Ochrobactrum intermedium DN2烟碱降解酶的分离纯化及酶产量的优化研究
|
摘要
从降低烟草行业成本和减少环境污染的角度出发，降解上部烟叶和烟草废弃物中的烟碱已成为烟草行业的一个研究热点。本论文分离和纯化了一株新型的烟碱降解细菌中间苍白杆菌(Ochrobacterum intermedium)DN2所产的烟碱降解酶，对该酶的酶学性质进行了研究，并通过优化培养基和培养条件提高了酶产量。此外，还对烟碱降解酶在烟草薄片液中的应用进行了初步研究。论文的主要内容和结论如下：
烟碱降解酶粗酶液经硫酸铵沉淀，凝胶过滤层析和离子交换柱层析分离纯化，得到了电泳纯的烟碱降解酶。分离纯化过程中酶被提纯了35.24倍，酶活回收率为6.4％。烟碱降解酶的分子量为40.06kDa。其最适反应pH为6.0，在pH5.0-8.0范围内都很稳定。烟碱脱氢酶的最适反应温度为55℃，50℃下酶活力较稳定，保温2.5h后仍有80％以上酶活，在温度超过60℃后酶活下降较快。EDTA和Na_2MoO_4对酶活有一定的促进作用，CoCl_2、MnCl_2对酶活抑制作用较显著。酶的Km为0.13mmol／L。
采用Plackett-Burman设计法，对影响O．intermedium DN2产烟碱降解酶的20个因素进行了筛选。结果表明：影响该菌发酵产酶的显著因子为：烟碱、酵母膏、葡萄糖、Tween-80、初始pH、接种量、装液量和培养时间。在此基础上，采用响应曲面法分别对影响该菌发酵产酶的培养基组成和培养条件进行了优化。得到上述各显著因子的最佳水平为：烟碱2.183g／L、葡萄糖0.823g／L、酵母膏0.844g／L、Tween-80 0.976g／L、初始pH6.9、接种量8.6％、装液量413ml／1L三角瓶、培养时间32h。在此优化条件下得到实际酶活为9412U／L，与预测值9625U／L相近。
实验确定利用烟碱降解酶降解烟草薄片液中的烟碱的最适反应条件为：烟碱含量1.21g／L，粗酶含量6％，pH7.0，45℃下反应，添加EDTA至终浓度为1mM／L。在此最适条件下，12h的烟碱降解率达到88％，薄片液中的烟碱浓度从最初的1.21g／L降至0.15g／L，从而大大提高了薄片液的可用性。
Nicotine-degradation enzyme has attracted considerable research interest in recent yearsbecause of its potential application in the tobacco industry.
In the paper, nicotine-degradation enzyme was purified by ammonium sulfateprecipitation, gel filtration and ion exchange chromatography. PAGE electrophoresis of thepurified fraction mitigated a simple band which revealed this enzyme was purified .Themolecular weight of the enzyme was 40.06KDa by SDS-PAGE. The optimal temperatureand pH of the enzyme was 55℃and pH6.0, respectively. This enzyme was stable atpH5.0-8.0 below 50℃. Kinetic studies demonstrated that it was an enzyme with Km of0.13mmol/L. EDTA and Mo~(6+) Can activate the enzyme although Mn~(2+) and Co~(2+) inhibited it.
Plackett-Burman design was used to evaluate the importance of the selected twentyinternal and external factors on the activity of nicotine-degradation enzyme of DN2. Inaddition, the central composite design and response surface analysis were used to determinethe optimal levels of the main facters. The optimal levels of the main facters were asfollows: nicotine 2.183g/L、glucose 0.823g/L、Yeast extract 0.844g/L、Tween-80 0.976g/L、initial pH6.9、inoculum amount 8.6%、volume of medium 413ml/1L erlenmeyer、incubation time 32h. Under this condition, enzyme activity can reach to 9412U/L which isclose to the predicated value 9625U/L.
The optimal condition for the enzyme to degradate nicotine in reconstituted tobaccoliquid was: nicotine 1.21g/L, enzyme 6%, EDTA 1mM/L, pH7.0, 45℃. Under thiscondition nicotine degradation ratio can reach to 88% after 12h, and the final nicotineconcentration got down to 0.15g/L from 1.21g/L.
烟碱降解酶粗酶液经硫酸铵沉淀，凝胶过滤层析和离子交换柱层析分离纯化，得到了电泳纯的烟碱降解酶。分离纯化过程中酶被提纯了35.24倍，酶活回收率为6.4％。烟碱降解酶的分子量为40.06kDa。其最适反应pH为6.0，在pH5.0-8.0范围内都很稳定。烟碱脱氢酶的最适反应温度为55℃，50℃下酶活力较稳定，保温2.5h后仍有80％以上酶活，在温度超过60℃后酶活下降较快。EDTA和Na_2MoO_4对酶活有一定的促进作用，CoCl_2、MnCl_2对酶活抑制作用较显著。酶的Km为0.13mmol／L。
采用Plackett-Burman设计法，对影响O．intermedium DN2产烟碱降解酶的20个因素进行了筛选。结果表明：影响该菌发酵产酶的显著因子为：烟碱、酵母膏、葡萄糖、Tween-80、初始pH、接种量、装液量和培养时间。在此基础上，采用响应曲面法分别对影响该菌发酵产酶的培养基组成和培养条件进行了优化。得到上述各显著因子的最佳水平为：烟碱2.183g／L、葡萄糖0.823g／L、酵母膏0.844g／L、Tween-80 0.976g／L、初始pH6.9、接种量8.6％、装液量413ml／1L三角瓶、培养时间32h。在此优化条件下得到实际酶活为9412U／L，与预测值9625U／L相近。
实验确定利用烟碱降解酶降解烟草薄片液中的烟碱的最适反应条件为：烟碱含量1.21g／L，粗酶含量6％，pH7.0，45℃下反应，添加EDTA至终浓度为1mM／L。在此最适条件下，12h的烟碱降解率达到88％，薄片液中的烟碱浓度从最初的1.21g／L降至0.15g／L，从而大大提高了薄片液的可用性。
Nicotine-degradation enzyme has attracted considerable research interest in recent yearsbecause of its potential application in the tobacco industry.
