RESUMEN
Thermostability improvement of enzymes used industrially or commercially would develop their capacity andcommercial potential due to increased enzymatic competence and cost-effectiveness. Several stabilizing factorshave been suggested to be the base of thermal stability, like proline replacements, disulfide bonds, surface looptruncation and ionic pair networks creation. This research evaluated the mechanism of increasing the rigidity oforganophosphorus hydrolase enzyme by flexible loop truncation. Bioinformatics analysis revealed that themutated protein retains its stability after loop truncation (five amino acids deleted). The thermostability of thewild-type (OPH-wt) and mutated (OPH-D5) enzymes were investigated by half-life, DGi, and fluorescence andfar-UV CD analysis. Results demonstrated an increase half-life and DGi 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 DGi also increased. Based onthese findings, a strong case is presented for thermostability improvement of OPH enzyme by flexible looptruncation after bioinformatics analysis.
RESUMEN
Previous studies have verified the feasibility of using Escherichia coli systems that display organophosphorous hydrolase (OPH) on the cell surface as whole-cell catalysts. However, the inefficient display of the enzyme on cell surfaces remains unaddressed. In the present study, multiple optimization experiments on full-length and truncated ice nucleation protein anchors, E. coli host cells, culture media, and culture conditions were performed to optimize whole-cell OPH enzymatic activity. The results show that apart from the dramatic effect of isopropyl-b-d-thiogalactoside concentration and culture temperature, the coordination between the anchor protein, culture media, and host cells is essential for highly efficient OPH display. Under optimal conditions, namely, culturing in M9 medium, 20 °C induction temperature, 0.1 mmol l-1 IPTG, and 100 μmol l-1 Co2+, the engineered E. coli strain MB109-406 that expresses the fusion enzyme InaK-N-OPH exhibited a whole-cell OPH activity of 0.62 U mg-1 ?cell d.wt. This result is much higher than that of several currently available OPH-displaying systems, which shows the potential of the current system for further large-scale industrial or environmental applications.
RESUMEN
Bacterial organophosphate hydrolases (OPH) have been shown to hydrolyze structurally diverse group of organophosphate (OP) compounds and nerve agents. Due to broad substrate range and unusual catalytic properties, the OPH has successfully been used to develop eco-friendly strategies for detection and decontamination of OP compounds. However, their usage has failed to gain necessary acceptance, due to short half-life of the enzyme and loss of activity during process development. In the present study, we report a simple procedure for immobilization of OPH on biocompatible gelatin pads. The covalent coupling of OPH using glutaraldehyde spacer has been found to dramatically improve the enzyme stability. There is no apparent loss of OPH activity in OPH-gelatin pads stored at room temperature for more than six months. As revealed by a number of kinetic parameters, the catalytic properties of immobilized enzyme are found to be comparable to the free enzyme. Further, the OPH‑gelatin pads effectively eliminate OP insecticide methyl parathion and nerve agent sarin.