School of Chemistry, Bio21 Institute, The University of Melbourne, Melbourne, VIC 3010, Australia.
School of Science, College of Science, Engineering and Health, RMIT University, VIC 3001, Australia.
J Chem Phys. 2018 May 21;148(19):193838. doi: 10.1063/1.5010055.
There has been a substantial increase in enzyme applications within the biochemical and pharmaceutical industries, for example, as industrial biocatalysts. However, enzymes have narrow marginal stability which makes them prone to become inactive and/or denature with a slight change in the solvent environment. Typically industrial applications require harsher solvent environments than enzyme native environments, and hence there is a need to understand solvent-protein interactions in order to develop strategies to maintain, or enhance, the enzymatic activity under industrially relevant solvent conditions. Previously we have shown that protic ionic liquids (PILs) with water can have a stabilising effect on lysozyme, with a large variation dependent on which PIL ions are present, and the water concentration [E. C. Wijaya , Phys. Chem. Chem. Phys. (37), 25926-25936 (2016)]. Here we extend on this work using non-stoichiometric aqueous PIL solvents to investigate, and isolate, the role of pH and ionicity on enzymes. We have used the PILs ethylammonium nitrate (EAN) and ethanolammonium formate (EOAF) since our previous work has identified these as good solvents for lysozyme. Solvent libraries were made from these two PILs with an additional precursor acid or base to modify the acidity/basicity of the neutral stoichiometric PIL, and with water added, to have solutions with 4-17 mol. % of the PIL ions in water. Molar ratios of base:acid were varied between 1:1.05 and 2:1 for EAN and 1:1.25 and 2:1 for EOAF, which enabled from highly basic to highly acidic solutions to be obtained. This was to modify the acidity/basicity of the neutral stoichiometric PILs, without the addition of buffers. The structure and stability of hen egg white lysozyme (HEWL) were explored under these solvent conditions using synchrotron small angle X-ray scattering (SAXS), Fourier transform infrared (FTIR), and activity assays. The radius of gyration and Kratky plots obtained from the SAXS data showed little change with varying ionicity or acid:base ratio. FTIR showed that α-helix was maintained in all, except for the most acidic solvent conditions. The activity data show that HEWL was active between pH 0 and 11 for the EA:N-water system and pH 4.4 and 11 for the EOA:F-water system. This work indicates that ionic liquids have the potential to enable enzymes to maintain activity across a broader range of solvent conditions.
在生化和制药等行业中，酶的应用已经大大增加，例如作为工业生物催化剂。然而，酶的边际稳定性很窄，这使得它们在溶剂环境稍有变化时容易失活和/或变性。通常，工业应用需要比酶的天然环境更苛刻的溶剂环境，因此需要了解溶剂-蛋白质相互作用，以便制定在工业相关溶剂条件下保持或增强酶活性的策略。以前，我们已经表明，质子离子液体（PILs）与水结合可以对溶菌酶产生稳定作用，其变化幅度取决于存在哪种 PIL 离子以及水的浓度[E. C. Wijaya ，Phys. Chem. Chem. Phys.（37），25926-25936（2016）]。在这里，我们使用非化学计量比的含水 PIL 溶剂扩展了这项工作，以研究并分离 pH 值和离子强度对酶的作用。我们使用了乙基铵硝酸盐（EAN）和乙醇铵甲酸盐（EOAF）这两种 PIL，因为我们之前的工作已经确定它们是溶菌酶的良好溶剂。从这两种 PIL 中制备了溶剂库，并添加了额外的前体酸或碱，以改变中性化学计量 PIL 的酸度/碱度，并加入水，使溶液中 PIL 离子的摩尔分数为 4-17%。EAN 的碱基：酸摩尔比在 1:1.05 和 2:1 之间变化，EOAF 的碱基：酸摩尔比在 1:1.25 和 2:1 之间变化，从而可以获得从高碱性到高酸性的溶液。这是为了在不添加缓冲液的情况下改变中性化学计量 PIL 的酸度/碱度。使用同步加速器小角 X 射线散射（SAXS）、傅里叶变换红外（FTIR）和活性测定法，在这些溶剂条件下探索了鸡卵清溶菌酶（HEWL）的结构和稳定性。从 SAXS 数据获得的回转半径和 Kratky 图显示，离子强度或酸：碱比的变化对其影响很小。FTIR 表明，除了最酸性的溶剂条件外，所有溶剂条件下的α-螺旋都得以保持。活性数据表明，对于 EA:N-水体系，HEWL 在 pH 值 0 到 11 之间具有活性，对于 EOA:F-水体系，HEWL 在 pH 值 4.4 和 11 之间具有活性。这项工作表明，离子液体有可能使酶在更广泛的溶剂条件下保持活性。
