Catalytic and structural effects of flexible loop deletion in organophosphorus hydrolase enzyme: A thermostability improvement mechanism

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.