Amperometric microbial biosensor for direct determination of organophosphate pesticides using recombinant microorganism with surface expressed organophosphorus hydrolase.

An amperometric microbial biosensor for the direct measurement of organophosphate nerve agents is described. The sensor is based on a carbon paste electrode containing genetically engineered cells expressing organophosphorus hydrolase (OPH) on the cell surface. OPH catalyzes the hydrolysis of organophosphorus pesticides with p-nitrophenyl substituent such as paraoxon, parathion and methyl parathion to p-nitrophenol. The later is detected anodically at the carbon transducer with the oxidation current being proportional to the nerve-agent concentration. The sensor sensitivity was optimized with respect to the buffer pH and loading of cells immobilized using paraoxon as substrate. The best sensitivity was obtained using a sensor constructed with 10 mg of wet cell weight per 100 mg of carbon paste and operating in pH 8.5 buffer. Using these conditions, the biosensor was used to measure as low as 0.2 microM paraoxon and 1 microM methyl parathion with very good sensitivity, excellent selectivity and reproducibility. The microbial biosensor had excellent storage stability, retaining 100% of its original activity when stored at 4 degrees C for up to 45 days.     

Mutagenesis of organophosphorus hydrolase to enhance hydrolysis of the nerve agent VX

Organophosphorus hydrolase (OPH) is capable of hydrolyzing a wide variety of organophosphorus pesticides and chemical warfare agents. However, the hydrolytic activity of OPH against the warfare agent VX is less than 0.1% relative to its activity against parathion and paraoxon. Based on the crystal structure of OPH and the similarities it shares with acetylcholinesterase, eight OPH mutants were constructed with the goal of increasing OPH activity toward VX. The activities of crude extracts from these mutants were measured using VX, demeton-S methyl, diisopropylfluoro-phosphate, ethyl parathion, paraoxon, and EPN as substrates. One mutant (L136Y) displayed a 33% increase in the relative VX hydrolysis rate compared to wild type enzyme. The other seven mutations resulted in 55-76% decreases in the relative rates of VX hydrolysis. There was no apparent relationship between the hydrolysis rates of VX and the rates of the other organophosphorus compounds tested.     

Highly-sensitive organophosphorous pesticide biosensors based on nanostructured films of acetylcholinesterase and CdTe quantum dots

The optical transducer of CdTe semiconductor quantum dots (QDs) has been integrated with acetylcholinesterase enzyme (AChE) by the layer-by-layer (LbL) assembly technique, resulting in a highly sensitive biosensor for detection of organophosphorus pesticides (OPs) in vegetables and fruits based on enzyme inhibition mechanism. The detection limits of the proposed biosensors are as low as 1.05 × 10(-11) M for paraoxon and 4.47 × 10(-12) M for parathion, which are significantly better than those of the conventional GC/MS methods or amperometric biosensors (0.5 nM). These biosensors are used for quick determination of low concentrations of OPs in real vegetable and fruit samples and exhibit satisfactory reproducibility and accuracy. Moreover, the stock stability of the biosensors are very good due to the stabilizing environment for the enzyme in the nanostructures made by LbL technique. Many advantages provided by these biosensors, like fluorescent change recognized by naked eyes and mass production with low cost, will facilitate future development of rapid and high-throughput screening of OPs.     

Organophosphorus compound detection on a cell chip with yeast coexpressing hydrolase and eGFP

Organophosphorus compounds (OPs) such as pesticides, fungicides, and herbicides are highly toxic but are nevertheless extensively used worldwide. To detect OPs, we constructed a yeast strain that co-displays organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (EGFP) on the cell surface using a Flo1p anchor system. OP degradation releases protons and causes a change in pH. This pH change results in structural deformation of EGFP, which triggers quenching of its fluorescence, thereby making this cell useful for visual detection of OPs. Fluorescence microscopy confirmed the high-intensity fluorescence displayed by EGFP on the cell surface. The yeast strain possessed sufficient OPH hydrolytic activities for degrading OPs, as measured by incubation with 1 mM paraoxon for 24 h at 30°C. In addition, with 20 mM paraoxon at 30°C, fluorescence quenching of EGFP on the single yeast cell was observed within 40 s in a microchamber chip. These observations suggest that engineered yeast cells are suitable for simultaneous degradation and visual detection of OPs.     

