烟碱降解菌的分离鉴定、降解特性及其降解途径的初步研究
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摘要
烟碱俗称尼古丁,是烟草中的主要生物碱,过量吸入烟碱则会抑制中枢系统,麻痹心脏,甚至有生命危险;同时烟草加工过程中产生了含高浓度烟碱的废弃物,严重污染了环境;而且我国烟草上部烟叶烟碱含量普遍偏高,不能用于烟组配方造成了资源浪费和经济损失,因此有效地控制烟草和环境中的烟碱含量,对于维护人类健康和环境有着深远的意义。在工业上利用微生物及其酶来降解烟草中的烟碱,比起传统的物理化学法,不仅可以产生更少有害副产品,而且可以改进烟气品质,提高烟叶资源利用率,有重大的经济效益。
本研究以烟碱为唯一碳源,从湖北襄樊烟草种植基地的土壤根际土和烟叶废弃物中分离得到29株具有烟碱降解能力的菌株,降解率都在70%以上,其中19株烟碱降解率能达到90%以上。其中12株烟碱降解菌经常规的形态观察、生理生化分析和16S rDNA序列同源性分析,这12株烟碱降解菌分别属于节杆菌属、假单胞菌属、苍白杆菌属和根癌农杆菌属。
通过一系列实验,初步确定根癌农杆菌BKCER11菌株在烟碱浓度0.5g/L-5g/L内生长良好,培养基烟碱浓度大于5g/L时,BKCER11菌株生长受到严重抑制,也不能降解烟碱;该菌在温度20℃-37℃范围内可以生长良好并能降解烟碱,其最适生长温度为30℃;在pH5.5-8.0范围内BKCER11菌株可以生长良好并能降解烟碱,其最适pH为5.5-6.0;经过正交优化,BKCER11菌株烟碱降解的最适条件为:烟碱初始浓度为2g/L、温度为30℃、初始pH为5.5-6.0、以蛋白胨和牛肉膏各0.5%作为复合氮源、微量元素的浓度为5mL/L,降解率能达到96.63%。
利用GC-MS初步分析了根癌农杆菌BKCER11菌株降解烟碱的中间代谢产物,检测到有3-(2,3,4-三氢-5-吡咯基)-吡啶和可替宁两种新物质的积累。这与已知的微生物代谢烟碱的三种途径中所涉及到的物质均不一样。
Nicotine, is a major alkaloid in tobacco Plants. Excessively taking into nicotine will restrain central nervous system, paralyse the heart, and even cause death; In manuafeutring tobacco products, wastes with high content of nicotine as the main toxic compound are produced, which seriouly contaminate the environment; and the nicotine content in the upper tobacco leaf from our country are generally high so that unable to be used as tobacco blend, which caused waste of resource and economic losses. Thus exploring an effective way to reduce the nicotine content in cigarette and environment is of great significance to human health. Interestingly, microbe and its metabolized enzyme can degrade nicotine, which is not only helpful for human health, but also improve the quality of smoke and utility of tobacco leaves and bring a lot of economic benefits.
This study utilized nicotine as the sole source of carbon, 29 bacterial strains was isolated from soil and tobacco waste. The degradation efficiency test showed that all of the stains’degradation efficiency were more than 70%, and 19 strains among the isolates whose degradation efficiency were more than 90%. Further study was basing on 12 strains which have been isolated, by the experimental results of the morphological,physiological and biochemical analysis and the comparison of their 16S rDNA analysis, these 12 strains were identified as Arthrobacter sp., Pseudomonas sp., Ochrobactrum intermedium and Agrobacterium tumefaciens.
