Facile purification and immobilization of organophosphorus hydrolase on protein-inorganic hybrid phosphate nanosheets

Oriented immobilization of enzymes helps to maintain their native structure and proper orientation for high-performance engineering to meet extensive biocatalysis demands.However,the supporting materials used for orientated immobilization are usually costly or complicated in preparation,affecting their practical applications.In this work,a facile purification and immobilization method was proposed for enzyme immobilization based on organic-inorganic hybrid calcium phosphate nanocrystal(Ca Ps)induced by Cu^(2+) modified bovine serum albumin(BSA-Cu).Then,the as-prepared hybrid calcium phosphate nanosheet,BSA-Cu@Ca Ps,was utilized for one-pot purification and immobilization of His-tagged organophosphorus hydrolase(OPH)by metal-affinity binding to the incorporated BSA.BSA-Cu@Ca PsOPH exhibited enhanced p H stability and thermal stability compared to the free enzyme.Moreover,BSA-Cu@Ca Ps-OPH could retain more than 75%and 56%of initial activity after reuse 5 and 10 times,respectively.The results demonstrated that this facile strategy was promising for the effective biodegradation of organophosphorus pesticides with the immobilized enzyme.

Recognition mechanism and sequence optimization of organophosphorus pesticides aptamers for better monitoring contaminations in food

Aptamers as a kind of biological recognition element have shown great potential in monitoring and the rapid quantification of organophosphorus pesticides(OPPs). However, molecules of OPPs are structurally similar and original aptamers selected by systematic evolution of ligands by exponential enrichment are usually long-chain bases, which hamper the further application under OPPs-aptamer recognition. The aim of the research was to develop a new strategy to design oligonucleotide sequences for binding OPPs by combination of experimental and molecular modeling methods. 3D models of aptamers binding OPPs were constructed, and binding energy and the most probable binding site for the OPPs were then determined by molecular docking, and the binding sites were further confirmed by the results of 2-AP replaced experiments. Based on the docking results, a new aptamer for detection 4 representative OPPs with only 29 bases was designed by reasonable truncation and mutation of the reported aptamer(named S4-29). The interaction between this new aptamer and OPPs were analyzed by molecular docking, microscale thermophoresis, circular dichroism and fluorometric analysis. The results revealed that the new aptamer exhibit more superior recognition performance to OPPs, which can be promote the monitoring ability of OPPs contaminations in food.

Biochemical indicators and the Peradeniya Organophosphate Poisoning scale in prediction and prognosis of organophosphorus poisoning:An observational prospective study

Objective:To study the value of some biochemical indicators and Peradeniya Organophosphate Poisoning scale in prediction and prognosis of organophosphorus poisoning.Methods:This was a hospital-based prospective,observational study.Various biochemical tests viz.complete blood count,random blood sugar,liver and renal function tests,creatine phosphokinase,and electrolytes were performed.Patients were assessed based on the Peradeniya Organophosphate Poisoning scale.All the patients were followed till the end point like recovery/death.Results:Out of the 100 patients,72%were males and 28%were females.The majority of the patients were farmers and 21 to 30 years of age.Suicidal was the most common manner(92,92%).Based on the Peradeniya Organophosphate Poisoning scale,47%were mild,34%moderate,and 19%severe.Serum creatinine,creatine phosphokinase,serum glutamic-oxaloacetic transaminase,serum glutamic pyruvic transaminase,and alkaline phosphatase levels showed a significant correlation with severity.Conclusions:Some biochemical indicators such as creatine phosphokinase,alkaline phosphatase can be used as prognostic markers of organophosphorus poisoning.The Peradeniya Organophosphate Poisoning scale can be used for assessing severity of the poisoning.

