Organophosphorus Acid Anhydrolase Research Articles

Recombinant Organophosphorus acid anhydrolase (OPAA) enzyme-carbon quantum dot (CQDs)-immobilized thin film biosensors for the specific detection of Ethyl Paraoxon and Methyl Parathion in water resources

Organophosphates pesticide (OP) toxicity through water resources is a large concern globally among all the emerging pollutants. Detection of OPs is a challenge which needs to be addressed considering the hazardous effects on the health of human beings. In the current research thin film biosensors of recombinant, Organophosphorus acid anhydrolase (OPAA) enzyme along with carbon quantum dots (CQDs) immobilized in thin films were developed. OPAA-CQDs thin film biosensors were used for the specific detection of two OPs Ethyl Paraoxon (EP) and Methyl Parathion (MP) in river water and household water supply. Recombinant OPAA enzyme was expressed in E. Coli, purified and immobilized on the CQD containing chitosan thin films. The CQDs used for this purpose were developed by a one-pot hydrothermal method from phthalic acid and Tri ethylene diamine. The properties of CQDs, OPAA and thin films were characterized using techniques like XPS, TEM, XRD, enzyme activity and CLSM measurements. Biosensing studies of EP and MP were performed by taking fluorescence measurements using a fiber optic spectrometer. The analytical parameters of biosensing were compared against an estimation carried out using the HPLC method. The biosensing performance indicates that the OPAA-CQDs thin film-based biosensors were able to detect both EP and MP in a range of 0–100 μM having a detection limit of 0.18 ppm/0.69 ppm for EP/MP, respectively with a response time of 5 min. The accuracy of estimation of EP/MP when spiked in water resources lie in the range of ∼100–102% which clearly indicates the OPAA-CQD based thin film biosensors can function as a point-of-use method for the detection of OP pesticides in complex water resources.

A Novel Organophosphorus Acid Anhydrolase from Deep Sea Sediment with High Degradation Efficiency for Organophosphorus Pesticides and Nerve Agent.

Organophosphorus compounds (OPCs), including highly toxic nerve agents and pesticides, have been used widely in agricultural and military applications. However, they have aroused widespread concern because they persistently pollute the environment and threaten human life. Organophosphorus acid anhydrolase (OPAA) is a promising enzyme that can detoxify OPCs. Here, a novel OPAA (OPAA114644) was isolated and characterized from deep-sea sediment (−3104 m). It exhibited excellent alkaline stability, and the loss of activity was less than 20% in the pH range 5.0–9.0, even after being incubated for 30 d at 4 °C. It also exhibited high salt tolerance, and its enzymatic activity increased by approximately fourfold in the presence of 20% NaCl (w/v). Additionally, OPAA114644 exhibited high degradation efficiency for soman, dichlorvos, paraoxon, coumaphos, and chlorpyrifos with a concentration of up to 250 mg/L, with the degradation rate being 100%, 100%, 100%, 80% and 51%, respectively, in 20 min under optimal conditions. Notably, OPAA114644 dissolved in different solutions, such as 20% NaCl, 1 mM SDS, 0.05% soap, 10% methanol, and tap water, could efficiently decontaminate the residual paraoxon on the surfaces of glasses, cotton tissues, and apples. These results indicate that OPAA114644 has excellent potential for the biodegradation and bioremediation of OPCs pollution and represents a real application of OPAA in the decontamination and detoxification of foods and clothes, and in the remediation of sites such as floors. Deep-sea sediment might also be an abundant resource for various functional microorganisms and enzymes.

Enzymatic Degradation of Organophosphorus Pesticides and Nerve Agents by EC: 3.1.8.2

The organophosphorus substances, including pesticides and nerve agents (NAs), represent highly toxic compounds. Standard decontamination procedures place a heavy burden on the environment. Given their continued utilization or existence, considerable efforts are being made to develop environmentally friendly methods of decontamination and medical countermeasures against their intoxication. Enzymes can offer both environmental and medical applications. One of the most promising enzymes cleaving organophosphorus compounds is the enzyme with enzyme commission number (EC): 3.1.8.2, called diisopropyl fluorophosphatase (DFPase) or organophosphorus acid anhydrolase from Loligo Vulgaris or Alteromonas sp. JD6.5, respectively. Structure, mechanisms of action and substrate profiles are described for both enzymes. Wild-type (WT) enzymes have a catalytic activity against organophosphorus compounds, including G-type nerve agents. Their stereochemical preference aims their activity towards less toxic enantiomers of the chiral phosphorus center found in most chemical warfare agents. Site-direct mutagenesis has systematically improved the active site of the enzyme. These efforts have resulted in the improvement of catalytic activity and have led to the identification of variants that are more effective at detoxifying both G-type and V-type nerve agents. Some of these variants have become part of commercially available decontamination mixtures.

