Browsing by Subject "Enzyme"
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Item Application of enzymatic catalysis and galvanic processes for biosensor development(2011-08) Zaccheo, Brian Andrew; Crooks, Richard M. (Richard McConnell); Browning, Karen; Hoffman, David; Johnston, Keith P.; Stevenson, KeithMethods for integrating enzyme systems with electrochemical reactions having applications to diagnostic sensing are described. Diagnostic tests that include biological molecules can be classified as biosensors. Existing testing methods often require trained technicians to perform, and laboratory settings with complex infrastructure. The theme of this dissertation is the development of methods that are faster, easier to use, and more applicable for non-laboratory environments. These goals are accomplished in systems using enzymatic catalysis and galvanic processes. Two biosensors with specific model pathologies have been designed and demonstrated in this study. The first assay senses a DNA fragment representing the Epstein Barr virus and uses enzyme-mediated Ag deposition over a v microfabricated chip. The chip contains a specially designed pair of electrodes in an interdigitated array (IDA). Detection is signaled by a change in the resistance between the two electrodes. The second biosensor discussed in this study is targeted towards the digestive enzyme trypsin. It is selfpowered due to its construction within an open-circuit galvanic cell. In this system, a small volume of blood serum is introduced onto the device over barriers made of protein and Al that block the anode from solution. In the presence of trypsin, the protein gel is rendered more permeable to sodium hydroxide. Adding hydroxide initiates the dissolution of the Al layer, closing the cell circuit and illuminating a light-emitting diode (LED). A relationship was observed between LED illumination time and trypsin concentration. Biosensors that utilize enzymes to generate or amplify a detectable signal are widely used, and the final project of this study uses a nanoparticle based approach to protect the catalytic activity of alkaline phosphatase (AlkP) from hostile chemicals. By incubating Au colloid with AlkP overnight and adding Ag+, core@shell nanoparticles of Au@Ag2O can be isolated that show AlkP activity. The resulting enzyme-metal composite material was analytically characterized and demonstrated greater activity in the presence of organic inhibitors relative to either wild type vi or Au colloid-associated AlkP without the Ag2O shell. The stabilization procedure is complete in one day using a onepot synthesis. This method may provide opportunities to carry out biosensing chemistry in previously incompatible chemical environments.Item Degradation of Guar-Based Fracturing Gels: A Study of Oxidative and Enzymatic Breakers(2012-02-14) Sarwar, Muhammad UsmanUnbroken gel and residue from guar-based fracturing gels can be a cause for formation damage. The effectiveness of a fracturing treatment depends on better achieveing desired fracture geometry, proper proppant placement and after that, a good clean-up. The clean-up is achieved by reducing the fluid viscosity using chemical additives called "Breakers". There are many different types of breakers used in the industry, but they can be broadly divided into two categories: oxidizers and enzymes. Breaker perfromance depends on bottomhole temperature, breaker concentration and polymer loading. Different kind of breakers, used at different concentrations and temperatures, give different kind of "break" results. Therefore, the amount of unbroken gel and residue generated is also different. This project was aimed at studying basic guar-breaker interactions using some of the most common breakers used in the industry. The breakers studied cover a working temperature range of 75 degrees F to 300 degrees F. The effectiveness of each breaker was studied and also the amount of damage that it causes. Viscosity profiles were developed for various field concentrations of breakers. The concentrations were tested over temperature ranges corresponding to the temperatures at which each breaker is used in the field. The majority of these viscosity tests were 6 hours long, with a few exceptions. Early time viscosity data, for the intial 10 minutes of the test, was also plotted from these tests for fracturing applications where the breaker is required to degrade the fluid by the time it reached downhole. This was needed to prevent the damage to the pumping equipment at the surface yet still have almost water-like fluid entering into the formation. The study provides a better understanding of different breaker systems, which can be used in the industry, while designing fracturing fluid systems in order to optimize the breaker performance and achieve a better, cleaner