Role of penehyclidine in acute organophosphorus pesticide poisoning

BACKGROUND:Penehyclidine is a newly developed anticholinergic agent.We aimed to investigate the role of penehyclidine in acute organophosphorus pesticide poisoning(OP)patients.METHODS:We searched the Pubmed,Cochrane library,EMBASE,Chinese National Knowledge Infrastructure(CNKI),Chinese Biomedical literature(CBM)and Wanfang databases.Randomized controlled trials(RCTs)recruiting acute OP patients were identifi ed for meta-analysis.Main outcomes included cure rate,mortality rate,time to atropinization,time to 60%normal acetylcholinesterase(AchE)level,rate of intermediate syndrome(IMS)and rate of adverse drug reactions(ADR).RESULTS:Sixteen RCTs involving 1,334 patients were identifi ed.Compared with the atropineor penehyclidine-alone groups,atropine combined with penehyclidine significantly increased the cure rate(penehyclidine+atropine vs.atropine,0.97 vs.0.86,RR 1.13,95%CI[1.07–1.19];penehyclidine+atropine vs.penehyclidine,0.93 vs.0.80,RR 1.08,95%CI[1.01–1.15])and reduced the mortality rate(penehyclidine+atropine vs.atropine,0.015 vs.0.11,RR 0.17,95%CI[0.06–0.49];penehyclidine+atropine vs.penehyclidine,0.13 vs.0.08,RR 0.23,95%CI[0.04–1.28]).Atropine combined with penehyclidine in OP patients also helped reduce the time to atropinization and AchE recovery,the rate of IMS and the rate of ADR.Compared with a single dose of atropine,a single dose of penehyclidine also signifi cantly elevated the cure rate,reduced times to atropinization,AchE recovery,and rate of IMS.CONCLUSION:Atropine combined with penehyclidine benefi ts OP patients by enhancing the cure rate,mortality rate,time to atropinization,AchE recovery,IMS rate,total ADR and duration of hospitalization.Penehyclidine combined with atropine is likely a better initial therapy for OP patients than atropine alone.

Immobilization of organophosphorus hydrolase enzyme by covalent attachment on modified cellulose microfibers using different chemical activation strategies:Characterization and stability studies

The plant cellulose powder was activated by two different methods using 1,4-butanediol diglycidyl ether(BTDE)and 1,1′-Carbonyldiimidazole(CDI) as the chemical coupling agents.Organophosphorus hydrolase(OPH) from Flavobacterium ATCC 27551 was immobilized on any of activated support through covalent bonding.The optimal conditions of affecting parameters on enzyme immobilization in both methods were found, and it was demonstrated that the highest activity yields of immobilized OPH onto epoxy and CDI treated cellulose were 68.32%and 73.51%, respectively.The surface treatment of cellulose via covalent coupling with BTDE and CDI agents was proved by FTIR analysis.The kinetic constants of the free and immobilized enzymes were determined, and it was showed that both immobilization techniques moderately increased the Kmvalue of the free OPH.The improvements in storage and thermal stability were investigated and depicted that the half-life of immobilized OPH over the surface of epoxy modified cellulose had a better growth compared to the free and immobilized enzymes onto CDI treated support.Also, the pH stability of the immobilized preparations was enhanced relative to the free counterpart and revealed that all enzyme samples would have the same optimum pH value for stability at 9.0.Additionally, the immobilized OPH onto epoxy and CDI activated cellulose retained about 59% and 68% of their initial activity after ten turns of batch operation, respectively.The results demonstrated the high performance of OPH enzyme in immobilized state onto an inexpensive support with the potential of industrial applications.

Biochar-mediated regulation of greenhouse gas emission and toxicity reduction in bioremediation of organophosphorus pesticide-contaminated soils

Organophosphorus pesticides(OPPs) are a set of toxic persistent organic pollutants(POPs) present in the environment. Recently, biochar-mediated bioremediation has exhibited many advantages over conventional methods for the remediation of pesticide-contaminated soil. In the present study, biochar and nitrogen fertilizer(NH_4NO_3)were employed to remediate OPP-contaminated soil and the greenhouse gas(GHG) emission during 90 days of incubation was investigated. After thermal desorption treatment, the content of organophosphorus pesticides reduced from 175.61 μg·kg^(-1) to 62.68 μg·kg^(-1). The addition of NH_4NO_3 in the following bioremediation led to larger reduction(34.35%) of the pesticide concentration than that of biochar(31.90%) for the contaminated soils with thermal desorption treatment, while the simultaneous addition of biochar and NH_4NO_3 led to the largest reduction of pesticide concentration(11.07%) for the soil without thermal desorption treatment. The addition of biochar and NH_4NO_3 only slightly increased the emission rate of GHGs from the soil without thermal treatment,but remarkably increased the emission rate of GHGs from the soil after thermal treatment. In most cases, the addition of NH_4NO_3 is more effective than biochar to promote the degradation of pesticide, but also exhibited higher GHG emission. The microbial community analysis suggests that the enhanced degradation of pesticide is mainly owing to the increased activity of microorganism.

