Matching Items (3)
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- All Subjects: Enzymes
- Creators: Allen, James P.
- Creators: Chaput, John C
Description
The principle of Darwinian evolution has been applied in the laboratory to nucleic acid molecules since 1990, and led to the emergence of in vitro evolution technique. The methodology of in vitro evolution surveys a large number of different molecules simultaneously for a pre-defined chemical property, and enrich for molecules with the particular property. DNA and RNA sequences with versatile functions have been identified by in vitro selection experiments, but many basic questions remain to be answered about how these molecules achieve their functions. This dissertation first focuses on addressing a fundamental question regarding the molecular recognition properties of in vitro selected DNA sequences, namely whether negatively charged DNA sequences can be evolved to bind alkaline proteins with high specificity. We showed that DNA binders could be made, through carefully designed stringent in vitro selection, to discriminate different alkaline proteins. The focus of this dissertation is then shifted to in vitro evolution of an artificial genetic polymer called threose nucleic acid (TNA). TNA has been considered a potential RNA progenitor during early evolution of life on Earth. However, further experimental evidence to support TNA as a primordial genetic material is lacking. In this dissertation we demonstrated the capacity of TNA to form stable tertiary structure with specific ligand binding property, which suggests a possible role of TNA as a pre-RNA genetic polymer. Additionally, we discussed the challenges in in vitro evolution for TNA enzymes and developed the necessary methodology for future TNA enzyme evolution.
ContributorsYu, Hanyang (Author) / Chaput, John C (Thesis advisor) / Chen, Julian (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2013
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
The public has expressed a growing desire for more sustainable and green technologies to be implemented in society. Bio-cementation is a method of soil improvement that satisfies this demand for sustainable and green technology. Bio-cementation can be performed by using microbes or free enzymes which precipitate carbonate within the treated soil. These methods are referred to as microbial induced carbonate precipitation (MICP) and enzyme induced carbonate precipitation (EICP). The precipitation of carbonate is the formation of crystalline minerals that fill the void spaces within a body of soil.
This thesis investigates the application of EICP in a soil collected from the Arizona State University Polytechnic campus. The surficial soil in the region is known to be a clayey sand. Both EICP and MICP have their limitations in soils consisting of a significant percentage of fines. Fine-grained soils have a greater surface area which requires the precipitation of a greater amount of carbonate to increase the soil’s strength. EICP was chosen due to not requiring any living organisms during the application, having a faster reaction rate and size constraints.
To determine the effectiveness of EICP as a method of improving a soil with a significant amount of fines, multiple comparisons were made: 1) The soil’s strength was analyzed on its own, untreated; 2) The soil was treated with EICP to determine if bio-cementation can strengthen the soil; 3) The soil had sand added to reduce the fines content and was treated with EICP to determine how the fines percentage effects the strength of a soil when treated with EICP.
While the EICP treatment increased the strength of the soil by over 3-fold, the strength was still relatively low when compared to results of other case studies treating sandy soils. More research could be done with triaxial testing due to the samples of the Polytechnic soil’s strength coming from capillarity.
This thesis investigates the application of EICP in a soil collected from the Arizona State University Polytechnic campus. The surficial soil in the region is known to be a clayey sand. Both EICP and MICP have their limitations in soils consisting of a significant percentage of fines. Fine-grained soils have a greater surface area which requires the precipitation of a greater amount of carbonate to increase the soil’s strength. EICP was chosen due to not requiring any living organisms during the application, having a faster reaction rate and size constraints.
To determine the effectiveness of EICP as a method of improving a soil with a significant amount of fines, multiple comparisons were made: 1) The soil’s strength was analyzed on its own, untreated; 2) The soil was treated with EICP to determine if bio-cementation can strengthen the soil; 3) The soil had sand added to reduce the fines content and was treated with EICP to determine how the fines percentage effects the strength of a soil when treated with EICP.
While the EICP treatment increased the strength of the soil by over 3-fold, the strength was still relatively low when compared to results of other case studies treating sandy soils. More research could be done with triaxial testing due to the samples of the Polytechnic soil’s strength coming from capillarity.
ContributorsRoss, Johnathan (Author) / Kavazanjian, Edward (Thesis advisor) / Zapata, Claudia (Committee member) / Hamdan, Nasser (Committee member) / Arizona State University (Publisher)
Created2018