Isolation Of Chlorpyrifos-Degrading Bacteria,Clonging And Direct-Evolution Of Mpd Gene And Application Research

Chlorpyrifos [O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate] is a mid-toxic organophosphorus insecticide which has been widely used by many countries. Its vast use ultimately caused more serious environment pollution. Microbial remediation has been deemed to be much more advantageous method than the others. So it has been an important research item for us to exploit and utilize chlorpyrifos-degrading microbial resource to remove the environment pollution. This research aimed at isolating the bacteria that can use chlorpyrifos as sole carbon source, study their degrading characterizes of chlorpyrifos in different environments, and provided the theory base for the bioremediation of environment pollution with chlorpyrifos. At the same time, the degrading gene of chlorpyrifos was cloned and evolved, which would be helpful to research the gene function and establish the foundation of construct the genetic engineered strain by enhanced degrading capacity.Seven chlorpyrifos-degrading bacteria, named Dsp-1to Dsp-7were isolated from chlorpyrifos-contaminated samples using selective culture medium with chlorpyrifos as sole carbon source. These isolated strains were identified based on morphological, physiological and biochemical tests with reference to Bergey’s Manual of Determinative Bacteriology combined with16S rDNA sequence analysis. Based on these analyses, strains Dsp-2, Dsp-4, Dsp-6and Dsp-7were identified as Sphingomonas sp., Stenotrophomonas sp., Bacillus sp. and Brevundimonas sp., respectively, while all other strains were members of Pseudomonas sp. This is the first report of isolating chlorpyrifos-degrading bacteria from Sphingomonas sp.and Brevundimonas sp. Comparative studies were performed to study their phylogenetic relationship based on their16S rDNA sequence. ERIC-PCR fingerprints and the degrading capability were also compared. These results revealed high biodiversity of chlorpyrifos-degrading bacteria in many aspects. Degrading characteristics of seven degrading bacteria were investigated. However, significant differences in the ability of degrading chlorpyrifos and other organophosphate pesticides were observed among these strains. Strain Dsp-l-Dsp-4can degrade several organophosphate pesticides. Contrast to orther six strains, strain Dsp-2can degrade profenofos and had the fastest degrading rate in liquid medium,100mg-L" chlorpyrifos was degraded to an undetectable level within12h. Strain Dsp-1instead of Dsp-2had the highest degrading rate in soil. Strain Dsp-5and Dsp-7can not degrade any supplied pesticides except chlorpyrifos. All these isolates could utilize chlorpyrifos as sole carbon source with3,5,6-trichloro-2-pyridinol (TCP) as intermediate.The primers were constructed based on the published sequences of the organophosphorus hydrolase gene opd and mpd. The methylparathion hydrolase gene mpd was cloned by PCR from Dsp-l-Dsp-4. The analysis and alignment of the four organophosphate pesticides hydrolase genes revealed that this gene has996bases, which coding for the organophosphate pesticides hydrolase contained331amino acids. The four genes were dividing into three subgroups at the level of gene base sequences, the similarity values between them were99%. At the level of amino acid sequences, the four organophosphate pesticides hydrolase also were dividing into three subgroups, the similarity values between them were92%.Each mpd gene from the four strains was linked to the expression plasmid vector pET29a, the expression strains of the four genes were gained by transfer of the expression plasmid into strain BL21(DE3). SDS-PAGE analysis of the total proteins of the four the expression strains identified the hydrolase.Error Prone PCR was used to generate MPH variants improvement in hydrolysis of chlorpyrifos and five mutations were obtained. One variant, Mn7which had five site mutations (N271T, I275S, G277S, A280V, P304S), displayed a10-fold increase in Arcat and118-fold increase in kcat/Km values. It also exhibited increase special activity for methylparathion chlorpyrifos and triazophos compared to wild type-MPH. We obtained8site-mutation variants by site-directed mutagenesis. Study of their kinetic parameters for hydrolysis of chlorpyrifos was performed. Results showed that N271I exhibited a15-fold increase in kcat/Km values and A280V exhibited a15-fold increase in kcat/Km values. These results showed that these two sites are the key sites for improved hydrolysis of chlorpyrifos. F196I which showed one fold reduction in kcat/Km values may because that Phe196is the key acid amino that form an aromatic cluster at the entrance of the catalytic center. No noticeable difference between MPH from Mn7and that from M6was observed when analysis of enzymatic3D-structure. But secondary structure analysis showed that the content of a-helix and β-pleated sheet was increased, suggesting that is likely to contribute to stabilized the conformation, so that improved the efficiency of catalysis.The optimal growth conditions of strain Dsp-1were studied. The optimal growth temperature and initial pH of strain Dsp-1are30℃and7.0, respectively. The oxygen had little effect on the growth of strain Dsp-1. Strain Dsp-1could growth without NaCl and special growth factor. Glucose was the best carbon source and organic nitrogen was better nitrogen source. The optimal inorganic nitrogen was NH4NO3. Strain Dsp-1was sensitive to many antibodies. In the ferment medium, after16hours, Dsp-1cell could reach to1011mL-1. Addition of strain Dsp-1to three test soils (Red soil. Brown soiland Sandy soil) treated with chlorpyrifos resulted in a high degradation rate of chlorpyrifos. The highest degradation rate was showed in brown soil, which was netrual pH. The degradation effect was not satisfactory in red soil which was acidic pH and the physical degradation of chlorpyrifos was obvisely in sandy soil because of its alkaline pH. However, the soil pH had notability effect on degradation. The moderate pH, moisture and nutrients could promote the degradation. Strain Dsp-1worked well when the concentration of chlorpyrifos in soil ranged in1-100mg kg-1. The degradation rate reached to90%at low concentration. The fine characteristic of Dsp-1will help it to play well in different chlorpyrifos-contamination soil. The research showed that strain Dsp-1had the potential to cleanup chlorpyrifos contaminated environment. The degradation patterns of chlorpyrifos on pakchoi in the greenhouse were performed in spring and winter separately. When using1010CFU-mL-1zymolytic strain Dsp-1, the degradation rates of chlorpyrifos on pakchoi reached to80%after7days. The results suggested that the strain Dsp-1could efficiently remove or detoxify chlorpyrifos residues on pakchoi in the greenhouse, making sure that the residual values of chlorpyrifos on pakchoi were lower than the China MRL (0.1mg-kg-1).