The Metabolism And Regulation Of The Nitrite In Bacillus Amyloliquefaciens SYBC H47:A Primary Investigation

Nitrite is potentially strong carcinogen that is widely distributed in nature.Health of humen and animals is seriously influenced by excessive nitrite in food and fodder.Thus,discovering a safety bacteria is significant not only in degrading nitrite but also in taking full advantage of microbial diversity.Bacillus amyloliquefaciens SYBC H47 which could metabolize nitrite has been screened from honey.The characteristic of nitrite metabolism by the strain was determined in LB system.Moreover,the genes related to nitrite metabolism in B.amyloliquefaciens SYBC H47 were found.The genes encoding key enzymes were successfully cloned and expressed to determine the metabolism pathway.Then,the initial regulation and control were investigated with fermentation level,enzyme level,and transcription level.Main results are as follows:1)B.amyloliquefaciens SYBC H47 was able to convert nitrate to nitrite and then convert nitrite to ammonium.The strain was not able to utilize nitrate or nitrite but ammonium as the sole source of nitrogen.2)The effect of nitrite and speed on the growth and ability to degrade nitrite of B.amyloliquefaciens SYBC H47 were studied.In addition,the nitrite accumulation in sodium nitrate medium were studied.The tolerance nitrite of B.amyloliquefaciens SYBC H47 was up to 3 mmol·L-1 and the degradation rate was more than 99%.The optimum speed was 100 r·min-1.3)Eighteen candidate genes related to nitrite generation were acquired.Among them,6 genes were deemed to coded nitrate reductase;5 genes were related to regulation and 7 genes were related to nitrate transport.Twelve candidate genes related to nitrite degration were acquired.Among them,5 genes coded nitrite reductase;5 genes were related to regulation and 2 genes were related to transport.The nitrite can be converted to ammonium by B.amyloliquefaciens SYBC H47 via dissimilatory nitrate reduction to ammonium.4)The key genes were amplified by PCR and analyzed.Nar A was encoded by the genes of NO.1592,NO.1593,NO.1594 and NO.1595.Nir BD was encoded by the genes of NO.2555 and NO.2556.The nitrite-degrading ability of Escherichia coli BL21(DE3)was improved significantly by NirBD.5)The NirBD was purified by the affinity column.The characterization was determined and analyzed using purified enzyme.The prodiction of the reaction catalyzed by Nir BD was ammonium.The remaining activity was 50% following stirring under air for 6 h.The optimum temperature and pH of NirBD was 35 ℃ and 7.0 respectively.Besides,its apparent values of Km,Vmax and kcat/Km were 1.34 mmol·L-1,30.21 U·mg-1 and 0.63 L·mmol-1·s-1.6)The effect of speed,NaNO3 and Na NO2 on the transcription of narG and nir B was measured.The transcription of narG and nir B at 100 r·min-1 was higher than 200 r·min-1.Nitrate had little effect on the transcription of narG and nirB.Nitrite could promote the transcription of nirB.