CHARACTERIZATION OF A PHOSPHOTRIESTERASE FROM GENETICALLY-ENGINEERED ESCHERICHIA-COLI

A phosphotriesterase (PTE) capable of hydrolyzing organophosphate este rs was purified from Escherichia coli strain DH-Sa carrying a cloned o pd 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 experiment s showed that PTE could accommodate two equivalents of Co2+ or Zn2+ io ns. PTE protein was found to have higher affinity for Co2+. In additio n, 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 operat ion in a practical environment was performed in a system designed to m imic a continuously stirred tank reactor (CSTR) with different solutio n compositions in the flow reservoir. PTE was deactivated in 24 hours when the inflow solution contained 5% ethanol or 1 mM EDTA, while it r etained 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.