Bacillus thermocatenulatus lipase (BTL2), a member of the isolated lipase family known as thermoalkalophilic lipases, carries potential for industrial applications owing to its ability to catalyze versatile reactions under extreme conditions. This study investigates the molecular effects of distinct solvents on the stability of BTL2 at different temperatures, aiming to contribute to lipase use in industrial applications. Initially, molecular dynamic (MD) simulations were carried out to address for the molecular impacts of distinct solvents on the structural stability of BTL2 at different temperatures. Two lipase conformations representing the active and inactive forms were simulated in 5 solvents including water, ethanol, methanol, cyclohexane, and toluene. Low temperature simulations showed that polar solvents led to enhanced lid fluctuations compared with non-polar solvents reflecting a more dynamic equilibrium between active and inactive lipase conformations in polar solvents including water, while the overall structure of the lipase in both forms became more rigid in non-polar solvents than they were in polar solvent. Notably, the native lipase fold was maintained in non-polar solvents even at high temperatures, indicating an enhancement of lipase’s thermostability in non-polar organic solvents. Next, we conducted experiments for which BTL2 was expressed in a heterologous host and purified to homogeneity, and its thermostability in different solvents was assessed. Parallel to the computational findings, experimental results suggested that non-polar organic solvents contributed to BTL2’s thermostability at concentrations as high as 70% (v/v). Altogether, this study provides beneficial insights to the lipase use under extreme conditions.

Graphical Abstract

中文翻译：

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了解嗜热脂肪酶的热和有机溶剂稳定性：从计算预测和实验中得出的见解。

嗜热芽孢杆菌脂肪酶（BTL2）是被称为热嗜碱脂肪酶的分离脂肪酶家族的一员，由于其在极端条件下具有催化多种反应的能力，因此具有工业应用潜力。这项研究调查了不同溶剂对BTL2在不同温度下的稳定性的分子影响，旨在促进脂肪酶在工业应用中的使用。最初，进行分子动力学（MD）模拟以解决不同温度下BTL2的结构对不同溶剂的分子影响。在5种溶剂（包括水，乙醇，甲醇，环己烷和甲苯）中模拟了代表活性形式和非活性形式的两种脂肪酶构象。低温模拟表明，与非极性溶剂相比，极性溶剂导致盖波动更大，这反映了极性溶剂（包括水）中活性和非活性脂肪酶构象之间的动态平衡更高，而两种形式的脂肪酶的整体结构在非极性溶剂中都变得更加坚硬-极性溶剂比在极性溶剂中的极性溶剂要多。值得注意的是，即使在高温下，天然脂肪酶在非极性溶剂中的折叠倍数也保持不变，这表明脂肪酶在非极性有机溶剂中的热稳定性得到增强。接下来，我们进行了实验，将BTL2在异源宿主中表达并纯化至均一，并评估了其在不同溶剂中的热稳定性。与计算结果平行，实验结果表明，非极性有机溶剂在高达70％（v / v）的浓度下有助于BTL2的热稳定性。总之，这项研究为极端条件下脂肪酶的使用提供了有益的见解。

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