Computational interaction analysis of organophosphorus pesticides with different metabolic proteins in humans

Pesticides have the potential to leave harmful effects on humans, animals, other living organisms, and the environment. Several human metabolic proteins inhibited after exposure to organophosphorus pesticides absorbed through the skin, inhalation, eyes and oral mucosa, are most important targets for this interaction study. The crystal structure of five different proteins, PDBIDs： 3LII, 3NXU, 4GTU, 2XJ1 and 1YXA in Homo sapiens （H. sapiens）, interact with organophosphorus pesticides at the molecular level. The 3-D structures were found to be of good quality and validated through PROCHECK, ERRAT and ProSA servers. The results show that the binding energy is maximum -45.21 relative units of cytochrome P450 protein with phosmet pesticide. In terms of H-bonding, methyl parathion and parathion with acetylcholinesterase protein, parathion, methylparathion and phosmet with protein kinase C show the highest interaction. We conclude that these organophosphorus pesticides are more toxic and inhibit enzymatic activity by interrupting the metabolic pathways in H. sapiens.

Enzyme Inhibition Rate Method for Rapid Detection of Organophosphorus and Carbamate Pesticides in Cowpea

[Objectives ] The paper was to explore enzyme inhibition rate method for rapid detection of organophosphorus and carbamate pesticides in cowpea. [ Methods ] Acetylcholinesterase （ACHE） was added to cowpea extract, to determine the inhibition rate of extract against enzyme. The influences of different sampiing methods and sampling parts on detection results were compared. [ Results] The positive rate of standard sampling was 18.18% higher than that of non-stand- ard sampling, and the positive rate of samples collected from cowpea tail was 16.67% higher than that collected from other parts. [ Condmions] Enzyme inhibi- tion rate method is suitable for rapid detection of organophosphorus and carbamate pesticides in cowpea.

Determination of Eight Organophosphorus Pesticide Residues in Pepper by QuEChERS-Gas Chromatography

[ Objectives] This study was conducted to establish a QuEChERS-gas chromatography method for determination of eight kinds of organophosphorus pesticide residues in pepper. [Methods] The samples were extracted by acetonitrile, and anhydrous magnesium sulfate was used for salting out. The extracts were then purified by dispersive solid phase extraction combining with GCB, C18 and PSA. Samples were analyzed by FPD detector. External standard was used as quantitive method. [ Results] The detection limits were in the range of 0. 001 -0. 008 mg/kg, the average recoveries ranged from 83.5% to 101.2% , and the relative standard deviation （RSD） of the eight organophosphorus pesticide residues were below 5%. [ Conclusions] The method is simple, quick, easy and effective for the determination of pepper.

Carboxylic Esterase and Its Associations With Long-term Effects of Organophosphorus Pesticides

To examine a） the effect of organophosphorus pesticide exposure on activity of carboxylic esterases, namely butyrylcholinesterase （BChE）, carboxylesterase （CarbE） and paraoxonase （PonE）; and b） the association of polymorphisms of BChE and PonE with individual genetic susceptibility to organophosphorus pesticide exposure. Methods A cross-sectional study was conducted in 75 workers exposed to organophosphorus pesticides and 100 non-exposed controls. The serum activity of these enzymes was measured. Variant forms of BCHE-K, PON-192, and PON-55 were detected. A symptom score was developed as a proxy measure of clinical outcomes. Results Activities of both BChE and CarbE were lower in exposed workers （27.3±21.65 runol.hl.mL^-l and 235.6±104.03 nmol-min^-l.mL^-l） than in non-exposed workers （78.313±30.354 nmol.h^-l.mL^-1 and 362.681_＋194.997 nmol.min^-1.mL^-1）. The activity of PonE was not associated with exposure status. The AChE activity in the exposed workers with BCHE-K genotype UU （61 cases）, genotype UK （12 cases） and genotype KK （2 cases） was 105.05, 84.42 and 79.00 mmol-h^-1.mL^-1, respectively and the accumulative symptom scores were 3.74, 9.17, and 12.50 accordingly. The AChE activity in the exposed workers with PON-192 genotype BB （37）, genotype AB （27） and genotype AA （11） was 116.8, 91.2, and 72,3 mmol-h^-1.mL^-1, respectively and the symptom scores were 2.00, 6.74, and 9.73 accordingly. The AChE activity in those with PON-55 genotype LL （70） and genotype LM （5） was 102.4 and 82.8 mmol-h^-1.mL^-1 and the symptom scores were 4.53 and 9.20. The symptom score was the highest in individuals with abnormal homozygote for each of the three gene loci. Condusions Long-term exposure to organophosphorus pesticides can inhibit BChE and CarbE activity, but exerts no inhibitory effect on PonE activity. Different genotypes of BCHE-K, PON-192, and PON-55 may be related to the severity of adverse health effects of organophosphorus pesticide exposure. Implications of potentially higher susceptibility of workers with mutant homozygotes should be evaluated to reduce health risks.

