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- Creators: Barrett, The Honors College
Description
Due to a continued interest in the fundamental properties of dihydrofolate reductase (DHFR) and its enzymatic activities, this study employed the use of six fluorescent tryptophan derivatives, for single site amino acid replacements. The two positions 30 and 47 within DHFR were studied to discover the rate at which these larger tryptophan analogues may be incorporated. Additionally, it was to be determined how much activity the mutated DHFR’s could retain when compared to their wild type counterpart. Through a review of literature, it was shown that previous studies have illustrated successful incorporation and toleration of unnatural amino acids.
Each of the six analogues A through F were relatively efficiently incorporated into the enzyme and well tolerated. Each maintained at least a third of their catalytic activity, measured through the consumption of β-nicotinamide adenine dinucleotide phosphate. Primarily, derivatives B, C, and D were able to retain the highest amount of activity in each position; B and D were the most tolerated in positions 30 and 47 with respective values of 68 ± 6.1 and 80 ± 12. The findings in this study illustrate that single tryptophan derivatives are able to be incorporated into Escherichia coli DHFR while still allowing the maintenance of a significant portion of its enzymatic activity.
Each of the six analogues A through F were relatively efficiently incorporated into the enzyme and well tolerated. Each maintained at least a third of their catalytic activity, measured through the consumption of β-nicotinamide adenine dinucleotide phosphate. Primarily, derivatives B, C, and D were able to retain the highest amount of activity in each position; B and D were the most tolerated in positions 30 and 47 with respective values of 68 ± 6.1 and 80 ± 12. The findings in this study illustrate that single tryptophan derivatives are able to be incorporated into Escherichia coli DHFR while still allowing the maintenance of a significant portion of its enzymatic activity.
ContributorsBaldwin, Edwin Alexander (Author) / Hecht, Sidney (Thesis director) / Chen, Shengxi (Committee member) / Barrett, The Honors College (Contributor) / W. P. Carey School of Business (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Quercetin 2,3-dioxygenase from Bacillus subtilis has been identified and characterized as the first known prokaryotic quercetinase. This enzyme catalyzes the cleavage of the O-heteroaromatic ring of the flavonol quercetin to the corresponding depside and carbon monoxide. The first quercetinase was characterized from a species of Aspergillus genus, and was found to contain one Cu2+ per subunit. For many years, it was thought that the B. subtilis quercetinase contained two Fe2+ ions per subunit; however, it has since been discovered that Mn2+ is a much more likely cofactor. Studies of overexpressed bacterial enzyme in E. coli indicated that this enzyme may be active with other metal ions (e.g. Co2+); however, the production of enzyme with full metal incorporation has only been possible with Mn2+. This study explores the notion that metal manipulation after translation, by partially unfolding the enzyme, chelating the metal ions, and then refolding the protein in the presence of an excess of divalent metal ions, could generate enzyme with full metal occupancy. The protocols presented here included testing for activity after incubating purified quercetinase with EDTA, DDTC, imidazole and GndHCl. It was found that the metal chelators had little to no effect on quercetinase activity. Imidazole did appear to inhibit the enzyme at concentrations in the millimolar range. In addition, the quercetinase was denatured in GndHCl at concentrations above 1 M. Recovering an active enzyme after partial or complete unfolding proved difficult, if not impossible.
ContributorsKrojanker, Elan Daniel (Author) / Francisco, Wilson (Thesis director) / Allen, James P. (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-05
Description
This dissertation investigates the potential for enzyme induced carbonate cementation as an alternative to Portland cement for creating building material from sand aggregate. We create a solution of urease enzyme, calcium chloride (CaCl2), and urea in water and added sand. The urease catalyzes the synthesis of carbonate from urea, and the carbonate then bonds with a dissociated calcium ion and precipitates from the solution as calcium carbonate (CaCO3). This precipitate can form small crystal bridges at contacts between sand grains that lock the sand grains in place. Using enzyme induced carbonate precipitation we created a cemented sand sample with a maximum compressive strength of 319 kPa and an elastic modulus of approximately 10 MPa. Images from the SEM showed that a major failure mechanism in the cemented samples was the delamination of the CaCO3 from the sand grains. We observed that CaCO3 cementation did not when solutions with high concentrations of CaCl2 and urea were used.
