Detoxification of organophosphate nerve agents by immobilized dual functional biocatalysts in a cellulose hollow fiber bioreactor

A whole-cell technology for detoxification of organophosphates based on genetically engineered Escherichia coli cell expressing both cellulose-binding domain (CBD) and organophosphorus hydrolase (OPH) onto cell surface was reported recently (Wang et al., 2002). This study reports the application of these biocatalysts when immobilized in a cellulose hollow fiber bioreactor (HFB) for the biodetoxification of a model organophosphate, paraoxon, in a continuous flow mode. In 24 h, 0.79 mg wet cell/cm2 fiber surface were immobilized onto cellulose fibers specifically and strongly through the cellulose binding domain, forming a monolayer demonstrated by Scanning Electronic Micrograph, and essentially no cell was washed away by washing buffer. The immobilized biocatalyst had a high performance of detoxifying paraoxon solution of 5,220 mumol/h x L reactor or 990 mumol/h x m2 reactor. The immobilized biocatalysts maintained a stable degradation capacity for 15 uses over a period of 48 days with only 10% decline in degradation efficiency under operating and storage conditions. In addition, the bioreactor was easily regenerated by washing with 1% sodium dodecyl sulfate (SDS), with 86.7% immobilization capacity and 93.9% degradation efficiency recovery. This is the first report using the HFB in a non-traditional way, immobilizing whole-cell biocatalysts by specific adhesion thus rendering the catalysis operation the advantages of low pressure drop, low shear force, and low energy requirement. The successful application of this genetically engineered dual functional E. coli strain in a model bioreactor shows its promise in large-scale detoxification of organophosphate nerve agents in bulk liquid phase.     

Specific adhesion to cellulose and hydrolysis of organophosphate nerve agents by a genetically engineered Escherichia coli strain with a surface-expressed cellulose-binding domain and organophosphorus hydrolase

A genetically engineered Escherichia coli cell expressing both organophosphorus hydrolase (OPH) and a cellulose-binding domain (CBD) on the cell surface was constructed, enabling the simultaneous hydrolysis of organophosphate nerve agents and immobilization via specific adsorption to cellulose. OPH was displayed on the cell surface by use of the truncated ice nucleation protein (INPNC) fusion system, while the CBD was surface anchored by the Lpp-OmpA fusion system. Production of both INPNC-OPH and Lpp-OmpA-CBD fusion proteins was verified by immunoblotting, and the surface localization of OPH and the CBD was confirmed by immunofluorescence microscopy. Whole-cell immobilization with the surface-anchored CBD was very specific, forming essentially a monolayer of cells on different supports, as shown by electron micrographs. Optimal levels of OPH activity and binding affinity to cellulose supports were achieved by investigating expression under different induction levels. Immobilized cells degraded paraoxon rapidly at an initial rate of 0.65 mM/min/g of cells (dry weight) and retained almost 100% efficiency over a period of 45 days. Owing to its superior degradation capacity and affinity to cellulose, this immobilized-cell system should be an attractive alternative for large-scale detoxification of organophosphate nerve agents.     

Specific Adhesion to Cellulose and Hydrolysis of Organophosphate Nerve Agents by a Genetically Engineered Escherichia coli Strain with a Surface-Expressed Cellulose-Binding Domain and Organophosphorus Hydrolase

A genetically engineered Escherichia coli cell expressing both organophosphorus hydrolase (OPH) and a cellulose-binding domain (CBD) on the cell surface was constructed, enabling the simultaneous hydrolysis of organophosphate nerve agents and immobilization via specific adsorption to cellulose. OPH was displayed on the cell surface by use of the truncated ice nucleation protein (INPNC) fusion system, while the CBD was surface anchored by the Lpp-OmpA fusion system. Production of both INPNC-OPH and Lpp-OmpA-CBD fusion proteins was verified by immunoblotting, and the surface localization of OPH and the CBD was confirmed by immunofluorescence microscopy. Whole-cell immobilization with the surface-anchored CBD was very specific, forming essentially a monolayer of cells on different supports, as shown by electron micrographs. Optimal levels of OPH activity and binding affinity to cellulose supports were achieved by investigating expression under different induction levels. Immobilized cells degraded paraoxon rapidly at an initial rate of 0.65 mM/min/g of cells (dry weight) and retained almost 100% efficiency over a period of 45 days. Owing to its superior degradation capacity and affinity to cellulose, this immobilized-cell system should be an attractive alternative for large-scale detoxification of organophosphate nerve agents.     

