Screening, Identification Of Nicotine Degrading Bacterium Pusillimonas Sp. T2 And The Preliminary Study On Its Degradation Characteristics And Metabolic Pathway

Nicotine is a dependence-producing or even addictive alkaloid that can cause a variety of diseases when entering the human body, including lung cancer, oral cancer, esophagus cancer, cardiovascular disease and gastric dysfunction. The massive amounts of smokers and the complexity of tobacco production and processing industry in our country leaded to the vast accumulations of tobacco waste containing high concentration of nicotine, which exerted great negative impact on the environment. Microorganisms are the main force in the degradation of environmental pollutants, the application of which in the environment improvement is an efficient, reliable and environmentally friendly processing way. Therefore, to find out such microorganisms that owns the ability to degrade nicotine, to study its degradation characteristics and mechanism and to explore the law of “changing useless to treasure”, are in line with the requirements of Chinese sustainable development strategy, which are of great theoretical significance and application value.In this study, we screened two nicotine-degrading strains from activated sludge of a pesticide factory in Hangzhou, and selected the degradation ability stronger as the researching object, named T2. According to the physiological-biochemical characteristics and 16 S rDNA(Gen Bank accession number is CCTCCM 2014272) gene homology analysis, the strain was identified as Pusillimonas. This study provides the first evidence that Pusillimonas bacteria participate in nicotine degradation. The optimum p H, temperature, inoculum and initial nicotine concentration were found to be 7.0, 30 °C,100 % and 500 mg/L.Six intermediates were detected and identified as 6-hydroxy-nicotine(6HN), 6-hydroxy-N-methylmyosmine(6HMM), 6-hydroxypseudooxynicotine(6HPON), 2,6-dihydroxypyridine(2,6-DHP), 6-hydroxy-3-succinoyl-pyridine(HSP) and 2,5-dihydroxypyridine(2,5-DHP) using the method of ultraviolet spectrophotometry(UV), high-performance liquid chromatography(HPLC), and liquid chromatography mass spectroscopy(LC-MS).The broth of nicotine, 6HPON or HSP degradation turned out to be blue, light blue and green according to the further degradation experiments. The activities of 6-hydroxy-3-succinoyl-pyridine hydroxylase(HSPH) and 2,6-dihydroxypyridine hydroxylase(2,6-DHPH) were determined in the cell extracts of strain T2, reaching 0.087 and 0.012 U/mg of protein, respectively. We therefore speculated that Pusillimonas sp. strain T2 could decompose nicotine via a unique nicotine-degrading pathway as follows: the upper part, nicotine was degraded to 6HN, 6HMM, 6HPON through pyridine pathway; the lower part, 6HPON could be degraded in two shunt pathways which contained the production of both 2,5-DHP and 2,6-DHP. That was a novel variant of the pyridine and pyrrolidine pathways.