Structural studies of Myceliophthora Thermophila Laccase in the presence of deep eutectic solvents
Structural studies of Myceliophthora Thermophila Laccase in the presence of deep eutectic solvents
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DOI：
10.1016/j.enzmictec.2021.109890
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发表时间：
2021-08-21
影响因子：
3.4
通讯作者：
Zhang, Xiao
中科院分区：
文献类型：
--
作者：
Chan, Jou Chin;Zhang, Bixia;Zhang, Xiao
本文通过晶体学和动力学分析阐明了嗜热毁丝霉漆酶与低共熔溶剂（DES）的相互作用。实验中使用了四种不同氢键受体（HBA）和氢键供体（HBD）的DES：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对漆酶活性的提高高达300%，而甘油：氯化胆碱和乳酸：氯化胆碱DES已经发现基于氯化胆碱的DES在相同浓度范围内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是一个S-抛物线-I-抛物线混合的非竞争性抑制剂，在那里可以发生构象变化。在1.6埃下获得了漆酶与乳酸：氯化胆碱DES（LCDES）的晶体结构。晶体学分析表明，LCDES的加入导致漆酶活性位点的变化，但在所得晶体中观察到的水分子的增加阻止了漆酶经历剧烈的结构变化。荧光光谱和圆二色谱也被用来确定DES对漆酶的结构构象的影响。结果表明，DES的存在可引起漆酶活性位点氨基酸的局部环境发生变化，从而影响漆酶的活性和稳定性。
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 noncompetitive inhibitor, where conformational changes can occur. The crystal structures of laccase with lactic acid: choline chloride DES (LCDES) were obtained at 1.6 angstrom. 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.