In the paper, nicotine-degradation enzyme was purified by ammonium sulfateprecipitation, gel filtration and ion exchange chromatography. PAGE electrophoresis of thepurified fraction mitigated a simple band which revealed this enzyme was purified .Themolecular weight of the enzyme was 40.06KDa by SDS-PAGE. The optimal temperatureand pH of the enzyme was 55℃and pH6.0, respectively. This enzyme was stable atpH5.0-8.0 below 50℃. Kinetic studies demonstrated that it was an enzyme with Km of0.13mmol/L. EDTA and Mo~(6+) Can activate the enzyme although Mn~(2+) and Co~(2+) inhibited it.
Plackett-Burman design was used to evaluate the importance of the selected twentyinternal and external factors on the activity of nicotine-degradation enzyme of DN2. Inaddition, the central composite design and response surface analysis were used to determinethe optimal levels of the main facters. The optimal levels of the main facters were asfollows: nicotine 2.183g/L、glucose 0.823g/L、Yeast extract 0.844g/L、Tween-80 0.976g/L、initial pH6.9、inoculum amount 8.6%、volume of medium 413ml/1L erlenmeyer、incubation time 32h. Under this condition, enzyme activity can reach to 9412U/L which isclose to the predicated value 9625U/L.
The optimal condition for the enzyme to degradate nicotine in reconstituted tobaccoliquid was: nicotine 1.21g/L, enzyme 6%, EDTA 1mM/L, pH7.0, 45℃. Under thiscondition nicotine degradation ratio can reach to 88% after 12h, and the final nicotineconcentration got down to 0.15g/L from 1.21g/L.
引文
1. Baitsch D, Sandu C, Brandsch R, et al. Gene cluster on pA01 of Arthrobacter nicotinoborans involved in degradation of the plantalkaloid nicotine: Cloning, purification, and characterization of 2,6-dihydroxypyridine 3-hydroxylase[J]. J Bacteriol, 2001, 183(18): 5262-5267
2. Bernauer H, Mauch L, Brandsch R. Interaction of the regulatory protein NicR1 with the promoter region of the pA01-encoded 6-hydroxy-D-nicotine oxidase gene of Arthrobacter oxidans[J], Molecular Microbiology, 1992,2: 1809-1820
3. Brandsch R, Baitsch D, Sandu C, et al. Igloi gene cluster on pA01 of Arthrobruter nicotiaovorarans involved in degradation of the Plant Alkaloid Nicotine: cloning, Purification, and characterization of 2,6-dihydroxypyridine 3-Hydroxylase[J]. J Bacteriology, 2001, 183: 5262-5267.
4. Brandsch R, Hinkkanen A E, Mauch L, et al. 6-Hydroxy-D-nicotine oxidase of Arthrobacter oxidans: Gene structure of the flavoenzyme and its relationship to 6-hydroxy-L-nicotine oxidase[J]. Eur J Biochem, 1987, 167: 315-320
5. Civilini M, Domenis C, Sebastianutto N, et al. Nicotine decontamination of tobacco agro-industrial waste and its degradationby micro-organisms. Waste Manage Res, 1997,15: 349-358
6. Coussirat J C. Influence of tobacco epiphytic bacteria on biodegradation and production of alkaloids[J]. A du Tabac Sect, 1978,2(15): 5-134
7. Decker K, Bleeg H. Induction and purification of steteospecificnicotine oxidizing enzymes from Arthrobacter oxidans[J]. Biochem Biopltys Acta, 1965, 105: 313-324
8. Detraglia M C, Tometsko A M. Separation of D-(+)-nicotine froma racemic mixture by stereospecific degradation of the L-(-)isomer with Pseudomonas putida[J]. App Environ Microbiol, 1980,39:1067-1069
9. Eberwein H, Gries F A, Decker K. Uber den Abbau des Nicotine durch Bakterienenzyme, II. Isolierung und Charakterisierung eines nicoti-abbauenden Boaenbakteriums[J]. Z Phys Chem. 1961, 323: 236-248
10. Edwards W B, McCuen R. Preparation deR-(+)-nicotine optiquement pure. Etudes surla degradation microbienne desnicotinoides[J]. J Org Chem, 1983,48(15): 2484-2487
11. Enders C, Windisch S. The decomposition of nicotine by yeast [J]. Biochem, 1947, 318: 54-62
12. Frankenburg W G. Transformation products of nicotine in fermented leaves[J]. Science, 1948, 107: 427-428
13. Freudenberg W, Koenig K, Andreesen J R. Nicotine dehydrogenase from Arthrobacter oxidans: A molybdenum-containing hydroxylase[J]. FEMS Microbiol Lett, 1988, 52: 13-18
14. Friesen J B, Leete E. Nicotine synthase-an enzyme from Nicotiana species which cataclyses the formation of (s)-nicotine from nicotinic acid and 1 -methyl-△'-pyrrolinium chloride[J]. Tetrahedr Lett, 1990,31:6295-6298
15. Geiss V L. Process for reduction of nicotine content of tobacco by microbial. United States Patent, 4,038,993. 1977
16. Giovannozzi-Sermanni G Industrial experiments of fermentation with addition of microbic cultures[J]. Tobacco, 1947, 51: 6-15
17. Giovannozzi-Sermanni G, a new species of Corynebacterium causing nicotine degradation[J]. Coersta, 1957, 3: 0605
18. Giovannozzi-Sermanni G Arthrobacter nicotianae,a new type of Arthrobacter causing nicotine degradationtJ]. Coersta, 1959,3: 2595
19. Gravely L E, Lawrence E, Geiss V L, et al. Process for reduction of nitrateand nicotine content of tobacco by microbial treatment. US Patent,4557280.1985-12-10