Biosensor for direct determination of organophosphate nerve agents. 1. Potentiometric enzyme electrode

A potentiometric enzyme electrode for the direct measurement of organophosphate (OP) nerve agents was developed. The basic element of this enzyme electrode was a pH electrode modified with an immobilized organophosphorus hydrolase (OPH) layer formed by cross-linking OPH with bovine serum albumin (BSA) and glutaradehyde. OPH catalyses the hydrolysis of organophosphorus pesticides to release protons, the concentration of which is proportional to the amount of hydrolysed substrate. The sensor signal and response time was optimized with respect to the buffer pH, ionic concentration of buffer, temperature, and units of OPH immobilized using paraoxon as substrate. The best sensitivity and response time were obtained using a sensor constructed with 500 IU of OPH and operating in pH 8.5, 1 mM HEPES buffer. Using these conditions, the biosensor was used to measure as low as 2 microM of paraoxon, ethyl parathion, methyl parathion and diazinon. The biosensor was completely stable for at least one month when stored in pH 8.5, 1 mM HEPES + 100 mM NaCl buffer at 4 degrees C.     

Characterization and comparison of putative Stenotrophomonas maltophilia methyl parathion hydrolases

Three Stenotrophomonas maltophilia isolates, KKWT11, CBF10-1, TTF10, were collected from organophosphate (OP)-contaminated soil in the Houston metropolitan area. A conserved metallo-β-lactamase (MBL) enzyme purported to function as a methyl parathion hydrolase was identified and found to be distantly homologous to the characterized Pseudomonas sp. WBC-3 methyl parathion hydrolase and shared no significant homology with other organophosphate hydrolases. Following expression of MBL enzymes cloned from S. maltophilia strains KKWT11, CBF10-1, and TTF10, respectively, an enzymatic preference for paraoxon was observed, with concentrations of 70, 40, and 30 µM of p-nitrophenol (PNP) formed after 48 h. Comparatively limited hydrolysis against the phosphorothioate methyl parathion was recorded with concentrations of PNP ranging from 9.5 to 3.5 µM after 48 h. A coexpressive construct harboring a modified organophosphorus hydrolase enzyme and the CBF10-1 MBL enzyme yielded only a slight improvement in degradation of methyl parathion, resulting in 75 µM of PNP formed compared with 69 µM formed by the organophosphorus hydrolase (OPH) control over 48 h. These results suggest that S. maltophilia MBL enzymes are currently insufficient for broad-spectrum hydrolysis of phosphorothioate insecticides. Future studies will thus seek to elucidate their catalytic efficiency against other notable phosphotriester oxons, including chlorpyrifos oxon, and malaoxon.     

Whole cell-enzyme hybrid amperometric biosensor for direct determination of organophosphorous nerve agents with p-nitrophenyl substituent

In this paper, we reported the construction of a hybrid biosensor for direct, highly selective, sensitive, and rapid quantitative determination of organophosphate pesticides with p-nitrophenyl substituent using purified organophosphorus hydrolase (OPH) for the initial hydrolysis and Arthrobacter sp. JS443 for subsequent p-nitrophenol oxidation. The biocatalytic layer was prepared by co-immobilizing Arthrobacter sp. JS443 and OPH on a carbon paste electrode. OPH catalyzed the hydrolysis of organophosphorus pesticides with p-nitrophenyl substituent such as paraoxon and methyl parathion to release p-nitrophenol that was oxidized by the enzymatic machinery of Arthrobacter sp. JS443 to carbon dioxide through electroactive intermediates 4-nitrocatechol and 1,2,4-benzenetriol. The oxidization current of the intermediates was measured and correlated to the concentration of organophosphates. The best sensitivity and response time were obtained using a sensor constructed with 0.06 mg dry weight of cell and 965 IU of OPH operating at 400 mV applied potential (vs. Ag/AgCl reference) in 50 mM citrate-phosphate pH 7.5 buffer at room temperature. Using these conditions, the biosensor measured as low as 2.8 ppb (10 nM) of paraoxon and 5.3 ppb (20 nM) of methyl parathion without interference from phenolic compounds, carbamate pesticides, triazine herbicides, and organophosphate pesticides that do not have the p-nitrophenyl substituent. The biosensor had excellent operational life-time stability with no decrease in response for more than 40 repeated uses over a 12-h period when stored at room temperature, while its storage life was approximately 2 days when stored in the operating buffer at 4 degrees C.     