Studies on the Agrobacterium tumefaciens BKCER11 strain’s degradation capability and optimization of fermentation conditions were carried out. Finaly the results showed that the BKCER11 strain could tolerate high concentration of nicotine up to 5g/L, when the concentration of nicotine in the culture medium was greater than 5g/L, the BKCER11 strain’s growth was highly inhibited, and the nicotine in the medium could not be degraded. The BKCER11 strain not only has a wide tolerance to pH, but also possesses the ability to tolerate temperature. Lots of experiments showed that the BKCER11 strain was stable at pH5.5-8 and growed well, and the optimum pH for the strain’s growing and degradating nicotine was 5.5; moreover the BKCER11 strain was stable at 20℃-37℃and growed well, and the optimum temperature was 30℃. In order to define the strain’s optimum degradation condition orthogonal test was used in the next study. The results indicated that the BKCER11 strain degradation efficiency could reach to 96.63%, when the peptone and beef extract simultaneously used as the complex nitrogen sources of the medium and their content were 0.5% respectively; besides some microelement was at the content of 5mL/L and the medium’s pH was at the range of 5.5-6.0, moreover the most important is the original content of nicotine was 2g/L and kept a temperature at 30℃.
The intermediate metabolite was detected by using the method of GC-MS. Two novel intermediate metabolites, 3-(2,3,4-dihydro-3H-pyrrol-5-y1)-pyridine and cotinine were detected in the culture medium. They were both different from the substances involed in the three everkown pathways of microbial metabolism of nicotine.
本研究以烟碱为唯一碳源,从湖北襄樊烟草种植基地的土壤根际土和烟叶废弃物中分离得到29株具有烟碱降解能力的菌株,降解率都在70%以上,其中19株烟碱降解率能达到90%以上。其中12株烟碱降解菌经常规的形态观察、生理生化分析和16S rDNA序列同源性分析,这12株烟碱降解菌分别属于节杆菌属、假单胞菌属、苍白杆菌属和根癌农杆菌属。
通过一系列实验,初步确定根癌农杆菌BKCER11菌株在烟碱浓度0.5g/L-5g/L内生长良好,培养基烟碱浓度大于5g/L时,BKCER11菌株生长受到严重抑制,也不能降解烟碱;该菌在温度20℃-37℃范围内可以生长良好并能降解烟碱,其最适生长温度为30℃;在pH5.5-8.0范围内BKCER11菌株可以生长良好并能降解烟碱,其最适pH为5.5-6.0;经过正交优化,BKCER11菌株烟碱降解的最适条件为:烟碱初始浓度为2g/L、温度为30℃、初始pH为5.5-6.0、以蛋白胨和牛肉膏各0.5%作为复合氮源、微量元素的浓度为5mL/L,降解率能达到96.63%。
利用GC-MS初步分析了根癌农杆菌BKCER11菌株降解烟碱的中间代谢产物,检测到有3-(2,3,4-三氢-5-吡咯基)-吡啶和可替宁两种新物质的积累。这与已知的微生物代谢烟碱的三种途径中所涉及到的物质均不一样。
Nicotine, is a major alkaloid in tobacco Plants. Excessively taking into nicotine will restrain central nervous system, paralyse the heart, and even cause death; In manuafeutring tobacco products, wastes with high content of nicotine as the main toxic compound are produced, which seriouly contaminate the environment; and the nicotine content in the upper tobacco leaf from our country are generally high so that unable to be used as tobacco blend, which caused waste of resource and economic losses. Thus exploring an effective way to reduce the nicotine content in cigarette and environment is of great significance to human health. Interestingly, microbe and its metabolized enzyme can degrade nicotine, which is not only helpful for human health, but also improve the quality of smoke and utility of tobacco leaves and bring a lot of economic benefits.
This study utilized nicotine as the sole source of carbon, 29 bacterial strains was isolated from soil and tobacco waste. The degradation efficiency test showed that all of the stains’degradation efficiency were more than 70%, and 19 strains among the isolates whose degradation efficiency were more than 90%. Further study was basing on 12 strains which have been isolated, by the experimental results of the morphological,physiological and biochemical analysis and the comparison of their 16S rDNA analysis, these 12 strains were identified as Arthrobacter sp., Pseudomonas sp., Ochrobactrum intermedium and Agrobacterium tumefaciens.
