| Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile), a broad-spectrum chlorinated aromatic fungicide, is the second most widely used agricultural fungicide in the United States with 5 million kilograms applied annually. Chlorothalonil is highly toxic to fish, birds and aquatic invertebrates and is commonly detected in ecosystems. Bioremediation of CTN pollution is recognized as a cost-effective and reliable method. Therefore, it has been an important research item for us to isolate microbial resource of high efficiency chlorothalonil-degrading, explore the role of the degradation mechanism, and give full play to their degradation ability to remove the environment pollution. It has great theoretical significance and application value.This research aimed at isolating the bacteria that can be independent of other carbon sources and degrade chlorothalonil, study their degrading characteristics of chlorothalonil in different environments, and provided the theory base for the bioremediation of environment pollution with chlorothalonil; the same time, we cloned the key enzyme gene using the gene library and further researched on the expression, enzyme properties, catalytic mechanisms and so on, which would be helpful to illustrate the novel hydrolytic dehalogenation mechanism for the chlorinated aromatic compound.Fifteen chlorothalonil-degrading bacteria, named CTN-1 to CTN-15 were isolated from chlorothalonil-contaminated water and soil using selective culture medium with chlorothalonil as sole carbon source. These isolated strains were identified based on morphological, physiological, ERIC-PCR fingerprints and biochemical tests with reference to Bergey’s Manual of Determinative Bacteriology combined with 16S rRNA sequence analysis. Based on these analyses, the fifteen strains were identified as Ochrobactrum sp., Pseudoxanthomonas sp., Bordetella sp., Shinella sp., Caulobacter sp., Rhizobium sp. and Pseudomonas sp., respectively, while strain CTN-1 was identified as a novel member of the genus Lysobacter. Phylogenetic analysis based on the 16S rRNA gene sequences, DNA-DNA hybridization data and biochemical and physiological characteristics strongly supported the genotypic and phenotypic differentiation of strain CTN-1T from recognized species of the genus Lysobacter. Strain CTN-1T, therefore, represents a novel member of the genus Lysobacter, for which the name Lysobacter ruishenii sp. nov. is proposed. The type strain is CTN-1T(=DSM 22393T=CGMCC 1.10136T).Comparative studies were performed to study their phylogenetic relationship and the degrading capability. These results revealed high biodiversity of chlorothalonil-degrading bacteria in contaminated environment. Degrading characteristics of fifteen degrading bacteria were investigated. All these isolates could utilize chlorothalonil as sole carbon source with TPN-OH as intermediate. We choose eight different genus to be researched, however, significant differences in the ability of degrading chlorothalonil. The rate of degradation between 4 to 40 mg·l-1·d-1, the five strains isolated from liquid are faster than other three strains coming from soil. Contrast to other three strains, strain CTN-3 and CTN-14 have the fastest degrading rate in liquid medium,20 mg·l-1 chlorothalonil was degraded to an undetectable level within 12 h. All the results implied that the chlorothalonil degrading strains had the potential to clean up the chlorothalonil fungicide contaminated environment. This study confirms new resources could be used efficiently for remediation of chlorothalonil-contaminated environment.Method to extract high quality total DNA from strain CTN-3 was founded. Gene library of CTN-3 total DNA was constructed by the method of shotgun cloning. Two positive clones, designated as 1-10 and 2-1, were screened from a library containing approximately 15,000 transformants. The inserted fragments in the two positive clones had a 1,243 base pair (bp) overlap in sequence that nearly covered a 1,026-bp ORF. The 1,026-bp ORF was expressed in E. coli DH5a and the capacity to degrade chlorothalonil was confirmed. The sequence of Chd was compared with other known enzymes available in the NCBI database. Chd contains a putative conserved domain of the metallo-β-lactamase superfamily and shows the highest identity with several metallohydrolases, including a cyclase from Streptomyces coelicolor (29% identity), a Zn-dependent hydrolase from Stackebrandtia nassauensis (27% identity) and