20. Gutierrez R. Nicotine degradation by bacteria. 2. Enterobacter cloacae [J]. An INIA Ser agric, 1983,22:85-98
21. Hinkkanen A, Lilius E M, Nowack J, et al. Purification of the flavopreitens 6-hydroxy-D-and 6-hydroxy-L-nicotine oxidase using hydrophobic affinity chromatography [J]. Anal Biochem, 1983, 132: 202-208
22. Hochstein L I, Dalton B P. The purification and properties of nicotine oxidase[J]. Biochem Biophys Acta, 1967,139:50-68
23. Hochstein L I, Rittenberg S C. The bacterial oxidation of nicotine.I. nicotine oxidation by cell-free preparation[J]. J Biol Chem, 1959,234:151-155
24. Holmes P E, Rittenberg S C. The bacterial oxidation of nicotine.VII. Partial purification and properties of 2,6-dihxdroxypyridine oxidation[J]. J Biol Chem, 1972a, 247: 7628-7633
25. Holmes P E, Rittenberg S C. The Bacterial oxidation of nicotine.VIII.Synthesis of 2,3,6-trihydroxy pyridine and accumulation andpartial characterization of the product of 2,6-dihydroxypyridine Oxidase[J]. J Biol Chem, 1972b, 247: 7622-7627
26. Hylin J W. The microbial degradation of nicotine. II The mode of action of Achromobacte nicotinophagum[J]. Arch Biochem Biophy, 1959, 83: 528-537
27. Izquierdo T A, Ruiz G V. Etudes sur la flore bacterienne du tabac.VI. Role des bacterienne Durant le processes de fermentation du tabac et de degradation de la nicotine[J].An Inst Nac Investig Agrar, Seragricola,1982,18:117- 142.(En espagnol)
28. Lawrence E, Gravely, Louisville K Y, et al. Process for reduction of nitrate and nicotine content of tobacco by microbial treatment. US Patent, 4557280.1978
29. Maeda S, Kisaki T. Microbial degradation of nicotine-1'-N-oxide[J]. Agric Biol Chem, 1981,45(3): 565-569
30. Maeda S, Matsushita H, Mikami Y, et al. Structural changes of N-methyl-myosmine based on pH[J]. Agric Biol Chem, 1980, 44: 1643-1645
31. Meher K K, Panchwagh A M, Rangrass S, et al. Biomethanation of tobacco waste[J]. Environmental Pollution, 1995, 90(2): 199-202
32. Nagel M, Koenig K, Andreesen J R. Bactopterin as component of eubacterial dehydrogenases involved in hydroxylation reactions initiatinf the segradation of nicotine, nicotinate, and 2-furan-carbosylate[J]. FEMS Mictrobiology Letters, 1989,60: 323-326
33. Newton, Richard P, Geiss, et al. Process for reduction of nicotinecontent of tobacco by microbial treatment. US Patent, 4037609.1977-07-26
34. Richard P, Newton V L, Geiss V L, et al. Lowering the nicotine content of tobacco by microbial treatment. Gre Patent. 2634186.1977
35. Ruan A D, Min H, Peng X H, et al. Isolation and characterization of pseudomonas sp. Strain HF-l,capable of degrading nicotine[J]. Res Micobiol, 2005,156: 700-706
36. Schenk S, Hoelz A, Kraub B, et al. Gene structures and properties of enzymes of the plasmid-encoded nicotine catabolism of Arthrobacter nicotinovorans[J]. J Mol Biol, 1998, 284:1323-1339
37. Sguros P L. Microbial transformation of the tobacco alkaloids I. cultural and morphological characteristics of a nicotinophile[J], J Bacteriol, 1955, 69:28-37
38. Sindelar R D, Rosazza J P, Barfknecht C F. N-Demethylation of nicotine and reduction of nicotine-1'-N-oxide by Microsporumgypseum[J]. Appl Environ Microbio 1, 1979, 38:836-839
39. Spann B M. Reconstituted tobacco from venezue lanby products by enzyme conversion. Philip Morris Research Center, 1965
40. Sponza D T. Toxicity studies in a tobacco industry biological treatment plant, water, air, and soil pollution[J]. Environmental Pollution, 2002, 134:137-164
41. Tabuchi T, Microbial degration of nicotine and nicotinic acid I.Isolation of nicotine-decomposing bacteria and these morphological and physiological properties[J], J Agr Chem Soc Japan, 1954, 28: 807-810
42. Takahashi T. Studies on the control of nicotine in tobacco[R].3rd World Tobacco Congress,Salisburg,So,Rhodesia, 1963
43. Tashiro A, Yuji K, Yonemura C, et al. Pure isolation of nicotine- degrading microbes[J]. Kyushu Kyoritsu Daigaku Kenkyu Hokoku Kogakubu, 1996, 20:147-155
44. Tso T C, Lowe R H, Dejomg D W. Homogenied Leaf curing.I.Theoretical basis and preliminarg results. Beitrage zur Tobakforschung. 1975,8:44-51
45. Uchida S, Maeda S, Kisaki T. Conversion of nicotine intonomicotine and N-methyl myosmine by fungi[J].Agric Biol Chem, 1983, 47:1949-1953
46. Uchida S, Maeda S, Masubuchi E, et al. Isolation of nicotine- degrading bacteria and degradation of nicotine in shredded tobacco and tobacco extract[J]. Sci Pap, 1976, 118:197-201
47. US Environmental Protection Agency. Emergency planning and community right-to-know section 313: list of toxic chemicals EPA745-B-96-002. Washington D C: Environmental Protection Agency, 1996, 44
48. Wada E, Yamasaki K. Mechanism of microbial degredation of nicotine[J]. Science, 1953, 117: 152-153
49. Ward E. Microbial degradation of the tobacco alkaloids and some related compounds[J]. Arch biochem biophys, 1958, 72(1): 145-162.