Studies on the Agrobacterium tumefaciens BKCER11 strain’s degradation capability and optimization of fermentation conditions were carried out. Finaly the results showed that the BKCER11 strain could tolerate high concentration of nicotine up to 5g/L, when the concentration of nicotine in the culture medium was greater than 5g/L, the BKCER11 strain’s growth was highly inhibited, and the nicotine in the medium could not be degraded. The BKCER11 strain not only has a wide tolerance to pH, but also possesses the ability to tolerate temperature. Lots of experiments showed that the BKCER11 strain was stable at pH5.5-8 and growed well, and the optimum pH for the strain’s growing and degradating nicotine was 5.5; moreover the BKCER11 strain was stable at 20℃-37℃and growed well, and the optimum temperature was 30℃. In order to define the strain’s optimum degradation condition orthogonal test was used in the next study. The results indicated that the BKCER11 strain degradation efficiency could reach to 96.63%, when the peptone and beef extract simultaneously used as the complex nitrogen sources of the medium and their content were 0.5% respectively; besides some microelement was at the content of 5mL/L and the medium’s pH was at the range of 5.5-6.0, moreover the most important is the original content of nicotine was 2g/L and kept a temperature at 30℃.
The intermediate metabolite was detected by using the method of GC-MS. Two novel intermediate metabolites, 3-(2,3,4-dihydro-3H-pyrrol-5-y1)-pyridine and cotinine were detected in the culture medium. They were both different from the substances involed in the three everkown pathways of microbial metabolism of nicotine.
引文
[1]张槐苓,葛翠英,穆怀静,等.烟草分析与检验.郑州:河南科学技术出版社,1994,158-170
[2]阎克玉.烟草化学.郑州:郑州大学出版社,2002年6月. 112
[3]肖协忠.烟草化学.北京:中国农业科技出版社,1997,161. 180
[4] Wada E, Kisaki T. Autoxidation of Nicotine[J]. Arch Biochem Biophys, 1959, 79: 124-130
[5]于川芳,王兵,罗登山,等.国产白肋烟与津巴布韦、马拉维及美国白肋烟的分析比较[J].烟草科技,1999,(4):6-8
[6] Wada E, Yamaaski K. Degradation of nicotine by soil bacteria. J Am Chem Soc, 1954, 76: 155-157
[7] Wada E. Microbial degradation of the tobacco alkaloids and some related compounds[J]. Arch. Biochem. biophys, 1958, 72(1): 145-162
[8] Newton, Richard P, Geiss, et al. Process of rerduction of nicotinecontent of tobacco by microbial treatment. US Patent,4037609, 1977-07-26
[9] Edwards W B, MccuenR. PreParation of optically Puer(R) - (+)-nicotine: Studies on the micorbial degradation of ncotinoids. J Ogr Chem, 1983, 48: 2484-2487
[10] Thaeker R, Rovrig O, Kahlon, P, et al.NIC, a conjugative nioctine-nioctinate degardative plasmid in Pseudomonas convexa. J Bacteriol, 1978, 135: 289-290
[11] Sguors PL. Microbial tarnsformations of the tobacco alkaloidsⅠ. Cultural and morphological characteristics of a nicotinophile. J Baeteriol, 1955, 69: 28-37
[12] W.G. Frankenburg, A.M. Cottscho,A.A. Vaitekunas. Biochemical conversions of some tobacco alkaloids[J]. Tobacco Science, 1958, 2: 2-13
[13] Decker K, Bleeg H. Induction and purification of steteospecificnicotine oxidizing enzymes from Arthrobacter oxdains. Biochem BioPhys Acta, 1965, 105: 313- 324
[14] Brandsch R, Hinkkanen A E, Decker K. Plasmid-mediated nicotine degradation in Arthrobacter oxidans[J]. Arch Microbiol, 1982, 132: 26-30
[15] Uchida, Maeda, Masubuchi, et al. Isolation of nicotine-degrading bacteria anddegradation of nicotine in shredded tobacco and tobacco extract[J]. Sci. Pap., 1976, 118: 197-201. [ 16 ] Kolenbrander PE, Weinbegrer. 2-Hydroxypyridine metabolism and pigment formation in three Athrobacter species. J Bacteriol, 1977, 132: 51-59
