Hydrolysis Of Methyl Parathion Research Articles

Zwitterionic polymer-mediated immobilization of organophosphorus hydrolase enhances hydrolysis of methyl parathion by substrate enrichment

Organophosphorus pesticides (OPPs) degradation is highly demanded due to the toxic nature of the pesticides that have caused serious environmental concerns. Biocatalytic hydrolysis using organophosphorus hydrolase (OPH) is an important way to OPPs degradation, but the poor stability of OPH severely hindered its extensive applications in environmental bioremediation. It is well known that immobilization is an efficient way of enzyme stabilization, so this work has proposed to immobilize OPH onto mesoporous silica nanoparticles coated with a zwitterionic polymer containing short hydrophobic chains. It is found that the zwitterionic polymer coating significantly modified the microenvironment, leading to the stabilization of the immobilized enzyme while keeping its activity toward methyl parathion hydrolysis. More importantly, the coating resulted in the substrate enrichment onto the carrier (capacity, 49.7 mmol·g−1; dissociation constant, 11.7 μM), so the immobilized OPH presented a much lower Michaelis constant (0.09 mM) than its free counterpart (0.34 mM) as revealed by kinetic studies. Consequently, the catalytic efficiency (kcat/Km) of the immobilized OPH was 2.41 times higher than free OPH. Thus, this work demonstrated the promise of the zwitterionic polymer-mediated immobilization strategy for OPH in enhancing the enzyme performance for the hydrolysis of OPPs that are of low concentration in open environments.

Structure-based redesign of the bacterial prolidase active-site pocket for efficient enhancement of methyl-parathion hydrolysis

Mutagenesis at four residues surrounding the active-site pocket of an old bacterial prolidase scaffold led to a 10 000-fold increase in methyl-parathion hydrolysis and broadening substrate specificity against organophosphorus compounds.

Molecular binding scaffolds increase local substrate concentration enhancing the enzymatic hydrolysis of VX nerve agent.

Kinetic enhancement of organophosphate hydrolysis is a long-standing challenge in catalysis. For prophylactic treatment against organophosphate exposure, enzymatic hydrolysis needs to occur at high rates in the presence of low substrate concentrations and enzymatic activity should persist over days and weeks. Here, the conjugation of small DNA scaffolds was used to introduce substrate binding sites with micromolar affinity to VX, paraoxon, and methyl-parathion in close proximity to the enzyme phosphotriesterase (PTE). The result was a decrease in KM and increase in the rate at low substrate concentrations. An optimized system for paraoxon hydrolysis decreased KM by 11-fold, with a corresponding increase in second-order rate constant. The initial rates of VX and methyl-parathion hydrolysis were also increased by 3.1- and 6.7-fold, respectively. The designed scaffolds not only increased the local substrate concentration, but they also resulted in increased stability and PTE-DNA particle size tuning between 25 and ~150 nm. The scaffold engineering approach taken here is focused on altering the local chemical and physical microenvironment around the enzyme and is therefore compatible with active site engineering via combinatorial and computational approaches.

Transcriptional analysis reveals the metabolic state of Burkholderia zhejiangensis CEIB S4-3 during methyl parathion degradation.

Burkholderia zhejiangensis CEIB S4-3 has the ability to degrade methyl parathion (MP) and its main hydrolysis byproduct p-nitrophenol (PNP). According to genomic data, several genes related with metabolism of MP and PNP were identified in this strain. However, the metabolic state of the strain during the MP degradation has not been evaluated. In the present study, we analyzed gene expression changes during MP hydrolysis and PNP degradation through a transcriptomic approach. The transcriptional analysis revealed differential changes in the expression of genes involved in important cellular processes, such as energy production and conversion, transcription, amino acid transport and metabolism, translation, ribosomal structure and biogenesis, among others. Transcriptomic data also exhibited the overexpression of both PNP-catabolic gene clusters (pnpABA′E1E2FDC and pnpE1E2FDC) present in the strain. We found and validated by quantitative reverse transcription polymerase chain reaction the expression of the methyl parathion degrading gene, as well as the genes responsible for PNP degradation contained in two clusters. This proves the MP degradation pathway by the strain tested in this work. The exposure to PNP activates, in the first instance, the expression of the transcriptional regulators multiple antibiotic resistance regulator and Isocitrate Lyase Regulator (IclR), which are important in the regulation of genes from aromatic compound catabolism, as well as the expression of genes that encode transporters, permeases, efflux pumps, and porins related to the resistance to multidrugs and other xenobiotics. In the presence of the pesticide, 997 differentially expressed genes grouped in 104 metabolic pathways were observed. This report is the first to describe the transcriptomic analysis of a strain of B. zhejiangensis during the biodegradation of PNP.

