Grantee Research Project Results
Final Report: Genetic Engineering of Enzymatic Cyanide Clearance
EPA Grant Number: R828598C789Subproject: this is subproject number 789 , established and managed by the Center Director under grant R828598
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Texas - Indiana Virtual STAR Center
Center Director: Gustafsson , Jan-Ake
Title: Genetic Engineering of Enzymatic Cyanide Clearance
Investigators: Benedik, Michael , Willson, Richard C.
Institution: University of Houston - University Park
EPA Project Officer: Aja, Hayley
Project Period: September 1, 2000 through August 31, 2004
RFA: Gulf Coast Hazardous Substance Research Center (Lamar University) (1996) RFA Text | Recipients Lists
Research Category: Hazardous Waste/Remediation , Targeted Research
Objective:
The primary deliverable for this project is an improved cyanidase preparation for waste remediation. The intermediate goals are as follows:
1. We will establish a high level expression systems for cyanide hydrolytic enzymes, and express several of the newly-described enzymes for characterization. We plan to utilize a strong promoter for efficient expression in E. coli and to fuse enzymes to a six-histidine tag for easy purification.
2. We will characterize each candidate for ease of production (i.e., low projected cost), resistance to inhibition by metals, and stability. Stability will be augmented if necessary by methods compatible with low-cost production, including cross-linking of cell mass with glutaraldehyde.
3. Finally, we will test the performance of promising candidates under realistic (soil, temperature, pH, salt, metals) conditions.
Summary/Accomplishments (Outputs/Outcomes):
We have an ongoing collaboration with a small Houston company, EcoShield, to test the use of our enzyme system to for the cleanup of cyanide contaminated waster water from industrial sites. If effective our process will both reduce the costs of waste water cleanup, reduce the danger of shipping such waste water for chemical remediation, and most importantly have resources available for the immediate cleanup of any environmental cyanide spills before significant spreading occurs.
In this final year we have essentially completed the 3rd objective listed above. We completed our analysis of the candidate enzymes with respect to their kinetic profiles and their response range to pH, temperature, metal inhibition and stability. This work has been submitted for publication and the manuscript has been accepted pending minor revisions.
Recombinant forms of three cyanide-degrading nitrilases, CynD from Bacillus pumilus C1, CynD from Pseudomonas stutzeri, and CHT from Gloeocercospora sorghi, were prepared after their genes were cloned with C-terminal hexahistidine purification tags and expressed in Escherichia coli, and the enzymes purified using nickel-chelate affinity chromatography. The enzymes were compared with respect to their pH stability, thermostability, metal tolerance, and kinetic constants. The two bacterial genes, both cyanide dihydratases, were similar with respect to pH range, metal tolerance and kinetic properties. However, the fungal CHT enzyme, in addition to having a significantly higher Km and Vmax; was less sensitive to Hg2+ and alkaline pH; but exhibited a lower thermal tolerance and was more sensitive to Pb2+ than the CynD enzymes.
We have initiated studies using contaminated water samples from a local electroplating firm. These samples have very high levels of cyanide as well as high levels of various metal contaminants. Our work to date suggest the bacterial cyanide dihydratases are very susceptible to inhibition by contaminants in the electroplating samples. If the wastewater is diluted about 10-fold and buffered then we can obtain enzyme activity about 10-30% of optimal. However the fungal cyanide hydratase is less inhibited and displays significantly higher activity on these samples. Hence we are pursuing further studies and characterization of cyanide hydratases.