Detoxification of the organophosphate nerve agent coumaphos using organophosphorus hydrolase immobilized on cellulose materials

Neurotoxic organophosphates (OPs) are widely used as pesticides and for public health purposes, as well as being nerve gases. As a result of the widespread use of these compounds for agriculture, large volumes of wastewater are generated. Additionally, there are large stockpiles of the nerve gases soman, sarin and VX in the United States and elsewhere around the world. Organophosphorus hydrolase (OPH) is an enzyme that catalyzes the hydrolysis of OP nerve agents. To date, however, the use of this enzyme in detoxification processes has been rather limited due to the high cost of its purification and short catalytic half-life. This paper reports the development of a cost-effective method for the production and immobilization of OPH in a pilot application in an enzyme bioreactor column for detoxification of paraoxon and coumaphos in contaminated wastewaters. A fusion between OPH and a cellulose binding domain that binds selectively to cellulose was generated to allow one-step purification and immobilization of OPH on cheap and abundantly available cellulose immobilization matrices. When packed in a column bioreactor, the immobilized fusion enzyme was able to completely degrade coumaphos up to a concentration of 0.2 mM. However, stirring of OPH immobilized on cellulose materials resulted in complete OP degradation of 1.5 mM coumaphos. The bioreactor column degraded the compounds tested at high concentration, rapidly, and without loss of process productivity for about 2 months.     

Detoxification of the organophosphate nerve agent coumaphos using organophosphorus hydrolase immobilized on cellulose materials

Neurotoxic organophosphates (OPs) are widely used as pesticides and for public health purposes, as well as being nerve gases. As a result of the widespread use of these compounds for agriculture, large volumes of wastewater are generated. Additionally, there are large stockpiles of the nerve gases soman, sarin and VX in the United States and elsewhere around the world. Organophosphorus hydrolase (OPH) is an enzyme that catalyzes the hydrolysis of OP nerve agents. To date, however, the use of this enzyme in detoxification processes has been rather limited due to the high cost of its purification and short catalytic half-life. This paper reports the development of a cost-effective method for the production and immobilization of OPH in a pilot application in an enzyme bioreactor column for detoxification of paraoxon and coumaphos in contaminated wastewaters. A fusion between OPH and a cellulose binding domain that binds selectively to cellulose was generated to allow one-step purification and immobilization of OPH on cheap and abundantly available cellulose immobilization matrices. When packed in a column bioreactor, the immobilized fusion enzyme was able to completely degrade coumaphos up to a concentration of 0.2 mM. However, stirring of OPH immobilized on cellulose materials resulted in complete OP degradation of 1.5 mM coumaphos. The bioreactor column degraded the compounds tested at high concentration, rapidly, and without loss of process productivity for about 2 months.

     

Detoxification of organophosphate pesticides using an immobilized phosphotriesterase from Pseudomonas diminuta

A purified phosphotriesterase was successfully immobilized onto trityl agarose in a fixed bed reactor. A total of up to 9200 units of enzyme activity was immobilized onto 2.0 mL of trityl agarose (65 mumol trityl groups/mL agarose), where one unit is the amount of enzyme required to catalyze the hydrolysis of one micromole of paraoxon in one min. The immobilized enzyme was shown to behave chemically and kinetically similar to the free enzyme when paraoxon was utilized as a substrate. Several organophosphate pesticides, methyl parathion, ethyl parathion, diazinon, and coumaphos were also hydrolyzed by the immobilized phosphotriesterase. However, all substrates exhibited an affinity for the trityl agarose matrix. For increased solubility and reduction in the affinity of these pesticides for the trityl agarose matrix, methanol/water mixtures were utilized. The effect of methanol was not deleterious when concentrations of less than 20% were present. However, higher concentrations resulted in elution of enzyme from the reactor. With a 10-unit reactor, a 1.0 mM paraoxon solution was hydrolyzed completely at a flow rate of 45 mL/h. Kinetic parameters were measured with a 0.1-unit reactor with paraoxon as a substrate at a flow rate of 22 mL/h. The apparent K(m) for the immobilized enzyme was 3-4 times greater than the K(m) (0.1 mM) for the soluble enzyme. Immobilization limited the maximum rate of substrate hydrolysis to 40% of the value observed for the soluble enzyme. The pH-rate profiles of the soluble and immobilized enzymes were very similar. The immobilization of phosphotriesterase onto trityl agarose provides an effective method esterase onto trityl agarose provides an effective method for hydrolyzing and thus detoxifyuing organophosphate pesticides and mammalian acetylcholinesterase inhinbitors.     

