Molecular binding of different classes of organophosphates to methyl parathion hydrolase from Ochrobactrum species.

Methyl parathion hydrolase (MPH) is an enzyme of the metallo-ß-lactamase superfamily, which hydrolyses a wide range of organophosphates (OPs). Recently, MPH has attracted attention as a promising enzymatic bioremediator. The crystal structure of MPH enzyme shows a dimeric form, with each subunit containing a binuclear metal ion center. MPH also demonstrates metal ion-dependent selectivity patterns. The origins of these patterns remain unclear but are linked to open questions about the more general role of metal ions in functional evolution and divergence within enzyme superfamilies. We aimed to investigate and compare the binding of different OP pesticides to MPH with cobalt(II) metal ions. In this study, MPH was modeled from Ochrobactrum sp. with different OP pesticides bound, including methyl paraoxon and dichlorvos and profenofos. The docked structures for each substrate optimized by DFT calculation were selected and subjected to atomistic molecular dynamics simulations for 500 ns. It was found that alpha metal ions did not coordinate with all the pesticides. Rather, the pesticides coordinated with less buried beta metal ions. It was also observed that the coordination of beta metal ions was perturbed to accommodate the pesticides. The binding free energy calculations and structure-based pharmacophore model revealed that all the three substrates could bind well at the active site. However, profenofos exhibit a stronger binding affinity to MPH in comparison to the other two substrates. Therefore, our findings provide molecular insight on the binding of different OP pesticides which could help us design the enzyme for OP pesticides degradation.

Higher-order epistasis shapes the fitness landscape of a xenobiotic-degrading enzyme.

Characterizing the adaptive landscapes that encompass the emergence of novel enzyme functions can provide molecular insights into both enzymatic and evolutionary mechanisms. Here, we combine ancestral protein reconstruction with biochemical, structural and mutational analyses to characterize the functional evolution of methyl-parathion hydrolase (MPH), an organophosphate-degrading enzyme. We identify five mutations that are necessary and sufficient for the evolution of MPH from an ancestral dihydrocoumarin hydrolase. In-depth analyses of the adaptive landscapes encompassing this evolutionary transition revealed that the mutations form a complex interaction network, defined in part by higher-order epistasis, that constrained the adaptive pathways available. By also characterizing the adaptive landscapes in terms of their functional activities towards three additional organophosphate substrates, we reveal that subtle differences in the polarity of the substrate substituents drastically alter the network of epistatic interactions. Our work suggests that the mutations function collectively to enable substrate recognition via subtle structural repositioning.

Dual-opposite multi-walled carbon nanotube modified carbon fiber microelectrode for microfluidic chip-capillary electrophoresis determination of methyl parathion metabolites in human urine.

Methyl parathion (MP) is a highly toxic organophosphate and its exposure may lead to substantial adverse effects to human health. The existence of 4-nitrophenol (4-NP) in the form of free phenol, glucuronide (4-NP-G) or as a sulfate ester (4-NP-S) can be used as biomarkers to assess the duration and extent of MP exposure. In this work, a MC-CE device incorporating post-CE amperometric detection using multi-walled carbon nanotubes (MWNTs) modified carbon fiber microelectrode (CFME) was fabricated and assessed for simultaneous determination of 4-NP, 4-NP-G, and 4-NP-S in human urine. The detection sensitivity and stability was greatly enhanced by the modification of MWNTs. The capability of the MC-CE device with dual MWNTs modified CFME for detecting impurity was assessed and reliability established by high recoveries from 95 to 97% for spiked MP biomarkers. The method developed is shown to provide a simple, sensitive, and reliable means for monitoring 4-NP, 4-NP-G, and 4-NP-S in human urine.

Characterization of methyl parathion degradation by a Burkholderia zhejiangensis strain, CEIB S4-3, isolated from agricultural soils.