[17] Ruiz Gutierrez. Nicotine degradation by bacteria Enterobacter cloacae as a nicotine[J]. Degrader(Spain), 1983, 22: 85-98
[18] Giovannozzi. Industrial experiments of fermentation with addition of microbic cultures[J]. Tobaco, 1947, 573(51): 6-15
[19]袁勇军,陆兆新,黄丽金,吕凤霞,别小妹.烟碱降解细菌的分离、鉴定及其降解性能的初步研究.微生物学报,2005,45 (2):181-182
[20] Hylin J W. The microbial degradation of nicotine,Ⅱ, The mode of action of Achromobacter nicotinophagum. Arch Biochem Biophy, 1959, 83: 528-537
[21] Mesnard F, Girard S, Fliniaux O, el al. Chiral specificity of the degradation of nicotine by Nicotiana plumbaginifolia cell suspension cultures. Plant Science, 2001, (161): 1011-1018
[22] Sindelar R D, Rosazza J P, Barfknecht C F.N-Demethyaltion of nicotine and reduction of nicotine-l’-N-oxide by Microsporum gypseum. Appl Environ Microbiol, 1979, 38: 836-839
[23] Uchida S., Maeda s., Kisaki T.Conversion of nicotine into nornicotine and N-methylmyosmine by fungi[J]. Agric.Bio1. Chem, 1983, 47(9): 1949-1953
[24] Eberhardt, Hans-Jochen. The biological degradation of nicotine by nicotinophilic microorganisms Beitr[J]. Tabakforsh Int, 1995, 16(3): 119-129
[25] Eberhardt, Hans-Jochen. The biological degradation of nicotine by nicotinophilic microorganisms Beitr[J]. Tabakforsh Int, 1995, 16(3): 119-129
[26] Baitsch D, Sandu C, Brandsch R, et al. A gene cluster on pA01 of Arthrobacter nicotinovorans involved in the degradation of the plant alkaloid nicotine: Cloning, purication and characterization of 2,6-dihydroxypyridine 3-hydroxylase[J]. J Bacterial, 2001, 183: 5262-5267.
[27]雷丽萍,夏振远,王弱,魏海雷,刘杳忠.尼古丁降解菌株Ll的分离与降解特性.农业生物技术学报,2007,15 (4):721～722
[28] Frankenburg W.G., and Vaitekunas A.A., Chemical studies on nicotine degradation by microorganism derived from the surface oftobacco seeds, Archives of Biochemistry and Biophysics, 1955,58(2): 509-512
[29]韩锦峰,叶波平.陈化发酵期间烤烟叶面微生物活性及其应用研究[J].中国烟草科学,1997,4:13-14
[30] Casida L.E.J., and Rosenfield R., Bacterial oxidation of nicofine formation ofγ-aminobutyric acid[J]. J.Bacteriol. April; 1957,75(4): 474-479
[31] Ward E., Microbial degradation of the tobacco alkaloids and some related compounds, Archives of Biochemistry and Biophysics, 1957, 72(1): 145-162
[32] Brandsch R, Hinkkanen A E, Decker K. Plasmid-mediated nicotine degradation in Arthrobacter oxidans[J]. Arch Microbiol, 1982, 132: 26-30 [ 33 ] Brandsch R, Bichler V, Nagursky H. Covalent flavinylation of 6-hydroxy-D-nicotine oxidase by partial deletions of the gene[J]. Eur J Biochem, 1987, 165: 559-564
[34] Benmauer H, Mauch L, Brandsch R. Interaction of the regulatory protein Nic Rl with the promoter region of the pAO1-encoded 6-hydroxy-D-nicotine oxidase gene of Arthrobacter oxidans[J]. Mol Microbiol,1992,6: 1809-1820 [ 35 ] Grether-Beck S, Igloi G L, Pust S, et al. Structural analysis and molybdenum-dependent expression of the pAO1-encoded nicotine dehydrogenase genes of Arthrobacter nicotinovorans[J]. Mol Microbiol, 1994, 13: 929-936.
[36] Schenk S, Hoelz A, Krau B, et al. Gene structure and properties of enzymes of the plasmid-encoded catabolism of Arthrobacter nicotinovorans [J]. J Mol Biol, 1998,284: 1323-1339
[37] Baitsch D, Sandu C, Brandsch R, et al. A gene cluster on pA01 of Arthrobacter nicotinovorans involved in the degradation of the plant alkaloid nicotine: Cloning, purication and characterization of 2,6-dihydroxypyridine 3-hydroxylase[J]. J Bacterial, 2001, 183: 5262-5267.