Thermally triggered purification and immobilization of elastin-OPH fusions

A bifunctional fusion protein consisting of organophosphorus hydrolase (OPH) and elastin-like polypeptide (ELP) was synthesized for the detoxification of organophosphorus compounds. ELPs undergo a reversible phase transition upon an increase in temperature, forming hydrophobic aggregates. This thermally triggered property of phase transition allows for a simple and rapid means of purifying the fusion protein. Over 1,300-fold purification was achieved after only 2 cycles of inverse phase transition. The purified fusion protein showed identical kinetic properties as the native OPH with only a modest 10% increase in K(m) and a 5% decrease of K(cat). The ability of the ELP domain to form collapsed aggregates also improved long-term stability of the fusion enzyme. Aggregated ELP-OPH retained nearly 100% activity over a span of three weeks. In addition to facilitating purification and stability, the ELP moiety served as a hydrophobic tag for one-step immobilization of the fusion protein onto hydrophobic surfaces. The ELP-OPH was capable of rapidly degrading paraoxon while immobilized. The protein also retained ELP functionality of reversible phase transition thereby allowing for the regeneration of the treated surface. This technology offers a swift and convenient means for purification, immobilization, and regeneration of OPH onto a variety of hydrophobic surfaces by simple environmental triggers.     

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.     

Biosensor system for continuous monitoring of organophosphate aerosols

An enzyme-based monitoring system provides the basis for continuous sampling of organophosphate contamination in air. The enzymes butyrylcholinesterase (BuChE) and organophosphate hydrolase (OPH) are stabilized by encapsulation in biomimetic silica nanoparticles, entrained within a packed bed column. The resulting immobilized enzyme reactors (IMERs) were integrated with an impinger-based aerosol sampling system for collection of chemical contaminants in air. The sampling system was operated continuously and organophosphate detection was performed in real-time by single wavelength analysis of enzyme hydrolysis products. The resulting sensor system detects organophosphates based on either enzyme inhibition (of BuChE) or substrate hydrolysis (by OPH). The detection limits of the IMERs for specific organophosphates are presented and discussed. The system proved suitable for detection of a range of organophosphates including paraoxon, demeton-S and malathion.     

Biodegradation of organophosphate pesticide using recombinant Cyanobacteria with surface- and intracellular-expressed organophosphorus hydrolase

The opd gene, encoding organophosphorus hydrolase (OPH) from Flavobacterium sp. capable of degrading a wide range of organophosphate pesticides, was surface- and intracellular-expressed in Synechococcus PCC7942, a prime example of photoautotrophic cyanobacteria. OPH was displayed on the cyanobacterial cell surface using the truncated ice nucleation protein as an anchoring motif. A minor fraction of OPH was displayed onto the outermost surface of cyanobacterial cells, as verified by immunostaining visualized under confocal laser scanning microscopy and OPH activity analysis; however, a substantial fraction of OPH was buried in the cell wall, as demonstrated by proteinase K and lysozyme treatments. The cyanobacterial outer membrane acts as a substrate (paraoxon) diffusion barrier affecting whole-cell biodegradation efficiency. After freeze-thaw treatment, permeabilized whole cells with intracellular-expressed OPH exhibited 14-fold higher bioconversion efficiency (Vmax/Km) than that of cells with surface-expressed OPH. As cyanobacteria have simple growth requirements and are inexpensive to maintain, expression of OPH in cyanobacteria may lead to the development of a lowcost and low-maintenance biocatalyst that is useful for detoxification of organophosphate pesticides.     

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.     

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.     

