| Nicotine, as the main alkaloid, is up to 95 percent in tobacco. Smoking for a long time will make one addictive to nicotine, and inhaling nicotine excessively will prevent central neurotransmitter from transferring, be paralytic to heart, and even be deathful. So exact nicotine content must be labeled on tobacco package by law in many countries, warning that "cigarette smoking is harmful to one's health". Interestingly, microbe and its metabolized enzyme can degrade nicotine, which is helpful for human health, improve the quality of smoke and utility of tobacco leaves and bring a lot of economic benefits.(1) Selection and identification of a microorganism effective to degrade nicotine : Some strains producing nicotine dehydrogenase (NDH) were isolated from soil by selection medium with nicotine as the sole carbon source, and a high nicotine degradation strain(Z3) was selected by experiments in shake flasks. The morphological , physiological characteristics and sequence of 16s rRNA of the strain showed that it was Arthrobacter nicotinovorans Z3, and the 16s rRNA of the strain and that of Arthrobacter nicotinovorans were 97.8% homogenous.(2) Optimization of NDH production by A. nicotinovorans Z3 in shake flask : Nicotine was the optimal carbon source, yeast extract and (NH4)2SO4 were the optimal nitrogen source. The activity of nicotine dehydrogenase was up to the greatest after culturing 48h in rotary shaker (220r/min) at 30 ℃, initial pH 7.0,5% inoculum culture.(3) Purification and characterization of NDH: NDH was purified from the culture filtrate ofA. nicotinovorans Z3 by ammonium sulfate precipitation, ion exchange chromatography and gel filtration. SDS-PAGE electrophoresis of the purified fraction mitigated a simple band, which revealed that this enzyme was purified and the molecular weight of this enzyme was 126 KDa. Results showed that optimal activity of NDH at pH 7.0, temperature 40℃. Dynamic studies demonstrated that it is an enzyme with Km of 7.694 × 10-4 mol/L and Vmax 9.95 × 10-4 mol/(min . mg). Mn2+ and Co2+ can activate the enzyme although Cu2+ inhibit it.(4) Cloning and expression of nicotine dehydrogenase gene of A. nicotinovorans: Escherichia coli engineered strain with high yield of NDH is obtained. Nicotine dehydrogenase gene of A. nicotinovorans was cloned and constructed into express vector PET-17B, recombinant PET-17B/ndh was transformed into E. coli BL21(DE3) pLYS and was highly expressed. The engineered strain could produce soluble and active recombinant NDH, and the yield of its NDH was as much 25 times as that of Z3 strain. Recombinant NDH was purified , and it is composed of three subunit(A, B and C) of Mr 87 KDa, 30 KDa and 15 KDa.(5) Condition of nicotine degradation by NDH and safety evaluation of degrading production: Nicotine was degraded by NDH with NAD, methylene blue, or 2,6-dichlorophenolindophenol supplementation in reaction system which was analyced by HPLC, nicotine decreasing 48.92%, 16.53%, 15.68%, respectively. But nicotine was not converted in absence of electron acceptors. Animal test andAmes assay indicate that the toxicity of degrading production of nicotine is less than that of nicotine.(6) Application of nicotine dehydrogenase in tobacco industry: The method of reconstituted tobacco and burley tobacco treated by NDH was researched. With the 2 % purified NDH (v/w, enzyme concentration 1.0 mg/ml) and overdose of NAD supplementation at 40℃ forl6 h, the decreasing rate of nicotine of reconstituted tobacco extract was 50.58%. In addition of purified nicotine dehydrogenase at 10 mg/kg, at 40℃ for 16 h, nicotine content of burley tobacco degreased from 5.82% to 2.85%. The quality of reconstituted tobacco and burley tobacco can be improved by NDH.
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