50. Yoshida D. Mechanisms of tobacco plants containing lower alkaloid[J]. Bull Hatano, Tob Exp Sta, 1973, 73:245-257
51．戴冕．烟草科技论文选集[M]．广州：少东科技出版社，1997：35-50
52．金博闻，戴亚．烟碱的合成、代谢和作用[J]．烟草科技，1995，(1)：14-16
53．金闻博，戴亚．烟草化学IM]．北京：清华大学出版社，1994：35-66
54．金闻博，刘春祥．被动吸烟[M]．北京：轻工业出版社，1994：15-26
55．刘绚霞，刘朝侠．影响烟叶烟碱含显的因素分析[J]．甘肃农业科技，1996，(7)：39-40
56．齐群钢，郭月清，韩锦峰．植物激素和无机营养元素对烟草根系内烟碱生物合成调节机理的研究[J]．河南农业大学学报，1990，24(3)：32-39
57．孙君社，马林，武仪，等．利用微生物降解烟草烟碱的方法[P]．CN1465300．2004
58．王伯毅．烟碱的形成及提高烟碱量的栽培措施[[J]．烟草科技，1984，2：22-24．
59．王革．一种微生物(NO．1)及其制备方法[P]．CN1439715．2003a
60．王革．一种微生物(NO．31)及其制备方法[P]．CN1439713．2003b
61．王革．一种微生物(NO．4)及其制各方法[P]．CN1439714．2003c
62．王革．一种微生物(NO．J1)及其制备方法[P]．CN1439716．2003d
63．肖协忠．烟草化学[M]．北京：中国农业科技出版社，1997：161-80
64．阎玉秋，方智勇，王智宇，等．试论烟草中烟碱含量及其调节因素[[J]．烟草科技，1996，6：31-34．
65．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，1999a，(4)：6-8
66．于川芳，王兵，罗登山，等．国产混合型卷烟与国外知名品牌混合型卷烟的分析比较及发展思路[J]．中国烟草学报，1999b，5(1)：17
67．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，2003，197：3-7
1. Baitsch D, Sandu C, Brandseh R, et al. Gene Cluster on pAO1 of Arthrobacter nicotinovorans involved in degradation of the plant alkaloid nicotine: cloing, purification, and characterization of 2,6-dihydroxypyridine 3-hydroxylase[J]. J Bacteriol,2001,183( 18):5262-5267.
2. Brandsch R, Decher K. Isolation and partial characterization of plasmid DNA from Arthrobacter oxydans[J]. Arch Microbiol, 1984,138:15-17.
3. Decker K, Bleeg H. Induction and purification of stereospecific nicotine oxidizine enzyme from Arthrobacter Oxidans[J]. Bioehimica Biophy Aeta, 1965,105:313-324.
4. Freudenberg W, Koning K, Andreesen JR. Nicotine dehydrogenase from Arthrobacter oxidans:A molybdenum-containing hydroxylase[J]. FEMS Microbiol Lett, 1988,52(1-2): 13-18.
5. Grether-Beck S.Structural analysis and molybdenum-dependent expression of the PAO1-encoded nicotine dehydrogenase genes of Arthrobacter nicotinovorans.Mol Microbiology, 1994,13(5):929-936
6. Laemmli U K. Cleavage of structural protein during the assembly of the head of bacteriophage T4[J].Nature, 1970,227:680-686
7. Robert DS, John PR, Charles FB. N-demethylation of nicotine and reduction ofnicotine-1'-N-oxide by Microsporum gypseum[J]. Appl Environ Mierobiol, 1970,38(5):836-839.
8. Schenk S, Hoelz A, Krauβ B, et al.Gene structures and properties of enzymes of the plasmid-encoded nicotine catabolism of Arthrobacter nicotinovorans[J]. J Mol Biol, 1998,284(5): 1323-1339.
9. Setsuko U, Susumu M, Takuro K. Conversion of nicotine into nornicotine and N-methylmyosmine by fungi[J]. Agr Biol Chem, 1982, 47(9): 1949-1953.
10. Thacker R, Rorvig O, Kahlon P, et al.. NIC, a conjugative nicotinate degradative plasmid in pseudomonas convexa[J]. J Bacteriol, 1978,135(1):289-290.
11. Yuan Y J, Lu ZX, Huang LJ, et al. Optimization of a medium for enhancing nicotine biodegradation by Ochrobactrum intermedium DN2[J].Appl microbiol, 2006,101 (3):.691-697.
12. Yuan YJ, Lu ZX, Wu N, et al. Isolation and preliminary characterization of a novel nicotine-degrading bacterium, Oehrobactrurn intermedium DN2[J].Inter Biodeter & Biodegr, 2005,56:45-50.