break to minimize the formation damage caused by polymer degradation.Item Enzymatic treatment of pharmaceuticals and personal care products (PPCPs) in municipal wastewater(2013-05) Sharkey, Margaret E; Lawler, Desmond F.; Kinney, Kerry A.Conventional wastewater treatment plants do not effectively remove pharmaceuticals and personal care products (PPCPs). As a result, PPCPs enter the environment via treated wastewater discharge. Enzymatic treatment, using the laccasemediator system, is a novel biochemical process that has been shown to effectively treat some PPCPs. This study investigates the efficacy of the laccase-mediator system to treat PPCPs using a process that can be easily implemented at an existing wastewater treatment plant. Enzymatic treatment will be most beneficial after primary sedimentation and before conventional biological treatment, where unoxidized PPCPs and byproducts could have the opportunity for further degradation in biological treatment. In this work, two enzymatic treatment configurations were studied. A step-wise optimization process was used that alternately varied treatment conditions: pH, enzyme activity, mediator concentration, and reactor detention time. In the optimization process of each configuration, successful oxybenzone removal (~90%) was achieved in municipal primary effluent. In a direct comparison of treatment configurations, both resulted in vi similar percent removals of oxybenzone. Therefore, the configuration with the simpler operation and reactor design was chosen for further study. During the optimization process, several noteworthy conclusions were made that might have full-scale enzymatic treatment implications. Specifically, successful oxybenzone removal occurred at unadjusted pH and without aeration, but increased biological oxygen demand of the wastewater increased the required mediator concentration. While the first finding would decrease enzymatic treatment costs, the latter would increase the costs associated with the mediator. Thus, an alternative mediator source, specifically one high in phenolic compounds, is desired. The use of wine, as a surrogate of winery wastewater, was in investigated and proved ineffective. Further investigation of alternative mediator sources is required. Treatment of another PPCP, sulfamethoxazole, was less efficient (65% removal) than that of oxybenzone, but nevertheless, the substantial removal might indicate that other PPCPs can be treated with the laccase-mediator system. The most promising result of this work was the simultaneous treatment of multiple PPCPs, oxybenzone and sulfamethoxazole. Simultaneous treatment proved to be as effective as when each PPCP was treated individually.Item Enzyme-based detoxification of organophosphorus neurotoxic pesticides and chemical warfare agents(2009-05-15) Kern, Rory JamesThere are some 15,000 known organophosphorus chemicals. Some of these OP?s, including VX and paraoxon, demonstrate an acute neurotoxicity due to the inhibition of cholinergic enzymes. Organophosphorus chemical warfare agents and pesticide neurotoxins are subject to hydrolysis by OP degrading enzymes. To be useful as a bioremediation or anti-chemical warfare agent, the enzyme must be tailored for, and integrated into, a practical application platform. Several studies have established enzyme-based countermeasures, describing such diverse applications as decontaminating foams for surface remediation, encapsulating enzyme with liposome for in vivo therapy, enzyme attachments to surfaces for biosensors and development of a corn expression system for large-scale enzyme production. The goal of the research described here is to select, investigate and improve the operational potential of organophosphate-degrading enzymes including Organophosphorus Hydrolase (OPH, 3.1.8.1) and Organophosphorus Acid Anhydrolase (OPAA, 3.1.8.2). Using saturation kinetics, the catalytic efficiencies of these two major detoxification enzymes were characterized with substrates representing each class of OP neurotoxin, phosphotriester, phosphothioate and phosphofluoridate. OPH presents superior kinetic parameters with each OP class tested. Variants of OPH were created to increase the operational effectiveness of OP hydrolytic enzymes against phosphorothioates. An H254S/H257L mutation in the active site resulted in an improvement in the kinetics (kcat/KM) for the phosphorothioate, demeton-S. To screen potential vascular protection therapies, an in vitro protocol was developed to predict enzymatic effectiveness for protection of acetylcholinesterase from acute OP-inhibition. The protection abilities of the enzymes were directly related to their second order rate constants as inhibitory levels of OP are below the KM of the enzymes. Consideration of contaminant nature concentration and enzyme kinetic