Isolation And Identification Of Dichloromethane-degrading Bacterium, Characteristics Of Biodegradation, Cloning And Expression Of Key Enzyme Genes

In this thesis, two dichloromethane-degrading bacterial were:isolated by using traditional incubation method. Their identification were based on standard morphological and physiological properties, cellular fatty acid composition, mol% G+C and nucleotide sequence analysis of enzymatically amplified 16S ribosomal deoxyribonucleic acid. The factors influencing growth of dichloromethane-degrading bacteria and degradation of dichloromethane, the cloning and expression of dichloromethane degrading gene of two strains, were studied in this thesis. The results were expected to supply useful reference for building up alert index systems in transformation and biodegradation mechanism of xenobiotics, and for environmental quality evaluation and for bioremediation of halogenated hydrocarbon pollution.Here are presented the main results of this study:1. Two strains which could use dichloromethane as sole carbon source were isolated from halogenated hydrocarbon contaminated sample. Their identification were based on standard morphological and physiological properties, G+C content and nucleotide sequence analysis of enzymatically amplified 16S ribosomal deoxyribonucleic acid. Strain named WZ-12 (GenBank accession no.EF100968) was isolated and identified as Bacillus circulans and strain named wh22 (GenBank accession no. FJ418643) as Lysinibacillus sphaericus which were the first representative of Bacillus circulans and Lysinibacillus sphaericus able to degrade dichloromethane very fast at high experimental concentration.2. Both WZ-12 and wh22 has wide temperature, pH range for growth and degradation, and could tolerate high concentration of dichloromethane. The optimal growth conditions of strain WZ-12 (pH6.0, 37℃,degradation rate 85%) and wh22 (pH7.0, 30℃,degradation rate 80%), respectively. Addition of yeast extraction, peptone or glucose could promote the growth and dichloromethane degradation ability of both WZ-12 and wh22 to different degree. Biodegradation of DCM followed the modified Gompertz model. WZ-12 degrade CH₂C1₂,CH₂BrCl,C₂H₄C1₂ and C₂H₂C1₂ efficiently in the medium containing NaCl at concentrations of 15 g L^-1 in 72 h. Kinetic analysis revealed that there was an inverse relationship between the velocity of the degradation reaction and salt concentration over the range between 5 and 60 g NaCl L^-1 and a linear reciprocal relationship (R²=from 0.85 to 0.94) was observed.3. The strain wh22 harbored a novel degradative plasmid, pRC11 (48.8 kilobases). The genes coding for the metabolism of dichloromethane were found to be plasmid-borne, and a physical map of the plasmid has been established. The purified plasmid was transformed to dcm^- Escherichia coli DH5 at a rate of 1.65×10⁵.The transformed cells were able to grow on dichloromethane at concentration of 5-16 mM, and can be further used as a excellent source for genetic manipulations leading to construction of genetically modified microbial strains or genetically engineered microorganisms.4. Response surface methodology (RSM) was employed to evaluate the optimum aerobic biodegradation of dichloromethane (DCM) in batch culture. The parameters investigated include the initial DCM concentration, glucose as an inducer and hydrogen peroxide as terminal electron acceptor (TEA). Maximum aerobic biodegradation efficiency was predicted to occur when the initial DCM concentration was 380 mg/Lwith the glucose of 13.72 mg/L and the H₂O₂ of 115 mg/L.Under these conditions the aerobic biodegradation rate reached up to 93.2%, which was significantly higher than that obtained under original conditions. Without additives, the degradation efficiencies≤80% were obtained with the DCM concentrations < 326 mg/L. In order to achieve an 80% or higher biodegradation efficiency, DCM concentrations should be lower than 350 mg/L and the addition of glucose is necessary. When concentrations of DCM were more than 480 mg/L, the addition of H₂O₂ did not significantly contribute to increase DCM degradation efficiency. When DCM concentrations was increased from 240 mg/L to 480 mg/L, the overall DCM degradation efficiency decreased from 91% to 60% as the presence of H₂O₂ for 120mg/L.5. Dichloromethane dehalogenase from B. circulans WZ-12 was purified to 8.27-fold with a yield of 34.83%. The electrophoretically homogeneous-purified enzyme exhibited a specific activity of 118.82 U/mg. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified DCM dehalogenase gave a distinct band with an estimated molecular mass of 31 kDa. Enzyme activity was optimized at 30℃to 35℃and pH from 6.5 to 7.0. The enzyme was stable in the pH range of 5.0-8.0. The reaction was apparently accelerated by addition of Mg²⁺and Ca²⁺,causing an 22 to 27% stimulation, whereas it was inhibited by the addition of Hg²⁺,Cu²⁺,Zn²⁺ and Ba²⁺.DCM dehalogenase exhibited extremely broad substrate and the apparent Km value at 30℃(pH7.0) for DCM was 5.25×10^-3 mol·L^-1. Vmax was 3.67×10^-4mol·L^-1/min and K_cat was 6.97×10⁴ S^-1,respectively.6. The gene dcmR encoding a novel dichloromethane dehalogenases(DehalA), has been cloned from strain WZ-12. Its accession number in Genbank was FJ418643. The open reading frame of dcmR, spanning 864 bp, encoded a 288-amino-acid protein. A homology search with the BLAST program revealed that the nucleotide sequence of the dcmR gene was almost identical (98.6%) to Methylobacterium sp DM4.7. The gene dcmR of strain WZ-12 was recombined and expressed in E.coli BL21(DE3) successfully. A high level of soluble dehalogenases (DehalA) was expressed in E.coli BL21(DE3) from a pET expression system(pET21a and pET15b) and the activity of recombined enzyme protein expressed by recombinant obtained was much more than that by the original WZ-12 strain in primary detection. The gene dcmR was subcloned into pET21a vector at Nde I and Xho I sites with no any tag, the gene dcmR was subcloned into pET21a vector at Nde I and Xho I sites with his-tag and the gene dcmR was subcloned into pET15b vector at Nde I and BamH I sites with his-tag and LVPRGS thrombin. The recombined vectors were confirmed by DNA sequencing and transformed into the E. coli strain ArcticExpress^TM (DE3) RP for expression optimization. The expressions were analyzed by SDS-PAGE followed by Coomassie blue staining. There were obvious expression band and there were soluble fusion protein when induced at low temperature.8. Then plasmid pET-15b-dcmR with his-tag and LVPRGS thrombin was introduced into Escherichia. coli BL21(DE3). Expression was induced by IPTG, and the enzyme activity reached 25.78 U/mL, the specific enzyme activity reached 88.86 U/mg protein. The periplasmic and cytoplasmic enzyme activity reached 2.92 U/mL and 22.86 U/mL respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified recombinant DCM dehalogenase gave a distinct band with an estimated molecular mass of 33±1kDa. All results analysis demonstrated that the E.coli. strain carrying the dcmR gene could produce dichloromethane dehalogenase efficiently. The growth characteristics of dcmR-l was compared with the original strain, and the result showed that there was no difference,A₆₀₀ nm of dcmR-1 in LB medium could reach about 2.4 in logarithmic period, which was the same as that of the original strain. The recombinant strain dcmR-1 showed the higher degrading ability than B. circulans WZ-12 and with more than 90% removal efficiency of 120 mM CH₂C1₂ in 25 h.All these results indicated that recombinant strain dcmR-1 was a promising strain in bioremediation of CH₂Cl₂ contaminated environment.