A novel biosensor for the detection of organophosphorus (OP)-based pesticides using organophosphorus acid anhydrolase (OPAA)-FL variant.

Indiscriminate use of organophosphorus (OP)-based insecticides is a great concern to human health because of bioaccumulation-induced health hazards. Potentially fatal consequences and limited treatment methods of OP poisoning necessitate the need for the development of reliable, selective, cost-effective, and sensitive methods of OP detection. To tackle this issue, the development of effective devices and methods is required to sensitively detect as well as degrade OPs. Enzymatic sensor systems have gained popularity due to high catalytic activity, enhanced detection limits, and high sensitivity with the environmentally benign operation. Organophosphorus acid anhydrolase (OPAA) from Alteromonas sp. JD6.5 is capable of hydrolyzing the P-F, P-O, P-S, and P-CN bonds, in OPs, including nerve agents of the G/V-series. Several mutants of OPAA are reported which have greater activity against various OPs. In this study, recombinant expression of the OPAA-FL variant in Escherichia coli was performed, purified, and subsequently tested for activity against ethyl paraoxon. OPAA-FL variant showed its optimum activity at pH 8.5 and 50 °C. Colorimetric and fluorometric assays were used for estimation of ethyl paraoxon based on p-nitrophenol and fluorescein isothiocyanate (FITC) fluorescence intensity, respectively. Colorimetric and fluorometric assay estimation indicates that ethyl paraoxon can be estimated in the linear range of 0.01 to 1 mM and 0.1 to 0.5 mM, with LOD values 0.04 mM and 0.056 mM, respectively. Furthermore, the OPAA-FL variant was immobilized into alginate microspheres for colorimetric detection of ethyl paraoxon and displayed a linear range of 0.025 to 1 mM with a LOD value of 0.06 mM. KEY POINTS: • Biosensing of paraoxon with purified and encapsulated OPAA-FLvariant. • Colorimetric and fluorometric biosensing assay developed using OPAA-FLvariant for paraoxon. • First report on alginate encapsulation of OPAA-FLvariant for biosensing of paraoxon. Graphical abstract.

OPAA/fluoride biosensor chip towards field detection of G-type nerve agents

Sensor technology, particularly disposable, field-deployable devices offer considerable promise for addressing major security demands, in general, and in particular the urgent needs for selective and rapid field detection of highly toxic G-type organophosphate (OP) nerve agents. Herein, we report on the first example of a disposable electrochemical biosensor chip for decentralized field detection of diisopropyl fluorophosphate (DFP), as the simulant compound of G-type nerve agents (e.g., Sarin and Soman), based on the integration of an all-solid-state fluoride potentiometric transducer with the enzyme organophosphorus acid anhydrolase (OPAA) in connection to a hand-held potentiometric analyzer. Such solid-contact fluoride ion-selective electrodes were realized by comparing different fluoride ionophores to select the bis(fluorodioctylstannyl)methane molecule, along with systematic optimization of the experimental parameters. This first solid-contact fluoride detection chip displayed a favorable analytical performance, with near-Nernstian behavior at neutral pH in different buffers, high selectivity against various anions along with excellent reproducibility. Combining such desirable merits of the new fluoride sensor with the selective biocatalytic hydrolysis via an immobilized OPAA, to break the PF bond of G-type neurotoxins and release detectable fluoride ions, thereby enabled an attractive analytical performance. Both the fluoride and the nerve-agent recognition layers have thus been systematically optimized. The resulting biosensor chip thus displays a fast, sensitive and selective response towards DFP (including efficient discrimination against OP pesticides), along with a wide dynamic range of 250–3000 μM. Key challenges towards developing the nerve-agent detection chip were carefully addressed, including the pH incompatibility of the enzymatic and detection processes. By providing effective discrimination against common OP pesticides and minimizing related false alarms, the resultant disposable potentiometric biochip platform is expected to provide an instantaneous reliable warning of chemical attack and exposure towards a timely countermeasure action, and hence, would dramatically advance the protection of our military personnel.

