James R. Wild

The development of a new biosensor based on recombinant E. coli for the direct detection of organophosphorus neurotoxins

A new biosensor for the direct detection of organophosphorus (OP) neurotoxins has been developed utilizing cryoimmobilized, recombinant E. coli cells capable of hydrolyzing a wide spectrum of OP pesticides and chemical warfare agents. The biological transducer was provided by the enzymatic hydrolysis of OP neurotoxins by organophosphate hydrolase which generates two protons through a reaction in which P-O, P-F, P-S or P-CN bonds are cleaved, and the proton release corresponded with the quantity of organophosphate hydrolyzed. This stoichiometric relationship permitted the creation of a potentiometric biosensor for detection of OP neurotoxins and a pH-based assay was developed as a direct function of the concentration of OP neurotoxins and the immobilized biomass. In these studies utilizing paraoxon as the substrate, neurotoxin concentration was determined with two different types of measuring units containing immobilized cells: (1) a stirred batch reactor; and (2) a flow-through column minireactor. A pH glass electrode was used as the physical transducer. The linear detection range for paraoxon spanned a concentration range of 0.25-250 ppm (0.001-1.0 mM). The response times were 10 min for the batch reactors and 20 min for the flow-through systems. It was possible to use the same biocatalyst repetitively for 25 analyses with a 10 min intermediate washing of the biocatalyst required for reestablishing the starting conditions. The cryoimmobilized E. coli cells exhibited stable hydrolytic activity for over 2 months under storage in 50 mM potassiumphosphate buffer at +4 degrees C and provide the potential for the development of a stable biotransducer for detecting various OP neurotoxins.

Biotransformation Patterns of 2,4,6-Trinitrotoluene by Aerobic Bacteria

Abstract. 2,4,6-Trinitrotoluene (TNT), a toxic nitroaromatic explosive, accumulates in the environment, making necessary the remediation of contaminated areas and unused materials. Although bioremediation has been utilized to detoxify TNT, the metabolic processes involved in the metabolism of TNT have proven to be complex. The three aerobic bacterial strains reported here (Pseudomonas aeruginosa, Bacillus sp., and Staphylococcus sp.) differ in their ability to biotransform TNT and in their growth characteristics in the presence of TNT. In addition, enzymatic activities have been identified that differ in the reduction of nitro groups, cofactor preferences, and the ability to eliminate-NO2 from the ring. The Bacillus sp. has the most diverse bioremediation potential owing to its growth in the presence of TNT, high level of reductive ability, and capability of removing-NO2 from the nitroaromatic ring.

Enzymatic hydrolysis of the chemical warfare agent VX and its neurotoxic analogues by organophosphorus hydrolase

Organophosphorus hydrolase (OPH) is a bacterial enzyme that hydrolyzes a variety of organophosphorus (OP) neurotoxins, including many widely used pesticides and chemical warfare agents containing P-O, P-F, P-CN and P-S bonds. It has extremely high efficiency in hydrolysis of many different phosphotriester and phosphothiolester pesticides (P-O bond) such as paraoxon (kcat3800s−1) and coumaphos (kcat = 800s−1) or phosphonate (P-F) neurotoxins such as DFP (kcat = 350s−1) and the chemical warfare agent sarin (kcat = 56s−1). In contrast, the enzyme has much lower catalytic capabilities for phosphonothioate neurotoxins such as acephate (kcat = 2.8 s−1) or the chemical warfare agent VX [O-ethyl S-(2-diisopropyl-aminoethyl) methylphosphonothioate] (kcat = 0.3s−1). This lower specificity for VX and its analogues are reflected by the specificity constants (kcat/Km values) for VX = 0.75 × 103 M−1 s−1 compared to 5.5 × 107M−1 s−1 for paraoxon. Different metal-associated forms of the enzyme demonstrated significantly ...

A New Approach For Discriminative Detection of Organophosphate Neurotoxins in the Presence of Other Cholinesterase Inhibitors

Abstract A radically new approach for the discriminative determination of various neurotoxins has been developed. This novel biosensor combines a highly sensitive acetylcholinesterase (AChE) biosensor with immobilized organophosphate hydrolase (OPH). The value of the new concept was demonstrated by the discrimination between carbamate and organophosphorus pesticides. It was shown that the response of traditional AChE-based biosensor to mixed samples containing paraoxon and carbofuran was not simply additive, and the measured concentrations of these pesticides were very different from their real concentrations. This combined OPH/AChE system was able to improve the accuracy of the AChE-based biosensor and to uniquely distinguish paraoxon in mixed solutions containing carbofuran. The presented approach promises a new perspective for “real world” analyses and opens a new area of discriminative determination of various species in multicomponent solutions.