Simultaneous degradation of organophosphorus pesticides and p-nitrophenol by a genetically engineered Moraxella sp. with surface-expressed organophosphorus hydrolase.

Moraxella sp., a native soil organism that grows on p-nitrophenol (PNP), was genetically engineered for the simultaneous degradation of organophosphorus (OP) pesticides and p-nitrophenol (PNP). The truncated ice nucleation protein (INPNC) anchor was used to target the pesticide-hydrolyzing enzyme, organophosphorus hydrolase (OPH), onto the surface of Moraxella sp., alleviating the potential substrate uptake limitation. A shuttle vector, pPNCO33, coding for INPNC-OPH was constructed and the translocation, surface display, and functionality of OPH were demonstrated in both E. coli and Moraxella sp. However, whole cell activity was 70-fold higher in Moraxella sp. than E. coli. The resulting Moraxella sp. degraded organophosphates as well as PNP rapidly, all within 10 h. The initial hydrolysis rate was 0.6 micromol/h/mg dry weight, 1.5 micromol/h/mg dry weight, and 9.0 micromol/h/mg dry weight for methyl parathion, parathion, and paraoxon, respectively. The possibility of rapidly degrading OP pesticides and their byproducts should open up new opportunities for improved remediation of OP nerve agents in the future.     

Bacterial cell surface display of organophosphorus hydrolase for selective screening of improved hydrolysis of organophosphate nerve agents

Organophosphorus hydrolase (OPH) is a bacterial enzyme that has been shown to degrade a wide range of neurotoxic organophosphate nerve agents. However, the effectiveness of degradation varies dramatically, ranging from highly efficient with paraoxon to relatively slow with methyl parathion. Sequential cycles of DNA shuffling and screening were used to fine-tune and enhance the activity of OPH towards poorly degraded substrates. Because of the inaccessibility of these pesticides across the cell membrane, OPH variants were displayed on the surface of Escherichia coli using the truncated ice nucleation protein in order to isolate novel enzymes with truly improved substrate specificities. A solid-phase top agar method based on the detection of the yellow product p-nitrophenol was developed for the rapid prescreening of potential variants with improved hydrolysis of methyl parathion. Two rounds of DNA shuffling and screening were carried out, and several improved variants were isolated. One variant in particular, 22A11, hydrolyzes methyl parathion 25-fold faster than does the wild type. Because of the success that we achieved with directed evolution of OPH for improved hydrolysis of methyl parathion, we believe that we can easily extend this method in creating other OPH variants with improved activity against poorly degraded pesticides such as diazinon and chlorpyrifos and nerve agents such as sarin and soman.     

Improved degradation of organophosphorus nerve agents and p-nitrophenol by Pseudomonas putida JS444 with surface-expressed organophosphorus hydrolase

Pseudomonas putida JS444, isolated from p-nitrophenol (PNP) contaminated waste sites, was genetically engineered to simultaneously degrade organophosphorus pesticides (OP) and PNP. A surface anchor system derived from the ice-nucleation protein (INP) from Pseudomonas syringae was used to target the organophosphorus hydrolase (OPH) onto the surface of Pseudomonas putida JS444, reducing the potential substrate uptake limitation. Engineered cells were capable of targeting OPH onto the cell surface as demonstrated by western blotting, cell fractionation, and immunofluorescence microscopy. The engineered P. putida JS444 degraded organophosphates as well as PNP rapidly without instability problems associated with the engineered Moraxella sp. The initial hydrolysis rate was 7.90, 3.54, and 1.53 micromol/h/mg dry weight for paraoxon, parathion, and methyl parathion, respectively. The excellent stability in combination with the rapid degradation rate for organophosphates and PNP make this engineered strain an ideal biocatalyst for complete mineralization of organophosphates.     

