建立苯環化合物分解菌中苯環加氧與切割

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林美鳳(Meng-Fong Lin) 查詢紙本館藏

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生命科學系

論文名稱

建立苯環化合物分解菌中苯環加氧與切割

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摘要(中)

隨著人類的活動及科技的發展，為我們帶來工業的發展，但是石油、石化工業所生產的苯環化合物，卻造成了環境的污染。苯環類化合物之所以難分解是由於其具有穩定的苯環結構，因此會一直長期存在環境中，目前已知環境中許多細菌具有分解苯環類化合物的能力，其分解途徑與?的特性已被研究；本研究則以一本土性之苯環化合物分解菌(Pseudomonas putida SH1)為材料，此菌可以?、酚、鄰甲基苯酚、間甲基苯酚、對甲基苯酚、菲、芘、苯並芘為單一碳源生長，尤其以?或酚為單一碳源時生長狀況最好。當我們以不同的苯環化合物為唯一碳源培養P. putida SH1，已純化出四種苯環切割?(aromatic ring-cleavage dioxygenases)，其皆具鄰苯二酚加氧?之活性。在生化特性及NH2端胺基酸序列上皆顯示四個為不同之?，由?所誘發生成之catechol 2,3-dioxygenase (簡稱C23O)命名為C23Onap(SH1)，由酚誘發的有兩個分別為C23OpheI(SH1)及C23OpheII(SH1)，而以鄰甲基苯酚為唯一碳源生長時P. putida SH1可生成另一個C23O，命名為C23Oo-cre(SH1)。因之我們以其他已知之C23Os基因核?酸序列具高相似度的片段設計成引子，以聚合?連鎖反應選殖P. putida SH1中不同C23Os的基因，所放大的片段再經由pUC18-T載體接合，經由轉型至E. coli JM109並以C23O活性篩選和雜交兩種方法進行篩選SH1菌株中之C23O基因，所得兩個具有活性之片段與本研究室之前所選殖到的相同，故尚未發現其他C23O基因。
本論文另一個主題為利用苯環化合物分解菌中雙加氧?在基因上的相似性設計了幾組引子以偵測已知分解菌如可以分解?的P. putida G7及P. putida NCIB 9816-4，具分解甲苯之P. putida F1、P. putida mt-2及P. mendocina KR1，另外尚有能分解聯苯之P. putida LB400、能分解PAH之Sphingomonas B1及本實驗室篩出之菌株。PCR所用引子的設計為可偵測aromatic ring-hydroxylationg dioxygenase的ISP-F/ISP-R，是以此?中之終端加氧分子即iron-sulfur protein中a次單位的與輔因子( [2Fe-2S] Rieske center)結合的保守序列為主要偵測的位置；C23O-1F/C23O-1R、C23O-2F/C23O-2R與C23O-2’F/C23O-2R則是用在偵測間位苯環切割?的存在與否；另外還有針對?雙加氧?所設計的NahAc-F/NahAc-R及為偵測甲苯雙加氧酵?所設計的TodC1-F/TodC1-R用意在區別單和雙環苯環化合物之分解菌。在實驗中發現引子C23O-1F/C23O-1R的結果正確性很高，但是精確度不足；反之引子C23O-2F/C23O-2R及C23O-2’F/C23O-2R即是精確度很高但正確性不足，對於引子C23O-1F/C23O-1R的缺點，可以探針C23O-1經南方雜合加以相互印證；另外在偵測苯環化合物加氧?的實驗中，所設計的引子ISP-F/ISP-R的專一性很好，但是卻無法偵測到聯苯分解菌中的aromatic ring-hydroxylationg dioxygenase；專為?雙加氧?所設計的引子NahAc-F/NahAc-R可以成功的區分出?雙加氧?與單環的加氧?，但是卻無法偵測到同為?分解菌之P. putida SH1中之?雙加氧?，再經由探針NDO所進行南方雜合之結果發現P. putida SH1確實存在著一個NahAc-F/NahAc-R無法偵測到之?雙加氧?。以上引子並應用於未知代謝途徑之Triton X-100分解菌及6個經石油污染之土壤樣品中是否含有上述之?的存在，以初步鑑定出含有上述基因之存在，以供進一步快速偵測和研究之參考。

