Journal of Biotechnology & Biomaterials
Open Access

Abstract

Organophosphate pesticides (OPs) constitute the largest class of worldwide-employed insecticides but some of them are potent nerve agents. Enzymes showing phosphotriesterase activity are ideal candidate to be used as bioscavengers and biodecontaminants. Some years ago, looking for a stable OPs catalyst, a hyperthermophilic phosphotriesterase (SsoPox) was isolated from the archaeon Sulfolobus solfataricus . SsoPox is a noncovalent dimer, with a primary lactonase activity and a promiscuous phosphotriesterase activity. Many mutants of SsoPox have been generated in order to increase the phosphotriesterase activity, with the aim of evolving its ancillary hydrolytic capability on nerve agents and to reach the same level of activity as the related pPTE from Pseudomonas diminuta (k cat /KM of 4 x 10 7 M -1 s -1 ). Here it is reported the production of a triple mutant of SsoPox, obtained by a directed evolution strategy, involving the residues C258, I261, W263. The triple mutant was over-expressed in Escherichia coli , purified to homogeneity and characterized. The enzyme is thermostable, has an optimal temperature for catalysis of 65°C, and possesses degradative activity on different pesticides, such as paraoxon. The specificity constant (k cat /K M ) for the paraoxon was higher than those reported for the wild type enzyme. Furthermore, our data showed that SDS induces the triple mutant activation. Then, decontamination experiments of surfaces, such as glass or cotton layers, showed the ability of such enzyme to hydrolyze total amounts of paraoxon. Further in vitro evolution of the triple mutant is currently ongoing in order to optimize its characteristics.

Biography

Immacolata Del Giudice has completed her PhD in ?Industrial and Molecular Biotechnology? at the age of 28 years from University Federico II of Naples (Italy). After PhD completion she has obtained a post-doctoral fellowship at CNR-Institute of Protein Biochemistry (Naples, Italy). She is now involved in a project regarding to the isolation of recombinant enzymes to use in the decontamination of pesticides.