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.     

Overexpression of methyl parathion hydrolase and its application in detoxification of organophosphates

The coding region of mpd gene corresponding to mature methyl parathion hydrolase (MPH) was heterologously overexpressed in Escherichia coli BL21 (DE3) by using pET expression system. The lactose-induced expression yield of MPH is increased 2-fold compared with IPTG as inducer. Furthermore, it was found that specific activity of MPH increased 48% by reducing the induction temperature to 22°C. The addition of 25 mM lactose at 22°C, the MPH activity of fermentation broth had a specific activity of 1.4 × 10⁴ U/mg protein. Plasmid was no significant decrease in the modified medium. The optimal pH and temperature of MPH were 8.0 and 30°C, respectively. Over a period of 5 months, the dried cells showed no significant decrease in the activity of the detoxifying enzymes. The crude enzymes in 50 mM citrate-phosphate buffer (pH 8.0) were able to degrade about 98% of the organophosphate pesticides sprayed on cabbage. The detoxification efficiency was superior to that of the treatments of water, detergent, and a commercially available enzyme product. Additionally, the products of pesticide hydrolysis generated by treatment with the enzyme extract were determined to be virtually nontoxic.     

Inactivation and conformational changes in methyl parathion hydrolase in 2,2,2-trifluoroethanol solutions: Inactivation kinetics and molecular dynamics simulation

Many improvements have been made in the understanding of functional and structural characteristics of proteins in a denaturant-based microenvironment. This study reports the chemical denaturation of methyl parathion hydrolase (MPH, EC 3.1.8.1) using 2,2,2-trifluoroethanol (TFE). MPH is an important enzyme that catalyzes the hydrolysis of organophosphorus agents. However, the regulation of MPH activity and structural changes during unfolding are not well studied, particularly for TFE unfolding. We investigated MPH unfolding with TFE for the first time. In this study, changes in enzymatic activity and unfolding of MPH at different TFE concentrations were investigated by enzyme activity measurements, intrinsic fluorescence and by 1-anilino-8-naphthalenesulfonate (ANS) fluorescence emission spectral scans. The results showed TFE inactivated MPH in a dose-dependent manner. A Lineweaver–Burk plot analysis revealed that the type of inhibition was reversible noncompetitive inhibition. Intrinsic fluorescence and ANS-binding fluorescence showed that TFE induced obvious tertiary structural changes in MPH by exposing hydrophobic groups. Furthermore, we conducted a docking simulation between MPH and TFE. The computer simulation successfully showed the binding structure and we estimated stability by calculating the binding energy (lowest binding energy: -3.18 kcal/mol). The results demonstrate that MPH can be inactivated by TFE, and provide new insights into the mechanism of TFE-induced unfolding of MPH and inhibition of ligand binding.     

甲基对硫磷水解酶OPHC2的序列分析及结构预测

将氨基酸的多序列局部比对、基于结构的序列比对和氨基酸替换忍耐性分析等方法联合起来对甲基对硫磷水解酶OPHC2进行了序列分析,确定出4组保守性功能序列。根据其高级结构发现这类酶具有典型的α/β（TIM）折叠桶结构,由8个折叠结构构成活性中心区域,并且这4组保守序列都位于这8个折叠结构上。通过对这4组保守序列的进一步分析发现当一个序列中同时含有它们时,这个序列为甲基对硫磷水解酶的可能性大于60％。     

