一株新的尼古丁降解菌的分离鉴定及降解特性
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摘要
针对降解高毒、高危害的尼古丁微生物种类较少的实际情况筛选出具有高尼古丁降解活性的菌株,探究其降解条件及降解特性,为尼古丁的生物降解提供新的微生物资源。从烟草种植大田土壤分离纯化得到一株具有高尼古丁降解能力的菌株,对其形态学、生理生化特征、16S rRNA基因序列同源性进行了分析鉴定,探究了不同条件下尼古丁降解特性并对其动态降解过程进行了HPLC分析。经鉴定命名为Stenotrophomonas sp. ZUC-3,适宜的发酵条件为:温度30℃、起始pH 7.0、接种量10%、培养转速180 r/min,此条件下尼古丁降解率可达到91.53%,HPLC分析显示了ZUC-3降解尼古丁过程的动态变化及其高效性。研究结果为尼古丁生物降解提供了新的微生物资源,对烟草行业可持续发展具有理论意义与实际应用价值。
The aim of this study was to isolate and identify a strain with high nicotine degradation activity in the situation where there are few kinds of nicotine-degrading microorganisms available, and to explore the conditions and characteristics of nicotine degradation, which will provide a new microbiological resource for the biodegradation of nicotine. A strain with high activity of nicotine degradation was obtained by isolation and purification from tobacco field soil. The strain was identified by morphological,physio-biochemical and 16 S rRNA gene analysis, and the characteristics of nicotine degradation under different conditions were investigated with dynamic process by HPLC analysis. The optimum condition of nicotine degradation by Stenotrophomonas sp.ZUC-3 was 30 ℃ with 10% inoculum at the initial pH 7.0 and 180 r/min, in which the rate of nicotine degradation by Stenotrophomonas sp. ZUC-3 reached 91.53%. HPLC analysis illustrated the dynamic process and high efficiency of nicotine degradation. The study provides a new microbiological resource for biodegradation of nicotine and has theoretical and practical significance in tobacco industry.
The aim of this study was to isolate and identify a strain with high nicotine degradation activity in the situation where there are few kinds of nicotine-degrading microorganisms available, and to explore the conditions and characteristics of nicotine degradation, which will provide a new microbiological resource for the biodegradation of nicotine. A strain with high activity of nicotine degradation was obtained by isolation and purification from tobacco field soil. The strain was identified by morphological,physio-biochemical and 16 S rRNA gene analysis, and the characteristics of nicotine degradation under different conditions were investigated with dynamic process by HPLC analysis. The optimum condition of nicotine degradation by Stenotrophomonas sp.ZUC-3 was 30 ℃ with 10% inoculum at the initial pH 7.0 and 180 r/min, in which the rate of nicotine degradation by Stenotrophomonas sp. ZUC-3 reached 91.53%. HPLC analysis illustrated the dynamic process and high efficiency of nicotine degradation. The study provides a new microbiological resource for biodegradation of nicotine and has theoretical and practical significance in tobacco industry.
引文
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[21]LIU Y,WANG L,HUANG K,et al.Physiological and biochemical characterization of a novel nicotine-degrading bacterium Pseudomonas geniculata N1[J].Plos One,2014,9(1):e84399.
[22]YU H,TANG H,ZHU X,et al.Molecular mechanism of nicotine degradation by a newly isolated strain,Ochrobactrum sp.strain SJY1[J].Applied&Environmental Microbiology,2015,81(1):272-281.
[23]SHANG C,CHEN A,CHEN G,et al.Microbial niofertilizer decreases nicotine content by improving soil nitrogen supply[J].Appl Biochem Biotechnol,2017,181(1):1-14.
[24]SU Y,HE X,SHI S,et al.Biodegradation of lignin and nicotine with white rot fungi for the delignification and detoxification of tobacco stalk[J].Bmc Biotechnology,2016,16(1):81.
[25]YAO Y,TANG H,SU F,et al.Comparative genome analysis reveals the molecular basis of nicotine degradation and survival capacities of Arthrobacter[J].Scientific Reports,2015,5:8642.
[26]TANG H,YAO Y,ZHANG D,et al.A novel NADH-dependent and FAD-containing hydroxylase is crucial for nicotine degradation by Pseudomonas putida[J].Journal of Biological Chemistry,2011,286(45):39179-39187.
[27]HUANG H,YU W,WANG R,et al.Genomic and transcriptomic analyses of Agrobacterium tumefaciens S33 reveal the molecular mechanism of a novel hybrid nicotine-degrading pathway[J].Scientific Reports,2017,7(1):4813.