Through the use of an enrichment technique, we isolated from the agricultural soils of Morelos in central México a strain of Burkholderia zhejiangensis identified as CEIB S4-3, it's could use the pesticide methyl parathion (MP) as the only source of carbon and degrade completely p-nitrophenol (PNP). For more efficient MP and PNP degradation by the CEIB S4-3 strain, the absence of an extra carbon source, a large inoculum and an MP concentration up to 50 mg/l are required. Sequence and annotation analysis of the draft genome, showed presence of mpd functional gene, which was expressed and its activity on the MP was confirmed. Additionally, the genes coding for enzymes in the benzoquinone pathway (conducted by Gram-negative bacteria) and the benzenotriol pathway (conducted by Gram-positive bacteria) were found, which was corroborated by identification of intermediary metabolites by HPLC. Thus, we propose that B. zhejiangensis CEIB S4-3 uses both degradation pathways.

Genetically engineered Pseudomonas putida X3 strain and its potential ability to bioremediate soil microcosms contaminated with methyl parathion and cadmium.

A multifunctional Pseudomonas putida X3 strain was successfully engineered by introducing methyl parathion (MP)-degrading gene and enhanced green fluorescent protein (EGFP) gene in P. putida X4 (CCTCC: 209319). In liquid cultures, the engineered X3 strain utilized MP as sole carbon source for growth and degraded 100 mg L(-1) of MP within 24 h; however, this strain did not further metabolize p-nitrophenol (PNP), an intermediate metabolite of MP. No discrepancy in minimum inhibitory concentrations (MICs) to cadmium (Cd), copper (Cu), zinc (Zn), and cobalt (Co) was observed between the engineered X3 strain and its host strain. The inoculated X3 strain accelerated MP degradation in different polluted soil microcosms with 100 mg MP kg(-1) dry soil and/or 5 mg Cd kg(-1) dry soil; MP was completely eliminated within 40 h. However, the presence of Cd in the early stage of remediation slightly delayed MP degradation. The application of X3 strain in Cd-contaminated soil strongly affected the distribution of Cd fractions and immobilized Cd by reducing bioavailable Cd concentrations with lower soluble/exchangeable Cd and organic-bound Cd. The inoculated X3 strain also colonized and proliferated in various contaminated microcosms. Our results suggested that the engineered X3 strain is a potential bioremediation agent showing competitive advantage in complex contaminated environments.

Chemiluminescence reaction kinetics-resolved multianalyte immunoassay strategy using a bispecific monoclonal antibody as the unique recognition reagent.

The multianalyte immunoassay (MIA) has attracted increasing attention due to its high sample throughput, short assay time, low sample consumption, and reduced overall cost. However, up to now, the reported MIA methods commonly require multiple antibodies since each antibody can recognize only one antigen. Herein, a novel bispecific monoclonal antibody (BsMcAb) that could bind methyl parathion and imidacloprid simultaneously was produced by a hybrid hybridomas strategy. A chemiluminescence (CL) reaction kinetics-resolved strategy was designed for MIA of methyl parathion and imidacloprid using the BsMcAb as the unique recognition reagent. Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) were adopted as the signal probes to tag the haptens of the two pesticides due to their very different CL kinetic characteristics. After competitive immunoreactions, the HRP-tagged methyl parathion hapten and the ALP-tagged imidacloprid hapten were simultaneously bound to the BsMcAb since there were two different antigen-binding sites in it. Then, two CL reactions were simultaneously triggered by adding the CL coreactants, and the signals for methyl parathion and imidacloprid detections were collected at 0.6 and 1000 s, respectively. The linear ranges for methyl parathion and imidacloprid were both 1.0-500 ng/mL, with detection limits of 0.33 ng/mL (S/N = 3). The proposed method was successfully used to detect pesticides spiked in ginseng and American ginseng with acceptable recoveries of 80-118%. This proof-of-principle work demonstrated the feasibility of MIA using only one antibody.

Secretory expression of organophosphorus hydrolase OPHC2 in Yarrowia lipolytica Polg.