摘要(英)

The second part of this study was designed to detect the aromatic ring-hydroxylating dioxygenase and aromatic ring-cleavage dioxygenase genes in bacteria by PCR. The PCR method for the detection of naphthalene dioxygenase, toluene dioxygenase and catechol 2,3-dioxygenase were performed. The first two dioxygenases are two archetypical ring-hydroxylation dioxygenase. The catechol 2,3-dioxygenase is involved in the cleavage of aromatic ring and shows high identify among C23O genes from many bacteria. Strains test in this study included naphthalene-degrading bacteria, P. putida G7 and P. putida NCIB 9816-4; toluene-degrading bacteria, P. putida F1, P. putida mt-2 and P. mendocina KR1; biphenyl-degrading bacteria, P. putida LB400; polycyclic aromatic hydrocarbons-degrading bacteria, Sphingomonas B1, and bacterial strains isolated by our laboratory. In addition to petrochemical hydrocarbons-degrading bacteria, bacteria degrading Triton X-100 were also included. From our preliminary study, primers C23O-1F/C23O-1R are not very specificity, but if combined with Southern hybridization by probe C23O can accurate to detect aromatic ring-cleavage dioxygenase genes in bacteria. Primers C23O-2F/C23O-2R and C23O-2’’F/C23O-2R have specificity, but they have low accuracy for detect aromatic ring-cleavage dioxygenase genes in bacteria. Primers NahAc-F/NahAc-R are very accurate for detect naphthalene dioxygenase genes, one exception is the naphthalene-degrading P. putida SH1. When we used Southern hybridization by probe NDO, which showed low sequence similarity with P. putida G7. Primers ISP-F/ISP-R are very specificity for detect aromatic ring-hydroxylating dioxygenase. Combined with Southern hybridization, we can get the powerful tool to detect these genes not only using conserved primer sequence but also using full sequence similarity. This detection method will be applied to pretiminary screen of the key degradation genes in unknown isolates as well as for detecting aromatic compound-degrading bacteria in the environment.

關鍵字(中)

★ 苯環加氧酵素
★ 苯環切割酵素

關鍵字(英)

論文目次

目錄--------------------------------------------------I
縮寫與全名對照表--------------------------------------III
圖目錄------------------------------------------------IV
表目錄------------------------------------------------VII
壹、緒論----------------------------------------------1
一、前言-------------------------------------1
二、苯環化合物之代謝途徑--------------------1
三、加氧酵素之介紹---------------------------3
四、研究背景與目的---------------------------10
貳、材料與方法----------------------------------------15
一、菌種及其保存-----------------------------15
二、培養基-----------------------------------15
三、菌種篩選、鑑定與生化特性-----------------16
四、DNA的製備--------------------------------19
五、聚合?連鎖反應---------------------------21
六、基因選殖---------------------------------24
七、單股構形多型性---------------------------26
八、變性梯度凝膠電泳法-----------------------27
九、南方雜合---------------------------------28
十、藥品-------------------------------------30
十一、儀器設備-------------------------------31
參、實驗結果------------------------------------------33
一、鄰苯二酚加氧酵素之基因選殖---------------33
二、以單股構形多型性分析來探討P. putida SH1中不同的鄰
苯二酚加氧酵素---------------------------33
三、聯苯分解菌之篩選、鑑定與特性分析---------34
四、苯環化合物分解菌中苯環切割?之基因偵測---35
五、苯環化合物分解菌中苯環加氧?之基因偵測---38
肆、討論----------------------------------------------40
伍、參考資料------------------------------------------43
圖----------------------------------------------------52
表----------------------------------------------------109