来源于Rhinocladiella mackenziei的玉米赤霉烯酮水解酶的表达纯化与酶学性质

【背景】玉米赤霉烯酮(Zearalenone,ZEN)是污染最广泛的霉菌毒素之一,对饲料行业和畜牧业造成了巨大的经济损失。目前研究最为广泛的玉米赤霉烯酮降解酶ZHD101因其热稳定性较差,无法满足工业应用上的要求。【目的】为实现玉米赤霉烯酮降解酶在工业上的应用,寻找酶学性质更突出的ZEN降解酶。【方法】基于对Gen Bank数据库的挖掘,发现一个来源于麦氏喙枝孢霉(Rhinocladiella mackenziei CBS 650.93)的Rmzhd基因,构建p ET-46-Rmzhd质粒。利用大肠杆菌表达体系和亲和层析、离子交换纯化体系对蛋白进行表达和纯化,通过高效液相凝胶色谱分析酶学性质。【结果】发现一个新的ZEN水解酶Rm ZHD,RmZHD在pH 8.6和45°C条件下的活性最高,而且具有较高的耐热性。结构分析表明,较高的盐桥数目和溶剂暴露脯氨酸含量可能是造成其高耐热性的原因。【结论】本研究为促进玉米赤霉烯酮降解酶在工业上的应用打下基础。     

Crystal structure of methyl parathion hydrolase from Pseudomonas sp. WBC-3

Methyl parathion hydrolase (MPH, E.C.3.1.8.1), isolated from the soil-dwelling bacterium Pseudomonas sp. WBC-3, is a Zn(II)-containing enzyme that catalyzes the degradation of the organophosphate pesticide methyl parathion. We have determined the structure of MPH from Pseudomonas sp. WBC-3 to 2.4 angstroms resolution. The enzyme is dimeric and each subunit contains a mixed hybrid binuclear zinc center, in which one of the zinc ions is replaced by cadmium. In both subunits, the more solvent-exposed beta-metal ion is substituted for Cd2+ due to high cadmium concentration in the crystallization condition. Both ions are surrounded by ligands in an octahedral arrangement. The ions are separated by 3.5 angstroms and are coordinated by the amino acid residues His147, His149, Asp151, His152, His234 and His302 and a water molecule. Asp255 and a water molecule serve to bridge the zinc ions together. MPH is homologous with other metallo-beta-lactamases but does not show any similarity to phosphotriesterase that can also catalyze the degradation of methyl parathion with lower rate, despite the lack of sequence homology. Trp179, Phe196 and Phe119 form an aromatic cluster at the entrance of the catalytic center. Replacement of these three amino acids by alanine resulted in a significant increase of K(m) and loss of catalytic activity, indicating that the aromatic cluster has an important role to facilitate affinity of enzyme to the methyl parathion substrates.     

Pharmacokinetics of methyl parathion: a comparison following single intravenous,oral or dermal administration

Assessment of the risks posed by the residential use of methyl parathion requires an understanding of its pharmacokinetics after different routes of exposure. Thus, studies were performed using adult female rats to define the pharmacokinetic parameters for methyl parathion after intravenous injection and to apply the described model to an examination of its pharmacokinetics after single oral or dermal exposure. The pharmacokinetics of methyl parathion after intravenous administration (1.5 mg/kg) were best described by a three-compartment model; the apparent volume of the central compartment was 1.45 liters/kg, clearance was 1.85 liters/h/kg and the terminal half-life was 6.6 h with an elimination constant of 0.50 h(-1). The apparent oral absorption coefficient for methyl parathion (1.5 mg/kg) was 1.24 h(-1), and its oral bioavailability was approximately 20%. The latter likely includes a significant first pass effect. Concentrations of methyl parathion increased during the initial 10-60 min and then declined during the next 15-36 h. After dermal administration (6.25-25 mg/kg), methyl parathion concentrations peaked within 12-26 h and then declined dose dependently. The apparent dermal absorption coefficient was approximately 0.41 h(-1), and only two pharmacokinetic compartments could be distinguished. In conclusion, the pharmacokinetics of methyl parathion are complex and route dependent. Also, dermal exposure, because of sustained methyl parathion concentrations, may pose the greatest risk.     

