Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The year's studies were focused on the preparation and characterization of stericaly stabilized liposomes (SLs) encapsulating a recombinant organophosphorus hydrolyzing phosphotriesterase (OPH) enzyme for the antagonism of organophosphorus intoxication. Earlier results indicate that the liposomal carrier system provides an enhanced protective effect against the organophosphorus molecule paraoxon, presenting a more effective therapy with less toxicity than the most commonly used antidotes. Physicochemical characterization of the liposomal OPH delivery system is essential in order to get information on its in vitro stability and in vivo fate. Osmolarity, pH, viscosity, and encapsulation efficiency of the SL preparation and the surface potential of the vesicles were determined. The membrane rigidity and the impact of OPH enzyme on it was studied by electron-paramagnetic resonance spectroscopy, using spin probes. The in vitro stability of the liposomal preparations, the vesicle size distribution, and its alteration during a 3-week storage were followed by dynamic light-scattering measurements. Further, the stability of encapsulated and nonencapsulated OPH was compared in buffer and plasma. Dissemination: Conference Presentations: 1) Catalytic Bioscavengers with Broad Specificity Against OP Nerve Agents" NIH-NINDS CounterAct Network Research Symposium, Washington D.C., April 2009. 2) Petrikovics, I., Wild, J.R., Budai, M., Grof, P., Zimmer, A., Klebovich, I., Chapela P. and Wales, M.E. Physico-chemical characterization of stealth liposomes encapsulating a hydrolyzing enzyme employed in organophosphorus antagonism. 47th Annual Meeting of Society of Toxicology, Seattle, WA, March 16-20, 2008. PARTICIPANTS: Participants: Drs. James Wild, Melinda Wales, Boris Novikov and Janet Grimsley. Graduate Students: Ph.D in Toxicology Rory Kern (Ph.D. in Toxicology. 2008) Undergraduate Students: Manoj Rajaure. Collaborators: 1) Dr. Alex Simonian, Auburn University. Auburn U. graduate students on this project include Madhumati Ramanathan. 2) Dr Ilona Petrikovics, Sam Houston State University Training or Professional Development: There have been approximately 1-2 undergraduate research assistants in the lab who were working on OPH-related research projects, as well as international scientist Professor Dayananda Siddavatam, University of University of Hyderabad, Hyderabad India, and an associated doctoral student Pandeeti Emmanuel Vijay Paul. Three Ph.D. Degrees have been awarded to students who participated in the overall research program. Interdisciplinary Opportunities: Students have the interdisciplinary opportunity to work in an optical engineering laboratory at Auburn University, Department of Mechanical Engineering) and a molecular genetics facility at Texas A&M University. Three graduate students have tale this route (R. Kern, L. Viveros, T. Reeves from A&M and Sheetal Patel from Auburn.).. TARGET AUDIENCES: This project is oriented toward developing enzyme-based technologies for the decontamination of organophosphate neurotoxins and pesticides. These projects are b y their nature quite interdisciplinary and draw the attention of DOD, DHS, and NIH.. PROJECT MODIFICATIONS: Encapsulated enzymes are effective as intravascular scavengers of OP neurotoxins in animal protection studies that extend residence time as determined by phamico-kinetic studies with both guinea pigs and mice.
Impacts
Protein immobilization on solid interfaces is a crucial aspect of their successful application in technologies such as biosensing, purification, separation, decontamination, etc. Although immobilization can improve the long-term and operational stability of proteins, this is often at the cost of significant losses in the catalytic activity of the tethered enzyme. Covalent attachment methods take advantage of reactive groups on the amino acid side chains. The distribution of the solvent exposed side chains on an enzyme's molecular surface often results in an ensemble of orientations when the protein is immobilized on a surface or in a matrix through these side chain linkages. Depending on the attachment mechanism and resulting orientation, access to and from the active site could be restricted. This study describes a methodology for the design and implementation of an orientation specific attachment of an enzyme to a surface plasmon resonance sensor surface. The enzyme, organophosphorus hydrolase, was structurally analyzed to identify surface resides as candidates for modification to optimize active site accessibility and, thus, sensitivity of detection. A single surface lysine on the active site face of the enzyme dimer was selected for elimination, thus allowing for the immobilization of the catalyst in the preferred orientation. Kinetic evaluation of the enzymes determined that the surface lysine-to-alanine variant retained 80% of the wild-type activity with the neurotoxin substrates, paraoxon and demeton-S. After immobilization, surfaces bearing the variant were determined to be more active even though the enzyme coverage on the sensor surface was reduced by 17%.
Publications
- Lysozyme-mediated formation of protein-silica nano-composites for biosensing applications." Madhumati Ramanathan, Heather R. Luckarift, Ainur Sarsenova, James R. Wild, Erlan K. Ramanculov, Eric V. Olsen, and Aleksandr L. Simonian. 2009. Colloids and Surfaces Biointerfaces 73:58-64.
- Orientation Specific Positioning of Organophosphorus Hydrolase on Solid Interfaces for Biosensor Applications. S. Paliwal, Tony E. Reeves, Melinda E. Wales, James R. Wild, and Aleksandr Simonian. 2009. Langmuir 25:9615-9618
- Enhanced stability of enzyme organophosphate hydrolase interfaced on the carbon nanotubes. V.A. Davis, R. L. Ramanculov, S. Paliwal, J.R. Wild, B. Shankar, and A.L.Simonian. 2009. Colloids and Surfaces Biointerfaces. 73:58-64.
- Biocatalytic Paints and Coatings. C.S. McDaniel, J. McDaniel, J.R. Wild, and M.E. Wales. ACS Symposium Series. Smart Coatings. 2009. 1002:239-247.
- Protection of Acetylcholinesterase from Organophosphates: Kinetic Insight into bioscavengers. M.E. Wales, E. Tiffany-Castiglioni, and J.R. Wild. 2009. In Toxicology of Chemical Warfare Agents. Ed. R.C. Gupta. 93:1041-1051.
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Progress 06/20/03 to 06/19/09
Outputs
OUTPUTS: The goals and objectives over the five year work plan were to fully optimize a variety of enzyme catalysts (OPH) for the detection and complete destruction of the most toxic OP neurotoxins. The specific objects were to develop new enzymes and enzyme variants for OP decontamination and deploy them in a variety of systems and materials for detection and remediation neurotoxic pesticides and other chemical threat agents. The outputs include design, execution and analysis of experiments in the following areas: (1) Development of new and improved medical countermeasures designed to prevent, diagnose, and treat the conditions caused by potential and existing neurotoxic chemical agents, through participation in the NIH CounterACT Research Network. This interaction has lead to moving our research from basic to translational in an effort to enhance medical response capabilities during a chemical threat response emergency. (2) New methodologies for orientation specific attachment of an enzyme to a surface for detection or encapsulation for stability and in vivo application. (3) International collaboration investigating horizontal transfer of genes through microbial communities, with a specific focus on those genes involved in decontamination of OP pesticides worldwide. Other outputs include: (1) Education and training of four new PhD scientist in the field; (2) training of an international doctoral student in an NSF-funded exchange; (3) training of undergraduate scientists in the field, including presentations at state and regional conferences; (4) mentoring of 10 undergraduate scientist in research and more than 200 through formal classroom interactions; (5) international collaboration with a scientist in India in pesticide remediation; and (6) consulting to a new start-up company developing novel additives for creation of surface functionalities for decontamination. Products include graduation of four new PhD scientists in the field, contributing in industrial, nonprofit research and educational institutions. DISSEMINATION: The dissemination activities for the life of the project are too numerous to list individually, but include (on average) three conference presentations annually by senior scientist or collaborators and two annually by students engaged in research on the project. Specific national and international dissemination for the final year of the project can be found in the abstract publications section. PARTICIPANTS: Senior scientists: Drs. James Wild, Melinda Wales, Boris Novikov and Janet Grimsley. Collaborators: 1) Dr. Alex Simonian, Auburn University; 2) Dr Ilona Petrikovics, Sam Houston State University; 3) Dr Douglas Cerasoli, US Army Medical Research Institute of Chemical Defense; 4) Dr Tony Reeves, Southwest Research Institute; 5) Dr Teresa Good, University Maryland-Baltimore (UMBC) Graduate students: Texas A&M University Doctoral: Tony E Reeves, David Armstrong, Rory J Kern, Leamon Viveros. Texas A&M University Master Student: Richa Paliwal. At collaborating institutions-- Auburn: Sheetal Paliwal, Balasubramanian Shankar, Madhumati Ramanathan, Rigved Epur. UMBC-- Momar Seck, James Henry. University of Hyderabad, Hyderabad India: Pandeeti Emmanuel Vijay Paul Undergraduate Students mentored on this project exceeds 26; those with a significant research role include Texas A&M University: Manoj Rajaure; Auburn: George Vallone, Sean Carroll; UMBC: Anton Geisz and Neezet Agular. TARGET AUDIENCES: This project is oriented toward developing enzyme-based technologies for the protection and recovery of personnel and property resources from contamination events involving organophosphate neurotoxins and pesticides. These projects are, by their nature, quite interdisciplinary and draw the attention of DoD, DHS, and NIH, as well as industrial partners interested in environmental protection. PROJECT MODIFICATIONS: It is anticipated that this project will continue as TEX09488, with a focus on translational studies in development of therapeutics and prophylactics for OP poisoning, and basic research in the development of transducers for detection of pesticides and other chemical agents.