[28]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001.DONG X Z,CAI M Y.System Identification Manual of Common Bacterial[M].Beijing:Science Press,2001.
[29]GURUSAMY R,NATARAJAN S.Current status on biochemistry and molecular biology of microbial degradation of nicotine[J].Scientific World Journal,2013(1):1653-1656.
[30]李晓华,梅枫,孔雯,等.尼古丁微生物降解代谢机制和应用的研究进展[J].中南民族大学学报(自然科学版),2017,36(4):27-35.LI X H,MEI F,KONG W,et al.Research development of nicotine degrading mechanism in microorganisms and their applications[J].Journal of South-Central University for Nationalities(Natural Science Edition),2017,36(4):27-35.
[31]王晓萍,石贤爱,王毓虹,等.尼古丁降解微生物的研究进展及潜在价值[J].福建师大学报(自然科学版),2017(3):109-116.WANG X H,SHI A X,WANG Y H,et al.Research development and potential value of nicotine degrading microorganisms[J].Journal of Fujian Normal University(Natural Science Edition),2017(3):109-116.
[32]TARARINA M A,JANDA K D,ALLEN K N.Structural analysis provides mechanistic insight into nicotine oxidoreductase from Pseudomonas putida[J].Biochemistry,2016,55(48):6595-6598.
[33]ZHANG K,WU G,TANG H,et al.Structural basis for the transcriptional repressor NicR2 in nicotine degradation from Pseudomonas[J].Molecular Microbiology,2016,103(1):165-180.
[34]BRANDSCH R.Microbiology and biochemistry of nicotine degradation[J].Applied Microbiology&Biotechnology,2006,69(5):493-498.
[35]LI H J,LI X M,DUAN Y Q,et al.Biotransformation of nicotine by microorganism:the case of Pseudomonas spp.[J].Appl Microbiol Biotechnol,2010,86(1):11-17.
[36]IGLOI G L,BRANDSCH R.Sequence of the165-kilobase catabolic plasmid pAO1 from Arthrobacter nicotinovorans and identification of a pAO1-dependent nicotine uptake system[J].Journal of Bacteriology,2003,185(6):1976-1986.
[37]RAMOS P L,VAN T S,THOMPSON F L,et al.Screening for endophytic nitrogen-fixing bacteria in Brazilian sugar cane varieties used in organic farming and description of Stenotrophomonas pavanii sp.nov[J].Int J Syst Evol Microbiol,2011,61(4):926-931.
[38]RAMAN G,MOHAN K,MANOHAR V,et al.Biodegradation of nicotine by a novel nicotine-degrading bacterium,Pseudomonas plecoglossicida TND35 and its new biotransformation intermediates.[J].Biodegradation,2014,25(1):95-107.
[39]WANG S N,XU P,TANG H Z,et al.Biodegradation and detoxification of nicotine in tobacco solid waste by a Pseudomonas sp[J].Biotechnology Letters,2004,26(19):1493-1496.
[40]RUAN A,MIN H,ZHU W.Studies on biodegradation of nicotine by Arthrobacter sp.strain HF-2[J].J Environ Sci Health B,2006,41(7):1159-1170.
[41]MA Y,WEI Y,QIU J,et al.Isolation,transposon mutagenesis,and characterization of the novel nicotine-degrading strain Shinella,sp.HZN7[J].Applied Microbiology&Biotechnology,2014,98(6):2625-2636.
[42]YU H,TANG H,LI Y,et al.Molybdenum-containing nicotine hydroxylase genes in a nicotine degradation pathway that is a variant of the pyridine and pyrrolidine pathways[J].Applied&Environmental Microbiology,2015,81(24):8330-8338.
[2]EDWARDS S H,ROSSITER L M,TAYLOR K M,et al.Tobacco-Specific nitrosamines in the tobacco and mainstream smoke of U.S.commercial cigarettes[J].Chemical Research in Toxicology,2017,30(2):540-551.
[3]ZHONG W,ZHU C,SHU M,et al.Degradation of nicotine in tobacco waste extract by newly isolated Pseudomonas,sp.ZUTSKD[J].Bioresource Technology,2010,101(18):6935-6941.
[4]MA Y,WEN R,QIU J,et al.Biodegradation of nicotine by a novel strain Pusillimonas[J].Research in Microbiology,2015,166(2):67-71.
[5]YAO Y,TANG H,REN H,et al.Genome Sequence of a Nicotine-Degrading Strain of Arthrobacter[J].Journal of Bacteriology,2012,194(20):5714-5.