Application of Current Hapten in the Production of Broad Specificity Antibodies Against Organophosphorus Pesticides

Diethylphosphono acetic acid （DPA） was used as a current hapten to generate broad specificity polycolonal antibodies against a group of organophosphorus pesticides. Six New Zealand white rabbits were immunized with immunogens synthesized by the active ester method （AEM） or 1-ethyl-3-（3-dimethylaminopropyl）-carbodimide method （EDC）. The titers of antisera reached 25 600 by AEM and 6 400 by EDC, respectively. Polyclonal antibodies raised against DPA were screened and selected for the competitive indirect enzyme-linked immunosorbent assay （CI-ELISA）. A CI-ELISA for DPA was developed with a detection limit of 3.536 ng mL^-1and an I50 value of 0.182 μg mL^-1. The assay specificity was evaluated by obtaining competitive curves for several structurally related compounds as competitors. The antiserum showed high affinities to chlorpyrifos, diazinon, omethoate, parathion-ethyl and profenofos with I50 of 0.12, 0.15, 0.21, 0.88, 0.97 and 2.5 μg mL^-1, respectively. The results indicate that the assay could be a screening tool for quantitation and semiquantitation determination of the above former five organophosphorus pesticides.

Complete genome sequence of Bacillus amyloliquefaciens YP6, a plant growth rhizobacterium efficiently degrading a wide range of organophosphorus pesticides

Bacillus amyloliquefaciens YP6,a plant growth promoting rhizobacteria,is capable of efficiently degrading a wide range of organophosphorus pesticides(OPs).Here,we report the complete genome sequence of this bacterium with a genome size of 4009619 bp,4210 protein-coding genes and an average GC content of 45.9%.Based on the genome sequence,several genes previously described as being involved in solubilizing-phosphorus,OPs-degradation,indole-3-acetic acid(IAA)and siderophores synthesis.Interestingly,compared with the genomes of B.amyloliquefaciens species,strain YP6 had larger genome size and the most protein-coding genes.Moreover,the four categories of“cell envelope biogenesis,outer membrane(M),”“translation,ribosomal structure and biogenesis(J),”“transcription(K),”and“signal transduction mechanisms(T)”were fewer.These differences may be related to extensive environmental adaptability of the genus B.amyloliquefaciens.These results expand the application potential of strain YP6 for environmental bioremediation,provide gene resources involved in OPs degradation for biotechnology and gene engineering,and contribute to provide insights into the relationship between microorganism and living environment.

Multi-Residue Analysis of Organophosphorus Pesticides in Vegetable Using GC-MS

This study investigates the levels of pesticide residues in Cucumbers (Cucumis sativus), potatoes (Solanum tuberosum subsp. Tuberosum) and tomatoes (Lycopersicon esculentum). The samples were minced in a food processor initially subjected to extraction followed by cleanup using solid phase extraction (SPE) column. Separation was performed on a GC capillary column (Rtx-5 ms-30 m × 0.25 mm× 0.25 μm). The sample was injected by using splitless mode, helium as the carrier gas followed by quadrupole mass spectrometry detection. The mobile phase flow rates, column temperatures, and MS parameters were all optimized to reach high sensitivity and selectivity. Seven pesticides were detected in tomatoes sample, six in cucumber and four in potatoes samples. The findings indicate risks and concerns for public health.

Study on the Quantitative Relationship between the Structure and Toxicity of Organophosphorus Pesticide

The molecular electronegativity distance vector（MEDV） was applied to characterize the molecular structures of 30 organophosphorous compounds. Optimum MEDV descriptors were selected by using the variable selection and modeling method based on the prediction（VSMP） technique. The quantitative structure-toxicity relationship（QSTR） model was built for acute toxicity（96h pLC50） of organophosphorous compounds to steelhead. The developed QSTR model with strictly internal and external validations presents relatively high correlation coefficient（R2） of 0.9518, leave-one-out（LOO） cross-validated correlation coefficient（Q2LOO） of 0.9355, and leave-many-out（LMO） cross-validated correlation coefficient（Q2LMO） of 0.9290. The robustness of the model was confirmed by the y-randomization test（R2yrand = 0.0772 and Q2 yrand = –0.5313） and bootstrapping（R2bstr = 0.9502 and Q2 bstr = 0.9177） method. The result of external validation, Q2F1 = 0.9336, Q2F2 = 0.9336, Q2F3 = 0.9447, r2 m = 0.8120, and CCC = 0.9602, shows that the QSTR model has a high predictive ability.