Conclusions:
Our immediate plans will have a focus in 3 different areas. First we will continue our investigation into novel cyanide hydratases identified by genome sequencing. We have identified and are in the process of cloning and testing the genes from three fungal strains which have significant sequence similarity to known cyanide hydratases. Secondly we plan to continue our efforts in designing improved enzymes by directed evolution methods. We are developing and testing a new method for gene shuffling to generate candidate clones for high throughput testing. Thirdly we will improve our ability to express and rapidly purify, at low cost, the enzyme for production purposes by investigating the use of alternate affinity tags and confirming they are efficacious and have no effect on enzyme activity. Lastly we plan to continue our studies with industrial waste-water samples to develop improved protocols for enzymatic remediation and determine appropriate enzyme levels, buffering as necessary, temperature and pH adjustments that can be done at low cost to improve enzyme function.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
| Other subproject views: | All 5 publications | 3 publications in selected types | All 3 journal articles |
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| Other center views: | All 359 publications | 104 publications in selected types | All 90 journal articles |
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Jandhyala DM, Berman, M, Meyers PR, Sewell BT, Willson RC, Benedik MJ. CynD, the cyanide dihydratase from Bacillus pumilus:gene cloning and structural studies. Applied and Environmental Microbiology 2003;69(8):4794-4805. |
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Jandhyala DM, Willson RC, Sewell BT, Benedik MJ. Comparison of cyanide-degrading nitrilases. Applied Microbiology and Biotechnology 2005;68(3):327-335. |
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Sewell BT, Berman MN, Meyers PR, Jandhyala D, Benedik MJ. The cyanide degrading nitrilase from Pseudomonas stutzeri AK61 is a two-fold symmetric, 14-subunit spiral. Structure 2003;11(11):1413-1422. |
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Supplemental Keywords:
RFA, Scientific Discipline, INTERNATIONAL COOPERATION, Waste, POLLUTANTS/TOXICS, Hazardous, Remediation, Environmental Chemistry, Chemistry and Materials Science, Hazardous Waste, Chemicals, heavy metals, metal removal, enzymatic cyanide clearance, metal contamination, hazardous wate, metal remediation, metal extraction, metal ion removal, metal recovery, cyanide, extraction of metals, mining waste, genetic engineeringRelevant Websites:
None.
Progress and Final Reports:
Original AbstractMain Center Abstract and Reports:
R828598 Texas - Indiana Virtual STAR Center Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R822721C529 Environmentally Acceptable Endpoints: Risk Based Remediation Using Bioremediation
R822721C552 Degradative Solidification/Stabilization Technology for Chlorinated Hydrocarbons
R822721C569 Treatment and Product Recovery: Supercritical Water Oxidation of Nylon Monomer Manufacturing Waste
R822721C620 Colloidal Fouling of Membranes: Implications in the Treatment of Textile Dye Wastes and Water Reuse
R822721C626 Catalytic Hydroprocessing of Chlorinated Organics
R822721C627 The Interaction of Microbial Activity and Zero Valent Iron Permeable Barrier Technology
R822721C630 Microbial Cometabolism of Recalcitrant Chemicals in Contaminated Air Streams
R822721C633 Catalyst Lifetime Studies for Chlorocarbon Steam Reforming
R822721C635 Electrokinetic/Surfactant-Enhanced Remediation of Hydrophobic Pollutants in Low Permeability Subsurface Environments
R822721C636 Transformation Reactions of Nitroaromatic and Nitrogen Heterocyclic Compounds on Granular Activated Carbon (GAC) Surfaces: Enhancement of GAC Adsorption in Natural and Engineered Environmental Systems
