Applied Biotechnology Research Centre, Baqiyatallah University of Medical Sciences, Tehran, Iran.
J Biosci. 2020;45.
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, Delta Gi, and fluorescence and far-UV CD analysis. Results demonstrated an increase half-life and Delta Gi 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 Delta Gi also increased. Based on these findings, a strong case is presented for thermostability improvement of OPH enzyme by flexible loop truncation after bioinformatics analysis.
由于提高了酶的效能和降低了成本，因此提高工业或商业上使用的酶的热稳定性将提高其能力和商业潜力。已经提出了几种稳定因子作为热稳定性的基础，例如脯氨酸替换、二硫键、表面环截断和离子对网络的创建。本研究通过柔性环截断评估了提高有机磷水解酶刚性的机制。生物信息学分析表明，突变蛋白在环截断后保留其稳定性（删除了五个氨基酸）。通过半衰期、Delta Gi 和荧光和远紫外 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 Delta Gi 增加。这些结果通过外源性荧光和圆二色性（CD）光谱实验得到了证实，因此，在 OPHD5 中通过环截断增加刚性后，半衰期和 Delta Gi 也增加。基于这些发现，在生物信息学分析之后，通过柔性环截断提高 OPH 酶的热稳定性提出了强有力的案例。
