Function And Characterization Of Novel Genes From Pseudomonas Putida S16 In Nicotine Catabolism

China is largest producer and consumer of tobacco in the world, with 1/3 proportion of the global tobal consumption. While, 25% of tobacco upper leaves, dust, and other wastes were discarded and upper tobacco leaves accumulated and formed an important source of tobacco waste. These wastes, with exceeding 0.05%（w/w） content of nicotine, have been classified as “toxic and hazardous” by European Union Regulations. As the contaminant in water, nicotine has potential harmful to environment and human if appropriate disposal of the toxic tobacco waste is not performed. Nicotine could be nitresated into highly carcinogenic N-nitrosamines, so removing environmental pollutants such as nicotine is necessary.In this paper Pseudomonas putida strain S16 with nicotine degradation ability was utilized for the study. Based on data of genome and proteome, nicA1, hspA, pps₄076（mfs）, pps₄079（sapd）, pps₄080（pnao） and pps₄081（nicA2） genes were analyzed by RT-qPCR. The results revealed that pps₄076（mfs）, pps₄079（sapd）, pps₄080（pnao） and pps₄081（nicA2） genes appeared to be up-regulated in nicotineinduced P. putida S16.According to results of RT-qPCR, locations and functions of two novel genes nicA2 and pnao were identified. The mutant S16dnicA2 lost the ability to metabolize nicotine, and mutant S16 dpnao could not degrade pseudooxynicotine. The nicA2 gene was expressed in E.coli BL21（DE3）, and the transconjugant obtained nicotine degradation ability and yielded an equimolar amount of pseudooxynicotine. This result proved that NicA2 was a nicotine oxidase. Resting-cells reaction of mutant S16 dpnao showed that the nicotine metabolism pathway was blocked at pseudooxynicotine. The complemented strain S16 dpnaoC recovered the ability to degrade pseudooxynicotine at the wild-type level.