Screening,Identification And Degradation Characteristics Of Nicotine Degradation Bacteria In Lasioderma Serricorne

Tobacco processing produces a large amount of nicotine containing waste,which seriously harms the environment and human health.It has become one of the urgent problems to be solved in today's society.Lasioderma serricorne can complete its life cycle by eating tobacco leaves,and can metabolize most of the nicotine in the body into other non-toxic alkaloids.There are many endophytes in its body,which play an important role in degrading nicotine.In this study,Lasioderma serricorne was used as the research object,and three nicotine degradation bacteria J1,J2 and YC7 were isolated and screened on the tobacco plate.Morphology and phylogenetic tree identification of 16S r RNA sequence showed that J1 and J2 belonged to Mixta;and YC7 belonged to Bacillus;Using pure nicotine and nicotine in tobacco leaves as two substrates,the nicotine degrading activity of the strain fermentation broth and extracellular crude enzyme solution of the screened high nicotine degradation bacteria was tested by UV spectrophotometer.Then use whole genome sequencing analysis and KEGG annotation to find key genes in the nicotine metabolic pathway.The nicotine dehydrogenase(NDH)in both J1 and YC7 in the pyridine pathway was selected for molecular docking and functional verification,which laid a theoretical foundation for the treatment of nicotine pollution in the environment.The main results of this study are as follows:1.Separation screening and identification results.Twelve high nicotine degradation bacteria were isolated and screened from Lasioderma serricorne by gradient dilution method.Four kinds of tobacco leaf culture medium,gradually increase the content of tobacco leaves in the medium,and the re-screening of high nicotine degradation bacteria were carried out.And the tobacco leaves with 1 g,2 g,4 g,8 g and 10 g in 100 m L medium(tobacco leaf medium d)had the highest content.When the highest concentration was 10 g/100 m L,three strains of J1,J2 and YC7 were screened.The identification of the phylogenetic tree constructed by morphology and 16S r RNA sequence shows that J1,J2 have the highest similarity with Mixta calida LMG 25383^T,respectively reached 99.80%and 99.73%,belonging to Mixta;YC7 has the highest similarity with Bacillus halotolerans ATCC 25096^T,reaching 99.73%,belonging to Bacillus.The results of different enzyme activities showed that J1 and J2 do not produce?-glucosidase,cellulase,protease,and amylase,while YC7 produces?-glucosidase,cellulase,protease,and amylase.There was almost no difference in the detection results of various activities of J1 and J2.The 16S r RNA sequence identified both belong to Mixta,so J1 and J2were considered to be duplicate strains,and J1 was selected as the test strain in subsequent studies.2.Test results of degrading nicotine activity.Using pure nicotine and nicotine in tobacco leaves as two substrates,the nicotine degrading activity of the strain fermentation broth and extracellular crude enzyme solution of the screened high nicotine degradation bacteria was tested by UV spectrophotometer.The test results of the fermentation broth degrading pure nicotine showed that:at 37?and nicotine concentration of 1 g/L,the nicotine degradation rate of J1 was 64.60%at 15 min,91.60%at 4 h,and 95.80%at 8 h;the nicotine degradation rate of YC7 was 63.16%at 15 min,90.90%at 4 h,and 95.65%at 8 h;The nicotine degradation efficiency of J1 and YC7 has little significant difference.After treatment with J1 bacterial solution for 5 days,the nicotine content of the control was reduced from 4.15%to 3.57%,and the nicotine degradation rate of the tobacco leaves was 13.98%.After YC7 bacterial solution treated tobacco leaves for 5 days,the nicotine content of the control decreased from 4.15%to3.69%,and the nicotine degradation rate of tobacco