The present study aims at understanding the effect of organic solvents on the specific proteolytic activity and operational stability of asclepain cI in aqueous-organic media, using correlations between geometrical and structural parameters of asclepain cI. These correlations were determined by molecular dynamics (MD) simulations and the secondary structure of the enzyme validated by Fourier-transform Infrared (FTIR) spectroscopy. Asclepain cI exhibited significantly higher catalytic potential in 29 of the 42 aqueous-organic media tested, composed by 0.1 mM TRIS hydrochloride buffer pH 8 (TCB) and an organic solvent, than in buffer alone. Asclepain cI in water-organic miscible systems showed high FTIR spectral similarity with that obtained in TCB, while in immiscible systems the enzyme acquired different secondary structures than in buffer. Among the conditions studied, asclepain cI showed the highest catalytic potential in 50% v/v ethyl acetate in TCB. According to MD simulations, that medium elicited solvation and flexibility changes around the active center of asclepain cI and conducted to a new secondary structure with the active center preserved. These results provide valuable insights into the elucidation of the molecular mechanism of asclepain cI tolerance to organic solvents and pave the way for its future application for the synthesis of peptides in aqueous-organic media.

Graphical Abstract

中文翻译：

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使用 FTIR 和分子动力学模拟研究有机溶剂对 asclepain cI 的活性、稳定性和二级结构的影响

本研究旨在利用 asclepain cI 的几何和结构参数之间的相关性，了解有机溶剂对 asclepain cI 在水-有机介质中的特定蛋白水解活性和操作稳定性的影响。这些相关性是通过分子动力学 (MD) 模拟确定的，并通过傅里叶变换红外 (FTIR) 光谱验证酶的二级结构。在测试的 42 种水性有机介质（由 0.1 mM TRIS 盐酸缓冲液 pH 8 (TCB) 和有机溶剂组成）中，Asclepain cI 在 29 种中表现出比单独缓冲液更高的催化潜力。Asclepain cI 在水-有机混溶体系中表现出与 TCB 中获得的高度相似的 FTIR 光谱，而在不混溶体系中，该酶获得了与缓冲液中不同的二级结构。在研究的条件中，asclepain cI 在 TCB 中的 50% v/v 乙酸乙酯中显示出最高的催化潜力。根据 MD 模拟，该介质引发了 asclepain cI 活性中心周围的溶剂化和柔性变化，并形成了保留活性中心的新二级结构。这些结果为阐明asclepain cI对有机溶剂耐受的分子机制提供了有价值的见解，并为其未来在水-有机介质中合成肽的应用铺平了道路。

图形概要