[38] Igoli G L, Brandsch R. Sequence of the 165-kilobase catabolic palsmid pAO1 from Arthrobacter nicotinovorans and identification[J]. J Bcteriol, 2003, 185: 1976-1986
[39]金闻博,戴亚.烟草化学.北京:清华大学出版社,1994,149.
[40]东秀珠,蔡妙瑛.常见细菌系统鉴定手册.北京:科学出版社.2001,349-398
[41] Weisburg W G, Barns S M, Pelletier D A, et al. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol, 1991, 173(2): 697-703
[42]魏群.分子生物学实验.北京:高等教育出版社. 2004,53-55
[2]阎克玉.烟草化学.郑州:郑州大学出版社,2002年6月. 112
[3]肖协忠.烟草化学.北京:中国农业科技出版社,1997,161. 180
[4] Wada E, Kisaki T. Autoxidation of Nicotine[J]. Arch Biochem Biophys, 1959, 79: 124-130
[5]于川芳,王兵,罗登山,等.国产白肋烟与津巴布韦、马拉维及美国白肋烟的分析比较[J].烟草科技,1999,(4):6-8
[6] Wada E, Yamaaski K. Degradation of nicotine by soil bacteria. J Am Chem Soc, 1954, 76: 155-157
[7] Wada E. Microbial degradation of the tobacco alkaloids and some related compounds[J]. Arch. Biochem. biophys, 1958, 72(1): 145-162
[8] Newton, Richard P, Geiss, et al. Process of rerduction of nicotinecontent of tobacco by microbial treatment. US Patent,4037609, 1977-07-26
[9] Edwards W B, MccuenR. PreParation of optically Puer(R) - (+)-nicotine: Studies on the micorbial degradation of ncotinoids. J Ogr Chem, 1983, 48: 2484-2487
[10] Thaeker R, Rovrig O, Kahlon, P, et al.NIC, a conjugative nioctine-nioctinate degardative plasmid in Pseudomonas convexa. J Bacteriol, 1978, 135: 289-290
[11] Sguors PL. Microbial tarnsformations of the tobacco alkaloidsⅠ. Cultural and morphological characteristics of a nicotinophile. J Baeteriol, 1955, 69: 28-37
[12] W.G. Frankenburg, A.M. Cottscho,A.A. Vaitekunas. Biochemical conversions of some tobacco alkaloids[J]. Tobacco Science, 1958, 2: 2-13
[13] Decker K, Bleeg H. Induction and purification of steteospecificnicotine oxidizing enzymes from Arthrobacter oxdains. Biochem BioPhys Acta, 1965, 105: 313- 324
[14] Brandsch R, Hinkkanen A E, Decker K. Plasmid-mediated nicotine degradation in Arthrobacter oxidans[J]. Arch Microbiol, 1982, 132: 26-30
[15] Uchida, Maeda, Masubuchi, et al. Isolation of nicotine-degrading bacteria anddegradation of nicotine in shredded tobacco and tobacco extract[J]. Sci. Pap., 1976, 118: 197-201. [ 16 ] Kolenbrander PE, Weinbegrer. 2-Hydroxypyridine metabolism and pigment formation in three Athrobacter species. J Bacteriol, 1977, 132: 51-59
[17] Ruiz Gutierrez. Nicotine degradation by bacteria Enterobacter cloacae as a nicotine[J]. Degrader(Spain), 1983, 22: 85-98
[18] Giovannozzi. Industrial experiments of fermentation with addition of microbic cultures[J]. Tobaco, 1947, 573(51): 6-15
[19]袁勇军,陆兆新,黄丽金,吕凤霞,别小妹.烟碱降解细菌的分离、鉴定及其降解性能的初步研究.微生物学报,2005,45 (2):181-182
[20] Hylin J W. The microbial degradation of nicotine,Ⅱ, The mode of action of Achromobacter nicotinophagum. Arch Biochem Biophy, 1959, 83: 528-537
[21] Mesnard F, Girard S, Fliniaux O, el al. Chiral specificity of the degradation of nicotine by Nicotiana plumbaginifolia cell suspension cultures. Plant Science, 2001, (161): 1011-1018
[22] Sindelar R D, Rosazza J P, Barfknecht C F.N-Demethyaltion of nicotine and reduction of nicotine-l’-N-oxide by Microsporum gypseum. Appl Environ Microbiol, 1979, 38: 836-839
[23] Uchida S., Maeda s., Kisaki T.Conversion of nicotine into nornicotine and N-methylmyosmine by fungi[J]. Agric.Bio1. Chem, 1983, 47(9): 1949-1953
[24] Eberhardt, Hans-Jochen. The biological degradation of nicotine by nicotinophilic microorganisms Beitr[J]. Tabakforsh Int, 1995, 16(3): 119-129
[25] Eberhardt, Hans-Jochen. The biological degradation of nicotine by nicotinophilic microorganisms Beitr[J]. Tabakforsh Int, 1995, 16(3): 119-129
[26] Baitsch D, Sandu C, Brandsch R, et al. A gene cluster on pA01 of Arthrobacter nicotinovorans involved in the degradation of the plant alkaloid nicotine: Cloning, purication and characterization of 2,6-dihydroxypyridine 3-hydroxylase[J]. J Bacterial, 2001, 183: 5262-5267.
