Browsing by Subject "Olefin"

Now showing 1 - 4 of 4
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    Addressing unanswered questions in bacterial hydrocarbon biosynthesis
    (2017-10) Jensen, Matthew
    Modern society relies heavily on hydrocarbons. Because they are used as liquid transportation fuels, cosmetics, waxes, and food coatings, hydrocarbons are important components of most aspects of daily life. The majority of hydrocarbon products are extracted or derived from cude oil. Energy costs, fuel demand, and environmental concerns involving the non-renewable nature of petroleum-derived compounds have sparked recent interest in microbial hydrocarbon production. Engineering the diversity of microbial metabolic pathways to produce biofuels and chemicals represents a renewable alternative to fossil fuels. One pathway of interest is bacterial long-chain olefin biosynthesis. Divergent bacterial species have been shown to synthesize these waxy hydrocarbons using four enzymes: OleABCD. Recent investigations have aimed to understand how these enzymes work in concert to produce valuable hydrocarbon intermediates and products. These findings will be useful for future pathway engineering for renewable, bacterial olefin production. The first three chapters of this dissertation deal with elucidating the catalytic mechanism of the first enzyme in the olefin biosynthesis pathway, OleA. Chapters 2, 3, and 4 each investigate a separate amino acid necessary for OleA β-keto acid formation using methods of site-directed mutagenesis, biochemistry, and X-ray crystallography. In Chapter 2, the unique substrate binding channel architecture of OleA is directly demonstrated by trapping substrates and intermediates within Cys143 mutated enzymes. The role of Glu117 as the catalytic base needed to prime condensation through deprotonation of the second acyl-CoA substrate is established in Chapter 3. This represents the first dimeric thiolase superfamily enzyme that uses an active site base donated from the second monomer. It also provides evidence for the unique mechanistic strategy of OleA compared to other thiolases. Chapter 4 investigates the role His285 plays in positioning substrate and intermediates for productive condensation by OleA. It is also shown that His285 plays a role in protecting the Cys143 thiolate from oxidative damage. The dissertation concludes with the investigation of the catalytic function of OleC and the characterization of the multienzyme assembly formed by OleB, OleC, and OleD. In Chapter 6, OleC is demonstrated to produce β-lactones from β-hydroxy acids. This is the first example of a β-lactone synthetase, a novel enzyme function. It is also shown that OleC is homologous to amino acid sequences encoded in known β-lactone-producing natural product pathways, suggesting a common mechanism for β-lactone formation. Chapter 7 details the formation of an assembly consisting of OleBCD. Following co-expression of OleABCD, OleBCD are found to co-elute over nickel-affinity, anti-FLAG, and size-exclusion chromatographic purifications. These assemblies form ~2 MDa structures that produce cis-olefin following the addition of OleA and acyl-CoA. Negative stain transmission electron microscopy reveals a mixture of assemblies ranging from 24-40 nm in diameter. It is proposed that these assemblies are necessary for protecting the cell from the highly-reactive β-lactone intermediate.
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    Kinetics and mechanisms of methanol to hydrocarbons conversion over zeolite catalysts
    (2013-05) Hill, Ian Michael
    The methanol-to-hydrocarbons (MTH) process over zeolite catalysts is the final step in the synthesis of commodity chemicals and fuels from alternative carbon sources via synthesis gas intermediates. Emerging research has shown that olefins and aromatics are critical intermediates, acting as scaffolds for the addition of methyl groups from methanol or dimethyl ether (DME) in an indirect "hydrocarbon pool" mechanism. Outstanding questions in this research pertain to (i) the quantitation of reaction rates for C1 homologation and (ii) the mechanism of activating methanol or DME for the formation of carbon-carbon bonds. This research reports rate constants and activation energies of olefin and aromatic methylation steps over zeolites of different pore sizes and geometries from steady-state methylation reactions, as well as isotopic and post-reaction titration studies to determine mechanistic details regarding the structure of the active zeolite surface species. Specifically, isolated steady-state methylation of C2 to C4 olefins over zeolites at differential conditions have shown that reactions producing higher degrees of substitution of intermediate carbocations have rate constants that are an order of magnitude higher than less substituted intermediates. Benzene and toluene methylation reactions show similar kinetic behavior to propylene and linear butene, respectively, over H-ZSM-5. Pressure-dependent studies show a first-order rate dependence on olefin or aromatic pressures which is invariant of DME partial pressures, indicating a surface saturated in DME-derived species reacting with a gas phase co-reactant in the rate-limiting step. These surface species have been identified as methoxides, as observed using post-reaction titration and isotopic studies. The methylation of para- and ortho-xylene with DME at low conversions showed linear dependence of the reaction rate at low pressures of xylene, but the reaction rate became zero-order at higher xylene pressures over H-ZSM-5. The reaction rate remained zero-order in DME pressure, and when taken in conjunction with results from isotopic studies and surface titrations, indicates that the surface methoxides become saturated in adsorbed xylene isomers. A reduction in the critical diffusion length by a factor of >150 did not increase the reaction rate, indicating that the effect is adsorption and not one of transport limitations. Arguments based on derived rate equations modeling observed trends in kinetic, isotopic, and titration studies set a basis for building a microkinetic model for MTH reactions over H-ZSM-5, which can predict expected product distributions for a given set of reaction conditions.
