Physical Sciences and Mathematics Commons^™

Activity And Selectivity Of Class B Sortase Enzymes, Sophie Jackson

WWU Graduate School Collection

Gram-positive bacteria attach many proteins to their cell walls via sortase enzymes. Sortases are cysteine transpeptidases and are grouped into 6 classes, A-F. Sortase enzymes, particularly sortase A from Staphylococcus aureus, have been used extensively for in vitro protein ligations. Here, we investigate substrate-binding in sortase A from Streptococcus pyogenes. In addition, class B sortases are typically overlooked for research and development due to low in vitro activity and incomplete knowledge of substrate specificity. Here, we investigate the activity of class B sortases from Bacillus anthracis (baSrtB), Clostridioides difficile (cdSrtB), Listeria monocytogenes (lmSrtB), and Staphylococcus aureus (saSrtB). Of these, …

Capillary Electrophoresis: Method Development And Application In Biomolecule Analysis, Lindsay Marie Veltri

Graduate Theses, Dissertations, and Problem Reports

Abstract

Capillary Electrophoresis: Method Development and Application in Biomolecule Analysis

Lindsay Veltri

Biomolecule analysis is essential in disease diagnostics and the development of therapeutic agents. There is a vast array of methods available for biomolecule analysis, however, capillary electrophoresis is well-suited due to minimal sample consumption (~nanoliters of solution) and fast analysis times. In this report, capillary electrophoresis was used to analyze glycosylation of therapeutic proteins. Additionally, a capillary electrophoresis-mass spectrometry method is reported for the analysis of proteins.

Post-translation protein modifications like glycosylation, play an important in protein structure and function. Modifications of protein glycosylation can lead to improved …

Development Of Bioaffinity Based Assays For Forensic Applications, Juliana Maria Agudelo Cano

Legacy Theses & Dissertations (2009 - 2024)

In the field of forensic science, body fluids are a major contributor of evidence in investigations. However, body fluids are usually used only for their genetic content, overlooking its unique composition of proteins and low-molecular-weight compounds that often serve as biomarkers in clinical diagnostics. This investigation aimed to develop simple, effective, and economical models that can be used to identify different attributes of the originator. The focus of this research was to develop biocatalytic cascades that could analyze body fluids by using UV-Vis spectroscopy. These models could be implemented in forensic settings, as well as in biometric authentications. The central …

Two Dimensional Nanoparticles/Nanozymes For Biosensing Augmented By Machine Learning, Nidhi Chintan Nandu

Legacy Theses & Dissertations (2009 - 2024)

Biosensing is an ever-evolving field with many resources devoted towards new approaches in sensing and refining the existing methods. For a long time, such approaches involved use of state-of-the-art instruments and were often proved to be time consuming and expensive. Nanoparticles with their unique properties, inexpensive synthesis and ease of manipulation have found purpose in myriad fields including biosensing. Different nanoparticles based on their composition, morphology, dimensionality, and surface modifications have already gained foot hold in the biosensor ranks. The unique opto-electric properties due to the quantum effect in the nanoparticles have also made them one of the leading alternatives …

Employing Exoglycosidase And Transferase Enzymes In Capillary Nanogel Electrophoresis For The Determination Of N-Glycan Linkages And Enzyme Michaelis-Menten Constants., Lloyd Bwanali

Graduate Theses, Dissertations, and Problem Reports

As a post translational modification protein glycosylation plays a crucial role in protein signaling, binding, kinetics and folding. In disease diagnosis and prognosis, monitoring glycosylation has been identified as a biomarker. Sialylation and sialic acid linkage in N-glycans are markers of cancers including liver, pancreatic and kidney cancer. Quantification of sialic acid linkage is analytically challenging because of the diverse linkages and the presence of heterogenous branching. A capillary electrophoresis method is reported that integrates a unique combination of enzymes and lectins to modify sialylated asparagine-linked glycans (N-glycans) in real time in the capillary so that N-glycan structures containing α2–6-linked …

Nmr Based Studies Of Interactions Between Ribosomes And Glycolytic Enzymes, Devin Bryan Busch

Legacy Theses & Dissertations (2009 - 2024)

Recent research has been performed to better understand what implications ribosome-mediated quinary interactions have on cellular growth. These studies demonstrated that ribosomal binding to enzymes, and their cofactors, can cause significant changes in their enzymatic activity leading to a proposal that ribosome surfaces play a greater regulatory in cellular growth than thought of in the past. To widen the scope of investigations performed on these ribosomal-mediated quinary interactions, the regulatory enzymes associated in glycolysis (hexokinase, phosphofructokinase, and pyruvate kinase) were titrated into a solution of 70S E. coli ribosome and analyzed via 1H-15N-HSQC NMR. The perturbations of the peaks associated …