參考文獻

林春志. 1997. Pseudomonas putida SH1分解芳香族碳氫化合物之研究. 國立中央大學生命科學研究所碩士論文.
楊璧如. 1997. 鄰苯二酚加氧酵素的基因選殖與分析. 國立中央大學生命科學研究所碩士論文.
姜福慧. 1998. Pseudomonas putida SH1中鄰苯二酚加氧酵素的純化與特性分析. 國立中央大學生命科學研究所碩士論文.
李祖霖. 1999. Pseudomonas putida SH1中鄰苯二酚加氧酵素的純化與特性分析(II). 國立中央大學生命科學研究所碩士論文.
羅淑如. 1999. Pseudomonas putida SH1中誘發苯環化合物代謝之研究. 國立中央大學生命科學研究所碩士論文.
楊淑君. Unpublished data.
Aemprapa, S. and P. A. Williams. 1998. Implications of the xylQ gene of TOL plasmid pWW102 for the evolution of aromatic catabolic pathway. Microbiol. 144:1387-1396.
Ahel, M, D. Hrsak and W. Giger. 1994. Aerobic transformation of short-chain alkylphenol polyethoxylate by mixed bacterial cultures. Arch. Environ. Contam. Toxicol. 26:540-548.
Brenner, V., J. J. Arensdorf and D. D. Focht. 1994. Genetic construction of PCB degraders. Biodegradation. 5:359-377.
Butler, C. S. and J. R. Mason. 1997. Structure-function analysis of the bacterial aromatic ring-hydroxylating dioxygenases. Adv. Microbiol. Physiol. 38:47-84
Caldwell, D. R. 1995. Microbial physiology and metabolism. Wm. C. Brown publishers.
Cerdan, P., M. Rekik, and S. Harayama. 1995. Substrate specificity differences between two catechol 2,3-dioxygenase encoded by the TOL and NAH plasmid from Pseudomonas putida. Eur. J. Biochem. 229:113-118.
Chandler L. J. and N. G. Nordoff. 1999. Identification of genetic variation among St. Louis encephalitis virus isolates, using single-strand conformation polymorphism analysis. J. Virol. Methods. 80:169-178.
Corcia, A. D. 1998. Characterization of surfactants and their biointermediates by liquid chromatography-mass spectrometry. J. Chromatography 794:165-185.
Desai, J. D. and I. M. Banat. 1997. Microbial production of surfactants and their commercial potential. Microbiol. Mol. Biol. Rev. 61:47-64.
Eltis, L. D. and J. T. Bolin. 1996. Evolutionary relationships among extradiol dioxygenase. J. Bacteriol. 17:5930-5937.
Ensley, B. D. and D. T. Gibson. 1983. Naphthalene dioxygenase: Purification and properties of a terminal oxygenase component. J. Bacteriol. 155:505-511.
Erickson, B. D. and F. J. Mondello. 1992. Nucleotide sequencing and transcriptional mapping of genes encoding biphenyl dioxygenase, a multicoponent polychlorinated-biphenyl-degrading enzyme in Pseudomonas strain LB400. J. Bacteriol. 174:2903-2912.
Ghosal, D., I.-S. You and I. C. Gunsalus. 1987. Nucleotide sequence and expression of gene nahH of plasmid NAH7 and homology with gene xylE of TOL pWWO. Gene. 55:19-28.
van Ginkel, C. G. 1996. Complete degradation of xenobiotic surfactants by consortia of aerobic microorganisms. Biodegradation. 7:151-164.
Haigler, B. E. and D. T. Gibson. 1990a. Purification and properties of NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816. J. Bacteriol. 172:457-464.
Haigler, B. E. and D. T. Gibson. 1990b. Purification and properties of ferredoxinNAP, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816. J. Bacteriol. 172:465-468.
Hamann, C., J. Hegemann and A. Hildebrandt. 1999. Detection of polycyclic aromatic hydrocarbon degradation genes in different soil bacteria by polymerase chain reaction and DNA hybridization. FEMS Microbiol. Letter. 173:255-263.
Hammel, K. E. 1995. Organopollutant degradation by ligninolytic fungi. pp.331-346. In Microbial transformation and degradation of toxic organic chemicals. Gerniglia Young and Carl E. Cerniglia(ed.). Wiley-Liss, D. C.
