Thermostability improvement of enzymes used industrially or commercially would develop their capacity and commercial potential due to increased enzymatic competence and cost-effectiveness. Several stabilizing factors have been suggested to be the base of thermal stability, like proline replacements, disulfide bonds, surface loop truncation and ionic pair networks creation. This research evaluated the mechanism of increasing the rigidity of organophosphorus hydrolase enzyme by flexible loop truncation. Bioinformatics analysis revealed that the mutated protein retains its stability after loop truncation (five amino acids deleted). The thermostability of the wild-type (OPH-wt) and mutated (OPH-D5) enzymes were investigated by half-life, ΔGi, and fluorescence and far-UV CD analysis. Results demonstrated an increase half-life and ΔGi in OPH-D5 compared to OPH-wt. These results were confirmed by extrinsic fluorescence and circular dichroism (CD) spectrometry experiments, therefore, as rigidity increased in OPHD5 after loop truncation, half-life and ΔGi also increased. Based on these findings, a strong case is presented for thermostability improvement of OPH enzyme by flexible loop truncation after bioinformatics analysis.

中文翻译：

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有机磷水解酶柔性环缺失的催化和结构效应：热稳定性改善机制

由于酶促能力和成本效益的提高，工业或商业上使用的酶的热稳定性改进将开发它们的能力和商业潜力。一些稳定因素被认为是热稳定性的基础，如脯氨酸置换、二硫键、表面环截断和离子对网络的形成。本研究评估了柔性环截断增加有机磷水解酶刚性的机制。生物信息学分析表明，突变蛋白在环截断（五个氨基酸缺失）后仍保持其稳定性。通过半衰期、ΔGi、荧光和远紫外 CD 分析研究了野生型 (OPH-wt) 和突变型 (OPH-D5) 酶的热稳定性。结果表明，与 OPH-wt 相比，OPH-D5 的半衰期和 ΔGi 增加。这些结果通过外在荧光和圆二色性 (CD) 光谱实验证实，因此，随着环截断后 OPHD5 中的刚度增加，半衰期和 ΔGi 也增加。基于这些发现，在生物信息学分析后，通过灵活的环截断为 OPH 酶的热稳定性提高提供了强有力的案例。