Researchs On Degradation Of Pahs By Comamonas Testosteroni

Polycyclic aromatic hydrocarbons (PAHs) are among the most prevalent and persistent pollutants in the environment. Contaminated soil and sediment commonly contain a mixture of PAHs. These PAHs will ultimately endanger human health through the food chain enrichment.Comamonas testosteroni is gram-negative, ubiquitous and strict aerobes in diverse habitats. It is able to utilize PAHs as the sole carbon and energy source. To achieve the purpose of the biodegradation of PAHs pollution, we take the ability of recombinant Comamonas testosteroni engineered bacteria degradation of PAHs mode compounds such as naphthalene and phenanthrene into account. It should provide a theoretical basis for the relevant microbial environmental pollution remediation, local ecological environment improvement, and ultimately solving environmental problems or eliminating PAHs pollution.We used the UV-Vis spectrophotometer to measure the absorbance of naphthalene and phenanthrene in the MS liquid inorganic salt medium. The concentration of naphthalene and phenanthrene was calculated by the standard curve method which can analyze the ability of degradation of naphthalene and phenanthrene by Comamonas testosteroni engineered bacteria. To compare the ability of degradation with the wild strain Comamonas testosteroni, we found that the Comamonas testosteroni engineered bacteria has higher degradation efficiency.We inoculated the Comamonas testosteroni engineered bacteria on MS medium containing2mmol/L phenanthrene to culture the Arabidopsis. To observe the growth conditions of Arabidopsis as well as measured the content of Super Oxide Dismutase (SOD) and Malondialdehyde (MDA), we can analyze the effect of physiological response of Arabidopsis stressed in phenanthrene by Comamonas testosteroni engineered bacteria. Compared with the wild strain Comamonas testosteroni, the results show that engineering bacteria can partly reduce the poisoning to the Arabidopsis and much more prominent than the wild bacteria. Therefore, engineering bacteria may have the actual ability to repair environmental pollution. 3a-Hydorxysteorid dehydorgenase/carbonyl reductase (3a-HSD/CR) from Comamonas testosteroni is a key enzyme involved in the degradation of PAHs. Total DNA was extracted from Comamonas testosteroni,3α-HSD/CR gene was amplified by PCR. It was cloned to pMD18-T vector, and then3α-HSD/CR gene fragment was obtained. It was subcloned to pPROEXHTb vector for expression; recombinant expression plasmid ppPROEXHTb-3a was constructed. The recombinant expression plasmid was transformed into E.coli DH5α, and then was induced to express the recombinant protein3α-HSD/CR by IPTG in the Prokaryotic expression system of the E. coli, the expressed protein was identified by SDS-PAGE, and electrophoresis showed that the length of recombinant protein is the same as expected.The recombinant protein which induced the expression in the Prokaryotic expression system of the E. coli mixed with Incomplete Freund’s adjuvant, acquired the polyclonal antibody after the immunization of rabbits three times. With the polyclonal antibody, the total cell lysate of Comamonas testosteroni and engineered bacteria were extracted to detect the quantity of3α-HSD/CR using ELASA. The result indicated that engineered bacteria could express3α-HSD/CR gene evidently without the presence of substrates such as cholesterol. All of the studys above benefit for applying genetically engineered bacteria of Comamonas testosteroni to assimilate PAHs effectively.