POLAR SUBSTITUENT EFFECTS OF ORGANOPHOSPHORUS ESTERS IN STRUCTURE-REACTIVITY STUDIES

In order to study the polar substituent effect almost with no steric effect in organophosphorus chemistry,a series of 4-substituted-2,6,7-trioxa-1-phosphabicyclo[2.2.2]octane 1-oxides(1)and p-substituted phenylphosphonates(2)and corresponding mono hexyl esters(3)as model were synthesized.^(31)P-NMR chemical shifts of compounds(1)in methanol and deuterioacetone were determined.The Δδ values reflect 4-sbstituent po- lar effect.The ^(31)P-NMR of compounds(2)and(3)and the pKa of compounds(3)were also measured.The results showed that the trend of polar effects of substituents in both carboxylic and organophosphates chemistry are similar,and the change of polar effects in variation with the alkyl groups is not significant.

Electrochemical Biosensors for Determination of Organophosphorus Compounds: Review

In last few decades there is exponential increase in use of organophosphorus (OP) compounds as pesticides and insecticides leading to adverse effect on human population and live stock. There is a great need to develop portable analytical tools that are amenable for remediation and bioremediation process monitoring, where rapid analysis of large number of samples is essential. Determination of various organophosphorus compounds has been achieved by integrating biocomponents with different transducers. The close integration of the biological events with the generation of a signal offers the potential for fabricating compact and easy-to-use analytical tools of high sensitivity and specificity. With the availability of new materials, associated with new sensing techniques has led to remarkable innovations in the design and construction of organophosphorus biosensors. The present review describes the specifications of most of the electrochemical Organophosphorus biosensors reported till date.

Rapid Screening of 26 Organophosphorus Pesticides in Fresh Milk by Ultra Liquid Chromatography Coupled With Quadrupole-Time of Flight Mass Spectrometry

[Objectives]A rapid screening and analysis method for 26 organophosphorus agrochemicals in fresh milk was established using ultra performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry.[Methods]Raw milk was extracted with acetonitrile solution containing 0.2%formic acid by volume,and purified with a Dikma ProElut QuECHERS solid phase extraction cartridge.Target compounds were separated on a Waters ACQUITY UPLC HSS T3 chromatographic column(2.1 mm×50 mm,1.8μm)with methanol-water solution as a mobile phase for gradient elution,and through scanning with an electrospray ion source in positive ion mode,26 kinds of organophosphorus agrochemicals could be accurately qualitatively determined within 10 min.[Results]When using formic acid acetonitrile with a volume fraction of 0.2%,there were more types of detected compounds and a greater recovery;and using B cartridge could effectively eliminate the interference of non-polar substances such as phospholipids,achieve higher number of detected compounds than those of A and C,and well separate the 26 kinds of agrochemical residues.[Conclusions]This study provides a reference method for the rapid screening of agrochemical residues in dairy cows in the future.

Study of multiresidue analytical method for organonitrogen and organophosphorus pesticides in soil and water

A gas chromatographic method without derivatization was developed for the residue analysis of 10 organonitrogen and 9 organophosphorus pesticides in soil and water. The samples were blended or shaken with acetone for extraction. The extracts were cleaned up by coagulation, then, re-extracted with three 50 ml portions of dichloromethane. The final residue was detected by gas chromatography equipped with NPD. All of the 19 pesticides were completely separated at a constant temperature. The method described above was applicable to the simultaneous determination of 10 organonitrogen and 9 organophosphorus pesticides .in soil and water with the satisfactory recovery (from 82.42% to 103.57%), coefficient of variance (from 0.17% to 12.57%) and limit of detection (from 0.0006 ppm to 0.058 ppm).

The Influencing Factors of Disposable Acetylcholinesterase Biosensor for in Situ Detection of Organophosphorus Pesticide

An amperometric biosensor based on acetylcholinesterase (ACHE) is assembled by simple adsorption of the AChE on 7,7,8,8-tetracyanoquinodimethane (TCNQ) modified screen-printed electrodes.This biosensor is used to detect the inhibitory effect of organophosphorus pesticides on AChE activity.The detection of organophosphorus pesticides is done with acetylthiocboline chloride (ATCh) as substrate.In order to obtain the optimized response to substrate,the influencing factors of the biosensor are investigated,including temperature,pH,incubation time,substrate concentration and AChE concentration. The measurements were performed after inhibition by immersing the enzyme electrode into the parathion methyl solution. Under the optimized conditions,that is,500 U/ml AChE concentration,pH 7.2,10 min incubation time,2 mmol/L substrate concentration and temperature of 37℃,from 5×10^(-8) mol/L to 5×10^(-5) mol/L is close to linear (regression equation: y(%)=124.055+15.7991gx,R^2=0.99644),which corresponds to 8.7%～56.1% AChE inhibition.With the optimized conditions, the lowest detectable amount of parathion methyl is 13×10^(-9).