Development Of Novel Peroxidase-Tio2/Zno Hybrid Catalysts For The Degradation Of Emerging Pollutants, Rana Fawzi Mohamed Mohamed Morsi

Theses

Water pollution by different organic compounds is one of the most important environmental issues that is attracting the attention of many scientists due to its direct potential bad effect on human health. These organic pollutants include pesticides, pharmaceuticals, industrial wastes, and personal care products that are released in water bodies from domestic and industrial discharge. Various chemical, physical and biological approaches have been proposed to degrade these contaminants from the polluted water. In the present study, we immobilized versatile thermostable enzymes, Soybean peroxidase (SBP) and Horseradish peroxidase on functionalized photocatalysts - TiO2 and ZnO to create novel bio-composite catalysts (SBP-TiO2, …

Integrating Microscale Enzyme And Lectin Reactions Using Nanogel Assisted Capillary Electrophoresis, Srikanth Gattu

Graduate Theses, Dissertations, and Problem Reports

Protein glycosylation is one of the most prevalent post-translational modifications which plays an important role in determining the structure, stability, and function of proteins. Changes in glycosylation patterns are a hallmark of cancer and other diseases. Characterizing glycosylation is difficult because these structures vary in their linkage and monomer sequence, which gives rise to vast microheterogenity that could affect the function of the molecule. A novel method using capillary gel electrophoresis by integrating microscale enzymes and lectin reactions in the capillary was developed to verify glycan structure by analyzing monosaccharides. Capillary electrophoresis (CE) improves upon conventional methods of glycan structure …

Comparative Degradation Of Emerging Pollutants Using Chemical And Enzymatic Approaches, Khadega Abobaker Abudullarhman Al-Maqdi

Theses

Organic pollutants, especially those found in water bodies; pose a direct threat to various aquatic organisms as well as humans. A variety of different remediation approaches, including chemical and biological methods have been developed for the degradation of these organic pollutants. However, comparative mechanistic studies of pollutant degradation by these different systems are almost non-existent. In this study, the degradation of two emerging pollutants an antibiotic pollutant, Sulfamethoxazole (SMX) and a model thiazole pollutant, Thioflavin T (ThT), was carried out using advanced oxidation process (AOP), using UV+H2O2 or a peroxidase enzyme system. Optimization conditions for Sulfamethoxazole degradation by peroxidase enzyme …

Quef And Quef-Like: Diverse Chemistries In A Common Fold, Adriana Bon Ramos

Dissertations and Theses

The tunneling fold (T-Fold) superfamily is a small superfamily of enzymes found in organisms encompassing all kingdoms of life. Seven members have been identified thus far. Despite sharing a common three-dimensional structure these enzymes perform very diverse chemistries.

QueF is a bacterial NADPH-dependent oxidoreductase that catalyzes the reduction of the nitrile group of 7-cyano-7-deazaguanine (preQ₀) to a primary amine (preQ₁) in the queuosine biosynthetic pathway. Previous work on this enzyme has revealed the mechanism of reaction but the cofactor binding residues remain unknown. The experiments discussed herein aim to elucidate the role of residues lysine 80, …

Classical And Ab Initio Qm/Mm Simulations Of Bacterial Enzymes, Sajeewa Walimuni Dewage

Wayne State University Dissertations

This thesis describes analyses performed on three enzyme systems. Chapter 2, 3, and 4 involve studies carried out on the GatCAB enzyme of H. pylori and S. aureus. Since information at the electronic level was not required for these studies, sampling of the configuration space carried out at the molecular mechanics level was adequate. In Chapter 2, the snapshots sampled using MD were used as input for average correlation difference calculations and average RMSD difference calculations to ascertain the existence of a communication pathway between two subunits of GatCAB. Experimental and computational results obtained, suggest the existence of a communication …

Initial Characterization Of A Conserved Active Site Residue For The Cdc34 Ubiquitin Conjugating Enzyme, Arvin Akoopie

Honors College Theses

Ubiquitin-conjugating enzymes (E2s) covalently modify protein substrates with ubiquitins. The active site cysteine residues on E2s are essential for catalyzing the transfer of ubiquitin from the E2 active site onto the protein substrate, however there is a limited amount of information available concerning additional active site residues for E2s that may also participate in catalysis. Cdc34 is an essential E2 that has merited the lion’s share of attention for biochemical analysis of the E2 family. Previous phylogenetic analysis of Cdc34 amino acid sequences has identified an invariably conserved histidine residue close to the active site cysteine in the primary structure, …

The Development Of Capillary Electrophoresis Assays To Study Enzyme Inhibition, Sherrisse Kelly Bryant