In the present study, recombinant organophosphorus hydrolase OPHC2 was successfully produced by Yarrowia lipolytica and purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses showed a major polypeptide band of 36 kDa. The purified enzyme was optimally active at 65°C and pH 8.5 and also displayed good thermal and pH stability using methyl parathion (O,O-dimethyl-O-4-p-nitrophenyl phosphorothioate) as a substrate. Moreover, as Y. lipolytica is a non-pathogenic, generally regarded as safe (GRAS) yeast, the cell culture supernatant can be used directly on vegetables and fruits that are contaminated by organophosphorus pesticides.

OxyR-dependent expression of a novel glutathione S-transferase (Abgst01) gene in Acinetobacter baumannii DS002 and its role in biotransformation of organophosphate insecticides.

While screening a genomic library of Acinetobacter baumannii DS002 isolated from organophosphate (OP)-polluted soils, nine ORFs were identified coding for glutathione S-transferase (GST)-like proteins. These GSTs (AbGST01-AbGST09) are phylogenetically related to a number of well-characterized GST classes found in taxonomically diverse groups of organisms. Interestingly, expression of Abgst01 (GenBank accession no. KF151191) was upregulated when the bacterium was grown in the presence of an OP insecticide, methyl parathion (MeP). The gene product, AbGST01, dealkylated MeP to desMeP. An OxyR-binding motif was identified directly upstream of Abgst01. An Abgst-lacZ gene fusion lacking the OxyR-binding site showed a drastic reduction in promoter activity. Very low ß-galactosidase activity levels were observed when the Abgst-lacZ fusion was mobilized into an oxyR (GenBank accession no. KF151190) null mutant of A. baumannii DS002, confirming the important role of OxyR. The OxyR-binding sites are not found upstream of other Abgst (Abgst02-Abgst09) genes. However, they contained consensus sequence motifs that can serve as possible target sites for certain well-characterized transcription factors. In support of this observation, the Abgst genes responded differentially to different oxidative stress inducers. The Abgst genes identified in A. baumannii DS002 are found to be conserved highly among all known genome sequences of A. baumannii strains. The versatile ecological adaptability of A. baumannii strains is apparent if sequence conservation is seen together with their involvement in detoxification processes.

Burkholderia jiangsuensis sp. nov., a methyl parathion degrading bacterium, isolated from methyl parathion contaminated soil.

A methyl parathion (MP) degrading bacterial strain, designated MP-1(T), was isolated from a waste land where pesticides were formerly manufactured in Jiangsu province, China. Polyphasic taxonomic studies showed that MP-1(T) is a Gram-stain-negative, non-spore-forming, rod-shaped and motile bacterium. The bacterium could grow at salinities of 0-1 % (w/v) and temperatures of 15-40 °C. Strain MP-1(T) could reduce nitrate to nitrite, utilize d-glucose and l-arabinose, but not produce indole, or hydrolyse gelatin. Phylogenetic analysis based on 16S rRNA gene sequences demonstrated that MP-1(T) belongs to the genus Burkholderia, showing highest sequence similarity to Burkholderia grimmiae DSM 25160(T) (98.5 %), and similar strains including Burkholderia zhejiangensis OP-1(T) (98.2 %), Burkholderia choica LMG 22940(T) (97.5 %), Burkholderia glathei DSM 50014(T) (97.4 %), Burkholderia terrestris LMG 22937(T) (97.2 %) and Burkholderia telluris LMG 22936(T) (97.0 %). In addition, the gyrB and recA gene segments of strain MP-1(T) exhibited less than 89.0 % and 95.1 % similarities with the most highly-related type strains indicated above. The G+C content of strain MP-1(T) was 62.6 mol%. The major isoprenoid quinone was ubiquinone Q-8. The predominant polar lipids comprised phosphatidyl ethanolamine, phosphatidyl glycerol, aminolipid and phospholipid. The principal fatty acids in strain MP-1(T) were C18 : 1ω7c/C18 : 1ω6c (23.3 %), C16 : 0 (16.8 %), cyclo-C17 : 0 (15.0 %), C16 : 1ω7c/C16 : 1ω6 (8.5 %), cyclo-C19 : 0ω8c (8.1 %), C16 : 1 iso I/C14 : 0 3-OH (5.7 %), C16 : 0 3-OH (5.6 %) and C16 : 02-OH (5.1 %). The DNA-DNA relatedness values between strain MP-1(T) and the three type strains (B. grimmiae DSM 25160(T), B. zhejiangensis OP-1(T) and B. glathei DSM 50014(T)) ranged from 24.6 % to 37.4 %. In accordance with phenotypic and genotypic characteristics, strain MP-1(T) represents a novel species of the genus Burkholderia, for which the name Burkholderia jiangsuensis sp. nov. is proposed, the type strain is MP-1(T) (LMG 27927(T) = MCCC 1K00250(T)).