Impacts
OUTCOMES/ IMPACTS: Change in knowledge: The single most significant finding over the term of the project has occurred within the last 2 years, and that is a demonstration of complete in vivo protection, using a guinea pig model system, against the neurotoxic chemical warfare agents GA (tabun) and GB (sarin). Complete protection was demonstrated at molar ratios of 1:9,000 (OPH:GA) and 1:300 (OPH:GB). This exceeded the currently available technology, butryrlcholinesterase (BChE), which is stoichiometric and offers in vivo protection at 1:1 molar ratios. This represents a change in knowledge as prior to this project success the bias of the field was that catalytic molecules of foreign (i.e, non-host) origin would not be effective in vivo due to immunological and associated pharmacokinetic (PK) constraints. Other significant outputs over the term of the project include: (1) development of encapsulation procedures that enhance the enzyme's pharmacokinetic profile; (2) development of new production methods for cost effective production of enzyme therapeutics and prophylaxes. A change in action has occurred with a DoD core unit with the CounterAct Research Network. Prior to the discoveries made under this project, the core research unit focused exclusively on human stoichiometric and catalytic proteins. The performance metrics of bacterial-derived enzymes from our program outperformed so significantly that the new unit, currently under review by NIH, has adopted our technology and approach. The focus over the next 3 years will be FDA approval for the first bacterial-derived enzymatic therapeutic and prophylactic for organophosphorus poisoning.
Publications
- Novikov BN, Grimsley JK, Kern RJ, Wild JR, Wales ME. 2010. Improved pharmacokinetics and immunogenicity profile of organophosphorus hydrolase by chemical modification with polyethylene glycol. J Control Release. 15;146(3):318-25.
- Ramanathan M, Wang L, Wild JR, Meyeroff ME, Simonian AL. 2010. Monitoring of diisopropyl fluorophosphate hydrolysis by fluoride-selective polymeric films using absorbance spectroscopy. Anal Chim Acta. 667(1-2):119-22.
- Budai M. et al. 2009. Physico-chemical characterization of stealth liposomes encapsulating an organophosphate hydrolyzing enzyme. J. Liposome Res. 2:1-6.
- Catalytic Bioscavengers with Broad Specificity Against OP Nerve Agents. CounterAct Network Research Symposium, San Francisco, April 2010.
- Petrikovics, I., Kern, R., Wild, J.R., Wales, M.E. Hydrolyzing Enzymes in OP Antagonism: Cholinesterase Inhibition as Indicator of OP Intoxication. XII. International Congress of Toxicology, July 19-23, 2010, Barcelona, Spain;
- Petrikovics, I., Kern, R., Wild, J.R., Wales, M.E. Nano-captured Enzymes in OP Antagonism: Cholinesterase Inhibition as Indicator of OP Intoxication. 3rd International Conference on Nanotoxicology, June 2010, Edinburg, UK;
- Kuzmitcheva, G., Martin, S., Petrikovics, I. Study OPH Activity Packed in Minicapsules Against Paraoxon. 49th Annual Meeting of Society of Toxicology, 2010, Salt Lake City.
- Budai, M. et al. Liposomal Enzyme Encapsulation and Stability Studies with Liposomes. Pharmacokinetics and Drug Metabolism Symposium, April 2010 Galyatető, Hungary.
- Kern, Rory James Enzyme-based detoxification of organophosphorus neurotoxic pesticides and chemical warfare agents. Texas A&M University, December 2007.
- Armstrong, Charles David. 2007. Elucidating the chemical and thermal unfolding profiles of organophosphorus hydrolase and increasing its operational stability. Texas A&M University, Dissertation.
- Rodriguez Rodriguez, Mauricio 2005. Pyrimidine nucleotide de novo biosynthesis as a model of metabolic control. Texas A&M University, August 2005.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Research Objectives: 1) Enhance the catalytic properties available for the decontamination, destruction, and detection of O-P, and related O-F and O-CN, neurotoxic compounds utilizing both intelligent design and directed mutagenesis with the available bacterial OPHases. 2) New enzymes will be identified using bioinformatic approaches to gene discovery. 3) Utilizing new enzymes, develop chemical sensors for detection and discrimination of neurotoxic pesticides and other chemical threat agents. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Obj I Activities: The active site of OPH was re-designed for enhanced hydrolysis of some of the more recalcitrant organophosphorus compounds, such as VX. VX is the most toxic of chemical weapons in the United States arsenal, with an LD50 of 0.008 mg/kg, and remains a large component of the United States chemical weapons stockpile at 8,000 metric tons. RVX, an isomer of VX, is a major component of the Russian arsenal. In considering the general phosphotriester (P-O), phosphorofluoridate (P-F), and phosphorothioate (P-S) substrates, the P-S bond is hydrolyzed the slowest by any of the OP degrading enzymes, including OPH. VX and RVX are phosphonothioates with large aliphatic leaving groups and enzymes that do hydrolyze this class of compounds generally do so poorly, as demonstrated by the kcat for OPH of 0.3 sec-1 with VX. The sulfur of the P-S bond is larger than the oxygen or fluorine of the P-O and P-F bond substrates and the pKa of the P-S leaving group is high. The low electronegativity of the sulfur makes the phosphorus a weak electrophile, thus reducing the capacity of nucleophilic attack and bond hydrolysis. These characteristics contribute to the hydrolytic inefficiency of these substrates compared to the P-O and P-F substrates. Obj II Activities: A simple and fast procedure for the deposition of amorphous silica on a gold sensing surface was developed. The formation of the silica nanoparticles is triggered by lysozyme, which mediates the formation of a silica self-assembled monolayer through silicification. The silica layer significantly increases the surface area compared to the gold substrate and is directly compatible with a detection system. For the biosensor application, the silica matrix was shown to be a suitable method for immobilization of organophosphorus hydrolase (OPH) Paraoxon was used as a model substrate for the measurement of enzymatic activity. The silica forms at ambient conditions in a reaction that allows the encapsulation of OPH directly during silica formation. The detection limit for paraoxon and Km for the enzyme was found to be 20μM and 90μM respectively. In addition, OPH was immobilized covalently on functionalized planar waveguides. The patterning of the OPH along with a scaffold protein, in an array format on a single sensing waveguide, enabled simultaneous detection of multiple OPs. The Array biosensor platform, developed by the Naval Research Laboratories was adapted to our experimental needs and used to detect OPs.
Publications
- Reeves TE, Wales ME, Grimsley JK, Li P, Cerasoli DM & Wild JR (2008) Balancing the stability and the catalytic specificities of OP hydrolases with enhanced V-agent activities. Protein Eng Des Sel 21: 405-12
- Wild, J. R. Covalent Immobilization of Organophosphorus Hydrolase on Carbon Nanotubes for Biosensor Applications. Chemical and Biochemical Sensing Technologies, IMCS12. (2008).
- Wild, J. R. Detection of p-Nitrophenol and p-Nitrophenyl Substituent Organophosphates Based on Fluorescence Quenching, Sheetal Paliwal, 211th Meeting of the Electrochemical Society, May 6 - 10, 2007, Chicago, IL.
