Isolation And Caracteristic The Upstream Genes Of Carbazole-Degrader From Sphingomonas Sp. JS061

Carbazole, as a classic N-hydroaromatic compound , is often released into the environment to cause tremendous harm due to its toxic, carcinogenesis and mutagenic characteristics. The advantage of microorganism to treat the pollution can save money and energy. In this thesis, a Gram-negative bacterium isolated from carbazole- polluted soil, Sphingomonas sp. JS061, has been used to study the characteristic of car-degrading genes and enzymes in the process of carbazole degradation.The car-degrading gene cluster for the first three steps from Sphingomonas sp.JS061 genome DNA has been isolated by PCR. The up-stream genes degrading carbazole have been isolated successfully and the alignment of these genes as carAa→carB→carC→carAc。was also confirmed by and BLAST. Then updated the sequence to the GenBank and obtained the accession number EU854302. Southern blot has been performed to further confirm the car-cluster in the chromosomal DNA of JS061. The result indicated that there was one copy of car gene cluster in the genome.We have constructed carAa, carAc, carB and carC plasmids for gene expression in pET28a (+), which was expressed in BL21 (DE3.0) under the induction of IPTG. The result indicated that the molecular mass of these four proteins were 43 kDa, 15 kDa, 29 kDa (CarBa) & 14 kDa (CarBb) and 31 kDa, respectively. The molecular weight of CarB consisting of two components CarBa and CarBb was similar to CA10, but the quantity of CarBa and CarBb were different from other bacteria. Meanwhile, the substrate color reaction was employed to confirm the recombinant plasmid of pETWB. The CarB enzyme expressed in this study was of the bioactivity to degrade the aromatic ring.CarB and CarC proteins have been purified and the polyclonal antibodies have been made in female BALB/c mice, which were used to detect CarC expression level in Sphingomonas sp.JS061 under the various concentration of substrate and the same substrate concentration but different inducing time. This protein was expressed only when the substrate was existed and expression level was clear different when different induction time and substrate concentration were applied. The result confirmed that carbazole degrading proteins were inducible by carbazole.