LSU Doctoral Dissertations

New methods for studying enzymes and enzyme inhibition using capillary electrophoresis (CE) were developed and applied. Because CE is a separation technique, spectral interference is minimized by separation of substrates, products, inhibitors, and sample matrix components. Two types of CE enzyme assays were explored in this dissertation research. The first assay was based on optically gated vacancy capillary electrophoresis (OGVCE) with laser-induced fluorescence detection (LIF). This approach involves periodic photobleaching of fluorescent substrates and products. The initial goal of the study was to develop an assay for adenosine deaminase (ADA). A fluorescent ADA substrate was synthesized; however, the substrate was …

Synthesis Of Fluorogenic Substrates For The Enzymatic Characterization Of Rv0045c From M. Tuberculosis, Kelly Jo Mckenna

Undergraduate Honors Thesis Collection

Mycobacterium tuberculosis is the pathogenic bacterial agent commonly responsible for tuberculosis, or TB. Although treatment exists for the active form of tuberculosis, no method has been developed for eliminating M tuberculosis in its dormant state. One hypothesized method for the elimination of dormant TB is to develop an inhibitor specific for M tuberculosis esterases and lipases, as these esterases and lipases are essential to the survival of dormant TB infection. In this research, the substrate specificity of the Rv0045c esterase from M tuberculosis was studied due to the essential role of Rv0045c in TB metabolism and its dissimilarity to other …

Synthesis Of Nitrosyl Metallpoprphyrins, Thomas M. Braxton Jr.

McCabe Thesis Collection

Nitric oxide, NO, is a molecule with great versatility and importance. Made up of only a single atom of nitrogen and a single atom of oxygen, nitric oxide is perhaps one of the smallest paramagnetic molecules found in nature. The functions of NO in the body are not all known, but this molecule definitely plays a variety of roles. It is involved in the regulation of blood pressure, and it sometimes acts as a messenger for cells as well as a killer of them. It also is important in the defense of the body against foreign invaders. The significance of …

Determination Of Pancreatic And Salivary Amylase By Enzyme Immunoassay And Their Prevalence In Hyperamylasemic Patients, Sabdra Borgens Ward

Theses and Dissertations in Biomedical Sciences

Currently, amylase determinations are nonspecific for the organ source and are based entirely on the enzymatic properties of amylase to produce a measurable product or byproduct. The determination of pancreatic amylase is important in the diagnosis of acute pancreatitis. Most commercially available tests for amylase employ the measurement of the change in NADH absorbance at 280 nm or of the p-nitrophenol released from a maltotetrose substrate. These are nonspecific measurements of pancreatic amylase and often necessitate other tests to be run such as a serum lipase.

The two predominant isoenzymes of amylase are pancreatic (p-amylase) and salivary (s-amylase); the most …

A Comparative Investigation Of Mammalian Serine Transhydroxymethylases, Sanha Panichajakul

Theses and Dissertations

Serine transhydroxymethylase was purified one thousand-fold from bovine brain and one hundred and twenty-fold from bovine liver. Both enzymes were yellow due to the presence of the coenzyme pyridoxal 5'-phosphate. Polyacrylamide gel electrophoresis of the liver enzyme showed one major band containing 95% of the protein, while in the case of the brain enzyme two major bands of nearly equal intensity were observable. The specific activity of the crude liver enzyme was about sixty times higher than that of brain. The liver enzyme was also more heat stable than the brain enzyme. The purified enzymes exhibited L-allothreonine and L-threonine aldolase …

Studies On The Characterization And Activiation By Soluble Factor Of A Sodium-Activated, Magnesium-Dependent, Adenosinetriphosphatase In Rat Cerebral Cortex, Alan B. Forrest

University of the Pacific Theses and Dissertations

This study presented evidence for the existence of a membrane-bound, sodium-activated ATPase which does not require potassium ions for its activity. Furthermore, the data suggest the presence of a soluble factor which activates this enzyme and may play a regulatory role in sodium transport.

Molybdenum Chelates Of 8-Hydroxyquinoline-5-Sulfonic Acid, Ellis Ray Peterson

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Man has had an intense interest in the role of enzymes in biological reactions for many years. The basic question -- how enzymes are able to function so efficiently -- remains as yet largely unanswered.

Only in recent years has it been discovered that molybdenum is essential for certain biological processes. For example, traces of molybdenum added to certain soils have resulted in spectacular plant growth (1). Four enzymes are known to contain molybdenum: nitrate reductase (2), hydrogenase (3), xanthine oxidase (4), and aldehyde oxidase (5). In each of these enzymes riboflavin is also present as the coenzyme FAD (flavin …