R822721C640 Environmentally Friendly Organic Synthesis in Supercritical Fluids
R822721C645 Development and Evaluation of an Integrated Model to Facilitate Risk-Based Corrective Action at Superfund Sites
R822721C651 Adjustable Biopolymer Chelators for Cadmium, Lead and Mercury
R822721C653 New Electrochemically Smart Catalysts for Hazardous Waste Management and Development of Capillary Electrophoresis for Analysis of their Products
R822721C655 Soil Sampling in South Alabama Oil Fields
R822721C659 Subsurface Contamination Site Characterization via a Computer-Aided Visual Tool
R822721C661 New Insoluble supports for Protein Immobilization for Use in Metalloprotein Affinity Metal Chromatography
R822721C663 Soil Remediation with Ultra-High-Efficiency Hydrocyclones
R822721C669 Solid Acid Catalyzed Alkylation in Supercritical Fluids
R822721C679 Regeneration/Reactivation of Carbon Adsorbents by Radio Frequency (RF) Induction Heating
R822721C687 Improved Halogen Resistance of Catalytic Oxidation
R822721C696 Phytoremediation and Bioremediation of Land Contaminated By PAHs, PCBs, and TNT
R822721C697 Fundamental and Kinetic Investigation of Sorbent Technology for Optimum Mercury Emission Control
R822721C700 Effects of Natural and Cyclic Variations on Contaminant Fate and Transport
R822721C703 Enhancement of DNAPL Dissolution Rates by Dechlorinating Anaerobes
R826694C620 Colloidal Fouling of Membranes: Implications in the Treatment of Textile Dye Wastes and Water Reuse
R826694C625 Enhanced Treatment of DNAPLs Contaminated Soils and Groundwater Using Biosurfactants: In-Situ Bioremediation
R826694C626 Catalytic Hydroprocessing of Chlorinated Wastes
R826694C627 The Interaction of Microbial Activity and Zero Valent Iron Permeable Barrier Technology
R826694C629 Biofiltration of BTEX in Petroleum-Contaminated Soil Remediation Off-Gas
R826694C630 Microbial Cometabolism of Recalcitrant Chemicals in Contaminated Air Streams
R826694C633 Catalyst Lifetime Studies for Chlorocarbon Steam Reforming
R826694C635 Electrokinetic/Surfactant-Enhanced Remediation of Hydrophobic Pollutants in Low Permeability Subsurface Environments
R826694C636 Transformation Reactions of Nitroaromatic and Nitrogen Heterocyclic Compounds on Granular Activated Carbon (GAC) Surfaces: Enhancement of GAC Adsorption in Natural and Engineered Environmental Systems
R826694C640 Environmentally Friendly Organic Synthesis in Supercritical Fluids
R826694C645 Development and Evaluation of an Integrated Model to Facilitate Risk-Based Corrective Action at Superfund Sites
R826694C651 Adjustable Biopolymer Chelators for Cadmium, Lead, and Mercury Remeidation
R826694C659 Subsurface Contamination Site characterization Via a Computer-Aided Visual Tool
R826694C661 New Insoluble supports for Protein Immobilization for Use in Metalloprotein Affinity Metal Chromatography
R826694C669 Solid Acid Catalyzed Alkylation in Supercritical Reaction Media
R826694C679 Regeneration and Reactivation of Carbon Adsorbents by Radio Frequency Induction Heating
R826694C696 Phytoremediation and Bioremediation of Land Contaminated By PAHs, PCBs, and TNT
R826694C697 Fundamental and Kinetic Investigation of Sorbent Technology for Optimum Mercury Emission Control
R826694C700 Effects of Natural Cyclic Variations on Contaminated Fate and Transport
R826694C703 Enhancement of DNAPL Dissolution Rates by Dechlorinating Anaerobes
R826694C705 A Pilot Plant for Producing Mixed Ketones from Waste Biomass
R826694C722 The Effects of an Oily-Phase on VOC Emissions from Industrial Wastewater
R826694C724 Mercury Removal from Stack Gas by Aqueous Scrubbing
R826694C725 Transport, Fate and Risk Implications of Environmentally Acceptable Endpoint Decisions