From Biological Diversity to Structure-Function Analysis: Protein Engineering in Aspartate Transcarbamoylase

Aspartate transcarbamoylase (ATCase, EC 2.1.3.2) is a common enzyme which catalyzes the first unique step in pyrimidine biosynthesis in divergent biological systems; however, it possesses tremendous architectural variety from one organism to another. For example, the E. coli ATCase holoenzyme is comprised of two catalytic trimers and three regulatory dimers, while the mammalian enzyme is part of a multifunctional protein aggregate encoding the preceding and subsequent enzymes in pyrimidine biosynthesis. Despite extreme differences in quaternary architecture and enzymatic organization, protein engineering studies have demonstrated the existence of highly conserved units of protein structure that impart specific functional characteristics. 1 The largest of these units are discrete polypeptides or superdomains within multifunctional proteins which have been shown to be uniquely involved in specific catalytic steps within the CAD or CA complexes of eukaryotic pyrimidine biosynthesis. 2 The catalytic polypeptides of various ATCases are organized into two discrete and separable binding domains for its substrates, carbamoyl phosphate and aspartate. 3 The regulatory polypeptides of the enteric bacterial enzymes also contain two discrete tertiary domains, the Allosteric Binding Domain and Cys 4 coordinated Zinc Domain involved in the protein:protein interface between the regulatory and catalytic polypeptides of the holoenzyme. 4 There are sub-domain structural units within the various polypeptides which have a coordinated impact on specific catalytic and regulatory functions in the enzyme. 5 Finally, it has been possible to ascribe some individual function to specific amino acids relative to ligand binding, zinc coordination, protein:protein interactions, and the structural reorganizations in the T-R transition of the enteric holoenzymes.

Ancillary Function of Housekeeping Enzymes: Fortuitous Degradation of Environmental Contaminants

Most of the environmental contamination of greatest concern around the world today involves xenobiotic materials which have only been produced or used in industrial/agricultural production over the past several decades. These include various organic chemical solvents, heavy metals, neurotoxic pesticides, halogenated aromatic compounds, explosives and carcinogenic industrial chemicals which may or may not have natural counterparts. These have been synthesized with increasing diversity and volume over the last fifty to seventy years. Although their introduction into the environment is lamentable, and the effects they can have on the environment devastating, many different types of microbial systems have already acquired the ability to chemically modify many of these compounds. The likelihood that totally new enzymes with specific detoxification activities could emerge in such a brief period of time is problematic. It is much more likely that enzymes already possessed by microbial communities, possibly used for maintenance functions, can bee recruited to address the new challenge. For example, FMN oxidoreductases may serve to reduce trinitrotoluene and a human lipid-phosphoesterase or a bacterial peptidase can degrade organophosphate pesticides. Five categories of reactions wherein housekeeping enzymes may be able to catalyze ancillary reactions involving previously unknown chemicals include 1) general oxidoreductases, 2) denitrases, 3) phosphoesterases, 4) ring cleavage enzymes and 5) metal-sequestering proteins. Once a potential metabolic route is established, directed evolution based on random mutation or substitutional recombination may serve to enhance activity if a competitive metabolic edge could result.

Biodegradation of Hazardous Compounds Using Immobilized Microorganisms

Coumaphos, an organophosphate insecticide, is hydrolyzed to chlorferon and diethyl thiophosphate (DETP). In this research, two different consortia of microorganisms responsible for degrading either chlorferon or DETP were enriched from cattle dip solution. Both consortia of organisms were mostly rod type gram-negative bacteria. The enriched cultures were used as inocula to grow biomass for degradation studies. At concentrations greater than 50 mg/l, chlorferon inhibited DETP degrading organisms. From experiments using freely suspended cells, the optimum biomass concentration for chlorferon degradation was found to be 80 g/L, and pH 7.5 was selected as the optimum operating pH. Chlorferon degradation followed substrate inhibition kinetics. Parameters were estimated as Vm,C = 0.062 mg/g-biomass·h, Km,C = 20.634 mg/L, and KSi,C = 117.628 mg/L. For DETP degradation, the optimum biomass concentration was found to be 60 g/L and the operating reaction pH was in the range of 7.5 to 8. The DETP degradation reaction followed Monod kinetics with kinetic parameters estimated to be Vm,D = 1.523 mg/g-biomass·h and Km,D = 609.791mg/L. Chlorferon or DETP degrading organisms were separately immobilized in Ca-alginate beads. The optimum bead loading rate in a reactor was found to be 20% for chlorferon degradation and 30% for DETP degradation. The chlorferon degradation rate was significantly enhanced with immobilized cells. Both specific reaction rate and volumetric reaction rate were five times higher than that with freely suspended cells. This was attributed to the protection of cells from inhibitory ingredients in UCD solution by immobilization.