Harayama, S., A. Wasserfallen, P. Cerdan, and M. Rekik. 1992. Mutation modification of the substrate specificity of catechol 2,3-dioxygenase encoded by TOL plasmid pWW0 of Pseudomonas putida. pp. 223-230. In American society for microbiology. E. Galli, S. Silver, and B. Witholt(ed.). Washington, D. C.
Harayama, S. and M. Kok. 1992. Functional and evolutionary relationships among diverse oxygenase. Annu. Rev. Microbiol. 46:565-601.
Harayama, S. and M. Rekik. 1989. Bacterial aromatic ring-cleavage enzymes are classified into two different gene families. J. Biol. Chem. 264:15328-15333.
Henckel, T., U. Jackel, S. Schnell, and R. Conrad. 2000. Molecular analyses of novel methanotrophic communities in forest soil that oxidize atmospheric methane. Appl. Environ. Microbiol. 66:1801-1808.
Herrick, J. B., K. G. Stuart-Keil, W. C. Ghiorse and E. L. Madsen. 1997. Natural horizontal transfer of a naphthalene dioxygenase gene between bacteria native to a coal tar-contaminated field site. Appl. Environ. Microbiol. 63:2330-2337.
Hirose, J., A. Suyama, S. Hayashida and K. Furukawa. 1994. Construction of hybrid biohenyl (bph) and toluene (tod) genes for functional analysis of aromatic ring dioxygenases. Gene. 138:27-33.
Hiss, R. H., D. E. Norris, C. H. Diertrich, R. F. Whitcomb, D. F. West, C. F. Bosio, S. Kambhampati, J. Piesman, M. F. Antolin and W. C. Black IV. 1994. Molecular taxonomy using single-strand conformation polymorphism(SSCP) analysis of mitochondrial ribosomal DNA genes. Insect Mol. Biol. 3:171-182.
Huertas, M. J., E. Duque, S. Marques and J. L. Ramos. 1998. Survival in soil of different toluene-degrading Pseudomonas strains after solvent shock. Appl. Environ. Microbiol. 64:38-42.
Hurtubise, Y., D. Barriault and M. Sylvestre. 1998. Involvement of the terminal oxygenase b subunit in the biphenyl dioxygenase reactivity pattern toward chlorobiphenyls. J. Bacteriol. 180:5828-5835.
Jiang, H., R. E. Parales and D. T. Gibson. 1999. The a subunit of toluene dioxygenase from Pseudomonas putida F1 can accept electrons from reduced FerredoxinTOL but is catalytically inactive in the absence of the b subunit. Appl. Environ. Microbiol. 65:315-318.
John, D. M. and G. F. White. 1998. Mechanism for biotransformation of nonylphenol polyethoxylates to xenoestrogens in Pseudomonas putida. J. Bacteriol. 180:4332-4338.
Kauppi, B., K. Lee, E. Carredano, R. E. Parales, D. T. Gibson, H. Eklund and S. Ramaswamy. 1998. Structure of an aromatic ring-hydroxylating dioxygenase-naphthalene 1,2-dioxygenase. Structure. 6:571-586.
Kikuchi, M., K. Ohnishi, S. Harayama. 1999. Novel family shuffling methods for the in vitro evolution of enzymes. Gene. 236:159-167.
Kimura, N., A. Nishi, M. Goto and K. Furukawa. 1997. Functional analyses of a variety of chimeric dioxygenases constructed from two biphenyl dioxygenases that are similar structurally but different functionally. J. Bacteriol. 179:3936-3943.
Kita, A., Shin-ichi Kita, I. Fujisawa, K. Inaka, T. Ishida, K. Horiike, M. Nozaki and K. Miki. 1999. An archetypical extradiol-cleaving catecholic dioxygenase: the crystal structure of catechol 2,3-dioxygenase (metapyrocatechase) from Pseudomonas putida mt-2. Structure. 7:25-34.
Lau, P. C. K., H. Bergeron, D. Labbe, Y. Wang, R. Brousseau, and D. Gibson, T. 1994. Sequence and expression of the todGIH genes involved in the last three steps of toluene degradation by Pseudomonas putida F1. Gene. 146:7-13.