Enhancing methyl parathion degradation by the immobilization of Burkholderia sp. isolated from agricultural soils.

Organophosphate pesticides are of great interest for research because they are currently the most commonly used pesticides. In this study, a bacterial strain capable of completely degrading methyl parathion (MP) was isolated from agricultural soils in central Mexico. This strain was designated strain S5‐2 and was identified as Burkholderia cenocepacia. To increase degradation yields, cells were immobilized on three different supports: powdered zeolite and Opuntia sp. and Agave sp. fibers. The results indicated a significant increase in MP hydrolysis and p‐nitrophenol (PNP) degradation with immobilized cells compared to free cell cultures. Furthermore, immobilized cells were capable of withstanding and degrading higher concentrations of PNP compared to cell suspension cultures. The cell viability in the free cell cultures, as well as PNP degradation, was affected at concentrations greater than 25 mg/L. In contrast, cells immobilized on Opuntia sp. and Agave sp. fibers completely degraded PNP at concentrations of 100 mg/L. To verify that MP solution toxicity was decreased by B. cenocepacia strain S5‐2 via pesticide degradation, we measured the acetylcholinesterase activity, both before and after treatment with bacteria. The results demonstrate that the activity of acetylcholinesterase was unaffected after MP degradation by bacteria.

Microbial biosensor for detection of methyl parathion using screen printed carbon electrode and cyclic voltammetry

Whole cells of recombinant Escherichia coli were immobilized on the screen printed carbon electrode (SPCE) using glutaraldehyde. Recombinant E. coli was having high periplasmic expression of organophosphorus hydrolase enzyme, which hydrolyzes the methyl parathion into two products, p-nitrophenol and dimethyl thiophosphoric acid. Cells immobilized SPCE was studied under SEM. Cells immobilized SPCE was associated with cyclic voltammetry and cyclic voltammograms were recorded before and after hydrolysis of methyl parathion. Detection was calibrated based on the relationship between the changes in the current observed at +0.1 V potential, because of redox behavior of the hydrolyzed product p-nitrophenol. As concentration of methyl parathion was increased the oxidation current also increased. Only 20 μl volume of the sample was required for analysis. Detection range of biosensor was calibrated between 2 and 80 μM of methyl parathion from the linear range of calibration plot. A single immobilized SPCE was reused for 32 reactions with retention of 80% of its initial enzyme activity.

A colorimetric assay for determination of methyl parathion using recombinant methyl parathion hydrolase

A simple, rapid and sensitive colorimetric dipstick assay for the detection of the organophosphorous insecticide methyl parathion (MPT) residue in vegetables was developed. The assay was based on the hydrolysis of MPT by a recombinant methyl parathion hydrolase (recMPH), the encoding gene of which was isolated from Burkholderia cepacia, a soil bacterium indigenous to Thailand. This reaction generates protons leading to a change in pH that correlates with the amount of MPH present. Hence, the pH indicator bromothymol blue was used to monitor the MPH hydrolysis as the associated color changes can be observed by the naked eye. The recMPH was immobilized on a PVDF membrane to establish a dipstick assay format. The assays could detect MPT residues in spiked vegetable samples at the concentration of 1 mg/L without using analytical instrumentation. The test is reusable and stable for up to 3 months in the absence of any preservatives.

Removal of Methyl Parathion from Artificial Off-Gas Using a Bioreactor Containing a Constructed Microbial Consortium

Methyl parathion (MP), a highly toxic organophosphorus pesticide, was widely used for agriculture crop protection. During the production of MP and the process of MP-containing wastewater treatment, MP can release into the atmosphere and will do great harm to adjacent communities. A consortium comprised of an engineered microorganism and a natural p-nitrophenol (PNP) degrader was assembled for complete mineralization of MP. We genetically engineered Escherichia coli BL21 (DE3) enabling the overexpression of methyl parathion hydrolase (MPH). In addition, we isolated Ochrobactrum sp. strain LL-1 that utilized PNP, a product of MP hydrolysis, as the sole carbon, nitrogen, and energy source. The coculture effectively hydrolyzed 0.2 mM MP and prevented the accumulation of PNP in suspended culture. A laboratory-scale bioreactor containing the dual-species consortium was developed for the treatment of artificial off-gas containing MP. The bioreactor maintained over 98% of average MP removal efficiency over a 75 day period, and PNP produced from hydrolysis of MP was degraded completely, indicating that complete mineralization of MP was achieved. The strategy of linking degrading consortium to a bioreactor may provide an alternative to physicochemical abatement technologies for the treatment of waste-gas streams containing MP as well as other PNP-substituted organophosphates.