R826694C731 Development and Application of a Real-Time Optical Sensor for Atmospheric Formaldehyde
R826694C734 An Advanced System for Pollution Prevention in Chemical Complexes
R828598C001 Field Study Abstract: A Model of Ambient Air Pollution in Southeast Texas Using Artificial Neural Network Technology
R828598C002 Hollow Fiber Membrane Bioreactors for Treating Water and Air Streams Contaminated with Chlorinated Solvents
R828598C003 Fugitive Emissions of Hazardous Air Pollutants from On-Site Industrial Sewers
R828598C004 Biofiltration Technology Development
R828598C005 A Risk-Based Decision Analysis Approach for Aquifers Contaminated with DNAPLs
R828598C006 In-Situ Remediation for Contaminated Soils Using Prefabricated Vertical Drains
R828598C007 Membrane Technology Selection System for the Metal Finishing Industry
R828598C008 Sequential Environments for Enhanced Bioremediation of Chlorinated Aliphatic Hydrocarbons
R828598C009 Waste Minimization in the Magnetic Tape Industry: Waterborne Coating Formulations for Magnetic Tape Manufacture
R828598C010 Soil Remediation by Agglomeration with Petroleum Coke
R828598C011 Recovery of Dilute Phosphoric Acid in Waste Streams Using Waste Gas Ammonia: The Regenerative MAP/DAP Process
R828598C012 Stochastic Risk Assessment for Bioremediation
R828598C013 Selective Removal of Heavy Metals from Wastewater by Chelation in Supercritical Fluids
R828598C014 Optimization of Treatment Technologies for Detoxification of PCB Contaminated Soils
R828598C015 Wastewater Remediation by Catalytic Wet Oxidation
R828598C016 Permanence of Metals Containment in Solidified and Stabilized Wastes
R828598C017 Combustion Enhancement by Radial Jet Reattachment - Low Generation of Hazardous Gases and High Thermal Efficiency
R828598C018 A Process To Convert Industrial Biosludge and Paper Fines to Mixed Alcohol Fuels
R828598C019 Homogeneous Catalysis in Supercritical Carbon Dioxide
R828598C020 Ultrasonic Enhancement of the Removal of Heavy Metals
R828598C021 The Binding Chemistry and Leaching Mechanisms of Advanced Solidification/Stabilization Systems for Hazardous Waste Management
R828598C022 Development of an Air-Stripping and UV/H2O2 Oxidation Integrated Process To Treat a Chloro-Hydrocarbon-Contaminated Ground Water
R828598C023 A Comparative Study of Siting Opposition in Two Counties
R828598C024 Sonochemical Treatment of Hazardous Organic Compounds II: Process Optimization and Pathway Studies
R828598C025 Laser Diagnostics of the Combustion Process within a Rotary Kiln Incinerator
R828598C026 Use of Inorganic Ion Exchangers for Hazardous Waste Remediation
R828598C027 Kaolinite Sorbent for the Removal of Heavy Metals from Incinerated Lubricating Oils
R828598C028 Destruction of Chlorinated Hydrocarbons in Process Streams Using Catalytic Steam Reforming
R828598C029 Integrated Process Treatment Train (Bioremediation {Aerobic/Anaerobic} and Immobilization) for Texas Soils Contaminated with Combined Hazardous Wastes
R828598C030 Photo-Oxidation by H2O2/VisUV of Off-Gas Atmospheric Emissions from Industrial and Environmental Remediation Sources
R828598C031 Concentrated Halide Extraction and Recovery of Lead from Soil
R828598C032 Biodegradable Surfactant for Underground Chlorinated Solvent Remediation
R828598C033 A Software Guidance System for Choosing Analytical Subsurface Fate and Transport Models Including a Library of Computer Solutions for the Analytical Models
R828598C034 Hydrodynamic Modeling of Leachate Recirculating Landfill
R828598C035 Measurement of Oxygen Transfer Rate in Soil Matrices
R828598C036 Sorbent Technology for Multipollutant Control During Fluidized Bed Incineration
R828598C037 Pollution Prevention by Process Modification Using On-Line Optimization
R828598C038 Pollution Prevention by Process Modification
R828598C039 Water Solubility and Henry's Law Constant