ContributorsBull, Michael Ryan (Author) / Kavazanjian, Edward (Thesis director) / Chawla, Nikhilesh (Committee member) / Barrett, The Honors College (Contributor) / Materials Science and Engineering Program (Contributor)
Created2014-05
Description
The two chapters of this thesis focus on different aspects of DNA and the properties of nucleic acids as the whole. Chapter 1 focuses on the structure of DNA and its relationship to enzymatic efficiency. Chapter 2 centers itself on threose nucleic acid and optimization of a step in the path to its synthesis. While Chapter 1 discusses DNA and Uracil-DNA Glycosylase with regards to the base excision repair pathway, Chapter 2 focuses on chemical synthesis of an intermediate in the pathway to the synthesis of TNA, an analogous structure with a different saccharide in the sugar-phosphate backbone.
Chapter 1 covers the research under Dr. Levitus. Four oligonucleotides were reacted for zero, five, and thirty minutes with uracil-DNA glycosylase and subsequent addition of piperidine. These oligonucleotides were chosen based on their torsional rigidities as predicted by past research and predictions. The objective was to better understand the relationship between the sequence of DNA surrounding the incorrect base and the enzyme’s ability to remove said base in order to prepare the DNA for the next step of the base excision repair pathway. The first pair of oligonucleotides showed no statistically significant difference in enzymatic efficiency with p values of 0.24 and 0.42, while the second pair had a p value of 0.01 at the five-minute reaction. The second pair is currently being researched at different reaction times to determine at what point the enzyme seems to equilibrate and react semi-equally with all sequences of DNA.
Chapter 2 covers the research conducted under Dr. Chaput. Along the TNA synthesis pathway, the nitrogenous base must be added to the threofuranose sugar. The objective was to optimize the original protocol of Vorbrüggen glycosylation and determine if there were better conditions for the synthesis of the preferred regioisomer. This research showed that toluene and ortho-xylene were more preferable as solvents than the original anhydrous acetonitrile, as the amount of preferred isomer product far outweighed the amount of side product formed, as well as improving total yield overall. The anhydrous acetonitrile reaction had a final yield of 60.61% while the ortho-xylene system had a final yield of 94.66%, an increase of approximately 32%. The crude ratio of preferred isomer to side product was also improved, as it went from 18% undesired in anhydrous acetonitrile to 4% undesired in ortho-xylene, both values normalized to the preferred regioisomer.
Chapter 1 covers the research under Dr. Levitus. Four oligonucleotides were reacted for zero, five, and thirty minutes with uracil-DNA glycosylase and subsequent addition of piperidine. These oligonucleotides were chosen based on their torsional rigidities as predicted by past research and predictions. The objective was to better understand the relationship between the sequence of DNA surrounding the incorrect base and the enzyme’s ability to remove said base in order to prepare the DNA for the next step of the base excision repair pathway. The first pair of oligonucleotides showed no statistically significant difference in enzymatic efficiency with p values of 0.24 and 0.42, while the second pair had a p value of 0.01 at the five-minute reaction. The second pair is currently being researched at different reaction times to determine at what point the enzyme seems to equilibrate and react semi-equally with all sequences of DNA.
Chapter 2 covers the research conducted under Dr. Chaput. Along the TNA synthesis pathway, the nitrogenous base must be added to the threofuranose sugar. The objective was to optimize the original protocol of Vorbrüggen glycosylation and determine if there were better conditions for the synthesis of the preferred regioisomer. This research showed that toluene and ortho-xylene were more preferable as solvents than the original anhydrous acetonitrile, as the amount of preferred isomer product far outweighed the amount of side product formed, as well as improving total yield overall. The anhydrous acetonitrile reaction had a final yield of 60.61% while the ortho-xylene system had a final yield of 94.66%, an increase of approximately 32%. The crude ratio of preferred isomer to side product was also improved, as it went from 18% undesired in anhydrous acetonitrile to 4% undesired in ortho-xylene, both values normalized to the preferred regioisomer.
ContributorsTamirisa, Ritika Sai (Author) / Levitus, Marcia (Thesis director) / Stephanopoulos, Nicholas (Committee member) / Windman, Todd (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05