ABSTRACT
A heterologous phosphotriesterase (parathion hydrolase), previously cloned from a Flavobacterium species into Streptomyces lividans, was secreted at high levels and purified to homogeneity. N-terminal analysis revealed that it had been processed in the same manner as the native membrane-bound Flavobacterium hydrolase. The enzyme consisted of a single polypeptide with an apparent molecular weight of 35,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Substrate specificity studies showed Kms of 68 microM for parathion, 46 microM for O-ethyl O-p-nitrophenyl phenylphosphonothioate, 599 microM for methyl parathion, and 357 microM for p-nitrophenyl ethyl(phenyl)phosphinate. Temperature and pH optima were 45 degrees C and 9.0, respectively. The purified enzyme was inhibited by 1 mM dithiothreitol and 1 mM CuSO4. After chelation and inactivation by o-phenanthroline, however, activity could be partially restored by 1 mM CuCl or 1 mM CuSO4. The results showed that the purified recombinant parathion hydrolase has the same characteristics as the native Flavobacterium hydrolase. This system provides a source of milligram quantities of parathion hydrolase for future structural and mechanism studies and has the potential to be used in toxic waste treatment strategies.