Hydrolysis and enzymatic degradation of Novichok nerve agents

Several parameters of the Novichok nerve agents A230, A232 and A234 were determined. Hydrolysis rates were approximately one to three orders of magnitude slower than G-type nerve agents and approximately zero to two orders of magnitude slower than V-type nerve agents. A230 was the most labile Novichok compound followed by A232 then A234. Activation energies (Ea) and frequency factors (A) were determined for all three compounds. The organophosphorus acid anhydrolase (OPAA) enzyme had catalytic efficiencies on the Novichok compounds ranging between 104 and 105 M−1 min−1 with the highest kcat/Km value for A230, then A232 and lastly, A234.

Surface display of organophosphorus-degrading enzymes on the recombinant spore of Bacillus subtilis

Organophosphorus-degrading enzymes show high hydrolysis efficiency and provide an environmentally friendly solution to the pollution of organophosphorus compound. However, poor enzyme stability and tedious purification process have limited practical applications. Spore-based display system can provide many advantages, such as safety, low cost, easy preparation and high resistance to harsh conditions. Recently, we have constituted the recombinant spore displaying organophosphorus hydrolase and organophosphorus acid anhydrolase. In the spore display systems, recombinant spores could be reliably produced and normal sporulation was not affected; the activities of recombinant spores were 15.81 and 10.67 U/mg spores (dry weight) respectively; furthermore, the recombinant spores exhibited significantly enhanced resistance to various harsh conditions compared to free-form enzymes. These results indicated that the spore display could contribute to the practical application of organophosphorus-degrading enzymes and provide a promising solution to bioremediation of organophosphorus compounds.

Evaluation of the substitutions in 212, 342 and 215 amino acid positions in binding site of organophosphorus acid anhydrolase using the molecular docking and laboratory analysis.

OrganophosphorusAcid Anhydrolase (OPAA) is used as one of the most important enzymes in the decontamination process of organophosphate compounds. In this study, we aimed to evaluate the effects of amino acid substitution in OPAA's substrate-binding site on its catalytic activity using the rational engineering strategy. The native and three mutant forms of OPAA enzyme including 4ZWP, 4ZWU and Mut6 were studied using the docking technique toward parathion, paraoxon and R-VX compounds. Furthermore, enzyme assay was performed on the native OPAA and Mut6 toward parathion. Docking results showed a decreased catalytic activity of the mutant forms toward parathion and paraoxon. Furthermore, enzyme assay showed in accordance with docking results a decreased activity of Mut6 compared to the native form. The results of docking prediction for R-VX showed an increased catalytic activity of 4ZWP and 4ZWU. 4ZWU had the highest activity, while the activity of Mut6 was lower than the native form. Amino acid positions of 212 and 342 seem to be important sites in the small pocket of OPAA affecting the enzyme catalytic activity. Therefore, substitution of these sites with appropriate amino acids depending on the substrate structure, can affect the enzyme catalytic efficiency (Tab. 2, Fig. 3, Ref. 30).

English

Excessive use of pesticides has been known to be hazardous to the environment, affect soil fertility as well may impart toxicity in living beings. Presently there have been physical, chemical, biological and enzymatic approaches implicated to reduce pesticides. Although aimed to eradicate, physical and chemical methods are inefficient. Curiously, microbial pesticide remediation has been cost effective and thermodynamically more affordable, which may use any physical mater soiled with pesticide. Under favourable conditions microbes have been reported to use pesticides as source of carbon, sulphur and electron donor. Microbes; bacteria, actinomycetes and fungi have been found to help remove or detoxify chlorinated pesticides; polychlorinated diphenyl, polycyclic aromatic hydrocarbons, organophosphorus. Major bacterial genera includes; Bacillus, Pseudomonas, Flavobacterium, Moraxalla, Acinetobacter, Arthrobacter, Paracoccus, Aerobacter, Alkaligens, Burkholderia and Sphingomonas. Fungi with pesticide degradation potential includes;Fusarium, Aspergilus niger, Penicillium, Lentinulaedodes, Lecanicillium, Oxysporum. Among the Actinomycetes theStreptomycetes have been found to successfully detoxify pesticides. Persistent organic pollutants in the form of pesticides have also been reported to be taken care by the microbial enzymes viz-a-viz; dehydrogenase, ligninase, oxygenase, peroxidises, phosphotriesterase, hydrolases, dehalogenase, laccase and organophosphorus acid anhydrolase. Microbial strategies and tools; enzymes and genes involved in pesticide catabolism are reviewed. Key words: Chlorinated pesticides, organophosphors pesticides, bacteria, fungi, enzyme.

