Degradation Of Quinoline By Comamonas Sp.QYY And Cloning Of A Novel Gene In Quinoline-degradating Pathway

Quinoline is a typical of nitrogen heterocyclic aromatic compound, which is teratogenic, carcinogenic and mutagenic. It can accumulate in higher organisms including human through the food chain, which has a large potential hazards on human, animals and plants. Emissions of pollutants that containing quinoline will cause a serious threat on human health and the environment. Because of the wide application of quinoline, its consumption is increasing in recent years. Quinonline has become a common pollutant in the soil and water.For the problems of quinoline on environment, this research selected quinoline as the research object, isolated quinoline-degradating bacteria from activated sludge based on the biological methods, which is the most common type of quinoline wastewater treatment. And then studing the characteristics of the quinoline high efficient degradating bacteria under different conditions, analyzing the impact of foreign substances to the degrading bacteria for the degradation of quinoline. Finally, applicating molecular biology methods, cloning the novel gene of quinoline-degradating in the bacterium, clearing the degradating mechanism. The main research results are as follows:(1)A Comamonas testosteroni was isolated from the activated sludge of petrochemical wastewater, which could uitilize quinoline as the sole source of carbon, nitrogen and energy, named Comamonas sp. QYY. The NCBI registration number is KM246901. This bacteria is smooth and milky strains, its shape is convex lens and its edge is complete. Under the scanning electron microscopy(SEM) it is bacillus about 0.5×(1-2) μm. The bacteria has resistance to ampicillin, streptomycin, kanamycin, chloramphenicol. After 24 h cultivating, the liquid medium of quinoline inorganic salt changed from colorless to pink.(2)The quinoline-degradating process can be detected most obviously when the quinoline concentration is 70 mg/L. And when the quantity is 4%, the temperature is slightly higher than 30.00℃, the p H is slightly lower than 7.00, is the best conditions of quinoline degradation. The response surface interaction experiments confirm that when the temperature is 30.61 ℃, p H is 6.83, is the best degradating conditions for quinoline-degradating bacteria QYY. And when the quinoline was degradated completely, the accumulation of 2-hydroxyquinoline reached maximum, about two-thirds of the initial concentration of quinoline. When the quinoline and 2-hydroxyquinoline exist at the same time, the quinoline-degradating bacteria QYY prioritily use of quinoline.(3)The influence of exogenous substances on the quinoline-degradating process mainly include three points:promoting hydroxylation of quinoline; promoting the degradation of 2-hydroxyquinoline; producing toxic to quinoline-degradating bacteria QYY, inhibiting the growth of bacteria. Different exogenous organic and growth factors are often only has one of the three effects, but metal ion will produce different effects due to the different concentration, one of the effects is given priority in certain concentration range(4)The quinoline-degradating bacteria QYY contain a plasmid between 4000-6000 bp. The gene which controls 2-hydroxyquinoline into 2,8-hydroxyquinoline is on the genomic DNA of quinoline-degradating bacteria QYY. The gene fragment is between 250-500 bp, which most likely t control synthesis transportation enzyme sequence fragment which deliver 2-hydroxyquinoline to bacteria interior. SDS-PAGE electrophoresis showed that compared with the beef extract peptone medium, the number of stripe increased in quinoline medium. The extra two strips(43.0-66.2 k Da and 22.0-31.0 k Da) are likely to be the key protease of quinoline degradation.