Isolation And Purification Of Nicotine Demethylase From Aspergillus Oryzae And Nicotine-induced Transcriptome Analysis

As a major alkaloid in tobacco plants,nicotine plays a critical role in smoking addiction and is well known to be harmful to human beings because it easily crosses the blood-brain barrier and biological membranes.Nicotine is also an toxic substance produced during tobacco processing,and may cause serious environmental pollution.Therefore,it is very necessary to reduce nicotine content in tobacco and tobatto waste to decrease harmful health effects of cigarette smoking and ecotoxicity.Although a variety of physical and chemical methods have been employed to control the content of nicotine,negative impact on the quality of the aroma of cigarette products using these methods could not be avoid.Microorganisms have more obvious advantages in degrading nicotine with advantages such as increasing the incense smoke,reducing irritation,improving quality of flue gas,and decreasing health risks of smoking.Addition to their strong ability of nicotine degradation,microorganisms are very abundant in nature,which facilitate the use of microorganisms in the industrial production.In recent years,a lot of basic and applied studies on microbial degradation of nicotine has beed carried out.Those studies are mainly focused on bacteria,such as the genus Arthrobacter,and Pseudomonas spp.The studies of nicotine degradation in fungi are realitively rare,and nly the product of initial step of nicotine demethylation has been reported in Microsporum gypseum,Pellicularia filamentosa JTS-208 and Cunninghamella echinulata.Our group filled the gaps in nicotine degradation study by first proposed a nicotine degradation path way in Aspergillus oryzae 112822 and identified of some important metabolites.But the enzymies and molecular mechanism incolving in the nicotine degradation pathway are still unknown.In this paper,we worked on the first step of A.oryzae 112822 nicotine degradation path way-the nicotine demethylation reaction to explore the molecular biologic basis and the enzymology of fungal nicotine demethylase.So far,three cytochrome P450 monooxygenase enzymes in plant been successfully identified as nicotine demethylase,.We try to purify the nicotine demethlase from soluble supernatant and membrane protein components by ammonium sulfate precipitation,Source-15Q anion-exchange column chromatography,Gigapite K-100S column chromatography,and Superdex-200 gel filtration.Although we haven’t obtained electrophoresis pure nicotine demethylase after these purification steps,we have acquired a fragement with the active protion containing proteins between 50kDa～70kDa on SDS-PAGE which consist with the P450 predicted molecular weight.The protein bands ware isolated from SDS-PAGE and the protein identification was conducted by peptide mass fingerprinting.The fungal cytochrome P450 database(http://p450.ricebla st.snu.ac.kr/index.php?a=view)were used for data searching and comparison,and nine P450 proteins were successfully identified.In order to further study the genes involved nicotine metabolic pathway,A.oryzae 112822 was cultivated in medium containing nicotine and nicotine-free YD medium as control,respectively,and total RNA was extracted for constructing RNA library and RNA sequencing.By DEGseq software analysis,we found 4169 genes expressing differently between two culture conditions.Using fungal cytochrome P450 database for comparison analysis,we found 46 cytochrome P450 genes(cyp gene)display transcriptional differences,and 14 cytochrome P450 genes are up-regulate.By comparing the nicotine up-regulate cyp genes with the 9 cyp genes be were deduced through protein peptide mapping,we found that four cyp genes appeared in both conditions.The four cyp genes are CYP577A4,CYP55A5v2,CYP5113A1,and CYP531C3,with peptide mapping matching rates 10%,10%,15%,and 17%,respectively.The differences of the levels of transcription(log2 Ratio(NiC/YD))for the four cyp genes were 8.18621,1.86973,1.366636,and 1.305211,respectively.CYP577A4 has the highest level of transcriptional differences,suggesting it could be the nicotine demethylase gene in A.oryzae 112822.