published_or_final_version / Botany / Doctoral / Doctor of Philosophy

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

Phosphotriesters, 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.

Thesis (Ph. D.)--Oregon State University, 2002. / Typescript (photocopy). Includes bibliographical references. Also available online.

This thesis describes a way to achieve higher conversion rates of sugars from lignocellulosic biomass that can then be used for cellulosic ethanol production. Using oat hulls as the biomass, several chemical and physical pretreatment techniques were explored to overcome the recalcitrance and allow access to cellulose and hemicellulose. Experimentation with enzyme cocktails and dosing was done to obtain the highest conversions of cellulose and xylan to produce sugars. High solids-loading of the substrate, 14-16%, enabled higher conversion rates and would amount to lower cost of production in a commercial facility; however, end-product inhibition by the accumulation of inhibitors is also realized. To remove inhibition, a solid-liquid separation step was implemented which allowed enzymes to operate at a higher efficiency. The best combination of pretreatment and enzymatic hydrolysis led to a glucose of 89% and xylose yield of 84%, for trials conducted in a 20L bioreactor.