Melinda E. Wales

A novel layer-by-layer assembled multi-enzyme/CNT biosensor for discriminative detection between organophosphorus and non-organophosphrus pesticides

Organophosphate compounds are heavily used in agriculture and military activities, while non-organophosphate pesticides are mostly used in agriculture and home defense. Discriminative detection of such toxic compounds is very challenging and requires sophisticated and bulky instrumentation. Meanwhile, multi-enzyme biosensors may offer an effective solution to the problem and may become a versatile analytical tool for discriminative detection of different neurotoxins. In this study, we report for the first time a novel bi-enzyme biosensing system incorporating electrostatically interacted enzyme-armored MWCNT-OPH and MWCNT-AChE along with a set of cushioning bilayers consisting of MWCNT-polyethyleneimine and MWCNT-DNA on glassy carbon electrode for discriminative detection of organophosphorus (OP) and non-organophosphorus (non-OP) pesticides. LbL interfaces were characterized by surface plasmon resonance and electrochemical impedance spectroscopy, demonstrating stepwise assembly and electron conductivity studies. The detection limit was found to be ~0.5 for OP pesticide paraoxon and 1 μM for non-OP pesticide carbaryl, in a wide linear range. The biosensor performance was also validated using apple samples. Remarkable discriminative and straightforward detection between OP and non-OP neurotoxins was successfully achieved with cyclic voltammetry (CV) and UV-vis methods on the MWCNT-(PEI/DNA)2/OPH/AChE biosensor, showing great potential in large screening of OP and non-OP pesticides in practical applications.

Improved pharmacokinetics and immunogenicity profile of organophosphorus hydrolase by chemical modification with polyethylene glycol

A catalytic bioscavenger with broad substrate specificity for the therapeutic and prophylactic defense against recognized chemical threat agents has been a long standing objective of civilian and military research. A catalytic bioscavenger utilizing the bacterial enzyme organophosphorus hydrolase (OPH) is characterized in these studies, and has potential application for both military and civilian personnel in threat scenarios involving either nerve agents or OP pesticides. The present study examines the effects of PEGylation on the biochemical and pharmacological characteristics of OPH. The enzyme was conjugated with linear and branched methyl-PEO(n)-NHS esters of relatively small molecular mass from 333 to 2420Da. PEGylated OPH displayed a decreased maximal catalytic rate, though substantial activity was maintained against two tested substrates: up to 30% with paraoxon and up to 50-60% with demeton-S. The thermostability of the PEGylated enzymes ranged between 60 and 64 degrees C, compared to the unmodified OPH, which is approximately 67 degrees C. The enzyme conjugates revealed a significant improvement of pharmacokinetic properties in animal studies. The clearance from a guinea pigs blood stream significantly decreased relative to unmodified OPH, resulting in an increase of residence time and systemic availability. Evaluation of the humoral immune response indicated that the branched PEG-OPH conjugate significantly reduced production of anti-OPH antibodies, compared to the unmodified enzyme. The OPH-PEG conjugates with improved pharmacokinetic and immunogenicity properties, considerable catalytic activity and thermal stability provide a new opportunity for the in vivo detoxification of the neurotoxic OP compounds.

Balancing the stability and the catalytic specificities of OP hydrolases with enhanced V-agent activities.

Rational site-directed mutagenesis and biophysical analyses have been used to explore the thermodynamic stability and catalytic capabilities of organophosphorus hydrolase (OPH) and its genetically modified variants. There are clear trade-offs in the stability of modifications that enhance catalytic activities. For example, the H254R/H257L variant has higher turnover numbers for the chemical warfare agents VX (144 versus 14 s(-1) for the native enzyme (wild type) and VR (Russian VX, 465 versus 12 s(-1) for wild type). These increases are accompanied by a loss in stability in which the total Gibbs free energy for unfolding is 19.6 kcal/mol, which is 5.7 kcal/mol less than that of the wild-type enzyme. X-ray crystallographic studies support biophysical data that suggest amino acid residues near the active site contribute to the chemical and thermal stability through hydrophobic and cation-pi interactions. The cation-pi interactions appear to contribute an additional 7 kcal/mol to the overall global stability of the enzyme. Using rational design, it has been possible to make amino acid changes in this region that restored the stability, yet maintained effective V-agent activities, with turnover numbers of 68 and 36 s(-1) for VX and VR, respectively. This study describes the first rationally designed, stability/activity balance for an OPH enzyme with a legitimate V-agent activity, and its crystal structure.