Parameters controlling the gene-targeting frequency at the Sphingomonas species rrn site and expression of the methyl parathion hydrolase gene

AIMS: To investigate the key parameters controlling the exogenous methyl parathion hydrolase (MPH) gene mpd-targeting frequency at the ribosomal RNA operon (rrn) site of Sphingomonas species which has a wide range of biotechnological applications. METHODS AND RESULTS: Targeting vectors with different homology lengths and recipient target DNA with different homology identities were used to investigate the parameters controlling the targeting frequency at the Sphingomonas species rrn site. Targeting frequency decreased with the reduction of homology length, and the minimal size for normal homologous recombination was >100 bp. Homologous recombination could succeed even if there were 3-4% mismatches; however, targeting frequency decreased with increasing sequence divergence. The Red recombination system could increase the targeting frequency to some extent. Targeting of the mpd gene to the rrn site did not affect cell viability and resulted in an increase of MPH-specific activity in recombinants. CONCLUSIONS: Targeting frequency was affected by homology length, identity and the Red recombination system. The rrn site is a good target site for the expression of exogenous genes. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is useful as a foundation for a better understanding of recombination events involving homologous sequences and for the improved manipulation of Sphingomonas genes in biotechnological applications.     

Construction and application of a promoter-trapping vector with methyl parathion hydrolase gene mpd as the reporter

A facilitative and efficient promoter-trapping vector, pUC-mpd, was constructed with the promoterless methyl parathion hydrolase gene as the reporter. This reporter gene is easily used to clone promoters with different promoting strength on selective plates. Promoter regions of the ytkA and ywoF genes with strong promoting and signal peptide functions were cloned from the Bacillus subtilis 168 genomic promoter library with this vector.     

Characterization and comparison of putative Stenotrophomonas maltophilia methyl parathion hydrolases

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

Reductive transformation of parathion and methyl parathion by Bacillus sp.

Based on the results of phenotypic features, phylogenetic similarity of 16S rRNA gene sequences and BIOLOG test, a soil bacterium was identified as Bacillus sp. DM-1. Using either growing cells or a cell-free extract, it transformed parathion and methyl parathion to amino derivatives by reducing the nitro group. Pesticide transformation by a cell-free extract was specifically inhibited by three nitroreductase inhibitors, indicating the presence of nitroreductase activity. The nitroreductase activity was NAD(P)H-dependent, O₂-insensitive, and exhibited the substrate specificity for parathion and methyl parathion. Reductive transformation significantly decreased the toxicity of pesticides.     

A comparison of cholinesterase activity after intravenous, oral or dermal administration of methyl parathion

Time-dependent changes in blood cholinesterase activity caused by single intravenous, oral or dermal administration of methyl parathion to adult female rats were defined. Intravenous and oral administration of 2.5 mg/kg methyl parathion resulted in rapid (<60 min) decreases in cholinesterase activity which recovered fully in vivo within 30-48 h. In contrast, spontaneous reactivation of cholinesterase in vitro was complete within 6 h at 37 degrees C. Dermal administration of methyl parathion caused dose-dependent inhibition of cholinesterase activity which developed slowly (> or =6 h) and was prolonged (> or =48 h). Time- and route-dependent effects of methyl parathion on cholinesterase activity in brain and other tissues generally paralleled its effects on activity in blood. In conclusion, pharmacodynamics of methyl parathion differ substantially with route of exposure. Recovery of cholinesterase in vivo after intravenous or oral exposure may partially reflect spontaneous reactivation and suggests a rapid clearance of methyl parathion or its active metabolite methyl paraoxon. The more gradual and prolonged inhibition of cholinesterase caused by dermal administration is consistent with disposition of methyl parathion at a site from which it or methyl paraoxon is only slowly distributed. Thus, dermal exposure to methyl parathion may pose the greatest risk for long-term adverse effects.     

