School of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu Province 212013, China.
School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang, Jiangsu Province 212013, China.
Int J Biol Macromol. 2021 Nov 1;190:206-213. doi: 10.1016/j.ijbiomac.2021.08.221. Epub 2021 Sep 4.
Easy deactivation of free enzymes under non-native condition has become a stumbling block to the industrial application of biocatalysis. Natural deep eutectic solvent (NADES) has been exploited as a novel reaction medium for improving enzyme stability. The present work focused on preserving and enhancing the activity of carbonic anhydrase (CA) in a more economical and biocompatible NADES system. We synthesized six choline chloride/betaine-based NADES and analyzed the effects of compositions and concentrations of NADES on their physicochemical properties. The Bet-Gly (1: 2) NADES (55%) was proved to be more suitable as reaction medium for CA by analyzing enzyme activity in the presence of NADES. The enhancement in the stability of CA was found to be as a result of a three-dimensional hydrogen bonding network, rather than the individual or the synergistic effect of betaine and glyceride. The conformational change of CA to become more compact was confirmed both by fluorescence spectrum analysis and circular dichroism analysis. It is worth mentioning that a remarkable thermal stability was maintained when CA was incubated at temperature below 60 °C, and about 96% of activity was still restored in 55% NADES at 60 °C for 12 h.
在非天然条件下，使游离酶易于失活已成为生物催化工业应用的一个障碍。天然深共熔溶剂 (NADES) 已被开发为一种新型反应介质，以提高酶的稳定性。本工作重点是在更经济、更生物相容的 NADES 体系中保存和增强碳酸酐酶 (CA) 的活性。我们合成了六种氯化胆碱/甜菜碱基 NADES，并分析了 NADES 的组成和浓度对其物理化学性质的影响。通过分析 NADES 存在下的酶活性，证明 Bet-Gly (1:2) NADES (55%) 更适合用作 CA 的反应介质。发现 CA 稳定性的提高是由于形成了三维氢键网络，而不是甜菜碱和甘油酯的单独或协同作用。通过荧光光谱分析和圆二色性分析证实 CA 的构象变化变得更加紧凑。值得一提的是，当 CA 在低于 60°C 的温度下孵育时，保持了显著的热稳定性，并且在 60°C 下孵育 12 小时后，在 55%的 NADES 中仍有 96%的活性得到恢复。