- Wild, J. R. Advanced Enzyme Based Biosensors, Tutorial Session of 6th IEEE Sensors Conference, Atlanta, GA, October 28-31, 2007.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Activities: The study is an effort to develop a system for the selective and sensitive real-time detection of organophosphate neurotoxins. Instead of using the pH change associated with enzymatic hydrolysis of the OP substrate as an indicator of the presence of an OP compound, the method is based on the change in fluorescence of a competitive inhibitor of the OPH enzyme when the inhibitor is displaced by the OP substrate. The kinetic evaluation of OPH revealed the fluorescence compound 7-diethylamino-4-methylcoumarin (Coumarin1) as competitive inhibitor of the enzyme. This Coumarin derivative is a dye with fluorescence in the blue-green region of the spectra and bear similar structure to some OPs. Paraoxon having higher affinity for OPH displaces the fluorophore molecule from the active site of the enzyme and a change in fluorescence intensity is observed which is proportional to the paraoxon concentration in the range of 0.4 - 173 uM. Other substrates such as Parathion and
DFP were also investigated. The minimum parathion concentration detected was 0.7uM, and linearity (Y = 0.0069 + 0.00209X, R = 0.98575) was observed at concentrations up to 143 uM. These results provided evidence that this detection method will work for nitrophenyl substituent organophosphates. To determine if the detection was selective toward nitrophenyl substituent organophosphates as it is designed to be, non-pNP producing OPs like malathion and DFP were tested. A second approach investigated the use of enzyme-encapsulated silica monolayers for rapid functionalization of a gold surface. The formation of the silica nanoparticles is triggered by lysozyme. First a layer of lysozyme is deposited via non-specific binding to the gold. This lysozyme then mediates the formation of a silica self-assembled monolayer through silicification. Surface plasmon resonance (SPR) spectroscopy was used to characterize the silica formation at the gold surface. The silica layer significantly increases
the surface area compared to the gold substrate and is directly compatible with a detection system. For the biosensor application, the silica matrix was shown to be a suitable method for immobilization of the enzyme, organophosphorus hydrolase (OPH). Paraoxon was used as a model substrate for the measurement of enzymatic activity. The silica forms at ambient conditions in a reaction that allows the encapsulation of OPH directly during silica formation. The detection limit for paraoxon and Km for the enzyme was found to be 20 μM and 90 μM, respectively. Finally, wild type OPH was immobilized covalently on functionalized planar waveguides. OPH was labeled with a pH sensitive fluorophore (carboxynapthafluorescein, CNF) and patterned along with a scaffold protein in an array format, on a single sensing waveguide. This system enabled simultaneous detection of multiple OPs. The Array biosensor platform, developed by the Naval Research Laboratories, was adapted to our experimental
needs and used to detect OP concentrations as low as 1uM for paraoxon and 10uM for DFP and parathion.
PARTICIPANTS: Drs. James Wild, Melinda Wales, Boris Novikov and Janet Grimsley. Graduate Students: Ph.D students Tony Reeves,David Armstrong and Rory Kern. Master Student: Richa Paliwal. Collaborators: Dr. Alex Simonian, Auburn University, is a long and valued collaborator. Auburn U. graduate students on this project include Sheetal Paliwal, Balasubramanian Shankar (part time), Madhumati Ramanathan, Rigved Epur (part time). Undergraduate Students on this project include George Vallone, Sean Carroll.UMBC: Dr. Theresa Good. Graduate Students: Momar Seck, and James Henry Undergraduate Students:Anton Geisz and Neezet Agular. Training or Professional Development: This project has served to bridge the gap between engineering and biochemistry. Three different Departments are involved in this project: Materials Engineering at Auburn University, Biochemistry and Biophysics at Texas A&M University, and Chemical and Biochemical Engineering at University of Maryland Baltimore. At TAMU, Dr Tony
Reeves, Dr David Armstrong and Dr Rory Kern, all have been involved in the design and implementation of enzymes for the sensor platforms as graduate students, and have graduated. Dr Reeves is currently at USAMRICD as an NRC Postdoctoral Fellow, working on enzymatic decontamination of chemical warfare agents, and Dr Armstrong has just taken a position in industry developing on enzymes for the oil services industry. Dr Kern completed his degree in Dec 2007, and is currently in a Postdoctoral position at TAMU. In addition, each semester the TAMU lab teaches an undergraduate research experience course, which is 3-6 research hours per student each week. Over the course of this project, 1 or 2 students have been trained each semester in protein production and assays.
Impacts
Outcomes/Impacts: (3200 characters and spaces) The results report the development of an alternative method for the detection of p-nitrophenyl-substituted organophosphates using the fluorescence changes of a competitive inhibitor, coumarin1. This approach allows for the development of a simple, cost-effective and easy methodology for detection of p-nitrophenol and, by coupling with OPH, for detection of pNP-substituent organophosphates. It has been illustrated that this concept can be developed as a biosensor of paraoxon and parathion, it can also be applied to other nitrophenyl substituted OP pesticides like methyl parathion and fenitrothion. Further studies are underway to better characterize the critical components for sensitivity that include type of coumarin, binding characteristics of coumarin to the enzyme, and surface attachment methods. A second method utilized silica deposited on a surface to encapsulate OPH as the reactive component of an OP sensor. The
surface was quite stable under continuous flow conditions and a reactive surface for accurate and reproducible kinetic measurements. The silica-OPH deposited surface could be used continually for over 2 days, eventually losing activity due to the loss of silica from the surface. OPH is not directly attached to the gold surface, which may limit any restriction in the orientation of the active site, as often observed when enzymes are covalent attached to a surface. Further optimization in the deposition of silica could provide better enzyme stability and detection sensitivity. The final method, functionalized planar waveguides, enabled simultaneous detection of multiple OPs. and proved active for nearly 2 months.
Publications
- McDaniel, C.S., McDaniel, J., Wild, J.R., Wales, M.E. 2007. Biocatalytic Paints and Coatings. ACS Symposium Series: SmartCoatings 2006.
- Wales, M.E., McDaniel, C.S., Kern, R. and Wild, J.R. 2007. Enzyme Technology: Applications for the Decontamination of Organophosphorus Agents. OPCW Proceedings.
- Paliwal, S.; Wales, M.; Good, T.; Grimsley, J.; Wild, J.; and Simonian, A.. 2007. Fluorescence-Based Sensing of p-nitrophenol and p-nitrophenyl Substituent Organophosphates, Anal Chim Acta 596(1):9-15
- Petrikovics, I., Wales, M.E., Jaszberenyi, J.C., Budai, M., Baskin, S.I., Szilasi, M., Logue, B.A., Chapela, P., Wild, J.R. 2007. Enzyme-based intravascular defense against organophosphorus neurotoxins. Synergism of dentritic-enzyme complexes with 2-PAM and atropine. Nanotoxicology, 00: 1-9
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Progress 01/01/06 to 12/31/06
Outputs
Research Objectives Enhance the catalytic properties available for the decontamination, destruction, and detection of O-P, and related O-F and O-CN, neurotoxic compounds utilizing both intelligent design and directed mutagenesis with the available bacterial OPHases. New enzymes will be added using bioinformatic approaches to gene discovery. Utilizing the new OPHases, develop chemical sensors for detection and discrimination of neurotoxic pesticides and other chemical threat agents. Task 1: Develop New Enzyme Biocatalysts for Detection and Destruction of Pesticides and other Organophosphates. Since the common immobilization of an enzyme results in the random positioning of the protein's active side, this could restrict access to and from the active site of products and substrates and significantly reduce a sensitivity of the method. This study presents optimization of sensitivity and specificity for detection of OPs via an orientation specific attachment of an enzyme
using surface plasmon resonance (SPR) spectroscopy in order to optimize active site accessibility, and thus, sensitivity of the technology. OPH was genetically engineered by site-directed mutagenesis to replace the lysine (K175) from the face of the molecule with access to the active site. This allows for an orientation specific attachment, and thus better sensitivity, in contrast to the random attachment of the native OPH. Task 2: Develop Biosensing Systems for Discriminating Between Different Classes of Neurotoxins Based on Coupled AChE-OPH Enzyme Monitors. Current sensor technology suffers from a lack of specificity and sensitivity for real-time detection of different chemical and biological substances, including chemical/biological threat agents. Although a variety of approaches are used in different sensor systems, new revolutionary technologies are needed for any significant enhancement of performance. Noble metal nanoparticles have been recently shown to have an incredible
capability to amplify and boost signals in many biorecognition processes based on enzymes, DNA, antibodies and other systems. Here, the investigators have taken advantage of the surface modified fluorescence provided by a nanoparticle conjugated to organophosphate hydrolase (OPH) enzyme to develop an array of sensors that are specific for a number of organophosphate molecules. The studies performed in the reporting period demonstrated that when a system comprised of gold nanoparticles is conjugated to OPH and a fluorescent decoy, the specificity of enzyme-substrate interactions could be exploited for detection of target neurotoxins. The fluorescence intensity of the decoy was sensitive to the proximity of the gold nanoparticle. The different target neurotoxin concentrations were introduced to the OPH-nanoparticle-conjugate-decoy mixtures, and normalized ratio of fluorescence intensities were measured. The greatest sensitivity was obtained when decoys and OPH-gold nanoparticle
conjugates were present at near equimolar levels. The change in fluorescence intensity was correlated with concentration of OP presented in the solution.
Impacts
- This project is unique in that it provides both an original approach and a next-generation capability for detection and identification of chemical species in the presence of a cluttered background. This technology will have immediate impact in a) homeland security, b) environmental monitoring, and c) clinical analysis. The technology developed could be extended to other analytes of interest in the defense of homeland security or protection of the environment.