13．汪家政，范明．蛋白质技术手册[M]．北京：科学出版社，2000：．42-46．
1. Ambati P, Ayyanna C. Optimizing medium constituents and fermentation conditions for citric production from Palmyra jaggery using respponse surface method[J]. World J Microbiol Biotechnol, 2001, 17:331-335
2. Annadurai G. Design of optimum response surface experiments for adsorption of direct dye on chitosan[J]. Bioproc Eng, 2000, 23:451-455
3. Gouveia E R, Baptista-Neto A. Optimisation of medium composition for clavulanic acid production by Streptomyces clavuligerus[J]. Biotechnol Lett, 2001, 23:157-161
4. Hujanen M, Linko S, Linko Y Y, et al. Optimisation of medium and cultivation conditions for L(+)(S)-lactic acid production by Lactobacillus casei NRRL B-441[J]. Appl Microbiol Biotechnol, 2001, 56:126-130
5. Krishna S H, Sattur A P, Karanth N G. Lipae-catalyzed synthesis of isoamyl isobutyrate optimization of methyloleate by response surface methodology[J]. Bioproc Eng, 2000, 22: 35-39
6. Li C, Bai J, Cai Z. Optimization of a cultural medium for bacteriocin production by Lactobacillus lactis using responsee surface methodology[J]. J Biotechnol, 2002, 93:27-34
7. Oh S, Rheem S, Sim J, et al. Optimizing condition for the groth of Lactobacillus casei YIT 9018 in tryptone-glucose medium by using response surface methodology[J]. App Environ Microbiol, 1995, 61:3809-3814
8. Plackett R L, Burman J P. The design of optimum multifactorial experiments[J]. Biometrika, 1946, 33:305-325
9. Rastogi N K, Rashmi K R. Optimization of enzymatic liquefaction of mango pulp by response surface methodology[J]. Eur Food Technol, 1999, 21:497-503
10. Reddy P R M, Reddy G, Seenayya G. Production of thermostable pullulanase by Clostridium thermosulfurogenes SV2 in solid-state fermentation: optimization of nutrients levels using response surface methodology [J]. Bioproc Eng, 1999, 21:497-503
11. Triveni R, Shamala T R, Rastogi N K. Optinised production and utilisation of exopolysaccharide drom Agrobactedum radiobacter[J]. Proc Biochem, 2001, 36:787-795
12．陈洪，许平，马清仪，等．微生物酶法降解烟草总植物碱试验[J]．烟草科技，2004，4：12-16
13．何桢，潘越，刘子先，等．因子实验、RSM与田口方法的比较研究[J]．机械研究，1999，10：1-4
14．马林，武怡，曾晓鹰，等．降解烟碱微生物的筛选及其酶在烟草中的应用[J]．烟草科技，2005，9(218)：6-8
15．袁勇军，陆兆新，黄丽金，等．烟碱降解细菌的分离鉴定及其降解性能的初步研究[J]．微生物学报，2005，45(2)：181-184
1. Spann B.M. Reconstituted from venezue lanby products by enzyme conversion.Philip Morris Research Center, Mar.8,76,1965,
2. Tso T C, Lowe R H, Dejomg D W. Homogenied Leaf curing.I.Theoretical basis and preliminarg results[J] Beitrage zur Tobakforschung. 1975,8:44-51
3．陈洪，许平，马清仪，等．微生物酶法降解烟草总植物碱试验[J]．烟草科技，2004(4)：12-16
4．陈祖刚，蔡冰，王建新．国内外造纸法薄片工艺与品质比较[J]．烟草科技，2002(2)：4-10．
5．贺小琼，王琦，周建于．不同品牌卷烟烟焦油的致突变作用研究．昆明医学院学报，2004，(4)：43-46
6．李雪梅，杨伟祖，祝明亮，等．烟碱降解菌的选育及改善上部烟叶品质研究[J]．工业微生物，2006，36(1)：16-22
7．汪华文．造纸法薄片在卷烟工业中的应用效果分析[J]．烟草科技，2000，147：15-16
8．杨娟，张迪清，何照范，等．高效液相色谱法测定烟草中的烟碱含[J]．烟草科技，1999，(1)：27-28
9．郑勤安．造纸法再造烟叶生产过程中微生物增质剂的应用研究[J]．浙江工业大学学报，2004，32(4)：442-446，458
2. Bernauer H, Mauch L, Brandsch R. Interaction of the regulatory protein NicR1 with the promoter region of the pA01-encoded 6-hydroxy-D-nicotine oxidase gene of Arthrobacter oxidans[J], Molecular Microbiology, 1992,2: 1809-1820
3. Brandsch R, Baitsch D, Sandu C, et al. Igloi gene cluster on pA01 of Arthrobruter nicotiaovorarans involved in degradation of the Plant Alkaloid Nicotine: cloning, Purification, and characterization of 2,6-dihydroxypyridine 3-Hydroxylase[J]. J Bacteriology, 2001, 183: 5262-5267.
