| Summary: | This thesis describes the characterization of SsoPox, a lactonase with promiscuous phosphotriesterase activity from the hyperthermophilic archaeon, Sulfolobus solfataricus, and the potential of the phosphotriesterase from Brevundimonas diminuta (PTEBd) to function as an organophosphate sensor. Arg-223 and Tyr-99 of SsoPox are not essential for lactonase activity, however substitution of a phenylalanine in place of Tyr-97 abolished lactonase activity while reducing paraoxonase activity by 20-fold. Substrate specificity of SsoPox can be modulated through the partial blockage of the hydrophobic binding tunnel adjacent to the active site. The specificity constant for N-(3-oxo-decanoyl)-L-homoserine lactone decreased 37-fold when a phenylalanine was introduced in place of Leu-226. PTEBd was expressed and purified from Pseudomonas putida and, like SsoPox, can be immobilized to Disruptor paper. The immobilized enzyme can be used to detect five organophosphates at concentrations as low as 50 μM. Incubation of PTEBd-immobilized sensors at different temperatures proved that the enzyme is stable for at least 40 days at 23.5 degrees Celsius without any detectable change in activity.
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