aβ-lactamase-like protein from Candidatus Koribacter versatilis (24% identity), shows that Chd has a characteristic fold of metallo-β-lactamase. The characteristic metallo-β-lactamase fold consists of anαβ/βαsandwich consisting of a core ofβ-sheets surrounded by a-helices. Furthermore, His-X-His-X-Asp-His, the most characteristic signature of metallo-β-lactamase superfamily, was also found. However, the first histidine was replaced by serine. The primers were constructed based on the chd gene ORF. The chlorothalonil hydrolytic dehalogenase gene was cloned by PCR from strains CTN-1～CTN-15. The analysis and alignment of the fifteen chlorothalonil hydrolytic dehalogenase genes revealed that the gene is between 1002 and 1026 bp, there are 48 different base sites among them, the similarity values between them were about 99%.The metabolite was first identified by HPLC. The metabolite was further confirmed by MS/MS. NMR analysis was carried out to determine the position of the dehalogenated chlorine atom. On the other hand, we researched on the course of metabolism under aerobic and anaerobic conditions. According to the above data, the characterized enzyme was designated as a novel chlorothalonil hydrolytic dehalogenase for chlorinated aromatic compound (EC 3.8.1.12).In the characterization study of Chd which encode by chd structural gene and expression by the E. coli BL21. Enzymatic reaction system was established:50 mM PBS (pH7.0),0.2mM CTN,50μl enzyme,50℃,reaction 10 min, terminated reaction with 3ml CH2CI2 then immediate measure OD232. One unit of enzyme activity was defined as the amount of enzyme that catalytic convert lμM chlorothalonil per min.The enzyme was fairly stable at pH values between 6.0 to 8.0. The optimal pH of Chd was observed to be approximately 7.0 and the enzyme was fairly stable at pH values between 6.0 to 9.0. The enzyme retained more than 95% of its original activity after pre-incubation at that pH range for 30 min. The enzyme was stable over a range from 30℃to 70℃. Chd was fairly stable up to 40℃and retained more than 95% of its activity at 50℃for 10 min. Chd retained 18% of its residual activity at 50℃for 1 h and completely lost activity at 60℃for 1 h.The Chd activity was strongly inhibited by 1 mM Ag+, Al3+, Hg2+, Mn2+, Fe2+, Fe3+, 10 mM SDS,1 mM 1,10-phenanthroline,1 mM NBS and 0.5 mM DEPC; while 1 mM Cr3+, Co2+, Cu2+,10 mM Tween 80, Triton X-100, pCMB, PMSF, PAO and PGO showed only slight inhibition; in addition,1 mM Zn2+, Ca2+, Ni2+, Mg2+, Li+, Ba2+; 10 mM EDTA had little effect on the enzyme activity (less than 10% inhibition). Chd catalytic activity was completely inhibited by the Zn2+-chelating metalloprotease inhibitor 1,10-phenanthroline, and the catalytic activity of Chd was recovered by the subsequent supplementation of Zn2+. These data confirm that Chd is a Zn+-dependent metallohydrolase. Treatment of the Chd with DEPC resulted in the complete loss of catalytic activity that was fully regained by subsequent treatment with hydroxylamine, indicating the involvement of His residues in the active sites of the enzyme. Chemical modification of Chd with NBS resulted in the completely loss of catalytic activity and was prevented by the saturation of the active site with the product ligand 4-TPN-OH. These data provide evidence favoring an essential role of a tryptophan in the active site of Chd.The pI value of Chd was estimated to be 4.13. For chlorothalonil, the overall catalytic rate (kcat) value of Chd was 207 s-1 with a dissociation constant (Km) of 0.112 mM. The catalytic efficiency value (kcat/Km) under optimal conditions was 1.8±106M-1·s-1. These data indicate that chlorothalonil is a good substrate for Chd. The molecular mass of native Chd was estimated 33,886 Da, the determined molecular mass is in good agreement with the molecular mass deduced from the amino acid sequence (36,823 Da). These data indicate that Chd is a monomer. Site-directed mutagenesis was used to identify the specific residues essential for catalysis. As compared to the wild-type Chd, H38Q, H271Q, H326Q, D67A, D215A, D244A, D264A, D323A, C25A, W219F, W227F, W282F, W324F, H283Q, S81T, T150I and E203V did not significantly affect the catalytic activity of Chd. There is a little effect at position H63Q and D337A (30%-50%). H128Q, H157Q, D45A, D130A, D184A, W241F, S126H and G208A were found to completely lose their catalytic activities, indicating that these mutants residues are involved in catalysis. |
PDF Full Text Request