R828598C040 Transferring Technical Information on Hazardous Substance Research by Publishing on the World Wide Web
R828598C041 Stress Protein Responses to Multiple Metal Exposure in Grass Shrimp
R828598C042 Life-Cycle Environmental Costing for Managing Pollution Prevention in the Chemical and Petroleum Refining Industries: A Cross-Border Approach
R828598C687 Improved Halogen Resistance of Catalytic Oxidation Through Efficient Catalyst Testing
R828598C696 Phytoremediation and Bioremediation of Land Contaminated By PAHs, PCBs, and TNT
R828598C697 Fundamental and Kinetic Investigation of Sorbent Technology for Optimum Mercury Emission Control
R828598C700 Effects of Natural Cyclic Variations on Contaminated Fate and Transport
R828598C703 Enhancement of DNAPL Dissolution Rates by Dechlorinating Anaerobes
R828598C705 A Pilot Plant for Producing Mixed Ketones from Waste Biomass
R828598C722 The Effects of an Oily-Phase on VOC Emissions from Industrial Wastewater
R828598C724 Mercury Removal from Stack Gas by Aqueous Scrubbing
R828598C725 Transport, Fate and Risk Implications of Environmentally Acceptable Endpoint Decisions
R828598C731 Development and Application of a Real Time Optical Sensor for Atmospheric Formaldehyde
R828598C734 An Advanced System for Pollution Prevention in Chemical Complexes
R828598C743 Field Demonstration of Ultrasound Enhancement of Permeable Treatment Walls
R828598C744 Optical Fibers Coated With Titania Membrane/UV-Generating Crystal in a Distributed-Light Photoreactor for VOC Oxidation
R828598C749 Characterization and Modeling of Indoor Particulate Contaminants In a Heavily Industrialized Community
R828598C753 Adsolubilization and Photocatalysis in a Semiconducting Monolithic Reactor for Wastewater Treatment
R828598C754 Remote Detection of Gas Emissions in Industrial Processes
R828598C759 Searching for Optimum Composition of Phosphogypsum: Fly ash: Cement Composites for Oyster Culch Materials
R828598C761 Development of a Phytologically-Based Biosorptive Water Treatment Process
R828598C766 Chlorinated Solvent Impact and Remediation Strategies for the Dry Cleaning Industry
R828598C769 Soil/Sediment Remediation by Hot Water Extraction Combined with In-Situ Wet Oxidation
R828598C771 Fluoracrylate Polymer Supported Ligands as Catalysts for Environmentally Benign Synthesis in Supercritical Fluids
R828598C774 The Feasibility of Electrophoretic Repair of Impoundment Leaks
R828598C777 Surfactant Enhanced Photo-oxidation of Wastewaters
R828598C778 Stationary Power Generation Via Solid Oxide Fuel Cells: A Response to Pollution and Global Warming
R828598C786 Photocatalytic Recovery of Sulfur and Hydrogen From Hydrogen Sulfide
R828598C787 Biosurfactant Produced from Used Vegetable Oil for removal of Metals From Wastewaters and Soils
R828598C789 Genetic Engineering of Enzymatic Cyanide Clearance
R828598C791 Characterizing the Intrinsic Remediation of MTBE at Field Sites
R828598C799 Simultaneous Water Conservation/Recycling/Reuse and Waste Reduction in Semiconductor Manufacturing
R828598C801 Building Defined Mixed Cultures To Biodegrade Diverse Mixtures Of Chlorinated Solvents
R828598C802 Engineering of Nanocrystal Based Catalytic Materials for Hydroprocessing of Halogenated Organics
R828598C807 Commercial Demonstration of Hydrogen Peroxide Injection to Control NOx Emissions from Combustion Sources
R828598C809 Evaluating Source Grouting and ORC for Remediating MTBE Sites
R828598C810 Application of Total Cost Assessment To Process Design In the Chemical Industry
R828598C846 Quantitative Demonstration of Source-Zone Bioremediation in A Field-Scale Experimental Controlled Release System
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.
Project Research Results
3 journal articles for this subproject
Main Center: R828598
359 publications for this center
90 journal articles for this center