[6]CHEN C M,LI X M,YANG J K,et al.Isolation of nicotine-degrading bacterium Pseudomonas sp.Nic22,and its potential application in tobacco processing[J].International Biodeterioration&Biodegradation,2008,62:226-231.
[7]YUAN Y J,LU Z X,WU N,et al.Isolation and preliminary characterization of a novel nicotine-degrading bacterium,Ochrobactrum intermedium,DN2[J].International Biodeterioration&Biodegradation,2005,56(1):45-50.
[8]MENG X J,LU L L,GU G F,et al.A novel pathway for nicotine degradation by Aspergillus oryzae 112822isolated from tobacco leaves[J].Research in Microbiology,2010,161(7):626-633.
[9]CHEN C,LI X,YANG J,et al.Isolation of nicotine-degrading bacterium Pseudomonas sp.Nic22,and its potential application in tobacco processing[J].International Biodeterioration&Biodegradation,2008,62(3):226-231.
[10]ENAMORADO M F,ONDACHI P W,COMINS D L.AFive-Step Synthesis of(S)-Macrostomine from(S)-Nicotine[J].Organic Letters,2010,12(20):4513-4515.
[11]YU H,TANG H,XU P.Green strategy from waste to value-added-chemical production:efficient biosynthesis of 6-hydroxy-3-succinoyl-pyridine by an engineered biocatalyst[J].Scientific Reports,2014,4:5397.
[12]杨艳坤,席宇,张广乐,等.微生物降解尼古丁的分子生物学研究进展[J].中国烟草科学,2010,31(4):76-81.YANG Y K,XI Y,ZHANG G L,et al.Molecular biology progress in nicotine degradation by microorganisms[J].Chinese Tobacco Science,2010,31(4):76-81.
[13]LIU J,MA G,CHEN T,et al.Nicotine-degrading microorganisms and their potential applications[J].Appl Microbiol Biotechnol,2015,99(9):3775-3785.
[14]张彦东,罗昌荣,王辉龙,等.微生物降解烟碱研究应用进展[J].烟草科技,2003(12):3-7.ZHANG Y D,LUO R C,WANG H L,et al.Advances in microbial degradation of nicotine and its application[J].Tobacco Science&Technology,2003(12):3-7.
[15]MIHASAN M,BRANDSCH R.pAO1 of Arthrobacter nicotinovorans and the spread of catabolic traits by horizontal gene transfer in gram-positive soil bacteria[J].Journal of Molecular Evolution,2013,77(1-2):22-30.
[16]MALPHETTES L,WEBER C C,ELBABA M D,et al.A novel mammalian expression system derived from components coordinating nicotine degradation in Arthrobacter nicotinovorans p AO1[J].Nucleic Acids Research,2005,33(12):e107.
[17]LI H,XIE K,YU W,et al.Nicotine dehydrogenase complexed with 6-Hydroxypseudooxynicotine oxidase involved in the hybrid nicotine-degrading pathway in Agrobacterium tumefaciens S33[J].Appl Environ Microbiol,2016,82(6):1745-1755.
[18]WANG S,HUANG H,XIE K,et al.Identification of nicotine biotransformation intermediates by Agrobacterium tumefaciens strain S33 suggests a novel nicotine degradation pathway[J].Applied Microbiology&Biotechnology,2012,95(6):1567-1578.
[19]HU H,WANG W,TANG H,et al.Characterization of pseudooxynicotine amine oxidase of Pseudomonas putida S16 that is crucial for nicotine degradation[J].Scientific Reports,2015,5:17770.
[20]JIANG Y,TANG H,WU G,et al.Functional identification of a novel gene,moaE,for3-succinoylpyridine degradation in Pseudomonas putida S16[J].Scientific Reports,2015,5:13464.
[21]LIU Y,WANG L,HUANG K,et al.Physiological and biochemical characterization of a novel nicotine-degrading bacterium Pseudomonas geniculata N1[J].Plos One,2014,9(1):e84399.
[22]YU H,TANG H,ZHU X,et al.Molecular mechanism of nicotine degradation by a newly isolated strain,Ochrobactrum sp.strain SJY1[J].Applied&Environmental Microbiology,2015,81(1):272-281.
[23]SHANG C,CHEN A,CHEN G,et al.Microbial niofertilizer decreases nicotine content by improving soil nitrogen supply[J].Appl Biochem Biotechnol,2017,181(1):1-14.