parameters, kcat and KM, is critical to understanding decontamination and effective use of enzyme technology. These technologies continue to develop and provide promising new decontamination tools for OP compounds.Item Evolved enzymes for cancer therapeutics and orthogonal systems(2013-08) Lu, Wei-Cheng; Ellington, Andrew D.Directed evolution has been explored for a long time. Various ideas, methods, have been shown to be feasible and successful in the enzyme field. We were interested in evolving enzymes for applications. Therefore, we evolved human cystathionine gamma-lyase (hCGL) and E. coli biotin ligase for therapeutic and biotechnology applications. Wild-type human cystathionine gamma-lyase does not have any methionine-degrading activity, unlike the high methionine-degrading abilities of bacterial methionine gamma-lyase (MGL) found in Pseudomonas putida. The ability to engineer hCGL to breakdown methionine can be a potential cancer treatment by targeting the methionine-dependent cancer cells. However, the methionine-degrading activity of previously engineered hCGL has only shown 1% activity compared to MGL, too low to be useful in practical cancer therapeutics. By using a combination of protein design and phylogenetic analysis, we further evolved hCGL to achieve a higher methionine-degrading activity, with one variant displaying as much as 7% activity compared to bacterial MGL, making it a more likely candidate in cancer treatment.In addition, it has been shown that new orthogonal pairs of biotin protein ligase and biotin have many biotechnology applications. Therefore, we have developed selection scheme for directing the evolution of E. coli biotin protein ligase (BPL, gene: BirA) via in vitro compartmentalization, and have altered the substrate specificity of BPL towards the utilization of the biotin analogue desthiobiotin. Following just 6 rounds of selection and amplification several variants that demonstrated higher activity with desthiobiotin were identified. The best variants from Round 6, BirA₆₋₄₀ and BirA₆₋₄₇, showed 17-fold and 10-fold higher activity, respectively, their abilities to use desthiobiotin as a substrate. Further characterization of BirA₆₋₄₀ and the single substitution variant BirA[subscript M157T] revealed that they had 2- to 3-fold higher kcat values for desthiobiotin, and 3- to 4-fold higher K[subscript M] values. The k[subscript cat]/K[subscript M] values for these enzymes were around 0.7-fold that of BirA[subscript wt-]. It is interesting the selections did not lower the K[subscript M] for desthiobiotin and actually led to a less efficient enzyme. This is an example of how "you get what you select for". Because peptide:DNA conjugates were distributed such that there was on average one template or less per emulsion compartment there was selection only for the catalytic rate (k[subscript cat]) of desthiobiotinylation and not for turnover. Given these conditions, it might be anticipated that the peptide substrate, rather than desthiobiotin, should be bound better by the winning variants, and in fact BirA₆₋₄₀ showed a reduced K[subscript M] value for BAP.Item Fate of Alpha-Amylase Used to Degrade Starch in Water-Based Drilling Fluids(2014-12-11) Zhang, Jeffrey ZThe removal of water-based mudcakes (filter cakes) from horizontal sections is a difficult task. The use of the enzyme ?-amylase in enzymatic degradation of mudcake has proven conditionally effective in laboratory and in field treatments. Even so, the fate of the enzyme after treatment and the product distribution formed during treatment is unknown. This thesis presents a method of characterizing the mudcake-enzyme (specifically the starch-?-amylase) degradation system using analytical methods adopted from established biochemistry techniques. These methods were used to compare the effectiveness of the enzyme degradation system under various degradation conditions. The enzyme?s thermal tolerance under High Temperature/High Pressure (HPHT) conditions were determined using mud aging cells. Enzyme baseline activity (defined as rate of starch degradation) was established under well-mixed reaction condition. All enzyme treatment tests were performed in HPHT filter press to simulate downhole conditions. Retained permeability was determined for each test and concentrations of enzyme and unreacted starch was determined using UV-Vis spectroscopy. Starch degradation reaction product distribution was determined using high performance liquid chromatography coupled with refractive index detection (HPLC-RID). Experimental results show that the specific company provided enzyme is not effective in degrading filter cake at any of the tested temperatures and that the analytical methods developed were effective in characterizing the starch-?