Bacterial Cell Surface Display of Organophosphorus Hydrolase for Selective Screening of Improved Hydrolysis of Organophosphate Nerve Agents

Organophosphorus hydrolase (OPH) is a bacterial enzyme that has been shown to degrade a wide range of neurotoxic organophosphate nerve agents. However, the effectiveness of degradation varies dramatically, ranging from highly efficient with paraoxon to relatively slow with methyl parathion. Sequential cycles of DNA shuffling and screening were used to fine-tune and enhance the activity of OPH towards poorly degraded substrates. Because of the inaccessibility of these pesticides across the cell membrane, OPH variants were displayed on the surface of Escherichia coli using the truncated ice nucleation protein in order to isolate novel enzymes with truly improved substrate specificities. A solid-phase top agar method based on the detection of the yellow product p-nitrophenol was developed for the rapid prescreening of potential variants with improved hydrolysis of methyl parathion. Two rounds of DNA shuffling and screening were carried out, and several improved variants were isolated. One variant in particular, 22A11, hydrolyzes methyl parathion 25-fold faster than does the wild type. Because of the success that we achieved with directed evolution of OPH for improved hydrolysis of methyl parathion, we believe that we can easily extend this method in creating other OPH variants with improved activity against poorly degraded pesticides such as diazinon and chlorpyrifos and nerve agents such as sarin and soman.     

Altering the substrate specificity of organophosphorus hydrolase for enhanced hydrolysis of chlorpyrifos

Chlorpyrifos is one of the most popular pesticides used for agriculture crop protection, and widespread contamination is a potential concern. However, chlorpyrifos is hydrolyzed almost 1,000-fold slower than the preferred substrate, paraoxon, by organophosphorus hydrolase (OPH), an enzyme that can degrade a broad range of organophosphate pesticides. We have recently demonstrated that directed evolution can be used to generate OPH variants with up to 25-fold improvement in hydrolysis of methyl parathion. The obvious question and challenge are whether similar success could be achieved with this poorly hydrolyzed substrate, chlorpyrifos. For this study, five improved variants were selected from two rounds of directed evolution based on the formation of clear haloes on Luria-Bertani plates overlaid with chlorpyrifos. One variant, B3561, exhibited a 725-fold increase in the k(cat)/K(m) value for chlorpyrifos hydrolysis as well as enhanced hydrolysis rates for several other OP compounds tested. Considering that wild-type OPH hydrolyzes paraoxon at a rate close to the diffusion control limit, the 39-fold improvement in hydrolysis of paraoxon by B3561 suggests that this variant is one of the most efficient enzymes available to attack a wide spectrum of organophosphate nerve agents.     

Flow injection amperometric detection of OP nerve agents based on an organophosphorus-hydrolase biosensor detector

A flow-injection system with an organophosphorus-hydrolase (OPH)-biosensor detector has been developed and characterized for the rapid detection of organophosphorus (OP) nerve agents. The enzyme was immobilized onto a thin-film gold detector through a cystamine-glutaraldehyde coupling. Factors influencing the performance were optimized. The resulting flow system offered a fast, sensitive, selective, and stable response. The peak current increased linearly with the concentration of paraoxon and methyl parathion over the 1-10 microM range (sensitivity, 2.29 and 1.04 nA/microM, respectively). The OPH-biosensor flow injection systems offered low detection limits (e.g. 0.1 microM paraoxon), along with a good precision (R.S.D. of 3.6% for 20 successive injections of a 1.0 microM paraoxon solution). The OPH-biosensor flow detector offers great promise for rapid field screening of OP pesticides and nerve agents.     

Array biosensor based on enzyme kinetics monitoring by fluorescence spectroscopy: application for neurotoxins detection

The aim of the present work is to develop an evanescence wave array biosensor exploiting the “kinetic” approach of enzymatic reaction and further detection of the reaction products via pH sensitive fluorophore reporter. To demonstrate the feasibility of this approach, we have developed a biosensor array with the potential for direct detection of organophosphates using as a biorecognition element, an enzyme organophosphorus hydrolase (OPH), conjugated with a pH-sensitive fluorophore, carboxynaphthofluorescein (CNF). The presence of reference spots allows the discrimination of the enzymatic and non-enzymatic based pH changes; bovine serum albumin (BSA) was used as a non-enzymatic scaffold protein for CNF attachment at the reference spots. An array biosensor unit developed at the Naval Research Laboratories (NRL) was adopted as the detection platform and appropriately modified for enzyme-based measurements. A planar multi-mode waveguide was covered with an optically transparent TiO₂ layer to increase the surface area available for immobilization.

