| Value |
11700
Sec^-1
Range: ±580 Sec^-1
|
| Organism |
Bacteria Escherichia coli |
| Reference |
Mee-Hie Cho C, Mulchandani A, Chen W. Functional analysis of organophosphorus hydrolase variants with high degradation activity towards organophosphate pesticides. Protein Eng Des Sel. 2006 Mar19 (3):99-105PubMed ID16423845
|
| Method |
To determine the kinetic properties of the OPH variants, the OPH wild-type and the variants were expressed as histidine tag fusion and purified using an Ni affinity column. Plasmid-bearing bacterial cultures were grown in 200 ml of Terrific broth supplemented with ampicillin to a final concentration of 100 µg/ml and 10 µM of CoCl2 at 30°C. The kinetic constants for each variant were obtained by measuring the initial hydrolysis rate of the substrate at different concentrations using a constant enzyme concentration. The enzyme was diluted with citrate–phosphate buffer pH 8.0 containing 0.1 mM CoCl2 in the presence of 1 mg/ml bovine serum albumin (BSA), which was used to stabilize the diluted protein. The change in absorbance (412 nm, {epsilon}412 = 16 500 M^–1 cm–1 for p-nitrophenol) was measured in 50 mM phosphate–citrate buffer pH 8.0 containing either methyl parathion or paraoxon, 10% methanol and 0.1 mM CoCl2 in 1.5 ml disposable methacrylate cuvettes (Fisher) with a Beckman DU-60 spectrophotometer for 5 min at 37°C. All assays were performed in triplicate. The kinetic constants (Km and kcat) were obtained by fitting the data to Lineweaver–Burk reciprocal plot and Hanes–Woolf plot: kcat is calculated according to the equation kcat = Vmax/E, where E is the protein concentration used in the assay. |
| Comments |
Organophosphorus hydrolase (OPH, also known as phosphotriesterase) is a bacterial enzyme that is capable of degrading a wide range of neurotoxic organophosphate nerve agents. Directed evolution has been used to generate one variant (22A11) with up to 25-fold improved hydrolysis of methyl parathion. Paraoxon is an acetylcholinesterase inhibitor. |
| Entered by |
Uri M |
| ID |
104969 |