Publications
- Enzyme-based biosensors for the direct and discriminative detection of chemical warfare agents and related agricultural pesticides. A.L. Simonian, and J.R. Wild. 2006. In The Science of Homeland Security S.F. Amass, A.K. Bhumia, A.R. Chaturvedi, D.R. Dolk, S. Peeta and M.J. Atallah (eds.) Purdue University Press (Indiana), pp. 83-108.
- Enzyme-based additives for paints and coatings C.S. McDaniel, J. McDaniel, M.E. Wales and J.R. Wild. 2006. Prog. Org. Coat. 55(2):182-188.
- Degradation of organophosphorus neurotoxicity in SY5Y neuroblastoma cells by organophosphorus hydrolase (OPH) T.M. Cho, J.R. Wild, K.C. Donnelly, and E. Tiffany-Castiglioni. 2006. J. TOXICOLOGY ENVIRONMENTAL HEALTH-PART A-CURRENT ISSUES 69(15):1413-1429.
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Progress 01/01/05 to 12/31/05
Outputs
The main goal of this proposal is the development of new chemical and biochemical technologies that permit on-site detection and remediation of OP chemical contamination. OP compounds represent the single largest class of urban and rural pesticides used commercially in various locals throughout the world. The acute toxicity of these broad-range neurotoxins is of great environmental concern.
Impacts
We are in the process of producing industrial application for clean-up, detoxification, and detection of hazardous neurotoxins and pesticides in the U.S. Army Chemical Demilitarization Program and the Homeland Security activities as well as in-going programs to manage agricultural pesticides.
Publications
- Grimsley JK, Calamini B, Wild JR, Mesecar AD. Structural and mutational studies of organophosphorus hydrolase reveal a cryptic and functional allosteric-binding site. Arch Biochem Biophys. 2005 Oct 15;442(2):169-79. Epub 2005 Sep 6.
- Gold RS, Maxim J, Halepaska DJ Jr, Wales ME, Johnson DA, Wild JR. Electron beam irradiation as protection against the environmental release of ecombinant molecules for biomaterials applications. J Biomater Sci Polym Ed. 2005;16(1):79-89.
- Rodriguez M, Good TA, Wales ME, Hua JP, Wild JR. Modeling allosteric regulation of de novo pyrimidine biosynthesis in Escherichia coli. J Theor Biol. 2005 Jun 7;234(3):299-310.
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Progress 01/01/04 to 12/31/04
Outputs
The Research Objectives: 1. Enhance the catalytic properties available for the decontamination, destruction, and detection of O-P, and related O-F and O-CN, neurotoxic compounds utilizing both intelligent design and directed mutagenesis with the available bacterial OPHases. New enzymes will be added using bioinformatic approaches to gene discovery. 2. Utilizing the new OPHases, develop chemical sensors for detection and discrimination of neurotoxic pesticides and other chemical threat agents. The work in the previous year was focused primarily on Task 1. Task 1: Develop New Enzyme Biocatalysts for Detection and Destruction of Pesticides and other Organophosphates. Re-design of enzymes for surface attachment: A common mechanism by which proteins are incorporated onto sensor surfaces is crosslinking with amine reactive reagents. Virtually all proteins have lysine residues, and liphatic amines such as lysine's amino group are moderately basic and reactive with most
acylating reagents. There are 6 lysines in OPH, which provide multiple opportunities for attachment, yet can diminish activity by coupling in an unfavorable orientation. If the surface lysines are equally available for attachment, it is predicted that approximately one-third of the OPH molecules would be attached in a catalytically unfavorable orientation. By selectively eliminating the lysines on the active site face of the molecule, it may be possible to influence the coupling orientation so that more of the protein is attached in a catalytically favorable orientation. Surface lysine 175 was targeted in this study, due to its proximity to the active site. The replacement of Lysine 175 with an alanine eliminates the reactivity at that site in the surface attachment procedure. Evaluation of the activity of this variant verified that the catalytic activity was not significantly affected. Preliminary studies with surface attachment indicate that this strategy may be successful. SPR
analysis indicated that even though less protein bound to the surface, the surface exhibited greater activity at low substrate concentrations. Although these studies are ongoing, the results to date suggest that the ability to design protein ligands for specific surface orientation of OPH will provide an effective approach for tailoring enzymes for sensor platforms. Design of enzyme for probe attachment: A variety of different fluorescent probes are available for attachment via thiol groups, including the CYdyes from Amersham and the AlexaFluors from Molecular Probes. OPH has been re-designed with cysteine residues located near the active site to provide a thio group to test this strategy. Figure 5 below illustrates below the location of the cysteine residue at position 309. The mutant has been created and verified by sequencing, and the protein variant expressed. Assay to evaluated the activity of Y309C are underway, to be followed by attachment and monitoring of a fluorophore.
Impacts
This project is moving rapidly toward industrial application for clean-up, detoxification, and detection of hazardous neurotoxins and pesticides in the U.S. Army Chemical Demilitarization Program and the Homeland Security Activities as well as in-going programs to manage agricultural pesticides.
Publications
- Nanoparticle-based Optical Biosensors for the Direct Detection of Organophosphate Chemical Warfare Agents and Pesticides. 2004. A.L. Simonian, T. Good, S.-S. Wang, and J.R. Wild. Analytica Chima Acta. 534/1: 69-77.
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Progress 01/01/03 to 12/31/03
Outputs
There are four general classes of organophosphate hydrolases that have been identified as paraoxonase, somanases, phosphotriesterases, DFPases, etc. They all are OPA nhydrolases (E.C. 3.1.8.1). The most studied class (OPH-1) have been extensively studied for their ability to hydrolyze a large variety of organophosphate pesticides and neurotoxins including OP phosphotriesters, OP phosphothioates (including V-agents), phosphofluoridates (including G-agents), and the P-CN bonds of neurotoxins such as Tabun. The second class of enzymes (OPH-2) have been identified as OPAA although their hydrolytic capabilities are limited to P-F bonds (Soman, Sarin, and DFP). The third type of enzyme (OPH-3) is represented by the squid enzyme which is quite different than the OPH-2 class although it primarily is a DFPase and usually identified as such. The fourth class (OPH-4) is represented by the human paraoxonases (PON) that is reported to be a good paraoxonase with significant G-agent
hydrolysis. Genome-based gene discovery in our laboratory has led to the cloning and expression of additional OPH-1 and OPH-2 enzymes. In addition, it has been possible to identify and assemble three different human PON genes (OPH-4). It has been possible to compare the activities and protein natures of these enzymes with the intent to incorporate select enzymes into multifunctional active surfaces. We have established a new, collaborative NSF-MURI (Multiple University Research Initiative) with Auburn University and the University of Maryland, Baltimore Country. In addition, we have expanding research initiatives through an SBIR and STTR with LynnTech, Ltd. (College Station) and corporate funding with Reactive Surfaces, Ltd. (Austin). Furthermore, a collaboration with Dr. Cady Engler is addressing the potentials of bioremediation to manage agricultural pesticide persistence along the Gulf Coast.
Impacts
This project is moving rapidly toward industrial application for clean-up, detoxification, and detection of hazardous neurotoxins and pesticides in the U.S. Army Chemical Demilitarization Program and the Homeland Security Activities as well as in-going programs to manage agricultural pesticides.
Publications
- "Neurotoxicity induced in differentiated SK-N-SH-SY5Y human neuroblastoma cells by organophosphorus compounds." 2003. Hong, M.S., S.J. Hong, R. Barhoumi, R.C. Burghardt, K. C. Donnelly, J. R. Wild, V. Venkatraj, E. Tiffany-Castiglioni. Toxicol. Appl. Pharmacol., 186:110-118.
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Progress 01/01/02 to 12/31/02
Outputs
A large collection of native and mutant enzymes has been created. The kinetic characteristics have been evaluated with a variety of organophosphate neurotoxins (OP). Enzymes with enhanced rates of detoxification of select OPs have been identified. It is clear that there is a significant structural flexibility which can be utilized to dramatically improve the catalytic ability of organophosphate hydrolase (OPH). Specific amino acids have been targeted in a rational design approach, resulting in 25 new enzymes. In addition, to identify unpredictable structural changes that could lead to the enhancement of select OP destruction, a random mutagenic approach has also been used to create mutations in the enzyme. A collection of 67 of these new enzymes has been prepared and are being screened for enhanced hydrolysis. Through comparison of the X-ray crystallographic structures of existing genetic variants of OPH, we have developed a better understanding of the structure of
the active center of the enzyme. OPH and most of its genetic mutants have two metal molecules per monomer, but some of the mutants seem to have a single metal while some others may have three. In addition, CW agent studies have been performed with the native enzyme and select second generation enzymes against the V-agent neurotoxins (V-X and R-VX) which has verified a twenty-fold increase in hydrolysis rates (214 molecules/sec) over the native levels.Enzyme-based biosensors are being constructed to utilize an array of genetically modified enzymes which will allow discrimination between chemical warfare agents and other chemical neurotoxins. A new type of an optical, fluorimetric OPH-based chemical sensor utilizing existing enzymes has been constructed, and a two channel microfluidic system has been developed to provide a new, highly sensitive method for the detection of electroactive compounds such as those generated by the hydrolysis of P-neurotoxins. Ultimately, these sensors will
come together to produce a new generation of discriminating detectors that are designed for stability and environmental tolerance as well as chemical agent sensitivity. The new enzymes will be introduced into a variety of decontamination studies, including the formation of chemical decontamination wipes, in the formulation of chemical decontamination solutions, in enzyme-based protection filters, in bioreactors, etc. Studies on the effectiveness of enzyme-decorated cotton towelletes have been completed in collaboration with LynnTech INC. of College Station, Texas. The towelletes have been shown very effective at decontaminating dead skin and hard surfaces.
