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dc.contributor.advisorRaushel, Frank M
dc.creatorGithens, Tyler 1986-
dc.date.accessioned2013-03-14T16:22:32Z
dc.date.available2013-03-14T16:22:32Z
dc.date.created2012-12
dc.date.issued2013-01-17
dc.date.submittedDecember 2012
dc.identifier.urihttp://hdl.handle.net/1969.1/148353
dc.description.abstractPhosphotriesters, also known as organophosphates (OP), represent a class of toxic compounds first synthesized in Germany. Enzymatic removal of harmful insecticides and breakdown products is a promising alternative to skimming or dredging. Wild type bacterial phosphotriesterase (PTE) was screened against 7 agricultural organophosphates: coumaphos, chlorpyrifos, fenitrothion, temephos, profenofos, pirimiphosmethyl and diazinon. The initial results laid the groundwork for a mutagenesis study to investigate the determining factors in enzyme reactivity. Coumaphos is hydrolyzed more efficiently than any other target by the wild type cobalt enzyme (kcat/Km = 2 x 10^7 M^-1s^-1). Coumaphos, fenitrothion and chlorpyrifos had the lowest Km values from the initial screen and were targets for steady state kinetic characterization of active site mutants. Site directed mutagenesis of binding sites was conducted and the most reactive point mutants, F132G, F132V and S308G, were used as backgrounds for subsequent mutation. Seven active site double mutants: F132G/S308G, F132G/S308T, F132V/S308G, F132V/S308T, F132G/I106T, F132V/I106T and G308/W309 were purified to homogeneity for kinetic characterization. The double mutant G308/F132V enhanced chlorpyrifos reactivity relative to the wild type enzyme. This enhancement of reactivity is proposed to result from conformational rearrangement following substrate bond hydrolysis.en
dc.format.mimetypeapplication/pdf
dc.subjectenzymatic bioremediationen
dc.subjectorganophosphatesen
dc.subjectphosphotriesteraseen
dc.subjectPTEen
dc.titleImproving Reactivity Against Target Organothiophosphates via Active-Site Directed Mutagenisis of a Bacterial Phosphotriesteraseen
dc.typeThesisen
thesis.degree.departmentBiochemistry and Biophysicsen
thesis.degree.disciplineBiochemistryen
thesis.degree.grantorTexas A&M Universityen
thesis.degree.nameMaster of Scienceen
thesis.degree.levelMastersen
dc.contributor.committeeMemberRye, Hays
dc.contributor.committeeMemberBegley, Tadhg P
dc.type.materialtexten
dc.date.updated2013-03-14T16:22:32Z


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