Functional effects of amino acid substitutions within the large binding pocket of the phosphotriesterase OpdA from Agrobacterium sp. P230

The phosphotriesterase OpdA from Agrobacterium sp. P230 has about 10-fold higher activity for dimethyl organophosphate (OP) insecticides, than its homologue from Flavobacterium sp. ATCC27551, organophosphate hydrolase (OPH). OpdA shows about 10% amino acid sequence divergence from OPH and also has a 20 residue C-terminal extension. Here we show that the difference in kinetics is largely explained by just two amino acid differences between the two proteins. A truncated form of OpdA demonstrated that the C-terminal extension has no effect on its preference for dimethyl organophosphate substrates. Chimeric proteins of OPH and OpdA were then analysed to show that replacement of a central region of OpdA sequence, which encodes the residues in the large subsite of the active site, with the homologous region in OPH decreased the activity of OpdA towards dimethyl OPs, to values close to those for OPH. Site-directed mutagenesis in this region identified two differences between the proteins, Y257H and F272L (with the OpdA residues first) as being responsible for this reduction. These two differences were also responsible for the increased activity of OpdA towards the diisopropyl organophosphate, diisopropyl fluorophosphate, relative to OPH. Molecular modelling of triethyl phosphate in the active site of OpdA confirmed a reduction in the size of the large subsite relative to OPH.     

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.     

GFP-visualized immobilized enzymes: degradation of paraoxon via organophosphorus hydrolase in a packed column.

A versatile gene-fusion technique for immobilizing and visualizing biologically active enzymes which includes from the N to C-termini, an affinity histidine tag, the green fluorescent protein (GFP), a proteolytic enzyme (enterokinase, EK) cleavage site and the enzyme of interest, were developed. Specifically, the organophosphorus hydrolase was bound to the affinity (His(6))-reporter(GFP)-EK fusion elements. Organophosphorus hydrolase (OPH) is capable of degrading a variety of pesticides and nerve agents. In the case of immobilized OPH, paraoxon was rapidly degraded when pumped through a packed column. In reaction mixtures containing CHES buffer at pH 6.9, a continual decay in OPH activity was observed and importantly, this was monitored by GFP fluorescence. This decay in activity was fully restored, along with fluorescence, upon washing with PBS buffer. Many subsequent experiments were performed at varied pH and in different background buffer solutions. In all cases when there was OPH activity there was also marked fluorescence from the GFP fusion partner. Likewise, when OPH activity was lost, so was GFP fluorescence and, importantly, both were regenerated when washed in the presence of the kosmotropic salt, phosphate. Recently, Waldo et al. (1999) showed that GFP fluorescence from whole cells indicated the extent of proper folding of normally aggregated proteins designed via directed evolution. The present work demonstrates an application wherein GFP fluorescence indicates stability and activity of its fusion partner.     

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.     

Degradation of organophosphorous nerve agents by enzyme-polymer nanocomposites: efficient biocatalytic materials for personal protection and large-scale detoxification

The biocatalytic destruction of organophosphates has become an important focus area, as efficient "clean" technologies are sought for chemical weapons decommissioning, counteracting nerve agent attacks, and protecting against organophosphate pesticide poisoning. A novel method is advanced for immobilizing the broad-spectrum enzyme organophosphorous hydrolase (OPH) from Pseudomonas diminuta, based on the formation of nanocomposite protein-silicone polymers. The resulting materials are highly active, stable, and versatile biocatalysts for the liquid and gas phase detoxification of organophosphates, and can be fabricated as monoliths, sheets, thick films, granulates, or macroporous foams. This approach offers an efficient avenue to robust, high-performance biocatalytic OPH-containing polymers that outperform immobilized OPH catalysts reported to date. The method provides for the first time a route to biocatalytic materials that may be suitable for "active" protective wear, as well as bulk catalysts for the destruction of large volumes of organophosphates. The preparation of OPH-silicone biocomposites, their performances in the liquid and gas phase detoxification of paraoxon, dichlorvos, and diisopropyl fluorophosphate, and their features are discussed.     

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.     

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.     

Catalytic buffers enable positive-response inhibition-based sensing of nerve agents.

We report herein an efficient method to control pH in reactions catalyzed by hydrolytic enzymes, such as the degradation of paraoxon by phosphotriesterase (E.G. 3.1.8.1; OPH), using urease-catalyzed (E.G. 3.5.1.5) urea hydrolysis as a buffering agent. Given the distinct pH profiles of urease and OPH activities, urease produces base on demand in response to pH drop during paraoxon detoxification. As pH changes, the enzyme activities fluctuate to finally stabilize at a pH "set-point," where the rates of acid and base generation are equal. By varying the urease to OPH ratio, various pH "set-points" ranging between 6.5 and 8.5 were achieved within minutes and could be predicted theoretically. This dynamic approach for pH control was successfully applied to the development of a positive-response inhibition-based sensor.