Bioremediation of xenobiotic compounds including dioxins

研究了二苯并呋喃（DF）降解菌Terrabacter sp.菌株DBF63和咔唑（CAR）降解菌Pseudomonas resinovorans菌株CA10转化几种氯化DFs和二苯并对二恶英（dd）的能力。结果表明，菌株CA10的CAR - 1,9a-双加氧酶能催化本研究研究的所有单-至三-基-DF/ dd （CDF/ cdd）的角双氧化，而菌株DBF63的DF - 4,4a-双加氧酶不能转化2,7- dicdd。我们还尝试了利用土壤-浆液系统对实际二恶英污染土壤进行初步的生物修复。CA10和DBF63细胞单次接种7 d后，CDD和CDF同源物总量分别减少8.3和10%，表明菌株CA10和DBF63具有降解四氯化至七氯化同源物的能力，包括毒性最大的化合物2,3,7,8- tetracdd .[2]将荧光探针法、竞争性PCR法和内标细胞共萃取法相结合，建立了一种快速、灵敏、准确的CA10菌株靶基因carAa拷贝数和细胞数定量方法。此外，利用Tn5转座子对菌株CA10进行串联gfp基因的染色体标记。采用实时竞争PCR和GFP标记直接计数法对土壤中菌株CA10的carAa基因总数和细胞存活率进行了监测在CAR存在下，将CAR降解菌K23与苯酚降解菌DS1进行平板配对，获得了能够利用CAR的转接合物。转偶联物质粒pCAR2.[4]较大表达融合内质网信号肽的DbfB转基因植物不断从根部分泌重组蛋白到简单的水栽培养基中，分泌的DbfB蛋白保留了生物活性。

[1] Two kinds of bacteria, a dinenzofuran (DF)-degrader Terrabacter sp. strain DBF63 and a carbazole (CAR)-degrader Pseudomonas resinovorans strain CA10, were investigated for their ability to transform several chlorinated DFs and dibenzo-p-dioxins (DDs). It was shown that CAR 1,9a-dioxygenase from strain CA10 catalyzed angular dioxygenation of all mono- to tri-chioro-DF/DDs (CDF/CDDs) investigated in this study, but DF 4,4a-dioxygenase from strain DBF63 did not transform 2,7-diCDD. We also tried a preliminary bioremediation of the actual dioxin-contaminated soil using the soil-slurry system. The decrease of total CDD and CDF congeners by single inoculation with CA10 or DBF63 cells were 8.3 and 10%, respectively, after 7-day incubation, thus it was shown that strains CA10 and DBF63 had a potential to degrade tetra- to hepta-chlorinated congeners including the most toxic compound, 2,3,7,8-tetraCDD.[2] The fluorogenic probe assay, competitive PCR and co-extraction with internal standard cells were combined to develop a rapid, sensitive, and accurate quantification method for the copy number of a target carAa gene and the cell number of strain CA10. In addition, by using Tn5 transposon, strain CA10 was chromosomally marked with a tandem gfp gene. The real-time competitive PCR and direct counting using the GFP marker were employed to monitor the total number of carAa gene and survival of strain CA10 cells in the soil.[3] Following plate matings between CAR-degrader strain K23 and phenol-degrader strain DS1 in the presence of CAR, transconjugants able to utilize CAR were obtained. The transconjugants had large plasmid pCAR2.[4] Transgenic plants expressing DbfB fused to endoplasmic reticulum signal peptide continuously secrete recombinant proteins from their roots into a simple hydroponic medium, and the secreted DbfB proteins retained biological activity.