leaves was 11.08%.The degradation rate of nicotine in tobacco leaves treated with J1 bacterial solution was slightly higher than that degradation rate of nicotine in tobacco leaves treated with YC7.The test results of nicotine degradation activity of extracellular crude enzyme solution showed that J1 and YC7 showed the highest activity of nicotine degradation enzyme in crude enzyme solution with 80%ammonium sulfate saturation,J1 was 2600 U/m L,YC7 was 3430 U/m L,J1 was slightly less than YC7.Add 2 m L of ammonium sulfate 80%gradient precipitated crude enzyme solution to 1 g of cut tobacco(the concentration of crude enzyme solution is 4.360 mg/m L).After treating the tobacco leaves with J1 crude enzyme solution for 2 days,the nicotine content decreased from 4.57%of the control to 3.81%,and the nicotine degradation rate of the tobacco leaves was 16.63%,and compared with the control,there are significant differences.After treating tobacco leaves with YC7 crude enzyme solution for 2 days,the nicotine content in the control decreased from 4.57%to 3.87%,and the nicotine degradation rate of tobacco leaves was 15.32%.The nicotine degradation rate of tobacco leaves treated with crude enzyme solution of J1 was slightly higher than that nicotine degradation rate of tobacco leaves treated with crude enzyme solution of YC7,and compared with the control,there are significant differences.3.Whole genome sequencing analysis results.High nicotine endurance strains were functionally annotated through databases such as Uniprot,Refseq,Pfam,Nr,Tigrfams,GO,KEGG,COG and KEGG Pathway,and find the nicotine metabolic pathway and key genes or enzymes of nicotine metabolism in the strains.KEGG annotation results show that:J1 has nicotine dehydrogenase(NDH,nicotine dehydrogenase)in the pyridine pathway,3-succinoyl semialdehyde-pyridne dehydrogenase(SAPD,3-succinoyl semialdehyde-pyridne dehydrogenase)?N-formylmaleamate deformylase(NFO,N-formylmaleamate deformylase)?maleamate amidohydrolase(AMI,maleamate amidohydrolase)in the pyrrole pathway.YC7has nicotine dehydrogenase(NDH,nicotine dehydrogenase)in pyridine pathway,3-succinylpyridine monooxygenase(SPM,3-succinoylpyridine monooxygenase)?N-formylmaleamide deformylase(NFO,N-formylmaleamate deformylase)?maleamate amidohydrolase(AMI,maleamate amidohydrolase)in the pyrrole pathway.4.Degradation gene mining and functional verification results.Nicotine dehydrogenase(NDH)both in J1 and YC7 in the pyridine pathway was selected for molecular docking and functional verification.The binding ability of nicotine and nicotine degrading enzymes was investigated by molecular docking.The docking energy of nicotine dehydrogenase(NDH)of J1 with nicotine was-5.87 kcal/mol,on the A chain,87 site?285 site Ile(Isoleucine),280 site?284 site Asp(Aspartic acid),281 site Phe(Phenylalanine),282 site Leu(Leucine),283 site Lys(Lysine)can be docked with nicotine.The docking energy of nicotine dehydrogenase(NDH)of YC7 with nicotine was-5.51 kcal/mol,on the B chain,21 site?66 site?88 site Ile(Isoleucine),73 site Val(Valine),86 site Ala(Alanine),89 site Gly(Glycine),90 IEU(Leucine)can be docked with nicotine.1 g/L nicotine induced J1 for 24 h,the relative expression of ndh gene was up-regulated by 15.978 times compared with the control.1 g/L nicotine induced YC7 for 24 h,the relative expression of ndh gene was up-regulated by 11.672 times compared with the control.0.8%tobacco leaf extract induced J1 for 24 h,the relative expression of ndh gene was up-regulated by 1.117 times compared with the control.0.8%tobacco leaf extract induced YC7 for 24 h,the relative expression of ndh gene was up-regulated by 1.370 times compared with the control.The results of q PCR verification showed that the expression of ndh gene in J1 and YC7 was significantly up-regulated after nicotine induction,and ndh gene played an important role in the process of nicotine degradation.