[24]SU Y,HE X,SHI S,et al.Biodegradation of lignin and nicotine with white rot fungi for the delignification and detoxification of tobacco stalk[J].Bmc Biotechnology,2016,16(1):81.
[25]YAO Y,TANG H,SU F,et al.Comparative genome analysis reveals the molecular basis of nicotine degradation and survival capacities of Arthrobacter[J].Scientific Reports,2015,5:8642.
[26]TANG H,YAO Y,ZHANG D,et al.A novel NADH-dependent and FAD-containing hydroxylase is crucial for nicotine degradation by Pseudomonas putida[J].Journal of Biological Chemistry,2011,286(45):39179-39187.
[27]HUANG H,YU W,WANG R,et al.Genomic and transcriptomic analyses of Agrobacterium tumefaciens S33 reveal the molecular mechanism of a novel hybrid nicotine-degrading pathway[J].Scientific Reports,2017,7(1):4813.
[28]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001.DONG X Z,CAI M Y.System Identification Manual of Common Bacterial[M].Beijing:Science Press,2001.
[29]GURUSAMY R,NATARAJAN S.Current status on biochemistry and molecular biology of microbial degradation of nicotine[J].Scientific World Journal,2013(1):1653-1656.
[30]李晓华,梅枫,孔雯,等.尼古丁微生物降解代谢机制和应用的研究进展[J].中南民族大学学报(自然科学版),2017,36(4):27-35.LI X H,MEI F,KONG W,et al.Research development of nicotine degrading mechanism in microorganisms and their applications[J].Journal of South-Central University for Nationalities(Natural Science Edition),2017,36(4):27-35.
[31]王晓萍,石贤爱,王毓虹,等.尼古丁降解微生物的研究进展及潜在价值[J].福建师大学报(自然科学版),2017(3):109-116.WANG X H,SHI A X,WANG Y H,et al.Research development and potential value of nicotine degrading microorganisms[J].Journal of Fujian Normal University(Natural Science Edition),2017(3):109-116.
[32]TARARINA M A,JANDA K D,ALLEN K N.Structural analysis provides mechanistic insight into nicotine oxidoreductase from Pseudomonas putida[J].Biochemistry,2016,55(48):6595-6598.
[33]ZHANG K,WU G,TANG H,et al.Structural basis for the transcriptional repressor NicR2 in nicotine degradation from Pseudomonas[J].Molecular Microbiology,2016,103(1):165-180.
[34]BRANDSCH R.Microbiology and biochemistry of nicotine degradation[J].Applied Microbiology&Biotechnology,2006,69(5):493-498.
[35]LI H J,LI X M,DUAN Y Q,et al.Biotransformation of nicotine by microorganism:the case of Pseudomonas spp.[J].Appl Microbiol Biotechnol,2010,86(1):11-17.
[36]IGLOI G L,BRANDSCH R.Sequence of the165-kilobase catabolic plasmid pAO1 from Arthrobacter nicotinovorans and identification of a pAO1-dependent nicotine uptake system[J].Journal of Bacteriology,2003,185(6):1976-1986.
[37]RAMOS P L,VAN T S,THOMPSON F L,et al.Screening for endophytic nitrogen-fixing bacteria in Brazilian sugar cane varieties used in organic farming and description of Stenotrophomonas pavanii sp.nov[J].Int J Syst Evol Microbiol,2011,61(4):926-931.
[38]RAMAN G,MOHAN K,MANOHAR V,et al.Biodegradation of nicotine by a novel nicotine-degrading bacterium,Pseudomonas plecoglossicida TND35 and its new biotransformation intermediates.[J].Biodegradation,2014,25(1):95-107.
[39]WANG S N,XU P,TANG H Z,et al.Biodegradation and detoxification of nicotine in tobacco solid waste by a Pseudomonas sp[J].Biotechnology Letters,2004,26(19):1493-1496.
[40]RUAN A,MIN H,ZHU W.Studies on biodegradation of nicotine by Arthrobacter sp.strain HF-2[J].J Environ Sci Health B,2006,41(7):1159-1170.
[41]MA Y,WEI Y,QIU J,et al.Isolation,transposon mutagenesis,and characterization of the novel nicotine-degrading strain Shinella,sp.HZN7[J].Applied Microbiology&Biotechnology,2014,98(6):2625-2636.
[42]YU H,TANG H,LI Y,et al.Molybdenum-containing nicotine hydroxylase genes in a nicotine degradation pathway that is a variant of the pyridine and pyrrolidine pathways[J].Applied&Environmental Microbiology,2015,81(24):8330-8338.