4. Brandsch R, Hinkkanen A E, Mauch L, et al. 6-Hydroxy-D-nicotine oxidase of Arthrobacter oxidans: Gene structure of the flavoenzyme and its relationship to 6-hydroxy-L-nicotine oxidase[J]. Eur J Biochem, 1987, 167: 315-320
5. Civilini M, Domenis C, Sebastianutto N, et al. Nicotine decontamination of tobacco agro-industrial waste and its degradationby micro-organisms. Waste Manage Res, 1997,15: 349-358
6. Coussirat J C. Influence of tobacco epiphytic bacteria on biodegradation and production of alkaloids[J]. A du Tabac Sect, 1978,2(15): 5-134
7. Decker K, Bleeg H. Induction and purification of steteospecificnicotine oxidizing enzymes from Arthrobacter oxidans[J]. Biochem Biopltys Acta, 1965, 105: 313-324
8. Detraglia M C, Tometsko A M. Separation of D-(+)-nicotine froma racemic mixture by stereospecific degradation of the L-(-)isomer with Pseudomonas putida[J]. App Environ Microbiol, 1980,39:1067-1069
9. Eberwein H, Gries F A, Decker K. Uber den Abbau des Nicotine durch Bakterienenzyme, II. Isolierung und Charakterisierung eines nicoti-abbauenden Boaenbakteriums[J]. Z Phys Chem. 1961, 323: 236-248
10. Edwards W B, McCuen R. Preparation deR-(+)-nicotine optiquement pure. Etudes surla degradation microbienne desnicotinoides[J]. J Org Chem, 1983,48(15): 2484-2487
11. Enders C, Windisch S. The decomposition of nicotine by yeast [J]. Biochem, 1947, 318: 54-62
12. Frankenburg W G. Transformation products of nicotine in fermented leaves[J]. Science, 1948, 107: 427-428
13. Freudenberg W, Koenig K, Andreesen J R. Nicotine dehydrogenase from Arthrobacter oxidans: A molybdenum-containing hydroxylase[J]. FEMS Microbiol Lett, 1988, 52: 13-18
14. Friesen J B, Leete E. Nicotine synthase-an enzyme from Nicotiana species which cataclyses the formation of (s)-nicotine from nicotinic acid and 1 -methyl-△'-pyrrolinium chloride[J]. Tetrahedr Lett, 1990,31:6295-6298
15. Geiss V L. Process for reduction of nicotine content of tobacco by microbial. United States Patent, 4,038,993. 1977
16. Giovannozzi-Sermanni G Industrial experiments of fermentation with addition of microbic cultures[J]. Tobacco, 1947, 51: 6-15
17. Giovannozzi-Sermanni G, a new species of Corynebacterium causing nicotine degradation[J]. Coersta, 1957, 3: 0605
18. Giovannozzi-Sermanni G Arthrobacter nicotianae,a new type of Arthrobacter causing nicotine degradationtJ]. Coersta, 1959,3: 2595
19. Gravely L E, Lawrence E, Geiss V L, et al. Process for reduction of nitrateand nicotine content of tobacco by microbial treatment. US Patent,4557280.1985-12-10
20. Gutierrez R. Nicotine degradation by bacteria. 2. Enterobacter cloacae [J]. An INIA Ser agric, 1983,22:85-98
21. Hinkkanen A, Lilius E M, Nowack J, et al. Purification of the flavopreitens 6-hydroxy-D-and 6-hydroxy-L-nicotine oxidase using hydrophobic affinity chromatography [J]. Anal Biochem, 1983, 132: 202-208
22. Hochstein L I, Dalton B P. The purification and properties of nicotine oxidase[J]. Biochem Biophys Acta, 1967,139:50-68
23. Hochstein L I, Rittenberg S C. The bacterial oxidation of nicotine.I. nicotine oxidation by cell-free preparation[J]. J Biol Chem, 1959,234:151-155
24. Holmes P E, Rittenberg S C. The bacterial oxidation of nicotine.VII. Partial purification and properties of 2,6-dihxdroxypyridine oxidation[J]. J Biol Chem, 1972a, 247: 7628-7633
25. Holmes P E, Rittenberg S C. The Bacterial oxidation of nicotine.VIII.Synthesis of 2,3,6-trihydroxy pyridine and accumulation andpartial characterization of the product of 2,6-dihydroxypyridine Oxidase[J]. J Biol Chem, 1972b, 247: 7622-7627
26. Hylin J W. The microbial degradation of nicotine. II The mode of action of Achromobacte nicotinophagum[J]. Arch Biochem Biophy, 1959, 83: 528-537
27. Izquierdo T A, Ruiz G V. Etudes sur la flore bacterienne du tabac.VI. Role des bacterienne Durant le processes de fermentation du tabac et de degradation de la nicotine[J].An Inst Nac Investig Agrar, Seragricola,1982,18:117- 142.(En espagnol)
28. Lawrence E, Gravely, Louisville K Y, et al. Process for reduction of nitrate and nicotine content of tobacco by microbial treatment. US Patent, 4557280.1978
29. Maeda S, Kisaki T. Microbial degradation of nicotine-1'-N-oxide[J]. Agric Biol Chem, 1981,45(3): 565-569
30. Maeda S, Matsushita H, Mikami Y, et al. Structural changes of N-methyl-myosmine based on pH[J]. Agric Biol Chem, 1980, 44: 1643-1645
31. Meher K K, Panchwagh A M, Rangrass S, et al. Biomethanation of tobacco waste[J]. Environmental Pollution, 1995, 90(2): 199-202
32. Nagel M, Koenig K, Andreesen J R. Bactopterin as component of eubacterial dehydrogenases involved in hydroxylation reactions initiatinf the segradation of nicotine, nicotinate, and 2-furan-carbosylate[J]. FEMS Mictrobiology Letters, 1989,60: 323-326