Impacts
OP compounds represent the single largest class of urban and rural pesticides contributing over 100 different compounds for commercial use in various locals throughout the world. It has been estimated that over 1500 different OP neurotoxins have been synthesized during the past century. The acute toxicity of these broad-range neurotoxins is of great environmental concern, and stringent regulations have been implemented world-wide, limiting the agricultural use as well as defining the maximum levels of permissible residues on food for human and animal consumption. The main goal of this research project is the development of new chemical and biochemical technologies that permit on-site detection and remediation of chemical contamination associated with both the presence of organophosphate (OP) neurotoxins from agricultural and chemical warfare munitions waste and potential chemical threats against civilian and military populations (Homeland Security).
Publications
- A.L. Simonian, A. Revzin, J. R. Wild, J. Elkind, and M. V. Pishko. Characterization of Oxidoreductase/ Redox Polymer Electrostatic Film Assembly by Surface Plasmon Resonance. Spectroscopy, FTIR, and Ellipsometry on Gold. Anal.Chim.Acta, 2002, 466:201-212.
- Kim JW. Rainina EI. Mulbry WW. Engler CR. Wild JR. Enhanced-rate biodegradation of organophosphate neurotoxins by immobilized nongrowing bacteria. Biotechnology Progress. 18(3):429-36, 2002 May-Jun.
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Progress 01/01/01 to 12/31/01
Outputs
The dimeric bacterial enzyme Organophosphorus Hydrolase(OPH, EC 3.1.8.1) is capable of detoxifying many neurotoxic compounds by hydrolyzing a wide variety of phosphonate bonds(P-O, P-F, P-CN, P-S). Not all substrates are hydrolyzed with the same specificity and/or efficiency. A more detailed understanding of the individual residue contributions to stability and activity is necessary to rationally engineer the enzyme to modulate these physical parameters. Increasing catalytic efficiency and stabilizing the enzyme is not always achievable in a single enzyme variant. Using site-directed mutagenesis, we have been able to increase the substrate specificity for demetonS, an analog for the chemical warfare agent VX, 30-fold compared to the wild type enzyme, yet the overall conformational stability has been reduced based on thermal and chemical denaturation experiments. Rational design will yield enzymes with increased specificity and/or efficiency while maintaining stability
for applications under diverse environmental conditions and in detoxifying solutions, chemical degrading wipes and humoral protectants. A multidisciplinary team of research scientists at Texas A&M University has been formed to provide support for the development of new chemical and biochemical technologies which will permit on-site detection and remediation of chemical contamination that may be associated with the presence of chemical threat agents. As the result of positive developments during the first year of research activities, two additional research groups have been added to the program. Dr. Michael Pishko (Chemical Engineering) will be involved in developing an optical biosensor enzyme array, and Dr. Francois Gabbai (Chemistry) is constructing organometallic materials which are specifically designed to recognize chemical threat agents. Ongoing research studies in the laboratories of Dr. Richard Crooks (Chemistry, TAMU) and Dr. James Wild (Biochemistry, TAMU) have developed
and demonstrated prototype detectors, both chemical and enzyme-based biosensors, for the detection of chemical toxins, including organophosphate neurotoxins such as agricultural pesticides and chemical warfare agents. In addition, multiple enzyme biosensors have been shown to be very effective in discriminating between various classes of chemical neurotoxins. Extensive characterization of the broad spectrum organophosphate hydrolase (also called phosphotriesterase and organophosphorus acid anhydrolase, EC 3.1.8) which is the base for the biosensor has been ongoing in our laboratories since 1987. Recent extensions of these studies have shown that several other enzymes expand the biocatalyst capabilities of OPH enzyme systems. Finally, it has been possible to demonstrate that it is possible to utilize this enzyme in a variety of applications which should be able to protect and decontaminate both patients and medical personnel, sites and equipment
Impacts
a) This research has expanded the basic understanging of the mechanism of chemical hydrolysis of neurotoxins which has led to to the genetic engineering of enzymes to destroy chemical warfare agents. b) This research has led to the design of an enzyme-based biosensor that can discriminate between different classes of neurotoxins (e.g. heavy metals, carbamates, and organophosphates. c) A variety of decontamination technologies are being developed from this work including enzyme-based towellets, aerosol fogs, and detergent foams.
Publications
- Grimsley, J.K., Singh, W.P., Wild, J.R., and Giletto A., "A novel, Enzyme-based Method for the Wound-Surface Removal and Decontamination of Organophosphorus Nerve Agents." 2001. Bioactive Fibers and Polymers. 35-49.
- Simonian, A.L., Grimsley, J.K., Flounders, A.W., Schoeniger, J.S. Cheng, T.-C., DeFrank, J.J., and Wild, J.R., "An Enzyme-based Biosensor for the Direct Detection of Fluorine-containing organophosphates." 2001. Analytica Chimica Acta. 442:15-23.
- J.K. Grimsley, B. di-Sioudi, T.R. Holton, J.C. Sacchettini, A.L. Simonian, E.I. Rainina and J.R. Wild. "Active Site Modifications of OP Hydrolase for Improved Detoxification of OP Neurotoxins." 2000. Enzymes in Action. NATO-ASI pp223-242.
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Progress 01/01/00 to 12/31/00
Outputs
The central hypothesis of this project involves the use of natural and genetically engineered microbial strains for the development of a stable biological system to detoxify and biotransform OP neurotoxins to discardable biological products. OP neurotoxins comprise a unique class of chemical contaminants which, although they have low environmental persistence, have a high acute toxicity and a wide range of biological activities. The results thus far have demonstrated the potential for the development of the technology necessary for the detoxification and mineralization of neurotoxic organophosphates from agricultural and munitions wastes. Some parts of OP contamination contain mixtures of hazardous materials, similar to agricultural and industrial wastes, which provide novel toxicity problems of their own. The detoxification of such mixtures requires the integration of a number of different metabolic systems (different bacteria, purified enzymes, fungi, etc.). The
progress over the past year is summarized as follows: 1) We have four different OPH enzymes: OPH-1 from P. diminuta, OPAA-2 from a halophilic bacterium, OPH-2 from Aeromonas (probably), and paraoxonase from human liver. 2) OPH enzymes protected acetylcholine esterase against paraoxon as well as surrogates for Soman/Sarin and VX 3) OPH is a homodimeric metallo-enzyme with two cation binding centers per subunit. Its remarkable stability derives from hydrophobic interactions and is further enhanced by cross-linking or immobilization. The choice of cation influences catalytic rates for DFP, Soman, Sarin, Tabun and different V- agents. 4). Site-directed mutagenesis has produced enzymes with mono-metallic and bimolecular catalytic centers with dramatic shifts in specificity and stability. OPH linked to nano-porous membranes produces a sensitive enzyme-based biosensor. OPH is stable when bound within a PEG hydrogel. Furthermore a catalytically-active enzyme-sponge is formed when OPH is
co-polymerized with acrylamide. 5) In order to maximize the efficiency of the enzyme, a catalytic site must be finely tuned to a particular substrate. General protection against nerve agents requires engineering and deployment of a panel of highly specific OPH variants.
Impacts
(N/A)
Publications
- Grimsley, J.K., Di Sioudi, B. Holton, T.R., Sacchettini, J.C., Simonian, A.L., Rainina, E.I. and Wild, J.R.. 2000 Active Site Modifications of OP Hydrolase for Improved Detoxification of OP Neurotoxins." In Enzymes in Action: Green Solutions for Chemical Problems. B. Zwanenburg, M. Mikoajczyk and P. Kiebasiski, Eds. NATO Science Series. Kluwer Academic Publishers . pp. 223-242.
- Simonian, A.L., Rainina, E.I. and Wild, J.R. 2000 Microbial Biosensors Based on Potentiometric Detection. In Methods in Biotechnology: Enzyme and Microbial Biosensors: Techniques and Protocols. A. Mulchandani and K.R. Rogers. Eds. 6:237-248. Humana Press.