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    Kinetics, mechanisms, and site requirements
    (2016-08) DeWilde, Joseph
    We report the kinetics, mechanisms, and site densities of parallel ethanol dehydration and dehydrogenation over gamma-alumina (γ-Al2O3), a high surface area and thermally-stable metal oxide used both as a catalyst support and as a Lewis acid catalyst in industrial practice. We further extend our investigations to diethyl ether conversion over γ-Al2O3 to describe the reaction network for ethanol dehydration and dehydrogenation at conversions exceeding 10%. Steady state measurements demonstrate that unimolecular and bimolecular ethanol dehydration rates are inhibited by water-ethanol co-adsorbed complexes at 488 K. Reactive surface intermediates, rather than co-adsorbed complexes, inhibit the rates of ethanol dehydration and dehydrogenation at industrially-relevant temperatures (>623 K). Co-processing pyridine with ethanol/water feed mixtures results in a reversible inhibition of both unimolecular and bimolecular ethanol conversion pathways; the synthesis rates of ethylene and acetaldehyde are inhibited to a greater extent than diethyl ether synthesis rates, establishing that unimolecular reactions occur on a pool of catalytic sites separate from the pool for bimolecular dehydration reactions. An observed 1:1 ratio of acetaldehyde and ethane in the eluent verifies that ethanol dehydrogenation proceeds via a hydrogen transfer mechanism. We employ asymmetric ethers as probes to establish ether conversion on γ-Al2O3 occurs through a disproportionation pathway to form an olefin and an alcohol, rather than through a hydration pathway. Diethyl ether disproportionation rates were verified to (i) possess an intrinsic rate constant that is within a factor of two of that of unimolecular ethanol dehydration and (ii) be inhibited by pyridine to the same extent as ethylene synthesis rates from ethanol dehydration. These observations are consistent with a proposed mechanism in which ether disproportionation and unimolecular alcohol dehydration occur through a common alkoxide reaction intermediate and on a common pool of catalytic sites. Our combined investigations of alcohol and ether conversion establish the existence of two distinct pools of catalytic centers, verify all unimolecular pathways of alcohol dehydration, dehydrogenation, and ether disproportionation occur on a common set of active sites, and provide a rigorous kinetic description of these pathways.
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    Probing OleA Mechanism and Diversity Using Alternative Substrates
    (2021-08) Smith, Megan
    Various microorganisms posses the ability to create long chain olefinic hydrocarbons, which can be useful as biofuel precursors or for the creation of specialty chemicals. Additionally, the biosynthesis pathways for the production of these olefins can be used to create other high value compounds such as β-lactone therapeutics and surfactants. Our lab has identified hundreds of possible olefin and β-lactone biosynthetic gene clusters, however many of the end products are unknown. In order to test the diversity of olefins produced, we focused our efforts on the first enzyme in the pathway, OleA. OleA is a thiolase that condenses two fatty acyl-CoAs into a β-keto acid, which eventually becomes the olefin or β-lactone end-product. Earlier research has shown that OleA is responsible for the overall shape of the end-product, through its substrate specificity. Thus, we surmised that studying OleA would provide insight on the natural variance of Olefin and β-lactone compounds produced in nature. Through a collaboration with the Joint Genome Institute we have sampled a total of 72 OleA recombinant proteins using a new colorimetric assay that was developed through the use of inexpensive and readily available p-nitrophenyl compounds as substrate analogs. Our research has shown that many of these OleA proteins are able to accept a wide variety of compounds, many of which are not found in nature. In addition to the development of the screen, we discovered that the p-nitrophenyl ester could only participate as the first substrate of the Claisen condensation reaction of OleA. We then exploited this to produce the β-keto acid product using p-nitrophenyl esters, as well as further refine the OleA mechanism, by demonstrating the directionality of the Claisen condensation. Finally, we showed the addition of cetyltrimethylammonium bromide (CTAB), below critical micelle concentrations, can improve the yield of OleA, both when using native or alternative substrates. This improves the potential for OleA to become a biotechnologically important enzyme, and points to future ways we can improve the overall enzyme pathway to generate large amounts of desired product. This thesis represents a first step in a larger goal of producing industrially relevant β-lactone compounds and their derivatives.