Improving methyl parathion hydrolase to enhance its chlorpyrifos-hydrolysing efficiency.

UNLABELLED: Methyl parathion hydrolase (MPH) can degrade a wide range of organophosphorus compounds, but its efficiency in hydrolysing chlorpyrifos, one of the most popular pesticides applied for crop protection, is much lower than that in hydrolysing the preferred substrate methyl parathion. In this study, random mutagenesis was adopted to improve MPH to enhance its efficiency in hydrolysing the poorly hydrolysed substrate chlorpyrifos. Rapid screening of the improved MPH variants was carried out using Bacillus subtilis WB800 secretory expression system to investigate the distribution of improved MPH variants based on the size of clear haloes as a result of chlorpyrifos hydrolysis. Four improved MPH variants were isolated, and one variant K3, in particular, showed a 5-fold increase in kcat value for chlorpyrifos hydrolysis. Furthermore, most of the MPH variants obtained in this study possessed enhanced thermostability and pH stability. The approaches adopted in this study could be extended to create other MPH variants with increased activity for hydrolysing other poorly hydrolysed substrates. SIGNIFICANCE AND IMPACT OF THE STUDY: Chlorpyrifos is one of the toxic organophosphorus compounds (OP compounds) widely used for insecticides control. Water, soil and foodstuff have been contaminated seriously by chlorpyrifos in some areas. It is urgent to find effective methods to remove its contamination. This work contributes to improve methyl parathion hydrolase (MPH) to enhance its efficiency in hydrolysing the poorly hydrolysed substrate chlorpyrifos. Our study brings new insights for enzymatic strategy for the decontamination of toxic OP compounds.

Interfacial interaction between methyl parathion-degrading bacteria and minerals is important in biodegradation.

In the present study, the influence of kaolinite and goethite on microbial degradation of methyl parathion was investigated. We observed that the biodegradation process was improved by kaolinite and depressed by goethite. Calorimetric data further showed that the metabolic activities of degrading cells (Pseudomonas putida) were enhanced by the presence of kaolinite and depressed by the presence of goethite. A semipermeable membrane experiment was performed and results supported the above observations: the promotive effect of kaolinite and the inhibition of goethite for microbial degradation was not found when the bacteria was enclosed by semipermeable membrane and had no direct contact with these minerals, suggesting the important function of the contact of cellular surfaces with mineral particles. The relative larger particles of kaolinite were loosely attached to the bacteria. This attachment made the cells easy to use the sorbed substrate and then stimulated biodegradation. For goethite, small particles were tightly bound to bacterial cells and limited the acquisition of substrate and nutrients, thereby inhibiting biodegradation. These results indicated that interfacial interaction between bacterial cells and minerals significantly affected the biodegradation of pesticides.

Comparison of biosorption and phytoremediation of cadmium and methyl parathion, a case-study with live Lemna gibba and Lemna gibba powder.