- Cho, T.H., Wild, J.R., and Donnelly, K.C. 2000 Utility of OPH Enzyme for the Remediation of Mutagenicity of Methyl Parathion. Environ. Toxicol. Chem. 19:2022-2028.
- Gold, R. S., Wales, M.E., Grimsley, J.K., and Wild, J.R.. 2000 Ancillary Function of Housekeeping Enzymes: Fortuitous Degradation of Environmental Contaminants. In Enzymes in Action: Green Solutions for Chemical Problems. B. Zwanenburg, M. Mikoajczyk and P. Kiebasiski, Eds. NATO Science Series. Kluwer Academic Publishers. pp.263-286
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Progress 01/01/99 to 12/31/99
Outputs
The goal of this proposal is the development of an effective and complete biological remediation system capable of hydrolyzing organophosphorous neurotoxins from agricultural and chemical warfare munitions contamination and wastes. Specific microbial consortia based on cryoimmobilized recombinant E. coli and wild-type strain Pseudomonas putida will be employed for the mineralization of paraoxon, parathion, and methyl parathion. Bacteria will be isolated at ERDEC which utilize pinacolyl alcohol (PA) as the sold source of carbon.
Impacts
(N/A)
Publications
- Cho, T-H, Wild, J.R., Donnelly, K.C. (2000)"Utility of OPH Enzyme for the Remediation of Mutagenicity of Methyl Parathion." Accepted, Environ. Toxicology and Chemistry. (November 1999).
- J-W Kim, Rainina, E.I., Engler, C.R., Wild, J.R. (1999) "Processing Efficiency of Immobilized Non-Growing Bacteria: Biocatalytic Modeling and Experimental Analysis." Canadian J. Chem. Eng. 77:883-892. Simonian, A.L., B.D. diSioudi, and Wild, J.R."An enzyme-based Biosensor for the Direct Determination of Diisopropylfluorophosphate." Analytic Chimica Acta. 389:189-196.
- Simonian, A.L., Rainina, E.I., Fitzpatrick, P.F., and Wild, J.R. (1999) "Enhancement of the Specificity of an Enzyme-based Biosensor for L-Tryptophan." In Tryptophan: Basic Aspects and Practical Applications. Ed. T. Simat. Plenum Publishing Corporation, NY. In Press.
- Grimsley, J.K., B.D. diSoudi, and Wild, J.R. (1999) "Enzyme Engineering for the Improved Degradation of Organophosphorus Neurotoxins." J.K. Grimsley, B. D. diSoudi, and J.R. Wild. Biotechnology Intl. 2:235-242. B.D. diSioudi, Miller, C.L., Lai, K., Grimsley, J.K., and Wild. J.R. (1999) Rational Design of Organophosphorus Hydrolase for Altered Substrate Specificities." Chem. Biol. Interactions. 119:211-223.
- Wales, M.E. and Wild, J.R. (1999) "Aspartate Transcarbamoylase". M.E. Wales and J.R. Wild. In Encyclopedia of Molecular Biology. Ed. Tom Creighton. Publisher Academic Press, NY. p196-201.
- Zhang, Y., Autenreith, R.L., Bonner, J.S., Harvey, S.P., and Wild, J.R. (1999) "Biodegradation of Neutralized Sarin." 1999. Y. Zhang, R.L. Autenrieth, J.S. Bonner, S.P. Harvey, and J.R. Wild. Biotech. Bioengineering 64:221-231.
- B.D. diSioudi, Grimsley, J.K., Lai, K. and Wild, J.R.(1999) "Modification of Near-Active Site Residues in Organophosphorus Hydrolase Reduces Metal Stoichiometry and Alters Substrate Specificity." Biochemistry 38:2866-2872.
- Boronin, A.M., Ermakova, I.T., Sakharovsky, V.G., Grechkina, G.M., Starovoitov, I.I., Autenreith, R.L., Wild, J.R. "Ecologically Safe Destruction of Mustard-Lewisite Mixtures from the Russian Chemical Stockpile." 2000. J. Chemical Tech. Biotech. 75:1-7.
- Wales, Melinda E., Gold, Shane R., Grimsley, Janet K., Wild, James R. (2000) "Ancillary Function of Housekeeping Enzymes: Fortuitous Degradation of Environmental Contaminants." Enzymes in Heteroatom Chemistry. NATO-ASI Publication, In Press. Grimsley, J.K., B. di-Sioudi, Holton, T.R., Sacchettini, J.C., Simonian, A.L., Rainina, E.I., Wild, J.R. (2000) "Active Site Modifications of OP Hydrolase for Improved Detoxification of OP Neurotoxins." Enzymes in Heteroatom Chemistry. NATO-ASI Publication, In Press.
- Liu, L., Wales, M.E., Wild, J.R. (2000) "Allosteric Signal Transmission Involves Synergy between Discrete Structural Units of the Regulatory Subunit of Aspartate Transcarbamoylase." Arch. Biochem. Biophysics, In press. (Selected structural model chosen for journal cover.)
- Cunin, R., Rani, C.S., VanVliet, F., Wild, J.R., Wales, M.E. (1999) "Intramolecular Signal Transmission in Enterobacterial Aspartate Transcarbamylases (II): Engineering Cooperativity and Allosteric Regulation in the Aspartate Transcarbamylase of Erwinia herbicola." J. Mol. Biology, 294:1401-1411.
- Wales, M.E., Madison, L.L., Glaser, S.S., Wild, J.R. (1999) "Divergent Allosteric Patterns Establish a New Regulatory Paradigm for Aspartate Transcarbamoylase." 294:1387-1400. Russell, R.J., Pishko, Michael V., Simonian, A.L., Wild, J.R. (1999) "Poly(ethylene glycol) Hydrogel-Encapsulated Fluorophore-Enzyme Conjugates for Direct Detection of Organophosphorus Neurotoxins." Analytical Chemistry. 71:4909-4912.
- Flounders, A.W., Singh, A.K., Volponi, J.V., Carichner, S.C., Wally, K., Schoeniger, J.S., Simonian, A.L., Wild, J.R. (1999) "Development of Sensors for Direct Detection of Organophosphates: Sol-gel Modified Field Effect Transistor with Immobilized Organophosphate Hydrolase." Biosensors & Bioelectronics 14:713-720.
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Progress 01/01/98 to 12/31/98
Outputs
The objective of this research program is the development of a new type of robust biosensor for the direct detection and quantitation of organophosphate neurotoxinss, OPs. OP neurotoxins comprise a unique class of chemical contaminants which, although they have low environmental persistence, have a high acute toxicity and a wide range of biological activities. In the past year a new approach for discriminative determination of various classes of neurotoxins in natural samples has been implemented and a biosensor with the ability to distinguish between carbamate and organophosphorus pesticides has been developed. The progress over the past year is summarized as follows: 1. In collaboration with Sandia National Laboratories, we have developed a new biosensor based on the differential bioFET chip which can be used for "real world sample" measurements. Ongoing development includes using the multi-ISFET integrated device with various genetically engineered OPH enzymes
immobilized on each transistor. This array of biosensors will allow for the discrimination between various substrates in complex multicomponent samples. 2. A biosensor for the direct determination of DFP has been developed. A fluoride sensitive ion-selective electrode was exploited as the physical transducer for a batch-mode biosensor, and OPH enzyme immobilized on silica gel was used as a biological recognition element. The correlation between DFP concentration and hydrolyzed fluoride extended over a concentration range of 2.5 x 10-5 M to 5 x 10-3 M. 3) A new selective amperometric biosensor for reagentless L-tryptophan determination has been developed using immobilized tryptophan-2-monooxygenase, TMO, EC 1.13.12.3. This enzyme-based biosensor provides a rapid-response detection system for concentrations of L-tryptophan between 25 and 1000 mM in a batch mode system and between 100 and 50,000 mM in a flow-injection mode. The response time was 30 seconds, and the total analysis time
was less than 3 minutes. The biosensor retained catalytic activity and fidelity of phenylalanine and tryptophan response for greater than 4 months with repeated usage. The biosensor selectivity to L-tryptophan was dramatically increased relative to phenylalanine when a competitive inhibitor of TMO, indole acetamide, IA, was included. The biosensor was successfully used for L-tryptophan determination in nutrition broth, giving values identical to those determined by HPLC analysis.
Impacts
(N/A)
Publications
- LEJUNE, K.E., WILD, J.R., and RUSSELL, A.J., 1998. Nerve Agents Degraded by Enzymatic Foams. Nature 395:27-28.
- GRIMSLEY, J.K., RASTOGI, V.K., and WILD, J.R., 1998. Biological Detoxification of Organophosphorus Neurotoxins, In Bioremediation: Principles and Practice, S.K. Sikdar and R.L. Irvine,Eds., pp.577-613.Technomic Publishing Co., Inc.
