Characterization And Application Of A Nicotine Dehydrogenase From The Anthrobacter

Nicotine dehydrogenase have attracted considerable research interest in recent years because its potential application in the tobacco industry.In the paper, Nicotine dehydrogenase producing strains were isolated from soil by screening with nicotine as the sole carbon source, and among them a high nicotine degradation strain was selected by experiments in shake flasks. By the experimental results of the morphological, physiological and structural characteristics of bacterium strain and by the comparison of its whole sequence of 16s rRNA with that of the type strains of all known Arthrobacter in genebank, it was showed that the 16s rRNA of bacterium strain and that of Arthrobacter was 97.8% homogenous. Therefore bacterium strain was named as Arthrobacter sp..The effects of different factors on nicotine dehydrogenase production by Arthrobacter sp. were studied in shake flask condition. The better activity of nicotine dehydrogenase is reached after 48h of culturing in rotary shacker (220r/min) at 30℃, initial pH 7.0, 4% inoculum culture.Nicotine dehydrogenase was purified from the culture filtrate of Arthrobacter sp. by ammonium sulfate precipitation, ion exchange chromatography and gel filtration. SDS-PAGE electrophoresis of the purified fraction mitigated a simple band which revealed this enzyme was purified and the molecular weight of this enzyme was 126KDa.Result show that it had the higher activity at pH=6.85 and had the higher activity at temperature about 40℃ Dynamic studies demonstrate that it is an enzyme with Km of 7.694 ×10~-4 mol/L. Mn~2+ and Co~2+ can activate the enzyme although Cu~2+ and Fe~2+ inhibit it.The quality of reconstituted tobacco can be improved by adding 2% tobacco extract which is fermented by nicotine dehydrogenase.