Heavy metals and pesticides can be adsorbed by several biomasses such as living or non-living aquatic plants. In this study adsorption properties of live Lemna gibba and Lemna gibba powder were investigated with regard to cadmium and methyl parathion (MP). Toxicity data (IC50) on live L. gibba indicated that the period of four days was adequate for phytoremediation. Initial adsorption studies showed that both adsorbents were capable of removing cadmium and methyl parathion. Cadmium and methyl parathion adsorption onto L. gibba powder was fast and equilibrium was attained within 120min. The adsorption data could be well interpreted by the Freundlich model. The KF were: 7.8963 (Cd(2+)/ live Lemna); 0.7300 (MP/live Lemna); 11.5813 (Cd(2+)/Lemna powder); 1.1852 (MP/Lemna powder) indicating that Cd(2+) was more efficiently removed by both biosorbents than MP. Adsorption kinetics for cadmium and methyl parathion in both systems and rate constants were determined for each contaminant. It was found that the overall adsorption process was best described by pseudo-second-order kinetics. Boyd model and external mass-transfer expression were tested. It was concluded that cadmium and methyl parathion sorption onto Lemna powder is governed by film diffusion.

Improving the thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 using a computationally aided method.

Good protein thermostability is very important for the protein application. In this report, we propose a strategy which contained a prediction method to select residues related to protein thermal stability, but not related to protein function, and an experiment method to screen the mutants with enhanced thermostability. The prediction strategy was based on the calculated site evolutionary entropy and unfolding free energy difference between the mutant and wild-type (WT) methyl parathion hydrolase enzyme from Ochrobactrum sp. M231 [Ochr-methyl parathion hydrolase (MPH)]. As a result, seven amino acid sites within Ochr-MPH were selected and used to construct seven saturation mutagenesis libraries. The results of screening these libraries indicated that six sites could result in mutated enzymes exhibiting better thermal stability than the WT enzyme. A stepwise evolutionary approach was designed to combine these selected mutants and a mutant with four point mutations (S274Q/T183E/K197L/S192M) was selected. The Tm and T50 of the mutant enzyme were 11.7 and 10.2 °C higher, respectively, than that of the WT enzyme. The success of this design methodology for Ochr-MPH suggests that it was an efficient strategy for enhancing protein thermostability and suitable for protein engineering.

Mass spectrometric detection of CYP450 adducts following oxidative desulfuration of methyl parathion.

Cytochrome P450 (CYP)-mediated desulfuration of methyl parathion results in mechanism-based inhibition of the enzyme. Although previous data suggest that reactive sulfur is released and binds to the apoprotein, the identities of neither the adduct(s) nor the affected amino acid(s) have been clearly determined. In this study, nanospray tandem mass spectroscopy was used to analyze peptide digests of CYP resolved by SDS-PAGE from liver microsomes of male rats following incubation in the absence or presence of methyl parathion. Oxidative desulfuration was confirmed by measurement of methyl paraoxon, and inhibition of specific CYP isozymes was determined by measurement of testosterone hydroxylation. Total CYP content was quantified spectrophotometrically. Incubation of microsomes with methyl parathion decreased CYP content by 58%. This effect was not associated with a comparable increase in absorbance at 420 nm, suggesting the displacement of heme from the apoprotein. Rates of testosterone 2ß- and 6ß-hydroxylation, respectively, were reduced to 8 and 2%, implicating CYP3A and CYP2C11 in the oxidative desulfuration of methyl parathion. Mass spectrometric analysis identified 96 amu adducts to cysteines 64 and 378 of CYP3A1. In addition, a peptide containing cysteine 433 that coordinates with heme was possibly modified as it was detected in control, but not methyl parathion samples. A comparison of rat CYP3A1 with human CYP3A4 suggests that cysteines 64 and 378 reside along the substrate channel, remote from the active site. Alteration of these residues might modulate substrate entry to the binding pocket of the enzyme.

Optimization of methyl parathion biodegradation and detoxification by cells in suspension or immobilized on tezontle expressing the opd gene.