The biosensor enabled the detection of 2.5 μM paraoxon, and 10 μM DFP and parathion, respectively. Very short response time of 30 s can be achieved with a total analysis time of less than 2 min. When operated at room temperature and stored at 4 °C, the waveguide retained reasonable activity for greater than 45 days.     

Altering the Substrate Specificity of Organophosphorus Hydrolase for Enhanced Hydrolysis of Chlorpyrifos

Chlorpyrifos is one of the most popular pesticides used for agriculture crop protection, and widespread contamination is a potential concern. However, chlorpyrifos is hydrolyzed almost 1,000-fold slower than the preferred substrate, paraoxon, by organophosphorus hydrolase (OPH), an enzyme that can degrade a broad range of organophosphate pesticides. We have recently demonstrated that directed evolution can be used to generate OPH variants with up to 25-fold improvement in hydrolysis of methyl parathion. The obvious question and challenge are whether similar success could be achieved with this poorly hydrolyzed substrate, chlorpyrifos. For this study, five improved variants were selected from two rounds of directed evolution based on the formation of clear haloes on Luria-Bertani plates overlaid with chlorpyrifos. One variant, B3561, exhibited a 725-fold increase in the k_cat/K_m value for chlorpyrifos hydrolysis as well as enhanced hydrolysis rates for several other OP compounds tested. Considering that wild-type OPH hydrolyzes paraoxon at a rate close to the diffusion control limit, the 39-fold improvement in hydrolysis of paraoxon by B3561 suggests that this variant is one of the most efficient enzymes available to attack a wide spectrum of organophosphate nerve agents.     

A fluorogenic substrate for detection of organophosphatase activity

A new fluorogenic substrate for the specific detection of organophosphatase (OPase) activity has been designed and evaluated. Our results indicate that 7-diethylphospho-6,8-difluor-4-methylumbelliferyl (DEPFMU) is hydrolyzed specifically by the OPases, mammalian serum paraoxonase and bacterial organophosphorus hydrolase (OPH). The apparent K(m) of DEPFMU is 29 microM for OPH and 91 and 200 microM for the PON1 L(55)R(192) and PON1 L(55)Q(192) isoforms of human paraoxonase, respectively. DEPFMU-based assay systems are 10-100 times more sensitive for OPH and mammalian paraoxonase detection than existing methods. Importantly, DEPFMU is poorly hydrolyzed by both serum and cellular phosphatases and, therefore, may be used as part of a robust and sensitive assay for detecting not only purified, but also highly impure, preparations of OPase such as blood samples. The superior sensitivity of DEPFMU makes it potentially useful in the search for new enzymes that may hydrolyze nerve poisons such as sarin, soman, and VX, monitoring the decontamination of organophosphates (OPs) by OPH and determining serum paraoxonase activity which appears to be important for protection against atherosclerosis, sepsis, and OP toxicity.     

Enhanced biodegradation of toxic organophosphate compounds using recombinant Escherichia coli with sec pathway-driven periplasmic secretion of organophosphorus hydrolase

Although Escherichia coli can be genetically engineered to degrade environmental toxic organophosphate compounds (OPs) to nontoxic materials, a critical problem in such whole cell systems is limited substrate diffusion. The present work examined whether periplasmic expression of organophosphorus hydrolase (OPH) resulted in better whole cell enzymatic activity compared to standard cytosolic expression. Recombinant OPH periplasmic expression was achieved using the general secretory (sec) pathway with the pelB signal sequence. We found that while total OPH activity in periplasmic-expressing cell lysates was lower compared to that in cytosolic-expressing cell lysates whole cell OPH activity was 1.8-fold greater at 12 h post-induction in the periplasmic-expressing cells as a result of OPH translocation into the periplasmic space ( approximately 67% of whole cell OPH activity was found in the periplasmic fraction). These data suggest that E. coli engineered to periplasmically secrete OPH via the sec pathway may provide an improved whole cell biodegradation system for destruction of environmental toxic OPs.     

