Cloning And Characterization Of The Key Genes Responsible For Amides And Organophosphorus Pesticides Degradation From Strain Of Paracoccus Versutus M-1

Chemical pesticides play an irreplaceable role in agricultural production, but it also caused serious pesticide residues pollutions, which is stringent to be resolved. Microorganisms in situ remediation is considered to be an effective means to resolve this problem. Relevant theoretical and applied researches are hotspots always. Pesticide contamination is usually a mixture of pollution. Compared to single degrading strains, broad-spectrum pesticide degrading strains have more practical application in practical applications. Meanwhile, the molecular mechanisms of microbial degradation in pesticide residues will also provide theoretical guidance for microbial remediation.In our previous study, a broad-spectrum pesticide degrading strain M-1was separated in our laboratory. M-1could degrade organophosphorus pesticides such as dichlorvos, monocrotophos, phosphamidon as well as amide herbicides propanil including acetochlor and butachlor. The bacterium strains and their development has been applied for patent protection, and played an important role in the application. In this study, we try to use strain M-1for material and reveal the molecular mechanism in different pesticide residues degradation, and finally provide a theoretical reference to its application.1. Investigation of the substrate spectrum of M-1Substrate spectrum studies showed that Paracoccus versutus strain M-1had a wide-spectrum of pesticide-degradation. Strain M-1could degrade monocrotophos, dichlorvos, phosphamidon, propanil, acetochlor and butachlor, and our studies showed that strain M-1could also degrade methyl parathion, methyl paraoxon, acrylamide, benzamide and phenyl amide. When strain M-1was inoculated at concentration of1%in liquid minimal medium with glucose at30℃and pH7.5, the degradation rates of methyl parathion and methyl paraoxon were87.2%and93.4%within48h, respectively. Meanwhile, in liquid minimal medium, the degradation rates of acrylamide, benzamide and phenyl amide were96.3%,94.1%and89.20%within12h, respectively. 2. Cloning and characterization of a amidaseThe solubility of3,4-DCA is much higher than that of propanil in water; therefore, a clear transparent halo will be formed around the colonies due to the transformation of propanil to3,4-DCA in LB agar containing400mg L-1propanil. Using this approach, propanil amide hydrolase gene pamh was cloned from the library. The ORF is1434bp long and encodes a protein composed of477-amino acids with a calculated molecular mass of52,390Daltons (Da). A putative ribosomal binding site (AGGAGA) was found located5bp upstream of the start codon ATG. The G+C content of pamh gene sequence is49.30%, and no potential signal sequences were identified. The sequence of pamh was compared with other known enzymes available in the NCBI database. PamH shows the highest identity (31%identity) with a well-characterized amidase from Rhodococcus sp. N-771. The highly conserved catalytic triad of amidase signature (AS) enzyme family, Ser-Ser-Lys, was found in the amino acid sequence of Pamh. The site-directed mutagenesis confirmed that this motif was the catalytic site. Thus, we concluded that PamH from Paracoccus sp. M-1was a new member of the amidase signature family.The expression conditions of PamH in E. coli BL21(DE3) were investigated. PamH was only successfully expressed under the following conditions:200rpm/min shaking,0.005-0.01mM IPTG induction when the OD600nm was achieved at1.0within18-25℃in LBP media; otherwise, the expressed proteins formed inclusion bodies. After purified by ammonium sulfate fractionation, DEAE-Sephadex anion exchange column, DEAE-Cellulose chromatographic columns and dextran gel column, the enzyme yielded a single band with a molecular weight of52kDa on SDS-PAGE, which is in good agreement with the molecular mass deduced from amino acid sequence (52,390Da). The isoelectric point value of PamH was estimated to be5.13.PamH displayed the