- HONG, M.L., RAININA, E., GRIMSLEY, J.K., DALE, B.E., and WILD, J.R., 1998. Neurotoxic Organophosphate Degradation with Polyvinyl Alcohol Gel-Immobilized Microbial Cells. Bioremediation Journal 2,2: 145-157.
- SIMONIAN, A.L., RAININA, E, and WILD, J.R., 1998. "Microbial Biosensors Based on Potentiometric Detection" in "Enzyme and Microbial Biosensors. Techniques and Protocols", A. Mulchandani and K. Rogers Eds., Humana Press, Totowa, New Jersey, 237-248.
- ARAKELIAN, V.B., WILD, J.R, and SIMONIAN, A.L., 1998. "Investigation of Stochastic Fluctuations in the Signal Formation of Microbiosensors." Biosensors and Bioelectronics, 1998, Vol. 13, No.1, 55-59.
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Progress 01/01/97 to 12/31/97
Outputs
The objective of this research program is the development of a new type of robust biosensor. This biosensor is based on the hydrolytic capabilities of an organophosphate hydrolase (OPH) which can degrade a variety of organophosphorus (OP) neurotoxins including both agricultural pesticides and chemical warfare agents. OP neurotoxins comprise a unique class of chemical contaminants which, although they have low environmental persistence, have a high acute toxicity and a wide range of biological activities. This project is focussed on three converging areas of interest: 1) Despite shared neurotoxic effects, the OPs differ extensively in their chemical structure. Consequently, while OPH has been shown to hydrolyze many important agricultural insecticides (including parathion, methyl parathion, diazinon, dursban and coumaphos), the enzyme has relatively low specificities for chemical warfare agents such as soman, sarin and VX nerve agents. The modification of OPH using
mutagenesis techniques has produced enzymes with increased activity against soman and VX, thus demonstrating that the genetic engineering of OPH can result in the catalytic improvements necessary for environmental applications. (2) A variety of immobilization techniques have been investigated for the development of the biosensor. These include such diverged approaches as the covalent binding of purified enzyme to silica gel, covalent attachment to ISFET (ion selective field effect transitor) and the cryoimmobilization of intact microbial cells. While each of these approaches has its own characteristic advantages, the immobilization of purified enzyme to silica gel or ISFET transducer are currently being investigated. (3) A new approach for the discriminative determination of various neurotoxins involves the combination of an OP biosensor with a highly sensitive acetylcholinesterase-based biosensor. This biosensor is capable of discriminating between OP neurotoxins in a mixed solution
containing carbamate pesticides.
Impacts
(N/A)
Publications
- GRIMSLEY, J.K., RASTOGI, V.K. AND WILD, J.R. 1997. "Biological Detoxification of Organophosphorus Neurotoxins." in Sikdar, S.K. and Irvine , R.L. (eds.) Bioremediation: Principles and Practice--Vol II:Biodegradation Technology Developments. Lancaster, Pennsylvania, Technomic Publishing Company, pp 577-614.
- GRIMSLEY, J.K., SCHOLTZ, J.M., PACE, C.N. AND WILD, J.R. 1997. "Organophosphorus Hydrolase is a Remarkably Stable Enzyme that Unfolds Through a Homodimeric Intermediate" Biochemistry 36(47):14366-14374.
- SIMONIAN, A.L., RAININA, E.I. AND WILD, J.R. 1997. "A New Approach for Discriminative Detection of Organophosphate Neurotoxins in the Presence of Other Cholinesterase Inhibitors" Analytical Letters, 30(14):2453-2468.
- SIMONIAN, A.L., RAININA, E.I., FITZPATRICK, P.F. AND WILD, J.R. 1997. "A Tryptophan-2-monooxygenase Based Amperometric Biosensor for L-Tryptophan Determination. Use of a Competitive Inhibitor as a Tool for Selectivity Increase." Biosensors and Bioelectronics, 12(5):363-371.
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Progress 01/01/96 to 12/30/96
Outputs
Biodegradation can be defined as the biologically catalyzed reduction in complexity of chemicals leading to decreased biological toxicity. Recent years have witnessed an enormous growth in the controlled, practical use of microorganisms for the destrcution of chemical pollutants. These various technologies rely on the biodegradative activities of microorganisms, & they focus on enhancing existent but slow biodegradation processes in nature or by using technologies that direct chemicals into interactions with microorganisms in some types of reactor that allows for biotransformation. The objective of this research program is the development of a new type of robust biosensor based on a hydrolyitc enzyme which variably degrades many different OP neurotoxins, both agricultural pesticides and chemical warfare agents. Organophosphosphorus (OP) neurotoxins comprise an unique classof chemical contaminants which generally show low environmental persistence, but have a high
acute toxicity & wide range of biological activities. This project focussed on 3 areas of interest: (1) Because of ongoing protein engineering manipulations, it is possible to produce a series of enzymes with varying substrate specificity. (2) Enzymes will be immobilized by covalent binding to silica gelby techniques previously accomplished.
Impacts
(N/A)
Publications
- MASON, J. R., BRIGANTI, F., and WILD, J.R. 1996. "Protein engineering for Improved Biodegradation of Recalcitrant Pollutants." NATO SLI Series, Eds.
- GRIMSLEY, J.K., RASTOGI, V. K., and WILD, J.R. 1996. "Biological Systems for the Detoxification of Organophosphorus Neurotoxins: Potential for Environmental Remediation Applications." Technomic Publishing Co. Inc. In Press SHELLEY, M.
- D., DALE, B. E., AUTENREITH, R.L. and WILD, J.R. 1996. "Thermodynamic Analysis of Trinitrotoluene Biodegradation and Mineralization Pathways." 50: 198-205.
- RAININA, E.I., SIMONIAN, A.L., ELREMENCO, E. N., VARFOLOMEYEV, S.D. and WILD, J.R. 1996. "The Development of a New Biosensor Based on Recombinant E. coli for the Direct Detection of Organophosphorus Neurotoxins." Biosensors and Bioelectronic SIMONIAN, A.
- L., RAININA, E.I. and WILD, J.R. 1996. "Potentiometric Microbial Biosensors." In Press LAI, K.,and STOLOWICH, N.J. 1995. "Characterization of P-S bond Hydrolysis in Organophosphorothioate Pesticides by Organophosphorus Hydrolase." Arch. Biochem.Biophys. 318:59-64.
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Progress 01/01/95 to 12/30/95
Outputs
Environmental contamination by organophosphate pesticides and chemical warfare agents and the efficient disposal of these compounds are becoming increasingly urgent issues. The principal objective of this research is to integrate enzymatic, genetic and biochemical studies in order to better define the factors controlling the efficiency and rate of biodegradation of organophosphate compounds. The broad-spectrum organophosphate hydrolase (OPH) enzyme from Pseudomonas diminuta is capable of hydrolyzing organophosphorus neurotoxins ranging from insecticides (such as parathion) to mammalian neurotoxins (such as sarin and soman). This system has the potential, if properly exploited, to completely mineralize and detoxify these compounds. In order to achieve complete degradative ability, we must first understand and describe factors controlling the biodegradation of toxic wastes within this system. This project currently focuses on three areas of interest: 1) Evaluation of the
nature of the OPH active site. This involves site-directed modification and computer modeling of the native and modified enzymes. 2) Evaluation of the stability of native and mutant constructs utilizing CD, ultracentrifugation and urea denaturation. 3) Continued development of new genetic constructs designed to enhance stability and/or substrate specificity.
Impacts
(N/A)
Publications
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Progress 01/01/94 to 12/30/94
Outputs
Environmental contamination by organophosphate pesticides and chemical warfare agents and the efficient disposal of these compounds are becoming increasingly urgent issues. The principal objective of this research is to integrate enzymatic, genetic, and physiological studies in order to better define factors controlling the efficiency and rate of biodegradation of organophosphate compounds. The broad-spectrum organophosphorus hydrolase (OPH) enzyme from Pseudomonas diminuta is capable of hydrolyzing organophosphorus neurotoxins ranging from insecticides such as parathion to mammalian neurotoxins such as sarin and soman. This system has the potential, if properly exploited, to completely mineralize and detoxify model compounds. In order to ultimately achieve complete degradative capability, we must first understand and describe factors controlling the biodegradation of toxic wastes within this system. This project focusses on three areas of interest: 1) Evaluation of
the nature of the OPH active site; 2) Examination of the export capability of the OPH membrane signal sequence; and 3) Development of new genetic constructions. The knowledge gained from these studies will ultimately be used to evaluate organophosphorus degradation activity and stability under various environmental conditions.
