游离酶和酶-底物复合物结合。对接模拟表明，阳离子倾向于与活性位点附近的区域结合。随后，我们采用理性设计策略来提高laccase在[C2C1Im][OAc]中的活性。共进行了8个单氨基酸突变；然而，与野生型相比，突变体在[C2C1Im][OAc]中的活性没有显著提高。本研究结果揭示了酶-IL相互作用之复杂本质以及设计生物木质素增值化利用策略时所面临的挑战。