highest enzymatic activity at45℃and pH8.0, and was stable within a pH range of5.0-10.0. Most of the metal ions tested had no noticeable inhibitory effect on the amidase activity of PamH; however, Hg2+, Cu2+, and Co2+(1mM) were able to severely inhibit its activity, and the addition of Mg2+and Mn2+increased the enzymatic activity by1.3and1.1-fold, respectively. The enzyme was also seriously inactivated by PMSF, which is known to be an inhibitor of serine hydrolases. However, chelating agents EDTA and1,10-phenanthroline (10mM) only showed20-30%inhibition of PamH hydrolytic activity, suggesting that the activity of PamH was independent of metals. The thiol reagents pCMB and iodoacetamide (1mM) and the surfactants SDS, Tween-80, and Triton X-100(10mM) showed40-70%inhibition of PamH activity.A number of potential substrates were tested to investigate the substrate spectrum of PamH. PamH showed excellent activity toward the majority of aromatic and aliphatic amides, such as acetamide, propionamide, phenylacetamide, and benzamide. The production of acrylic acid by the enzymatic hydrolysis of acrylamide is important for the polymer manufacturing industry. PamH was able to catalyze the hydrolysis of acrylamide to acrylic acid and had a specific activity of14.33μmol min-1mg-1protein. Amino acid amides were also hydrolyzed by PamH. The amidase showed low activity towards asparagines (9%), L-glutamine (17%) and D-glutamine (13%) when compared with benzamide (100%). The anilide substrate range of PamH was very narrow, only propanil was a good substrate for PamH. For propanil, theat was2.8s-1and the Km was158μM with the catalytic efficiency value (kcat/km) of0.018μM-1s-1under optimal conditions.The relative acyl transferase activity of PamH (compared with acetamide) was found to be the most efficient with formamide (139%) and isobutyramide (178%). PamH also showed acyl transferase activity towards anilide substrates such as propanil (34%) and4-nitroacetanili (79%) when compared with acetamide. PamH was not active on amino acid amides and long-chain aliphatic amides (hexanamide).3. Cloning and characterization of a methyl paraoxon hydrolaseA novel methyl paraoxon hydrolase gene, designated as mpdp, was also cloned from Paracoccus sp. M-1. Sequence analysis indicated that the ORF encodes324amino acids with a calculated molecular mass of33,681Daltons (Da). The G+C content was59.08%. The deduced amino acid sequence of mpdp was compared with other known enzymes available in the NCBI database. Mpdp showed the highest identity with several metallohydrolases, including a Zn-dependent hydrolase from Magnetospirillum gryphiswaldense MSR-1(38%identity) and a methyl parathion hydrolase from Pseudomonas sp Wbc-3(33%identity). Furthermore, His-X-His-X-Asp-His, the most characteristic signature of metallo-(3-lactamase superfamily, was also found in the enzyme. Thus, Mpdp was a member of the metallo-β-lactamase superfamily.Paraoxon, methyl paraoxon and methyl parathion were hydrolyzed by Mpdp, and methyl paraoxon was a good substrate of Mpdp. The enzyme had a Km of286μM and a kcatat of14.8s-1for methyl paraoxon. The kcat/Km was0.052s-1μM-1. For paraoxon, the specific activity was0.81μmol per min per mg protein. For methyl parathion, the specific activity was158nmol per min per mg protein,2orders of magnitude lower than that was methyl paraoxon.Error Prone PCR was used to generate Mpdp variants to improve the hydrolysis of substrates and five mutations were obtained. The activity of the best variant Mppl on methyl paraoxon, ethyl paraoxon and methyl parathion were81.4,5.8,0.72U/mg, respectively, with11.2,7.1and4.5-fold activity improvement compared with the wild type Mpap. Site-directed mutagenesis suggested that the three amino acid residues Arg160, Arg176and Va1276were related to the methyl paraoxon activity of Mpdp, which exhibited a2.3,10.1and4.5-fold increase in kcat/km values, respectively. Mppl having all the three amino acid residues mutations (Arg160, Arg176and Va1276) exhibited a9.1and24.5-fold increase in kcat and kcat/km values, respectively.