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- Creators: Ghirlanda, Giovanna
- Creators: Kavazanjian, Edward
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
Enzymes which regulate the metabolic reactions for sustaining all living things, are the engines of life. The discovery of molecules that are able to control enzyme activity is of great interest for therapeutics and the biocatalysis industry. Peptides are promising enzyme modulators due to their large chemical diversity and the existence of well-established methods for library synthesis. Microarrays represent a powerful tool for screening thousands of molecules, on a small chip, for candidates that interact with enzymes and modulate their functions. In this work, a method is presented for screening high-density arrays to discover peptides that bind and modulate enzyme activity. A viscous polyvinyl alcohol (PVA) solution was applied to array surfaces to limit the diffusion of product molecules released from enzymatic reactions, allowing the simultaneous measurement of enzyme activity and binding at each peptide feature. For proof of concept, it was possible to identify peptides that bound to horseradish peroxidase (HRP), alkaline phosphatase (APase) and â-galactosidase (â-Gal) and substantially alter their activities by comparing the peptide-enzyme binding levels and bound enzyme activity on microarrays. Several peptides, selected from microarrays, were able to inhibit â-Gal in solution, which demonstrates that behaviors selected from surfaces often transfer to solution. A mechanistic study of inhibition revealed that some of the selected peptides inhibited enzyme activity by binding to enzymes and inducing aggregation. PVA-coated peptide slides can be rapidly analyzed, given an appropriate enzyme assay, and they may also be assayed under various conditions (such as temperature, pH and solvent). I have developed a general method to discover molecules that modulate enzyme activity at desired conditions. As demonstrations, some peptides were able to promote the thermal stability of bound enzyme, which were selected by performing the microarray-based enzyme assay at high temperature. For broad applications, selected peptide ligands were used to immobilize enzymes on solid surfaces. Compared to conventional methods, enzymes immobilized on peptide-modified surfaces exhibited higher specific activities and stabilities. Peptide-modified surfaces may prove useful for immobilizing enzymes on surfaces with optimized orientation, location and performance, which are of great interest to the biocatalysis industry.
ContributorsFu, Jinglin (Author) / Woodbury, Neal W (Thesis advisor) / Johnston, Stephen A. (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2010
Description
Enzymes keep life nicely humming along by catalyzing important reactions at relevant timescales. Despite their immediate importance, how enzymes recognize and bind their substrate in a sea of cytosolic small molecules, carry out the reaction, and release their product in microseconds is still relatively opaque. Methods to elucidate enzyme substrate specificity indicate that the shape of the active site and the amino acid residues therein play a major role. However, lessons from Directed Evolution experiments reveal the importance of residues far from the active site in modulating substrate specificity. Enzymes are dynamic macromolecules composed of networks of interactions integrating the active site, where the chemistry occurs, to the rest of the protein. The objective of this work is to develop computational methods to modify enzyme ligand specificity, either through molding the active site to accommodate a novel ligand, or by identifying distal mutations that can allosterically alter specificity. To this end, two homologues in the β-lactamase family of enzymes, TEM-1, and an ancestrally reconstructed variant, GNCA, were studied to identify whether the modulation of position-specific distal-residue flexibility could modify ligand specificity. RosettaDesign was used to create TEM-1 variants with altered dynamic patterns. Experimental characterization of ten designed proteins indicated that mutations to residues surrounding rigid, highly coupled residues substantially affected both enzymatic activity and stability. In contrast, native-like activities and stabilities were maintained when flexible, uncoupled residues, were targeted. Five of the TEM-1 variants were crystallized to see if the changes in function observed were due to architectural changes to the active site. In a second project, a computational platform using RosettaDesign was developed to remodel the firefly luciferase active site to accommodate novel luciferins. This platform resulted in the development of five luciferin-luciferase pairs with red-shifted emission maxima, ready for multicomponent bioluminescent imaging applications in tissues. Although the projects from this work focus on two classes of proteins, they provide insight into the structure-function relationship of ligand specificity in enzymes and are broadly applicable to other systems.
ContributorsKolbaba Kartchner, Bethany (Author) / Mills, Jeremy H (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Van Horn, Wade D (Committee member) / Arizona State University (Publisher)
Created2023
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
Description
In enzyme induced carbonate precipitation (EICP), calcium carbonate (CaCO3) precipitation is catalyzed by plant-derived urease enzyme. In EICP, urea hydrolyzes into ammonia and inorganic carbon, altering geochemical conditions in a manner that promotes carbonate mineral precipitation. The calcium source in this process comes from calcium chloride (CaCl2) in aqueous solution. Research work conducted for this dissertation has demonstrated that EICP can be employed for a variety of geotechnical purposes, including mass soil stabilization, columnar soil stabilization, and stabilization of erodible surficial soils. The research presented herein also shows that the optimal ratio of urea to CaCl2 at ionic strengths of less than 1 molar is approximately 1.75:1. EICP solutions of very high initial ionic strength (i.e. 6 M) as well as high urea concentrations (> 2 M) resulted in enzyme precipitation (salting-out) which hindered carbonate precipitation. In addition, the production of NH4+ may also result in enzyme precipitation. However, enzyme precipitation appeared to be reversible to some extent. Mass soil stabilization was demonstrated via percolation and mix-and-compact methods using coarse silica sand (Ottawa 20-30) and medium-fine silica sand (F-60) to produce cemented soil specimens whose strength improvement correlated with CaCO3 content, independent of the method employed to prepare the specimen. Columnar stabilization, i.e. creating columns of soil cemented by carbonate precipitation, using Ottawa 20-30, F-60, and native AZ soil was demonstrated at several scales beginning with small columns (102-mm diameter) and culminating in a 1-m3 soil-filled box. Wind tunnel tests demonstrated that surficial soil stabilization equivalent to that provided by thoroughly wetting the soil can be achieved through a topically-applied solution of CaCl2, urea, and the urease enzyme. The topically applied solution was shown to form an erosion-resistant CaCO3 crust on fine sand and silty soils. Cementation of erodible surficial soils was also achieved via EICP by including a biodegradable hydrogel in the stabilization solution. A dilute hydrogel solution extended the time frame over which the precipitation reaction could occur and provided improved spatial control of the EICP solution.
ContributorsHamdan, Nasser M (Author) / Kavazanjian Jr., Edward (Thesis advisor) / Rittmann, Bruce (Thesis advisor) / Shock, Everett (Committee member) / Arizona State University (Publisher)
Created2015