Enzyme-based intravascular defense against organophosphorus neurotoxins: Synergism of dendritic-enzyme complexes with 2-PAM and atropine

Novel, enzyme-complexed, nano-delivery systems have been developed to antagonize the lethal effects of organophosphorus (OP) molecules such as diisopropylfluorophosphate and paraoxon. Polymeric nanocapsules can be used to deliver metabolizing enzymes to the circulation, often increasing the enzymes efficacy by extending their circulatory life and, in some cases, enhancing their specific activity. The bacterial enzymes organophosphorus hydrolase (OPH) and organophosphorus anhydrolase (OPAA) were encapsulated within a nanocapsule, polyoxazoline-based dendritic polymer carrier and employed in combination with the OP antagonists pralidoxime (2-PAM) and atropine. The effective doses for OPH and OPAA, respectively, were 500–550 and 1500–1650 units/kg mice; the size of the entire complex is approximately 200 nm in diameter. These studies compare the efficacy of the two enzymes as prophylactic systems encapsulated within the dendritic polymer. When used in combination with 2-PAM and atropine, the dendritic encapsuled OPAA provided a 25×LD50 protection against DFP intoxication, while the similarly constructed OPH complex showed a more dramatic protection (780×LD50) against paraoxon intoxication in Balb/c mice. The studies demonstrate a synergistic enhancement of the antagonist, since the antidotal protection of 2-PAM+atropine against DFP and paraoxon is approximately 8 and 60×LD50, respectively.

Organophosphorus hydrolase as an in vivo catalytic nerve agent bioscavenger

The use of proteins as a treatment for organophosphorus intoxication has been investigated since A. R. Main demonstrated protective efficacy against parathion with an exogenously administered arylesterase in the late 1950s. His experiments spurred over 60 years of research and progress in the development of enzymes as potential bioscavengers of nerve agents and pesticides. Efforts have been made to broaden the specificity of enzymes to make a universal scavenger that would protect against multiple compounds, and an understanding of the differential isomer toxicity of these compounds has provided the impetus for rational and random mutagenic approaches in the stereospecific design of enzymes. As improved candidate enzymes are continually developed, our understanding of the contributions of the catalytic parameters (k(cat) , K(M) and catalytic efficiency) to efficacy expands. In addition to the scavenging properties of the proteins, another important aspect of development is the pharmacokinetic profile of the drug product. Immunogenicity, absorption, distribution and elimination contribute significantly to the level of protection afforded by the protein. A review of the development of organophosphorus hydrolase (OPH) for use as in vivo catalytic bioscavengers is presented here.

Molecular evolution of enzyme structure: construction of a hybrid hamster/Escherichia coli aspartate transcarbamoylase

