Biodegradation Of Nitrosamines And Purification Of Nitrate Reductase And Cloning Of The Encoding Gene

Tobacco-specific nitrosamines (TSNA) is a group of potential carcinogens which have been found in tobacco, tobacco products, and cigarette smoke only. The precursors are tobacco alkaloids, nitrate and nitrite. TSNA are formed and accumulated during curing, aging after harvesting. Tobacco alkaloids amines are abundant in leaves of tobacco and more than adequate for TSNA formation. Obviously, based on quantity, the major limiting factor for TSNA accumulation is nitrite. Nitrite is present in very small amount in a green leaf at harvest and during the early days of curing, but increases during tobacco curing and has been positively correlated with TSNA accumulation. The previous ways to reduce TSNA nearly based on agricultural methods and chemical medicines, but those had great restrictions. Using microbes is a new way to reduce TSNA.Denitrifying bacteria execute the function of denitrification depending on two substances, nitrate reductase and nitrite reductase, which were induced by nitrate and nitrite respectively. The denitrifying process must under the condition with no oxygen or little oxygen supplied. The method of Giltay medium was used to determine the intensity of denitrification. Dilution plate method was used to determine the optimum medium, aerobic characteristic and best grow temperature of screened bacterial strains. Two strains with high denitrification were screened out of 45 denitrifying bacteria. B88 and B237 degraded nitrate and nitrite much better, produced gas earlier and faster than other strains. Research results showed that without the execution of denitrifying process, B88 and B237 had aerobic characteristic and the growth was restricted by oxygen supply. The increase of CO₂ content had stronger inhibition on the growth of both strains. Different denitrifying bacteria had different growth speed in different medium, different temperatures. Aiming at three medium which often used on denitrifying bacteria: nitrate medium, denitrifying bacteria medium and YB medium, the optimum medium was YB medium. The best grow temperature of the two strains was about 30℃. Every strain had its highest density in certain medium, On rotary shaker in YB medium, the...