Voiland School of Chemical Engineering and Bioengineering - Washington State University, 2710 Crimson Way, Richland, WA, 99354, USA.
Department of Chemistry, Washington State University, Pullman, WA, 99164, USA.
Enzyme Microb Technol. 2021 Oct;150:109890. doi: 10.1016/j.enzmictec.2021.109890. Epub 2021 Aug 8.
In this work, we elucidated the interactions between Myceliophthora thermophila laccase and deep eutectic solvent (DES) by crystallographic and kinetics analyses. Four types of DESs with different hydrogen bond acceptor (HBA) and hydrogen bond donor (HBD), including lactic acid: betaine, glycerol: choline chloride, lactic acid: choline chloride and glycerol: betaine was used. The results revealed that different DES have different effects on laccase activity. Lactic acid-betaine (2:1) DES has shown to enhance laccase activity up to 300 % at a concentration ranged from 2% to 8% v/v, while glycerol: choline chloride and lactic acid: choline chloride DES choline chloride-based DES have found to possess inhibitory effects on laccase under the same concentration range. Detailed kinetic study showed that glycerol: choline chloride DES is a S-parabolic-I-parabolic mixed non-competitive inhibitor, where conformational changes can occur. The crystal structures of laccase with lactic acid: choline chloride DES (LCDES) were obtained at 1.6 Å. Crystallographic analysis suggested that the addition of LCDES causes changes in the laccase active site, but the increase in water molecules observed in the resulting crystal prevented laccase from experiencing drastic structural change. Fluorescence and circular dichroism spectroscopies were also applied to determine the effects of DES on the structural conformation of laccase. The results have confirmed that the presence of DES can trigger changes in the local environments of the amino acids in the active site of laccase which contributes to the changes in its activity and stability.
在这项工作中，我们通过晶体学和动力学分析阐明了嗜热毁丝霉漆酶与深共熔溶剂（DES）之间的相互作用。使用了四种不同氢键供体（HBD）和氢键受体（HBA）的 DES，包括乳酸：甜菜碱、甘油：氯化胆碱、乳酸：氯化胆碱和甘油：甜菜碱。结果表明，不同的 DES 对漆酶活性有不同的影响。乳酸-甜菜碱（2:1）DES 在 2%至 8% v/v 的浓度范围内显示出高达 300%的漆酶活性增强作用，而甘油：氯化胆碱和乳酸：氯化胆碱 DES 则在相同浓度范围内发现对漆酶具有抑制作用。详细的动力学研究表明，甘油：氯化胆碱 DES 是一种 S-抛物线-I-抛物线混合非竞争性抑制剂，其中可以发生构象变化。获得了与乳酸：氯化胆碱 DES（LCDES）结合的漆酶的晶体结构，分辨率为 1.6Å。晶体学分析表明，添加 LCDES 会导致漆酶活性部位发生变化，但在所得晶体中观察到的水分子增加阻止了漆酶经历剧烈的结构变化。荧光和圆二色性光谱也被应用于确定 DES 对漆酶结构构象的影响。结果证实，DES 的存在可以引发漆酶活性部位中氨基酸的局部环境变化，从而导致其活性和稳定性的变化。