The goal of this study was to optimize methyl parathion (O,O-dimethyl-O-4-p-nitrophenyl phosphorothioate) degradation using a strain of Escherichia coli DH5α expressing the opd gene. Our results indicate that this strain had lower enzymatic activity compared to the Flavobacterium sp. ATCC 27551 strain from which the opd gene was derived. Both strains were assessed for their ability to degrade methyl parathion (MP) in a mineral salt medium with or without the addition of glucose either as suspended cells or immobilized on tezontle, a volcanic rock. MP was degraded by both strains with similar efficiencies, but immobilized cells degraded MP more efficiently than cells in suspension. However, the viability of E. coli cells was much higher than that of the Flavobacterium sp. We confirmed the decrease in toxicity from the treated effluents through acetylcholinesterase activity tests, indicating the potential of this method for the treatment of solutions containing MP.

Biological responses to imazapic and methyl parathion pesticides in bioinspired lipid membranes and Tilapia fish.

Pesticide misuse has well-documented detrimental effects on ecosystems, with Nile tilapia (Oreochromis niloticus) being particularly vulnerable. The current study focuses on the impact of widely used sugarcane crop pesticides, Imazapic (IMZ) and Methyl Parathion (MP), on tilapia gill tissues and their lipid membranes. This investigation was motivated by the specific role of the lipid membrane in transport regulation. Bioinspired cell membrane models, including Langmuir monolayers and liposomes (LUVs and GUVs), were utilized to explore the interaction of IMZ and MP. The results revealed electrostatic interactions between IMZ and MP and the polar head groups of lipids, inducing morphological alterations in the lipid bilayer. Tilapia gill tissue exposed to the pesticides exhibited hypertrophic increases in primary and secondary lamellae, total lamellar fusion, vasodilation, and lifting of the secondary lamellar epithelium. These alterations can lead to compromised oxygen absorption by fish and subsequent mortality. This study not only highlights the harmful effects of the pesticides IMZ and MP, but also emphasizes the crucial role of water quality in ecosystem well-being, even at minimal pesticide concentrations. Understanding these impacts can better inform management practices to safeguard aquatic organisms and preserve ecosystem health in pesticide-affected environments.

On-site screening method for bioavailability assessment of the organophosphorus pesticide, methyl parathion, and its primary metabolite in soils by paper strip biosensor.

An important public concern worldwide is soil pollution caused by organophosphorus pesticides and their primary metabolites. To protect the public's health, screening these pollutants on-site and determining their soil bioavailability is important, but doing so is still challenging. This work improved the already-existing organophosphorus pesticide hydrolase (mpd) and transcriptional activator (pobR), and it first designed and constructed a novel biosensor (Escherichia coli BL21/pNP-LacZ) that can precisely detect methyl parathion (MP) and its primary metabolite p-nitrophenol with low background value. To create a paper strip biosensor, E. coli BL21/pNP-LacZ was fixed to filter paper using bio-gel alginate and sensitizer polymyxin B. According to the calibrations of the paper strip biosensor for soil extracts and standard curve, the color intensity of the paper strip biosensor collected by the mobile app may be used to compute the concentration of MP and p-nitrophenol. This method's detection limits were 5.41 µg/kg for p-nitrophenol and 9.57 µg/kg for MP. The detection of p-nitrophenol and MP in laboratory and field soil samples confirmed this procedure. Paper strip biosensor on-site allows for the semi-quantitative measurement of p-nitrophenol and MP levels in soils in a simple, inexpensive, and portable method.

Human hepatic cytochrome P450-specific metabolism of the organophosphorus pesticides methyl parathion and diazinon.