-amylase reaction system.Item Mechanism of Inhibition of Metallo-β-lactamase by Nucleic Acids(2011-05) Sims, Cynthe L.; Shaw, Robert W.; Nes, William David; Weber, JoachimSeveral mechanisms have been discovered by which bacteria acquire antibiotic resistance; however, the prevailing and most troubling resistance mechanism originates from the production of metallo-β-lactamases (Walsh et al., 2005). Metallo-β-lactamases are enzymes capable of efficiently catalyzing the hydrolysis of the cyclic amide bond of common β-lactam antibiotics rendering them ineffective as antibiotic agents in the treatment of bacterial infection. The metallo-β-lactamase from Bacillus cereus 5/B/6 is capable of efficiently catalyzing the hydrolysis of a variety of β-lactam substrates including those from the penicillin and cephalosporin classes (Myers and Shaw 1989; Shaw et al. 1991; Felici et al. 1993). The Bacillus cereus 5/B/6 metallo-β-lactamase has an active site capable of binding one or two zinc ions (de Seny et al. 2002; Llarull et al. 2008; Orellano, et al. 1998). The two metal sites are quite different in their amino acid constituents and binding affinities for zinc. Site 1 or the 3H site consists of a zinc ion bound to histidines 86, 88, and 149, and a water molecule in a tetrahedral geometry (Llarull et al. 2008). Site 2 or the DCH site contains a zinc ion bound in trigonal bipyramidal geometry to aspartic acid 90, cysteine 168 and histidine 210, a bridging H2O/OH-, and an additional water molecule (Llarull et al. 2008). A combinatorial method called SELEX (Systematic Evolution of Ligands by EXponential enrichment) (Ellington and Szostak, 1992) was used to derive a ten base oligonucleotide inhibitor of the metallo-β-lactamase from B. cereus 5/B/6. Steady-state kinetic experiments revealed a non-competitive inhibition pattern for this inhibitor (Kim, 2002, Kim, et al. 2009). To further investigate the details of the inhibition mechanism, metal-ion reconstitution studies with either cobalt or copper were performed. Like the native zinc containing enzyme, the inhibition patterns for inhibition by the oligonucleotide, for both the cobalt and copper reconstituted species were also non-competitive in nature. Visible spectroscopic studies with the cobalt-reconstituted enzyme reveal a change in the cysteine-associated site upon inhibitor binding. EPR studies of the mono-cobalt form of the enzyme reveal essentially no change in the coordination sphere of the histidine-containing site. EXAFS spectroscopy experiments for both the mono and di-zinc forms of the enzyme were also performed to study the interaction of the inhibitor with the enzyme. Results indicate in the di-zinc containing enzyme, perturbation in the primary coordination sphere of the DCH site upon inhibitor binding. X-ray diffraction analysis of crystals of the enzyme prepared with excess ss-DNA 10-mer at 1.5-angstrom resolution reveal that the active site is both devoid of metal ions and the inhibitor. Thus, the EXAFS (zinc enzyme) and visible spectroscopic studies (cobalt enzyme) indicate perturbation of the primary coordination sphere of the DCH site upon binding of the inhibitor. Visible spectroscopic studies of the copper enzyme with the substrate cefuroxime reveal changes in the coordination sphere of the metal ions upon substrate hydrolysis as well.Item Novel strategies towards engineering therapeutic enzymes with reduced immunogenicity for cancer therapy(2010-12) Cantor, Jason Robert; Georgiou, George; Iverson, Brent L.; Maynard, Jennifer A.; Mullins, Charles B.; Whitman, Christian P.Heterologous enzymes have been investigated for a variety of therapeutic applications, including the treatment of a number of cancers that are sensitive to the systemic depletion of specific amino acids. One such example is acute lymphoblastic leukemia (ALL) for which enzyme-mediated L-Asparagine (L-Asn) depletion by the Escherichia coli L-Asparaginase II (EcAII) has been proven critical for treatment. However, the repeated or prolonged therapeutic administration of such enzymes is restricted by their immunogenicity, which frequently results in the generation of anti-enzyme antibodies that may in turn mediate a variety of adverse hypersensitivity reactions and neutralization of the enzymes themselves. Thus, while the therapeutic efficacy of asparaginase is well established, a significant number of patients still develop adverse immune responses to the enzyme. Here, we have developed and explored novel strategies towards engineering an asparaginase with reduced immunogenicity for ALL therapy. First, we identified and investigated human enzymes that putatively shared functional similarity to asparaginase with the long-term aim of engineering such enzymes to acquire biochemical and pharmacological properties requisite for eventual therapeutic application. In one study, we described the