Label-free, multiplexed detection of bacterial tmRNA using silicon photonic microring resonators

This work describes the development of an automated flow-based biosensor that employs genetically modified acetylcholinesterase (AChE) enzymes B394, B4 and wild type B131. The biosensor was based on a screen printed carbon electrode (SPE) that was integrated into a flow cell. Enzymes were immobilised on cobalt (II) phthalocyanine (CoPC) modified electrodes by entrapment in a photocrosslinkable polymer (PVA-AWP). The automated flow-based biosensor was successfully used to quantify three organophosphate pesticides (OPs) in milk samples. The OPs used were chlorpyriphos-oxon (CPO), ethyl paraoxon (EPOx) and malaoxon (MOx). The total analysis time for the assay was less than 15 min. Initially, the biosensor performance was tested in phosphate buffer solution (PBS) using B394, B131 and B4 biosensors. The best detection limits were obtained with B394; therefore, this biosensor was used to produce calibration data in milk with three OPs in the concentration range of 5 × 10(-6)M to 5 × 10(-12)M. The limit of detection (LOD) obtained in milk for CPO, EPOx and MOx were 5 × 10(-12)M, 5 × 10(-9)M and 5 × 10(-10)M, respectively, with a correlation coefficient R(2)=0.9910. The automated flow-based biosensor successfully quantified the OPs in different fat-containing milk samples. There were no false positives or false negatives observed for the analytical figures of merit for the constructed biosensors. This method is inexpensive, sensitive, portable, non-invasive and provides real-time results. This analytical system can provide rapid detection of highly toxic OPs in food matrices such as milk.     

Neurotoxic Organophosphate Degradation with Polyvinyl Alcohol Gel-Immobilized Microbial Cells

A genetically engineered strain of Escherichia coli that expresses organophosphorus hydrolase (OPH) was immobilized in a polyvinyl alcohol (PVA) cryogel to form a porous biocatalyst that successfully degrades organophosphorus (OP) neurotoxins. The impacts of both diffusion and reaction on biocatalyst efficiency were determined to enable prediction and optimization of the biocatalyst performance. The kinetic rate parameters and activation energies of pure OPH, free cell suspensions, and the immobilized cell biocatalyst were compared. Diffusion was a determining factor for paraoxon hydrolysis because of the very rapid OPH kinetics for its model substrate. Both the paraoxon diffusion through the PVA matrix and the diffusion associated with microbial transport of paraoxon were shown to impact the biocatalyst reaction. However, the enhancement in storage stability resulting from diffusional limitations provides an advantage to diffusion-limited operation. This research may serve as a guide to define the influence of diffusion in biological reaction systems. The broad substrate specificity and hydrolytic efficiency of OPH coupled with the ability to genetically engineer the enzyme for specific target OP neurotoxins enhance the suitability of OPH-based technologies for detoxification of these compounds. Cryoimmobilization provides a suitable vehicle as a cost-effective, efficient technology for bioremediation of environmental media contaminated with OP compounds.     

Studies on acetylcholine sensor and its analytical application based on the inhibition of cholinesterase

Acetylcholine esterase electrodes, based on glass, Pd/PdO and Ir/IrO₂ electrodes as pH sensor, using the immobilized acetylcholine esterase in acrylamide-methacrylamide hydrazides prepolymer are reported and compared. New data on the analysis of nicotine, fluoride ion, and some organophosphorus compounds are reported using the present AChE sensor based on the inhibition of the immobilized acetylcholine esterase. Reactivation of immobilized AChE after inhibition with reversible inhibitor, i.e. nicotine and fluoride ion is carried out using a mixture of working buffer and acetylcholine, whereas reactivation after inhibition with irreversible inhibitor, i.e. organophosphorus compounds is carried out using a mixture of acetylcholine and pyridine-2-aldoxime methiodide (PAM). The detection limits for the nicotine and fluoride ion are found to be 10⁻⁵M whereas for paraoxon, methyl parathion and malathion are found to be 10⁻⁹ M and 10⁻¹⁰ M.