Organophosphorus pesticides (OPs) are a public health concern due to their worldwide use and documented human exposures. Phosphorothioate OPs are metabolized by cytochrome P450s (P450s) through either a dearylation reaction to form an inactive metabolite, or through a desulfuration reaction to form an active oxon metabolite, which is a potent cholinesterase inhibitor. This study investigated the rate of desulfuration (activation) and dearylation (detoxification) of methyl parathion and diazinon in human liver microsomes. In addition, recombinant human P450s were used to determine the P450-specific kinetic parameters (K(m) and V(max)) for each compound for future use in refining human physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) models of OP exposure. The primary enzymes involved in bioactivation of methyl parathion were CYP2B6 (K(m) = 1.25 µM; V(max) = 9.78 nmol · min(-1) · nmol P450(-1)), CYP2C19 (K(m) = 1.03 µM; V(max) = 4.67 nmol · min(-1) · nmol P450(-1)), and CYP1A2 (K(m) = 1.96 µM; V(max) = 5.14 nmol · min(-1) · nmol P450(-1)), and the bioactivation of diazinon was mediated primarily by CYP1A1 (K(m) = 3.05 µM; V(max) = 2.35 nmol · min(-1) · nmol P450(-1)), CYP2C19 (K(m) = 7.74 µM; V(max) = 4.14 nmol · min(-1) · nmol P450(-1)), and CYP2B6 (K(m) = 14.83 µM; V(max) = 5.44 nmol · min(-1) · nmol P450(-1)). P450-mediated detoxification of methyl parathion only occurred to a limited extent with CYP1A2 (K(m) = 16.8 µM; V(max) = 1.38 nmol · min(-1) · nmol P450(-1)) and 3A4 (K(m) = 104 µM; V(max) = 5.15 nmol · min(-1) · nmol P450(-1)), whereas the major enzyme involved in diazinon detoxification was CYP2C19 (K(m) = 5.04 µM; V(max) = 5.58 nmol · min(-1) · nmol P450(-1)). The OP- and P450-specific kinetic values will be helpful for future use in refining human PBPK/PD models of OP exposure.

Burkholderia zhejiangensis sp. nov., a methyl-parathion-degrading bacterium isolated from a wastewater-treatment system.

The taxonomic status of a methyl-parathion-degrading strain, OP-1(T), isolated from a wastewater-treatment system in China, was determined using a polyphasic approach. The rod-shaped cells were Gram-staining-negative, non-spore-forming and non-motile. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the novel strain belonged to the genus Burkholderia, as it appeared closely related to Burkholderia glathei ATCC 29195(T) (97.4 % sequence similarity), Burkholderia sordidicola KCTC 12081(T) (96.5 %) and Burkholderia bryophila LMG 23644(T) (96.3 %). The major cellular fatty acids, C(16:0), C(17:0) cyclo and C(18:1)ω7c, were also similar to those found in established members of the genus Burkholderia. The genomic DNA G+C content of strain OP-1(T) was 59.4 mol%. The level of DNA-DNA relatedness between the novel strain and the closest recognized species, Burkholderia glathei ATCC 29195(T), was only 30 %. Based on the phenotypic, genotypic and phylogenetic evidence, strain OP-1(T) represents a novel species of the genus Burkholderia, for which the name Burkholderia zhejiangensis sp. nov. is proposed. The type strain is OP-1(T) ( = CCTCC AB 2010354(T) = KCTC 23300(T)).

Selection of a whole-cell biocatalyst for methyl parathion biodegradation.

Whole-cell biocatalyst has the potential to become a cost-effective alternative to conventional enzyme methods for solving ecological and energy issues. However, cytosolic-expressing biocatalyst systems are critically disadvantaged due to the low permeability of the cell membrane. To overcome substrate transport barrier, periplasmic secretion and surface display biocatalysts were developed by expressing signal peptides or anchor proteins in Escherichia coli. In this work, six carriers were compared in regard to whole-cell activity of methyl parathion hydrolase (MPH). Our results indicate that the surface display systems yielded one to three times whole-cell activity than the periplasmic secretion systems. Although periplasmic secretion systems showed generally more stable than surface display systems, surface display appeared more suitable for whole-cell biocatalyst. It should note that the applicability of the DsbA/PhoA/AIDA-I leader to MPH expression is shown here for the first time. In addition, the result provided a useful reference for other whole-cell biocatalyst selection.