SummaryAspartate transcarbamoylase (ATCase, EC 2.1.3.2) is the first unique enzyme common to de novo pyrimidine biosynthesis and is involved in a variety of structural patterns in different organisms. InEscherichia coli, ATCase is a functionally independent, oligomeric enzyme; in hamster, it is part of a trifunctional protein complex, designated CAD, that includes the preceding and subsequent enzymes of the biosynthetic pathway (carbamoyl phosphate synthetase and dihydroorotase). The complete complementary DNA (cDNA) nucleotide sequence of the ATCase-encoding portion of the hamster CAD gene is reported here. A comparison of the deduced amino acid sequences of the hamster andE. coli catalytic peptides revealed an overall 44% amino acid similarity, substantial conservation of predicted secondary structure, and complete conservation of all the amino acids implicated in the active site of theE. coli enzyme. These observations led to the construction of a functional hybrid ATCase formed by intragenic fusion based on the known tertiary structure of the bacterial enzyme. In this fusion, the amino terminal half (the “polar domain”) of the fusion protein was provided by a hamster ATCase cDNA subclone, and the carboxyl terminal portion (the “equatorial domain”) was derived from a clonedpyrBI operon ofE. coli K-12. The recombinant plasmid bearing the hybrid ATCase was shown to satisfy growth requirements of transformedE. coli pyrB− cells. The functionality of thisE. coli-hamster hybrid enzyme confirms conservation of essential structure-function relationships between evolutionarily distant and structurally divergent ATCases.

Mineralization of Acephate, a Recalcitrant Organophosphate Insecticide Is Initiated by a Pseudomonad in Environmental Samples

An aerobic bacterium capable of breaking down the pesticide acephate (O,S-dimethyl acetyl phosphoramidothioic acid) was isolated from activated sludge collected from a pesticide manufacturing facility. A phylogenetic tree based on the 16 S rRNA gene sequence determined that the isolate lies within the Pseudomonads. The isolate was able to grow in the presence of acephate at concentrations up to 80 mM, with maximum growth at 40 mM. HPLC and LC-MS/MS analysis of spent medium from growth experiments and a resting cell assay detected the accumulation of methamidophos and acetate, suggesting initial hydrolysis of the amide linkage found between these two moieties. As expected, the rapid decline in acephate was coincident with the accumulation of methamidophos. Methamidophos concentrations were maintained over a period of days, without evidence of further metabolism or cell growth by the cultures. Considering this limitation, strains such as described in this work can promote the first step of acephate mineralization in soil microbial communities.

Physicochemical characterization of stealth liposomes encapsulating an organophosphate hydrolyzing enzyme

The present 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 puffer and plasma.

Nano-Intercalated Organophosphorus-Hydrolyzing Enzymes in Organophosphorus Antagonism

A dendritic poly(2-alkyloxazoline)-based polymer was studied as a new carrier system for the organophosphorus-hydrolyzing recombinant enzymes, organophosphorus acid anhydrolase and organophosphorus hydrolase. Paraoxon (PO) and diisopropylfluorophosphate (DFP) were used as model organophosphorus compounds. Changes in plasma cholinesterase activity were monitored. The cholinesterase activity was proportional to the concentrations of DFP or PO. Plasma cholinesterase activity was higher in animals receiving enzyme and oxime before the organophosphates than in the oxime-only pretreated groups. These studies suggest that cholinesterase activity can serve as an indicator for the in vivo protection by the nano-intercalated organophosphorus acid anhydrolase or organophosphorus hydrolase against organophosphorus intoxications. These studies represent a practical application of polymeric nano-delivery systems as enzyme carriers in drug antidotal therapy.

Thermodynamic prediction of hydrogen production from mixed-acid fermentations

The MixAlco™ process biologically converts biomass to carboxylate salts that may be chemically converted to a wide variety of chemicals and fuels. The process utilizes lignocellulosic biomass as feedstock (e.g., municipal solid waste, sewage sludge, and agricultural residues), creating an economic basis for sustainable biofuels. This study provides a thermodynamic analysis of hydrogen yield from mixed-acid fermentations from two feedstocks: paper and bagasse. During batch fermentations, hydrogen production, acid production, and sugar digestion were analyzed to determine the energy selectivity of each system. To predict hydrogen production during continuous operation, this energy selectivity was then applied to countercurrent fermentations of the same systems. The analysis successfully predicted hydrogen production from the paper fermentation to within 11% and the bagasse fermentation to within 21% of the actual production. The analysis was able to faithfully represent hydrogen production and represents a step forward in understanding and predicting hydrogen production from mixed-acid fermentations.