来源于嗜热氢化杆菌的新型对苯二甲酸双(羟乙)酯水解酶的表达纯化与酶学性质

石化来源的聚对苯二甲酸乙二酯(polyethylene terephthalate,PET)被广泛用于矿泉水瓶、食品包装和纺织品等领域,因其在自然界中不易分解,大量使用后的PET废弃物造成了严重的环境污染与资源浪费。使用生物酶法对PET废弃物进行解聚,并对解聚产物进行升级循环利用是进行塑料污染治理的重要方向之一,其中关键的是PET水解酶的解聚效率。对苯二甲酸双(羟乙基)酯(bis(hydroxyethyl)terephthalate,BHET)是PET生物酶解的中间产物,其累积是限制PET水解酶催化效率的一个重要因素,BHET水解酶和PET水解酶的联用能提升PET的整体水解效率。来源于嗜热氢化杆菌(Hydrogenobacter thermophilus)的双烯内酯酶(HtBHETase)对BHET有显著水解效果,将该酶在大肠杆菌(Escherichia coli)中进行重组表达并纯化后,对其酶学性质进行了研究。结果显示,HtBHETase对短碳链的酯类如对硝基苯酚乙酸酯催化活性较高,HtBHETase以BHET为底物时的最适反应pH值和最适反应温度分别为5.0和55℃;该酶有较好的热稳定性,经80℃的条件处理1 h仍能保持80%以上活性,显示出了良好的热稳定性,HtBHETase有在PET塑料生物解聚中使用的潜力,本研究为推动生物酶法降解PET提供了新的参考。     

Simultaneous biodegradation of methyl parathion and carbofuran by a genetically engineered microorganism constructed by mini-Tn5 transposon

A genetically engineered microorganism (GEM) capable of simultaneous degrading methyl parathion (MP) and carbofuran was successfully constructed by random insertion of a methyl parathion hydrolase gene (mpd) into the chromosome of a carbofuran degrading Sphingomonas sp. CDS-1 with the mini-transposon system. The GEM constructed was relatively stable and cell viability and original degrading characteristic was not affected compared with the original recipient CDS-1. The effects of temperature, initial pH value, inoculum size and alternative carbon source on the biodegradation of MP and carbofuran were investigated. GEM cells could degrade MP and carbofuran efficiently in a relatively broad range of temperatures from 20 to 30°C, initial pH values from 6.0 to 9.0, and with all initial inoculation cell densities (10⁵–10⁷ CFU ml⁻¹), even if alternative glucose existed. The optimal temperature and initial pH value for GEM cells to simultaneously degrade MP and carbofuran was at 30°C and at pH 7.0. The removal of MP and carbofuran by GEM cells in sterile and non-sterile soil were also studied. In both soil samples, 50 mg kg⁻¹ MP and 25 mg kg⁻¹ carbofuran could be degraded to an undetectable level within 25 days even if there were indigenous microbial competition and carbon sources effect. In sterile soil, the biodegradation rates of MP and carbofuran were faster, and the decline of the inoculated GEM cells was slower compared with that in non-sterile soil. The GEM constructed in this study was potential useful for pesticides bioremediation in natural environment.     

Affinity purification and characterization of a yeast epoxide hydrolase

Purification of the membrane-associated epoxide hydrolase from the yeast Rhodosporidium toruloides CBS 0349 to electrophoretic homogeneity was achieved in a single chromatographic step employing the affinity ligand adsorbent Mimetic Green. More than 68% of the total epoxide hydrolase activity present in the whole cells was recovered from the membrane fraction. The enzyme was purified 26-fold with respect to the solubilized membrane proteins and was obtained in a 90% yield. The purified epoxide hydrolase has an apparent monomeric molecular weight of 54 kDa, and a pI of 7.3. The enzyme was optimally active at 30–40 °C, and pH 7.3–8.5. The enzyme is highly glycosylated with a carbohydrate content >42%. The specific activity of the purified enzyme for (±)-1,2-epoxyoctane is 172 mol min^–1 mg protein^–1. The amino acid composition of the protein was determined. This is the first report of a yeast epoxide hydrolase purified to homogeneity in milligram amounts.     