33. Newton, Richard P, Geiss, et al. Process for reduction of nicotinecontent of tobacco by microbial treatment. US Patent, 4037609.1977-07-26
34. Richard P, Newton V L, Geiss V L, et al. Lowering the nicotine content of tobacco by microbial treatment. Gre Patent. 2634186.1977
35. Ruan A D, Min H, Peng X H, et al. Isolation and characterization of pseudomonas sp. Strain HF-l,capable of degrading nicotine[J]. Res Micobiol, 2005,156: 700-706
36. Schenk S, Hoelz A, Kraub B, et al. Gene structures and properties of enzymes of the plasmid-encoded nicotine catabolism of Arthrobacter nicotinovorans[J]. J Mol Biol, 1998, 284:1323-1339
37. Sguros P L. Microbial transformation of the tobacco alkaloids I. cultural and morphological characteristics of a nicotinophile[J], J Bacteriol, 1955, 69:28-37
38. Sindelar R D, Rosazza J P, Barfknecht C F. N-Demethylation of nicotine and reduction of nicotine-1'-N-oxide by Microsporumgypseum[J]. Appl Environ Microbio 1, 1979, 38:836-839
39. Spann B M. Reconstituted tobacco from venezue lanby products by enzyme conversion. Philip Morris Research Center, 1965
40. Sponza D T. Toxicity studies in a tobacco industry biological treatment plant, water, air, and soil pollution[J]. Environmental Pollution, 2002, 134:137-164
41. Tabuchi T, Microbial degration of nicotine and nicotinic acid I.Isolation of nicotine-decomposing bacteria and these morphological and physiological properties[J], J Agr Chem Soc Japan, 1954, 28: 807-810
42. Takahashi T. Studies on the control of nicotine in tobacco[R].3rd World Tobacco Congress,Salisburg,So,Rhodesia, 1963
43. Tashiro A, Yuji K, Yonemura C, et al. Pure isolation of nicotine- degrading microbes[J]. Kyushu Kyoritsu Daigaku Kenkyu Hokoku Kogakubu, 1996, 20:147-155
44. Tso T C, Lowe R H, Dejomg D W. Homogenied Leaf curing.I.Theoretical basis and preliminarg results. Beitrage zur Tobakforschung. 1975,8:44-51
45. Uchida S, Maeda S, Kisaki T. Conversion of nicotine intonomicotine and N-methyl myosmine by fungi[J].Agric Biol Chem, 1983, 47:1949-1953
46. Uchida S, Maeda S, Masubuchi E, et al. Isolation of nicotine- degrading bacteria and degradation of nicotine in shredded tobacco and tobacco extract[J]. Sci Pap, 1976, 118:197-201
47. US Environmental Protection Agency. Emergency planning and community right-to-know section 313: list of toxic chemicals EPA745-B-96-002. Washington D C: Environmental Protection Agency, 1996, 44
48. Wada E, Yamasaki K. Mechanism of microbial degredation of nicotine[J]. Science, 1953, 117: 152-153
49. Ward E. Microbial degradation of the tobacco alkaloids and some related compounds[J]. Arch biochem biophys, 1958, 72(1): 145-162.
50. Yoshida D. Mechanisms of tobacco plants containing lower alkaloid[J]. Bull Hatano, Tob Exp Sta, 1973, 73:245-257
51．戴冕．烟草科技论文选集[M]．广州：少东科技出版社，1997：35-50
52．金博闻，戴亚．烟碱的合成、代谢和作用[J]．烟草科技，1995，(1)：14-16
53．金闻博，戴亚．烟草化学IM]．北京：清华大学出版社，1994：35-66
54．金闻博，刘春祥．被动吸烟[M]．北京：轻工业出版社，1994：15-26
55．刘绚霞，刘朝侠．影响烟叶烟碱含显的因素分析[J]．甘肃农业科技，1996，(7)：39-40
56．齐群钢，郭月清，韩锦峰．植物激素和无机营养元素对烟草根系内烟碱生物合成调节机理的研究[J]．河南农业大学学报，1990，24(3)：32-39
57．孙君社，马林，武仪，等．利用微生物降解烟草烟碱的方法[P]．CN1465300．2004
58．王伯毅．烟碱的形成及提高烟碱量的栽培措施[[J]．烟草科技，1984，2：22-24．
59．王革．一种微生物(NO．1)及其制备方法[P]．CN1439715．2003a
60．王革．一种微生物(NO．31)及其制备方法[P]．CN1439713．2003b
61．王革．一种微生物(NO．4)及其制各方法[P]．CN1439714．2003c
62．王革．一种微生物(NO．J1)及其制备方法[P]．CN1439716．2003d
63．肖协忠．烟草化学[M]．北京：中国农业科技出版社，1997：161-80
64．阎玉秋，方智勇，王智宇，等．试论烟草中烟碱含量及其调节因素[[J]．烟草科技，1996，6：31-34．
65．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，1999a，(4)：6-8
66．于川芳，王兵，罗登山，等．国产混合型卷烟与国外知名品牌混合型卷烟的分析比较及发展思路[J]．中国烟草学报，1999b，5(1)：17
67．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，2003，197：3-7
1. Baitsch D, Sandu C, Brandseh R, et al. Gene Cluster on pAO1 of Arthrobacter nicotinovorans involved in degradation of the plant alkaloid nicotine: cloing, purification, and characterization of 2,6-dihydroxypyridine 3-hydroxylase[J]. J Bacteriol,2001,183( 18):5262-5267.
2. Brandsch R, Decher K. Isolation and partial characterization of plasmid DNA from Arthrobacter oxydans[J]. Arch Microbiol, 1984,138:15-17.
3. Decker K, Bleeg H. Induction and purification of stereospecific nicotine oxidizine enzyme from Arthrobacter Oxidans[J]. Bioehimica Biophy Aeta, 1965,105:313-324.
4. Freudenberg W, Koning K, Andreesen JR. Nicotine dehydrogenase from Arthrobacter oxidans:A molybdenum-containing hydroxylase[J]. FEMS Microbiol Lett, 1988,52(1-2): 13-18.
5. Grether-Beck S.Structural analysis and molybdenum-dependent expression of the PAO1-encoded nicotine dehydrogenase genes of Arthrobacter nicotinovorans.Mol Microbiology, 1994,13(5):929-936
6. Laemmli U K. Cleavage of structural protein during the assembly of the head of bacteriophage T4[J].Nature, 1970,227:680-686
7. Robert DS, John PR, Charles FB. N-demethylation of nicotine and reduction ofnicotine-1'-N-oxide by Microsporum gypseum[J]. Appl Environ Mierobiol, 1970,38(5):836-839.