Effect of Fe (III) on acid degradation of methylparathion

A study was undertaken to determine the transformation kinetic of methylparathion (O, O, -dimethyl O-4 nitrophenylphosphorotioate) in the presence of Fe(III) between pH 2 and 7. The Fe(III) was not electroactive under the conditions used in this study, and polarographic signals were exhibited by methylparathion and main degradation product only. Data suggest that hydrolysis of methylparathion in an acid medium is catalyzed by Fe(III) and the pesticide did not degrade in this medium without this cation. Methylparathion degradation was observed at all the pHs studied and was independent of the predominant chemical form of Fe(III) in the aqueous medium. The reaction was first-order with pH-dependent rate constant (k) values ranging from 3.3 × 10− 3 h− 1 to 7.0 × 10− 3 h− 1. The k values increased as pH decreased, suggesting that Fe(III) acted as an electrophile in the reaction mechanism.

Phospholipid‐Based Catalytic Nanocapsules

AbstractThe encapsulation and catalytic efficiency of organophosphate hydrolyzing enzymes in polymer‐stabilized nanocapsules is reported. Polymerized vesicles—derived from a headgroup‐polymerizable phospholipid, 1,2‐dipalmitoyl‐sn‐glycero‐3‐phospho‐N‐(2‐hydroxymethyl)‐3,5‐divinylbenzamide (DPPE‐DVBA)—containing enzymes were used as catalytic nanocapsules. Three enzymes, organophosphorus hydrolase (OPH), phosphotriesterase (PTE), and organophosphorus acid anhydrolase (OPAA), were encapsulated in vesicles by incubating them with freeze‐dried vesicles at 55 °C, followed by intermittent vortex‐mixing. Enzyme‐containing vesicles, collected after gel‐filtration, were stabilized by photopolymerization at 254 nm to yield crosslinked catalytic nanocapsules. The nanocapsules containing OPH and PTE showed specific activities of 0.36 and 1.74 μmol mg–1 min–1, respectively, against methyl parathion (MPT), and OPAA‐containing nanocapsules showed a specific activity of 57.1 μmol mg–1 min–1 against diisopropylfluorophosphate. Freeze‐dried, OPH‐ and PTE‐containing nanocapsules showed retentions of 83 % and 85 % specific activity, respectively, upon redispersion in buffer solution. Three‐week, room‐temperature storage of OPH‐containing nanocapsules showed a retention of 18 % enzyme activity. Hydrolysis of MPT in crosslinked DPPE‐DVBA/OPH vesicles showed that hydrophobic MPT permeated through the bilayer membrane of the freeze‐dried nanocapsules, releasing the hydrolysis product para‐nitrophenol, which permeated back to the exogenous dispersion medium leaving the enzymes free to react with freshly permeated MPT in the interior of the nanocapsules.

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.

Complete mineralization of methylparathion by Pseudomonas sp. A3.

Organophosphorus insecticides are widely used in agriculture. Despite their biodegradable nature, some are highly toxic and their residues are found in the environment. Reports on the mineralization of a spectrum of these insecticides by a single potential strain are scarce. We have isolated a soil isolate, Pseudomonas sp. A3, through enrichment technique, able to degrade methylparathion (MP), malathion, monocrotophos, and Diazinon. The potential of this strain to mineralize MP as a carbon and/or phosphorus source has been evaluated. On hydrolysis of MP, the aromatic portion (p-nitrophenol) was used as a carbon and energy source whereas the alkyl moiety (dithiomethylphosphorothioate) was broken down for the phosphorus source. The results from the experiments involving [U-14C]p-nitrophenol provided the evidence for incorporation of carbon into the cellular constituents and release of CO2 from this insecticide. During the breakdown of MP, nitrite was released as a catabolic by-product.

Hydrolysis of methyl parathion in a flooded soil.

Hydrolysis of methyl parathion in a flooded soil.

Hydrolysis of methylparathion in soils.

Hydrolysis of methylparathion in soils.