Nanosizing a Metal-Organic Framework Enzyme Carrier for Accelerating Nerve Agent Hydrolysis.

We report the synthesis and characterization of a water-stable zirconium metal-organic framework (MOF), NU-1003, featuring the largest mesoporous aperture known for a zirconium MOF. This material has been used to immobilize the nerve agent hydrolyzing enzyme, organophosphorus acid anhydrolase (OPAA). The catalytic efficiency of immobilized OPAA in nanosized NU-1003 is significantly increased compared to that of OPAA immobilized in microsized NU-1003 and even exceeds that of the free OPAA enzyme. This paper highlights a method for rapid and highly efficient hydrolysis of nerve agents using nanosized enzyme carriers.

Encapsulation of a Nerve Agent Detoxifying Enzyme by a Mesoporous Zirconium Metal–Organic Framework Engenders Thermal and Long-Term Stability

Immobilized enzymes typically have greater thermal and operational stability than their soluble form. Here we report that for the first time, a nerve agent detoxifying enzyme, organophosphorus acid anhydrolase (OPAA), has been successfully encapsulated into a water-stable zirconium metal-organic framework (MOF). This MOF features a hierarchical mesoporous channel structure and exhibits a 12 wt % loading capacity of OPAA. The thermal and long-term stabilities of OPAA are both significantly enhanced after immobilization.

Engineering the Organophosphorus Acid Anhydrolase Enzyme for Increased Catalytic Efficiency and Broadened Stereospecificity on Russian VX.

The enzyme organophosphorus acid anhydrolase (OPAA), from Alteromonas sp. JD6.5, has been shown to rapidly catalyze the hydrolysis of a number of toxic organophosphorus compounds, including several G-type chemical nerve agents. The enzyme was cloned into Escherichia coli and can be produced up to approximately 50% of cellular protein. There have been no previous reports of OPAA activity on VR {Russian VX, O-isobutyl S-[2-(diethylamino)ethyl] methylphosphonothioate}, and our studies reported here show that wild-type OPAA has poor catalytic efficacy toward VR. However, via application of a structurally aided protein engineering approach, significant improvements in catalytic efficiency were realized via optimization of the small pocket within the OPAA's substrate-binding site. This optimization involved alterations at only three amino acid sites resulting in a 30-fold increase in catalytic efficiency toward racemic VR, with a strong stereospecificity toward the P(+) enantiomer. X-ray structures of this mutant as well as one of its predecessors provide potential structural rationales for their effect on the OPAA active site. Additionally, a fourth mutation at a site near the small pocket was found to relax the stereospecificity of the OPAA enzyme. Thus, it allows the altered enzyme to effectively process both VR enantiomers and should be a useful genetic background in which to seek further improvements in OPAA VR activity.

Organophosphorus acid anhydrolase from Alteromonas macleodii: structural study and functional relationship to prolidases.

The bacterial enzyme organophosphorus acid anhydrolase (OPAA) is able to catalyze the hydrolysis of both proline dipeptides (Xaa-Pro) and several types of organophosphate (OP) compounds. The full three-dimensional structure of the manganese-dependent OPAA enzyme is presented for the first time. This enzyme, which was originally isolated from the marine bacterium Alteromonas macleodii, was prepared recombinantly in Escherichia coli. The crystal structure was determined at 1.8 Å resolution in space group C2, with unit-cell parameters a = 133.8, b = 49.2, c = 97.3 Å, β = 125.0°. The enzyme forms dimers and their existence in solution was confirmed by dynamic light scattering and size-exclusion chromatography. The enzyme shares the pita-bread fold of its C-terminal domain with related prolidases. The binuclear manganese centre is located in the active site within the pita-bread domain. Moreover, an Ni(2+) ion from purification was localized according to anomalous signal. This study presents the full structure of this enzyme with complete surroundings of the active site and provides a critical analysis of its relationship to prolidases.

