Background: The firefly luciferase enzyme is widely used in protein engineering and diverse areas of biotechnology, but the main problem with this enzyme is low-temperature stability. Previous reports indicated that surface areas of thermostable proteins are rich in arginine, which increased their thermal stability. In this study, this aspect of thermophilic proteins evaluated by mutations of surface residues to Arg. Here, we report the construction, purification, and studying of these mutated luciferases.

Methods: For mutagenesis, the QuikChange site-directed mutagenesis was used and the I108R, T156R, and N177R mutant luciferases were created. In the following, the expression and purification of wild-type and mutant luciferases were conducted and their kinetic and structural properties were analyzed. To analyze the role of these Arg in these loops, the 3D models of these mutants’ enzymes were constructed in the I-TASSER server and the exact situation of these mutants was studied by the SPDBV and PyMOL software.

Results: Overall, the optimum temperature of these mutated enzymes was not changed. However, after 30 min incubation of these mutated enzymes at 30°C, the I108R, T156R, N177R, and wild-type kept the 80%, 50%, 20%, and 20% of their original activity, respectively. It should be noted that substitution of these residues by Arg preserved the specific activity of firefly luciferase.

Conclusion: Based on these results, it can be concluded that T156R and N177R mutants by compacting local protein structure, increased the thermostability of luciferase. However, insertion of positively charged residues in these positions create the new hydrogen bonds that associated with a series of structural changes and confirmed by intrinsic and extrinsic fluorescence spectroscopy and homology modeling studies.

中文翻译：

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柔性环中精氨酸饱和度评估萤光素酶的热稳定性

背景：萤火虫荧光素酶被广泛用于蛋白质工程和生物技术的各个领域，但是这种酶的主要问题是低温稳定性。先前的报道表明，热稳定蛋白的表面富含精氨酸，从而提高了其热稳定性。在这项研究中，通过表面残基突变为Arg来评估嗜热蛋白的这一方面。在这里，我们报告这些突变的荧光素酶的构建，纯化和研究。

方法：为了诱变，使用QuikChange定点诱变，并创建了I108R，T156R和N177R突变体荧光素酶。在下文中，进行了野生型和突变型荧光素酶的表达和纯化，并分析了它们的动力学和结构性质。为了分析这些精氨酸在这些环中的作用，在I-TASSER服务器中构建了这些突变体酶的3D模型，并通过SPDBV和PyMOL软件研究了这些突变体的确切情况。

结果：总的来说，这些突变酶的最佳温度没有改变。但是，将这些突变的酶在30°C孵育30分钟后，I108R，T156R，N177R和野生型分别保持其原始活性的80％，50％，20％和20％。应该指出的是，这些残基被Arg取代保留了萤火虫荧光素酶的比活性。

结论：基于这些结果，可以得出结论，T156R和N177R突变体通过压缩局部蛋白质结构，增加了萤光素酶的热稳定性。但是，在这些位置插入带正电荷的残基会产生新的氢键，该氢键与一系列结构变化相关，并通过内在和外在荧光光谱法和同源性建模研究得到证实。