菌株DLL-1降解土壤和韭菜中甲基对硫磷的研究

施甲基对硫磷7．5、15和22．5kg·hm^-2(a．i．)时,韭菜中最终平均农药残留量为0．633、1．270和1．901mg·kg^-1,自然降解率分别为98．94％、96．44％和96．04％．施用高效农药残留降解菌剂能显著地降低农药残留的含量,施用75kg·hm^-2降解菌剂时,韭菜与土壤中平均农药残留量分别为0．269、0．099mg·kg^-1,与不施菌对照相比,能使农药进一步降低78．82％和98．68％．降解率随着菌剂用量增加而升高,当用量超过75kg·hm^-2时降解率不再提高．菌剂施用时间以施药后3d为最好．     

Biodegradation of methyl parathion in the presence of goethite: The effect of Pseudomonas sp. Z1 adhesion

In this research, the influence of goethite on biodegradation kinetic of methyl parathion was investigated in the presence of Pseudomonas sp. Z1. Semipermeable membrane experiments were performed to demonstrate the role of adhesion of degrading bacteria to surface of goethite in biodegradation of methyl parathion. Sorption of methyl parathion and bacteria onto goethite particles were also measured to assess the distribution of methyl parathion and bacteria between water and goethite surface. The first-order degradation rate constant of methyl parathion in different concentrations of goethite was in the order of 0.1 g L⁻¹ > 0.01 g L⁻¹ > 0 g L⁻¹ > 1 g L⁻¹ > 20 g L⁻¹, suggesting the presence of low concentrations of goethite accelerated the biodegradation of methyl parathion and high concentrations of goethite inhibited this biodegradation process. According to the result of semipermeable membrane experiment, when no bacterial attachment occurred in the system, the promotive effect of 0.1 g L⁻¹ goethite for microbial degradation was disappeared and the inhibition effect of 20 g L⁻¹ goethite increased. The results clearly demonstrated that the adhesion of bacteria to goethite was beneficial to the biodegradation of methyl parathion. The information obtained is of fundamental significance for the understanding of microbial degradation of organic pollution in soil.     

菌株DLL-1降解土壤和韭菜中甲基对硫磷的研究

施甲基对硫磷7.5、15和22.5kg·hm^-2(a.i.)时,韭菜中最终平均农药残留量为0.633、1.270和1.901mg·kg^-1,自然降解率分别为98.94%、96.44%和96.04%.施用高效农药残留降解菌剂能显著地降低农药残留的含量,施用75kg·hm^-2降解菌剂时,韭菜与土壤中平均农药残留量分别为0.269、0.099mg·kg^-1,与不施菌对照相比,能使农药进一步降低78.82%和98.68%.降解率随着菌剂用量增加而升高,当用量超过75kg·hm^-2时降解率不再提高.菌剂施用时间以施药后3d为最好.     

甲基对硫磷水解酶的重组表达及其纯化和性质研究

用PCR方法获得甲基对硫磷水解酶编码基因,构建了重组表达质粒pET29a_mpd,将其转化至Escherichia coli BL21(DE3)中,经IPTG诱导表达,得到C末端含有6个寡聚组氨酸的甲基对硫磷水解酶,用NiNTA亲和层析纯化得到具有活性的甲基对硫磷水解酶。测定了环境因素对酶活性的影响及酶动力学参数。甲基对硫磷水解酶水解甲基对硫磷时,最适pH86～8.8,最佳反应温度15℃;Mn2+、Zn2+、Cu2+可使酶活性增加15%～20%,Ca2+、Mg2+微弱地促进酶的作用,Ni2+对酶活性几乎无影响;1mmol/L EDTA·Na2+几乎不影响酶的活性,而10mmol/L EDTA·Na2+对甲基对硫磷水解酶有较强的抑制作用。甲基对硫磷水解酶水解乙基对硫磷时,最适pH86。25℃时,该酶对甲基对硫磷的米氏常数Km为（68.6 ± 5.1）μmol/L,kcat为（45 ± 6 ）S-1;对乙基对硫磷的米氏常数Km为（59.5 ± 6.0）μmol/L,kcat为（8 ± 1） S-1。Kcat/Km表明甲基对硫磷水解酶对甲基对硫磷的催化效率更高。