Impacts
(N/A)
Publications
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Progress 01/01/93 to 12/30/93
Outputs
Our studies on the plasmid-borne organophosphorus-degrading gene of Pseudomonas diminuta and its unique broad-spectrum organophosphate hydrolase have evaluated both the genetic organization and the enzymatic properties involved in this system. The bacterial gene encodes a single, broad spectrum organophosphorus hydrolase (OP anhydrase, EC 3.8.1), which is capable of a stereospecific hydrolysis of a wide range of organophosphorus neurotoxins ranging from insecticides such as parathion and diazinon to mammalian neurotoxins such as diisopropylfluorophosphate (DFP) and sarin (a chemical warfare neurotoxin). The organophosphorus degrading genes (opd) from two different plasmids in the soil bacteria P. diminuta and Flavobacterium have been sequenced and their structural organizations are being characterized. The cloned genes have been expressed in a number of biological systems from bacteria to insect tissue culture to native soil fungi, and the enzyme has been purified and
characterized from several different sources. Site-directed mutagenesis has been utilized to define the nature of amino-terminal signal sequence and the bimetallic binding site or sites. The derived understanding of the function of the various histidine and cysteine residues of the enzyme is currently being used to design regional mutagenic strategies with randomized oligonucleotides to further define the active site and manipulate the substrate specificities of the enzyme.
Impacts
(N/A)
Publications
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Progress 01/01/92 to 12/30/92
Outputs
Our studies on the plasmid-borne organophosphorus-degrading gene of Pseudomonas diminuta and its unique broad-spectrum organophosphate hydrolase have defined both the genetic organization and the protein chemistry involved in this system. The bacterial gene encodes a single, unique enzyme, a phosphotriesterase (organophosphorus hydrolase (anhydrase), which is capable of a sterespecific hydrolyzes of a wide spectrum of organophosphorus neurotoxins ranging from insecticides such as parathion, orthene, coumaphos and diazinon to mammalian neurotoxins such as diisopropylfluorophosphate (DFP), sarin, soman and mipafox. The organophosphorus degrading genes (opd) from two different plasmids in the soil bacteria P. diminuta and Flavobacterium have been sequenced and their structural organizations are being characterized. The cloned genes have been expressed in a number of biological systems from bacteria to insect tissue culture, and the enzyme has been purified and
characterized from several different sources. The catalytic reaction has been determined to involve a stereospecific mechanism which proceeds by the direct nucleophilic attack of an activated water at the reaction center. The reaction rate approaches a diffusion limited catalysis at 2100(mu)s and the enzyme is actively adsorbed to various column and particular matrices.
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Progress 01/01/91 to 12/30/91
Outputs
Recent molecular studies on a plasmid-borne, organophosphorus-degrading gene have defined both the genetic organization and the protein chemistry of the encoded OP Hydrolase. This unique, membrane-bound enzyme is capable of hydrolyzing a wide spectrum of organophosphorus insecticides such as parathion, orthene, coumaphos and diazinon as well as mammalian neurotoxins such as DFP, sarin and mipafox. The native protein is post-translationally processed with varying efficiency depending upon the host expression system. The structure of the active site of the OP Hydrolase and its membrane signal sequence are being evaluated by chemical and site-directed mutagenesis. It is possible to develop an understanding of the catalytic limitation of the enzyme as well as alter its physical and catalytic properties by genetic manipulations. A series of diverse genetic constructions have been designed to evaluate the heterologous expression and processing of the native protein. A
series of recent studies have begun with other investigators to evaluate the possible use of various molecular forms of the organophosphate hydrolase for bioremediation in soils or for the antagonism of neurotoxic exposure.
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Progress 01/01/90 to 12/30/90
Outputs
The OP anhydrases (phosphotriesterase) encoded by the opd genes isolated from large plasmids of Pseudomonas diminuta (pCS1,51kb) and Flavobacterium species ATCC27551 (43kb) have been shown to be able to hydrolyze parathion, paraoxon, DFP, sarin, soman, mipafox and other structurally related OP neurotoxins such as coumaphos, methyl-parathion, diazinon etc. (Chiange et al. 1985; McDaniel, 1985; McDaniel et al. 1988; Dumas et al. 1989; Dumas et al. 1989a,b,c). The mechanism used by the Pseudomonas enzyme to hydrolyze paraoxon and related substrates has been elucidated by Lewis et al. (1988) and involves a stereospecific attack of an activated water at the P-O bond which proceeded with a single inversion of the phosphate and did not involve a phosphorylated enzyme intermediate at the active center. The DNA sequences of the opd genes have been determined (McDaniel et al. 1988; Harper et al, 1988) and the genes have been expressed in heterologous bacterial hosts from
several host-specific promoters. In addition, it has been possible to produce native enzyme in insect tissue cultures derived from the Fall Armyworm, Spodoptera frugiperda (Sf9 cells) from the polyhedrin promoter of a baculoviral expression vector and selected for the production of OPA anhydrase activity. The Pseudomanas enzyme has been purified and shown to be able to detoxify a number of structurally related acetylcholinesterase inhibitors including the organophosphoro-fluoridate nerve agents, sarin and soman (Dumas et al.
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Progress 01/01/89 to 12/30/89
Outputs
As the concerns about the impact of environmental contamination by toxic chemicals increase, it is necessary to develop safe and efficient biodetoxification systems designed to address specific industrial or agricultural chemical wastes. The genetic/enzyme system being defined in this study is one of the few biodegradation systems which has been experimentally detailed to offer the potential for actual technology application in a number of important industrial and agricultural settings. Previous research with the organophosphate-degrading gene system of soil bacteria (the opd gene) and its broad-spectrum organophosphorus hydrolysing enzyme (OP-anhydrase, a phosphotriesterase) has defined a system that can be applied to the development of enzyme bioreactors, environmental decontaminating microorganisms and household OP-insecticide detoxifying additives for market-ready utility. The gene for the OP degradation has been cloned, sequenced and is being investigated by
site-directed mutagenesis in several different biological host systems. The enzyme mechanism and substrate spectrum have been determined for numerous phosphotriesters, thiolesters and fluorophosphate neurotoxins. In addition to the direct impact on OP-detoxification, the technology transfer systems described in this research provides a model for the future development of biological decontamination for other environmental hazards.
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Progress 01/01/88 to 12/30/88
Outputs
A number of significant goals in this collaborative study have been completed and several new directions have developed. The completed goals are represented by the accepted publications below and are summarized as: The reaction mechanism involved in the degradation of 4-nitrophenyl phosphate (paraoxon) by the bacterial organophosphate acid anhydrase under study in our laboratory has been shown to be a sterospecific, sincle in-line displacement hydrolysis resulting from the directed activation of a water molecule at the phosphorus center of the phosphotriester substrate. The bacterial genes (opd) involved in this system has been cloned and expressed in several different biological host systems including other bacteria, baculoviruses, and insect tissue cultures. The genes from two different bacterial sources have been sequenced and determined to encode identical polypeptides of 325 amino acids. The amino-terminal portion of this polypeptide contains classical membrane
signal sequences for bacterial, consistent with the membrane association of the native enzyme. New methodologies for identifying transgenic constructions have been developed in order to select transformed microorganisms, tissue cultures or organisms.
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Progress 01/01/87 to 12/30/87
Outputs
The opd (organophosphate degrading) genes of two different soil bacteria are similar and encode identical OP-phosphotriesterase enzymes (35, 418 d). The catalytic mechanism does not involve a phosophoenzyme intermediate and proceeds with inversion of configuration with chiral EPN. The purified enzyme from Pseudomas accepts a wide spectrum of phosphate and thiol esters, including DFP, which appears to be as effectively hydrolyzed a paraoxon, the chosen model substrate. Purification and amino acid sequencing of intragenic fusions with beta-galactosidase have verified the gene sequence; however, the hybrid protein shows excessive intracellular processing when expressed n E. coli. Purification studies have focused on the membrane-associated enzyme from native pseudomonad hosts. Overexpression of opd in enteric bacteria has been limited and outermembrane deposition of the enzyme does not occur. Expression studies have shifted to defined Pseudomonas species and
baculoviral insect tissue culture systems.
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Progress 01/01/86 to 12/30/86
Outputs
This has been a very productive year for this project in both the molecular definition of the genetic systems and the chemical analysis of two enzymes involved in the detoxification of a wide spectrum of organophosphorus pesticides. The opd gene has been isolated from two different bacterial plasmid sources and produced in E. coli on various phage and plasmid expression vectors. The genes show identical DNA sequences presumably encoding a protein of 282 aa. Protein characterization has shown that the enzyme appears to be active as a dimer with molecular weight of approximately 60,000 d. The enzyme appears to be partitioned between membrane vesicle association and the free dimer released as bacteria enter late growth. The substrate specificity is being analyzed against existing OPs obtained from the EPA and specific analogues synthesized in Chemistry. A major on-site experimental decontamination of coumaphos (Lo-Cal) being used in the tick fever eradication
program in Laredo, Texas was performed in collaboration with scientists from the USDA Pesticide Laboratory at Beltsville.
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