Catalytic mechanisms for phosphotriesterases

Phosphotriesters are one class of highly toxic synthetic compounds known as organophosphates. Wide spread usage of organophosphates as insecticides as well as nerve agents has lead to numerous efforts to identify enzymes capable of detoxifying them. A wide array of enzymes has been found to have phosphotriesterase activity including phosphotriesterase (PTE), methyl parathion hydrolase (MPH), organophosphorus acid anhydrolase (OPAA), diisopropylfluorophosphatase (DFP), and paraoxonase 1 (PON1). These enzymes differ widely in protein sequence and three-dimensional structure, as well as in catalytic mechanism, but they also share several common features. All of the enzymes identified as phosphotriesterases are metal-dependent hydrolases that contain a hydrophobic active site with three discrete binding pockets to accommodate the substrate ester groups. Activation of the substrate phosphorus center is achieved by a direct interaction between the phosphoryl oxygen and a divalent metal in the active site. The mechanistic details of the hydrolytic reaction differ among the various enzymes with both direct attack of a hydroxide as well as covalent catalysis being found. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.

In vitro release of organophosphorus acid anhydrolase from functionalized mesoporous silica against nerve agents

We report here that under different physiological conditions, biomolecular drugs can be stockpiled in a nanoporous support and afterward can be instantly released when needed for acute responses, and the biomolecular drug molecules can also be gradually released from the nanoporous support over a long time for a complete recovery. Organophosphorus acid anhydrolase (OPAA) was spontaneously and largely entrapped in functionalized mesoporous silica (FMS) due to the dominant electrostatic interaction. The OPAA–FMS composite exhibited a burst release in a pH 9.0 NaHCO3–Na2CO3 buffer system and a gradual release in pH 7.4 simulated body fluid. The binding of OPAA to NH2–FMS can result in less tyrosinyl and tryptophanyl exposure OPAA molecules to aqueous environment. The bound OPAA in FMS displayed lower activity than the free OPAA in solution prior to the enzyme entrapment. However, the released enzyme maintained the native conformational structure and the same high enzymatic activity as that prior to the enzyme entrapment. The in vitro results in the rabbit serum demonstrate that both OPAA–FMS and the released OPAA may be used as a medical countermeasure against the organophosphorus nerve agents.

Organophosphorus acid anhydrolase bio-template for the synthesis of CdS quantum dots

A direct conjugation of organophosphorus acid anhydrolase (OPAA) with CdS quantum dots was prepared via arrested precipitation within the enzyme matrix. The bio-conjugate was found not only to retain enzyme conformational structure but also to retain enzyme activity and be effective at detecting diisopropyl fluorophosphate (DFP) at the micro molar level.

Hydrolysis of organophosphorus compounds by microbial enzymes

There are classes of microbial enzymes that have the ability to degrade harmful organophosphorus (OP) compounds that are present in some pesticides and nerve agents. To date, the most studied and potentially important OP-degrading enzymes are organophosphorus hydrolase (OPH) and organophosphorus acid anhydrolase (OPAA), which have both been characterized from a number of organisms. Here we provide an update of what is experimentally known about OPH and OPAA to include their structures, substrate specificity, and catalytic properties. Current and future potential applications of these enzymes in the hydrolysis of OP compounds are also addressed.