bacterial expression and characterization of the human asparaginase-like protein 1 (hASRGL1). We presented evidence that hASRGL1 exhibited an activity profile consistent with enzymes previously designated as [Beta]-aspartyl peptidases, which had only been previously identified in plants and bacteria. Similar to non-mammalian [Beta]-aspartyl peptidases, hASRGL1 was revealed to be an N-terminal nucleophile (Ntn) hydrolase whereby Thr168 serves as the essential Ntn for both intramolecular processing and catalysis. In a second study, we described the optimized bacterial expression and biochemical characterization of the human N-terminal asparagine amidohydrolase 1 (hNTAN1). We demonstrated that hNTAN1 catalysis is dependent upon direct involvement of a thiol group, and subsequently identified Cys75 as an essential residue that may act as the catalytic nucleophile. Further, we presented the first description of hNTAN1 kinetics, secondary structure composition, and thermal stability. Second, we devised and validated a novel therapeutic deimmunization approach by combinatorial T-cell epitope removal using neutral drift. We showed that combinatorial saturation mutagenesis coupled with a robust neutral drift screen enabled the isolation of engineered EcAII variants that contained multiple amino acid substitutions yet exhibited catalytic efficiencies nearly indistinguishable to that of the parent enzyme. Three regions of EcAII were computationally identified as putative T-cell epitopes and then subjected to saturation mutagenesis at 4 positions (per region) believed to be critical for MHC-II binding. The resulting libraries were then sequentially subjected to a neutral drift FACS screen in order to isolate EcAII mutants that retained wild-type function. Pools of neutral drift variants were then computationally evaluated for MHC-II binding and those that displayed scores indicative of compromised binding were purified and biochemically characterized. Finally, T-cell activation assays and antibody titers in HLA-transgenic mice were used to evaluate T-cell epitope removal and immunogenicity, respectively. Ultimately, we revealed that mice immunized with an EcAII neutral variant containing 8 amino acid substitutions -- 3 of which were non-phylogenetically conserved -- within computationally predicted T-cell epitopes, displayed a significant 10-fold reduction in serum anti-EcAII IgG titer relative to mice similarly immunized with the parent enzyme.Item Unveiling the architectures of five bacterial biomolecular machines(2014-08) Fage, Christopher Dane; Keatinge-Clay, Adrian Tristan; Hoffman, David W; Whitman, Christian P; Appling, Dean R; Iverson, Brent L; Hackert, Marvin LNatural products represent an incredibly diverse set of chemical structures and activities. Given this fathomless, ever-evolving diversity, a reasonable approach to designing new molecules entails taking a closer look at the biochemistry that Nature has crafted over billions of years on Earth. In particular, much can be learned by unveiling the architectures of proteins, life’s molecular machines, through methods like X-ray crystallography. Acquiring the blueprints of an enzyme brings us closer to understanding the mechanism by which the enzyme transforms a simple substrate it into a complex product with biological function, and inspires us to engineer such systems to our own ends. With a focus on macromolecular structural characterization, this document elaborates on five Gram-negative bacterial biosynthetic enzymes from two categories: Cell-surface modifiers and polyketide synthases. Among the first category are the glycyl carrier protein AlmF and its ligase AlmE of Vibrio cholerae and the phosphoethanolamine transferase EptC of Campylobacter jejuni. These proteins are responsible for decorating cell-surface molecules (e.g., lipid A) of pathogenic bacteria with small functional groups to promote antibiotic resistance, motility, and host colonization. AlmE and EptC represent potential drug targets and their structures lay the groundwork for the design of therapeutics against food-borne illnesses. Included in the second category are the [4+2]-cyclase SpnF and two ketoreductase-linked dimerization elements, each from the spinosyn biosynthetic pathway in Saccharopolyspora spinosa. The former catalyzes a putative Diels-Alder reaction to form a tricyclic precursor of the insecticide spinosad, while the latter two organize ketoreductase domains within modules of a polyketide synthase. The second category also includes Ralstonia eutropha β-ketoacyl thiolase B, a substrate-permissive enzyme that can make or break carbon-carbon bonds with assistance from Coenzyme A or an analogous thiol. Each of these proteins exhibit intriguing structural features or catalyze reactions that show promise for biochemical engineering.