[27]雷丽萍,夏振远,王弱,魏海雷,刘杳忠.尼古丁降解菌株Ll的分离与降解特性.农业生物技术学报,2007,15 (4):721～722
[28] Frankenburg W.G., and Vaitekunas A.A., Chemical studies on nicotine degradation by microorganism derived from the surface oftobacco seeds, Archives of Biochemistry and Biophysics, 1955,58(2): 509-512
[29]韩锦峰,叶波平.陈化发酵期间烤烟叶面微生物活性及其应用研究[J].中国烟草科学,1997,4:13-14
[30] Casida L.E.J., and Rosenfield R., Bacterial oxidation of nicofine formation ofγ-aminobutyric acid[J]. J.Bacteriol. April; 1957,75(4): 474-479
[31] Ward E., Microbial degradation of the tobacco alkaloids and some related compounds, Archives of Biochemistry and Biophysics, 1957, 72(1): 145-162
[32] Brandsch R, Hinkkanen A E, Decker K. Plasmid-mediated nicotine degradation in Arthrobacter oxidans[J]. Arch Microbiol, 1982, 132: 26-30 [ 33 ] Brandsch R, Bichler V, Nagursky H. Covalent flavinylation of 6-hydroxy-D-nicotine oxidase by partial deletions of the gene[J]. Eur J Biochem, 1987, 165: 559-564
[34] Benmauer H, Mauch L, Brandsch R. Interaction of the regulatory protein Nic Rl with the promoter region of the pAO1-encoded 6-hydroxy-D-nicotine oxidase gene of Arthrobacter oxidans[J]. Mol Microbiol,1992,6: 1809-1820 [ 35 ] Grether-Beck S, Igloi G L, Pust S, et al. Structural analysis and molybdenum-dependent expression of the pAO1-encoded nicotine dehydrogenase genes of Arthrobacter nicotinovorans[J]. Mol Microbiol, 1994, 13: 929-936.
[36] Schenk S, Hoelz A, Krau B, et al. Gene structure and properties of enzymes of the plasmid-encoded catabolism of Arthrobacter nicotinovorans [J]. J Mol Biol, 1998,284: 1323-1339
[37] Baitsch D, Sandu C, Brandsch R, et al. A gene cluster on pA01 of Arthrobacter nicotinovorans involved in the degradation of the plant alkaloid nicotine: Cloning, purication and characterization of 2,6-dihydroxypyridine 3-hydroxylase[J]. J Bacterial, 2001, 183: 5262-5267.
[38] Igoli G L, Brandsch R. Sequence of the 165-kilobase catabolic palsmid pAO1 from Arthrobacter nicotinovorans and identification[J]. J Bcteriol, 2003, 185: 1976-1986
[39]金闻博,戴亚.烟草化学.北京:清华大学出版社,1994,149.
[40]东秀珠,蔡妙瑛.常见细菌系统鉴定手册.北京:科学出版社.2001,349-398
[41] Weisburg W G, Barns S M, Pelletier D A, et al. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol, 1991, 173(2): 697-703
[42]魏群.分子生物学实验.北京:高等教育出版社. 2004,53-55