Characterization of a phosphotriesterase from genetically-engineered Escherichia coli.
A phosphotriesterase (PTE) capable of hydrolyzing organophosphate esters was purified from Escherichia coli strain DH-5 alpha carrying a cloned opd gene from Flavobacterium. The effects of pH, temperature and metal ion concentrations on enzyme stability and activity were investigated. Optimum conditions for PTE's catalytic activity were determined to be 35 degrees C and pH 8.5. Protein-metal equilibrium binding experiments showed that PTE could accommodate two equivalents of Co2+ or Zn2+ ions. PTE protein was found to have higher affinity for Co2+. In addition, Co2+ was found to possess the most positive effects in maintaining and restoring PTE's stability and catalytic activity when compared to other divalent metal ions. Assessment of the feasibility of PTE operation in a practical environment was performed in a system designed to mimic a continuously stirred tank reactor (CSTR) with different solution compositions in the flow reservoir. PTE was deactivated in 24 hours when the inflow solution contained 5% ethanol or 1 mM EDTA, while it retained one third of its initial activity in a deionized water stream. When the inflow solution contained 1 mM Co2+, PTE was found to retain activity throughout the 24-hour experiment.