Study On The Acquisition And Characteristics Of Catechol-2,3-dioxygenase Based On Metagenomics

Polycyclic aromatic hydrocarbons(PAHs)are a class of toxic pollutants that are widely present in the environment,which have the characteristics of carcinogenicity,mutagenicity and teratogenicity.As people are increasingly aware of their various adverse effects on the ecosystem and human health,they have received extensive attention.The application of microorganisms has involved many fields,including the production of medical drugs,the restoration of pollutants in the environment,and industrial and agricultural production.Due to the diversity of its functions,it plays an important role in the degradation or repair of pollutant polycyclic aromatic hydrocarbon pollution and other environmental pollution problems.Therefore,screening PAHs high-efficiency degradation bacteria from the environment,exploring the relevant characteristics of PAHs degradation and discovering the key enzymes involved in the degradation process have profound and long-term significance for promoting the microbial remediation of pollutants.In this study,soil was collected from the oil-contaminated area of Baicheng,and a stable degrading bacterial flora was domesticated with high-concentration pyrene as the only carbon source.The composition of the flora under the selective pressure of pyrene and the degradation efficiency under different conditions were studied.Through orthogonal experiments,high performance liquid phase(HPLC)was used to detect the efficiency of bacterial colony degradation under different culture conditions,and the optimal conditions for bacterial colony culture were determined to be 30?,170 rpm,and pyrene concentration of 100 mg/L.about.The experimental results show that when the bacteria group uses pyrene as the only carbon source and energy source,the degradation efficiency of pyrene is98%when cultured at 30?,170 rpm,and 12 days.In order to further study the composition of domesticated flora,metagenomic sequencing technology was used to analyze the flora.The results showed that the flora was mainly composed of Methylobacterium sp.,Caulobacter sp.,Methylobacterium sp.,Achromobacter sp.The catechol-2,3-dioxygenase gene of Methylobacterium sp.Using PCR amplification technology,a nucleic acid fragment with a size of about 0.9 kb was amplified from the extracted bacterial colony DNA.The sequencing results showed that the length of the cloned gene fragment was 888 bp,which was consistent with the size in the metagenomic sequencing database.After that,the sequence was uploaded to NCBI for comparison.According to the comparison result,the highest similarity between the target sequence and the sequence collected in the database is only 82.03%,and it is necessary to conduct in-depth expression research on it.A prokaryotic expression system was constructed based on E.coli DH5?and E.coli BL21,and the catechol-2,3-dioxygenase gene was expressed using p ET28a vector.The results of polyacrylamide gel electrophoresis showed that the target protein was successfully expressed and was consistent with the theoretical molecular weight of 36.67k Da.The results of the study on the properties of the enzyme show that the optimum p H of catechol-2,3-dioxygenase is 8,the optimum temperature is 50?,and Cu²⁺can significantly increase the activity of the enzyme,while Ag⁺and Ca²⁺inhibit the enzyme's activity.Catalytic activity.The above results proved that the target gene was successfully expressed,and provided some information for exploring the metabolic mechanism of the degradation of polycyclic aromatic hydrocarbon pollution by the flora.