In search of a catalytic bioscavenger for the prophylaxis of nerve agent toxicity

A novel approach for treating organophosphorus (OP) poisoning is the use of enzymes, both stoichiometric and catalytic, as bioscavengers to sequester these compounds in circulation before they reach their physiological targets. Human serum butyrylcholinesterase and a recombinant form of this enzyme produced in the milk of transgenic goats have completed Phase I clinical trials as stoichiometric bioscavengers for the protection of humans against OP nerve agents. However, a major limitation of the first generation bioscavenger is the 1:1 stoichiometry between the enzyme and the OP. Therefore, efforts are underway to develop the second generation catalytic bioscavenger, which will neutralize/hydrolyze multiple OP molecules. To avoid any complications related to adverse immune reactions, three enzymes from human (Hu) sources are being considered for development as catalytic bioscavengers: (1) prolidase; (2) paraoxonase 1 (PON1); (3) senescence marker protein-30 (SMP-30). Towards this effort, native or recombinant (r) forms of candidate catalytic bioscavengers were isolated and characterized for their ability to hydrolyze G-type nerve agents at concentrations of 10 μM and 1 mM. Results show that mammalian enzymes were significantly less efficient at hydrolyzing nerve agents as compared to bacterial organophosphorus hydrolase (OPH) and organophosphorus acid anhydrolase (OPAA). Recombinant Hu prolidase was the most efficient and the only mammalian enzyme that hydrolyzed all four G-type nerve agents. On the other hand, both rHu PON1 and Mo SMP-30 showed 10-fold lower activity towards sarin compared to rHu prolidase and did not hydrolyze tabun. Based on these results, Hu prolidase appears to be the most promising candidate for further development: (1) it can be easily expressed in E. coli; (2) of the three candidate enzymes, it is the only enzyme that hydrolyzes all four G-type agents. Efforts to improve the catalytic efficiency of this enzyme towards OP nerve agents are underway.

Quantification of hydrolysis of toxic organophosphates and organophosphonates by diisopropyl fluorophosphatase from Loligo vulgaris by in situ Fourier transform infrared spectroscopy

The enzyme diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris effectively catalyzes the hydrolysis of diisopropyl fluorophosphate (DFP) and a number of organophosphorus nerve agents, including sarin, soman, cyclosarin, and tabun. Up to now, the determination of kinetic data has been achieved by techniques such as pH-stat titration, ion-selective electrodes, and fluorogenic substrate analogs. We report a new assaying method using in situ Fourier transform infrared (FTIR) spectroscopy with attenuated total reflection (ATR) for the real-time determination of reaction rates. The method employs changes in the P–O–R stretching vibration of DFP and nerve agent substrates when hydrolyzed to their corresponding phosphoric and phosphonic acids. It is shown that the Lambert–Beer law holds and that changes in absorbance can be directly related to changes in concentration. Compared with other methods, the use of in situ FTIR spectroscopy results in a substantially reduced reaction volume that adds extra work safety when handling highly toxic substrates. In addition, the new method allows the noninvasive measurement of buffered solutions with varying ionic strengths complementing existing methods. Because the assay is independent of the used enzyme, it should also be applicable to other phosphotriesterase enzymes such as organophosphorus hydrolase (OPH), organophosphorus acid anhydrolase (OPAA), and paraoxonase (PON).

Infrared Reflection−Absorption Spectroscopy and Polarization-Modulated Infrared Reflection−Absorption Spectroscopy Studies of the Organophosphorus Acid Anhydrolase Langmuir Monolayer

The secondary structure of the organophosphorus acid anhydrolase (OPAA) Langmuir monolayer in the absence and presence of diisopropylfluorophosphate (DFP) in the subphase was studied by infrared reflection-absorption spectroscopy (IRRAS) and polarization-modulated IRRAS (PM-IRRAS). The results of both the IRRAS and the PM-IRRAS indicated that the alpha-helix and the beta-sheet conformations in OPAA were parallel to the air-water interface at a surface pressure of 0 mN.m-1 in the absence of DFP in the subphase. When the surface pressure increased, the alpha-helix and the beta-sheet conformations became tilted. When DFP was added to the subphase at a concentration of 1.1 x 10(-5) M, the alpha-helix conformation of OPAA was still parallel to the air-water interface, whereas the beta-sheet conformation was perpendicular at 0 mN.m-1. The orientations of both the alpha-helix and the beta-sheet conformations did not change with the increase of surface pressure. The shape of OPAA molecules is supposed to be elliptic, and the long axis of OPAA was parallel to the air-water interface in the absence of DFP in the subphase, whereas the long axis became perpendicular in the presence of DFP. This result explains the decrease of the limiting molecular area of the OPAA Langmuir monolayer when DFP was dissolved in the subphase.