Isolation Identification Of A Pyrcne-degrading Bacteriμm And Cloning Of Catechol-2,3-dioxygenase Gene And Expression

The environment problems are more and more severe along with High-speedeconomic development．So，pollution remendiation is staring US in face．Polycyclicaromatic hydrocarbons are kinds of aromatic compounds，produced in many ways anddifficμlt to clean up．The elimination of polycyclic aromatic hydrocarbons draws theattention of the public because of its threaten for hμman’s health． Microbialdegradation is an important measure for polycyclie aromatic hydrocarbonsremendiation．As for this reason，getting high-effective degrading bacteria ofpolycyclic aromatic hydrocarbons， as well as doing rearch for its degradingcharacteristics and genes related with degradation and bring this fruits into thepractical application must be valueable．Polycyclic aromatic hydrocarbons degradation bacteria fromoil-contaminated soil.Using pyrene as the sole carbon and energy sorce and agra platesubliming method， a strain named B2was isolated from oil-contaminated soilsamples in Karamay， China. Based on16S rDNA gene sequence homology andphylogenetic analysis， B2was identified as genus Pseudomonas with highestsimilarity（99%）.Orthogonal design method was used to opitimize the The growthconditions， and mμlti-nonlinear model was constructed to determine the bestconditions of the strains were cμltured in37°C， pH7.0，150rpm pyreneconcentration of about50mg/L， liquid volμme of100ml/250ml.Experiments using orthogonal design of Pseudomonas genus B2pyrene-degrading conditions were optimized using DPS data processing systemanalysis of the data obtained， the establishment of B2degradation of pyreneregression equation to derive optimal degradation conditions. Y=16.864-41.214X₁+0.564X₂+22.355X₁²-0.003X₂²-0.048X₁X₂+7.016X₁X₃-0.0187X₂X₃. conditionsOD600=0.60，40°C， pH6.0The case can be completely degraded by theconcentration of38.214mg/L pyrene. Experimental verification of the above conditions， the actual measured maximμm degradation37.906mg/L Lpyreneprediction accuracy rate of prediction accuracy rate of99.19%.The plasmid elimination，such as PCR amplification methods Pseudomonas. B2of catechol-2，3-dioxygenase gene B2C23O is located on the bacterial chromosome.Strain B2was cloned from the size of the0.9kb gene fragment by PCR amplification.The resμlts of sequencing showed that，the gene is880bp in length，with a completeopen reading frame of catechol-2，3-dioxygenase gene（B2C23O） sequence，encoding246amino acids with catechol-2，3-dioxygenase. Has been included in theNCBI database and with catechol-2，3-dioxygenasenucleic acid and amino acidsequences. Catechol has been reported in catechol-2，3-dioxygenase nucleic acidsequences of B2catechol-2，3-dioxygenase gene and Pseudomonas bacteria catechol-2，3-dioxygenase gene most similar， which， with the P.putida W619homology ishighest，97%similarity with green; P.aerμginosa PAGI-6and Pseudomonasfluorescens F113was96%. The protein sequence of Vector NTI derives its code anddetailed analysis of its parameters.Mining construction as carrier by using a conventional method， the C23O genewas expressed by pET32a（+）vector， C23O enzyme activity test resμlts show，C23O in bacteria liquid on clear liquid not detected，indicating that the enzyme is notextracellμlar enzyme; enzyme in the inducing conditions next was broken on enzymeactivity in the Qing Dynasty with the inducing time growth increased， but when7.5-10h is induced in the process of enzyme activity growth phase when the slow.Protein electrophoresis resμlts show the fusion proteins， molecμlar weight of largeand small white is about30kDa， and the expected resμlts in27.35kDa. Throμgh thebiological test resμlts show， fusion base due to the expression， to further theenzyme gene transferred to the wild strains constructed gene engineering bacteria laidthe foundation...