8. Schenk S, Hoelz A, Krauβ B, et al.Gene structures and properties of enzymes of the plasmid-encoded nicotine catabolism of Arthrobacter nicotinovorans[J]. J Mol Biol, 1998,284(5): 1323-1339.
9. Setsuko U, Susumu M, Takuro K. Conversion of nicotine into nornicotine and N-methylmyosmine by fungi[J]. Agr Biol Chem, 1982, 47(9): 1949-1953.
10. Thacker R, Rorvig O, Kahlon P, et al.. NIC, a conjugative nicotinate degradative plasmid in pseudomonas convexa[J]. J Bacteriol, 1978,135(1):289-290.
11. Yuan Y J, Lu ZX, Huang LJ, et al. Optimization of a medium for enhancing nicotine biodegradation by Ochrobactrum intermedium DN2[J].Appl microbiol, 2006,101 (3):.691-697.
12. Yuan YJ, Lu ZX, Wu N, et al. Isolation and preliminary characterization of a novel nicotine-degrading bacterium, Oehrobactrurn intermedium DN2[J].Inter Biodeter & Biodegr, 2005,56:45-50.
13．汪家政，范明．蛋白质技术手册[M]．北京：科学出版社，2000：．42-46．
1. Ambati P, Ayyanna C. Optimizing medium constituents and fermentation conditions for citric production from Palmyra jaggery using respponse surface method[J]. World J Microbiol Biotechnol, 2001, 17:331-335
2. Annadurai G. Design of optimum response surface experiments for adsorption of direct dye on chitosan[J]. Bioproc Eng, 2000, 23:451-455
3. Gouveia E R, Baptista-Neto A. Optimisation of medium composition for clavulanic acid production by Streptomyces clavuligerus[J]. Biotechnol Lett, 2001, 23:157-161
4. Hujanen M, Linko S, Linko Y Y, et al. Optimisation of medium and cultivation conditions for L(+)(S)-lactic acid production by Lactobacillus casei NRRL B-441[J]. Appl Microbiol Biotechnol, 2001, 56:126-130
5. Krishna S H, Sattur A P, Karanth N G. Lipae-catalyzed synthesis of isoamyl isobutyrate optimization of methyloleate by response surface methodology[J]. Bioproc Eng, 2000, 22: 35-39
6. Li C, Bai J, Cai Z. Optimization of a cultural medium for bacteriocin production by Lactobacillus lactis using responsee surface methodology[J]. J Biotechnol, 2002, 93:27-34
7. Oh S, Rheem S, Sim J, et al. Optimizing condition for the groth of Lactobacillus casei YIT 9018 in tryptone-glucose medium by using response surface methodology[J]. App Environ Microbiol, 1995, 61:3809-3814
8. Plackett R L, Burman J P. The design of optimum multifactorial experiments[J]. Biometrika, 1946, 33:305-325
9. Rastogi N K, Rashmi K R. Optimization of enzymatic liquefaction of mango pulp by response surface methodology[J]. Eur Food Technol, 1999, 21:497-503
10. Reddy P R M, Reddy G, Seenayya G. Production of thermostable pullulanase by Clostridium thermosulfurogenes SV2 in solid-state fermentation: optimization of nutrients levels using response surface methodology [J]. Bioproc Eng, 1999, 21:497-503
11. Triveni R, Shamala T R, Rastogi N K. Optinised production and utilisation of exopolysaccharide drom Agrobactedum radiobacter[J]. Proc Biochem, 2001, 36:787-795
12．陈洪，许平，马清仪，等．微生物酶法降解烟草总植物碱试验[J]．烟草科技，2004，4：12-16
13．何桢，潘越，刘子先，等．因子实验、RSM与田口方法的比较研究[J]．机械研究，1999，10：1-4
14．马林，武怡，曾晓鹰，等．降解烟碱微生物的筛选及其酶在烟草中的应用[J]．烟草科技，2005，9(218)：6-8
15．袁勇军，陆兆新，黄丽金，等．烟碱降解细菌的分离鉴定及其降解性能的初步研究[J]．微生物学报，2005，45(2)：181-184
1. Spann B.M. Reconstituted from venezue lanby products by enzyme conversion.Philip Morris Research Center, Mar.8,76,1965,
2. Tso T C, Lowe R H, Dejomg D W. Homogenied Leaf curing.I.Theoretical basis and preliminarg results[J] Beitrage zur Tobakforschung. 1975,8:44-51
3．陈洪，许平，马清仪，等．微生物酶法降解烟草总植物碱试验[J]．烟草科技，2004(4)：12-16
4．陈祖刚，蔡冰，王建新．国内外造纸法薄片工艺与品质比较[J]．烟草科技，2002(2)：4-10．
5．贺小琼，王琦，周建于．不同品牌卷烟烟焦油的致突变作用研究．昆明医学院学报，2004，(4)：43-46
6．李雪梅，杨伟祖，祝明亮，等．烟碱降解菌的选育及改善上部烟叶品质研究[J]．工业微生物，2006，36(1)：16-22
7．汪华文．造纸法薄片在卷烟工业中的应用效果分析[J]．烟草科技，2000，147：15-16
8．杨娟，张迪清，何照范，等．高效液相色谱法测定烟草中的烟碱含[J]．烟草科技，1999，(1)：27-28
9．郑勤安．造纸法再造烟叶生产过程中微生物增质剂的应用研究[J]．浙江工业大学学报，2004，32(4)：442-446，458