Lee, Tsu-Lin, Fu-Hui Chiang and Shir-Ly Huang. 1998. Characterization of three catechol 2,3-dioxygenases from Pseudomonas putida SH1. Abstract of the 32nd Annual Meeting of The Chinese Society of Microbiology. pp.75.
Lee, Tsu-Lin, Yuan-Chang Hsu and Shir-Ly Huang. 1999. Characterization of four catechol 2,3-dioxygenases from Pseudomonas putida SH1. Abstract of the 33rd Annual Meeting of the Chinese Society of Microbiology. pp.68.
Lin, C.-C., F.-H. Chiang, and S.-H. Huang. 1997. Microbiological and biochemical studies of novel aromatic hydrocarbon-degrading Pseudomonas. Abstract of the Twelfth Joint Annual Conference of Biomedical Sciences. p.239. Apr. 19-20.
Maki, H., N. Masuda and Y. Fujiwara. 1994. Degradation of alkylphenol ethoxylates by Pseudomonas sp. strain TR01. Appl. Environ. Microbiol. 60:2265-2271.
Mesarch, M. B., C. H. Nakatsu and L. Nies. 2000. Development of catechol 2,3-dioxygenase-specific primers for monitoring bioremediation by competitive quantitative PCR. Appl. Environ. Microbiol. 66:678-683.
Meyer, S., R. Moser, A. Neef, U. Stahl and P. Kampfer. 1999. Differential detection of key enzymes of polyaromatic-hydrocarbon-degrading bacteria using PCR and gene probe. Microbiol. 145:1731-1741.
Michal, Gerhard. 1999. Amino acid and derivatives. pp.62. In Biochemical pathways: an atlas of biochemistry and molecular biology. Wiley, D. C.
Nakazawa, T., K. Furukawa, D. Hass, and S. Silver. 1996. Metabolic and regulatory check point in phenol degradation by Pseudomonas sp. strain CF600. pp. 153-175. In Molecular biology of Pseudomonads. V. Shingler(ed.), ASM Press, Washington, D. C.
Nelsen, A. T., W-T Liu, C. Filipe, L. Grady, JR., S. Molin and D. A. Stahl. 1999. Identification of a novel group of bacteria in sludge from a deteriorated biological phosphorus removal reactor. Appl. Environ. Microbiol. 65:1251-1258.
Nguyen, M. H. and J. C. Sigoillot. 1997. Isolation from coastal sea water and characterization of bacterial strains involved in non-ionic surfactant degradation. Biodegradation. 7:369-375.
Norbert, G. Swoboda-Colberg. 1995. Chemical contamination of the environment: sources, types, and fate of synthrtic organic chemicals. pp.27-74. In Microbial transformation and degradation of toxic organic chemicals. Gerniglia Young and Carl E. Cerniglia(ed.). Wiley-Liss, D. C.
Okuta, A., K. Ohnishi, and S. Harayama. 1998. PCR isolation of catechol 2,3-dioxygenase gene fragments from environmental samples and their assembly into functional genes. Gene. 212:221-228.
Smith, M. R. 1990. The biodegradation of aromatic hydrocarbons by bacteria. Biodegradation. 1:191-206.
Stanier, R. Y., G. Cohen-Bazire and W. R. Sistrom. 1957. Kinetics studies of pigment synthesis by non-sulfur purple bacteria. J. Cell. Comp. Physiol. 49:25.
Subramanian V., T.-N. Liu, W.-K. Yeh, M. Narro and D. T. Gibson. 1981. Purification and properties of NADH-ferredoxinTOL reductase. J. Biol. Chem. 256:2723-2730.
Subramanian V., T.-N. Liu, W.-K. Yeh, C. M. Serdar, L. P. Wackett and D. T. Gibson. 1985. Purification and properties of FerredoxinTOL. J. Biol. Chem. 260:2355-2363.
Suen, W.-C. and D. T. Gibson. 1993. Isolation and preliminary characterization of the subunits of the terminal component of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4. J. Bacteriol. 175:5877-5881.
Tanghe, T., W. Dhooge and W. Verstraete. 1999. Isolation of a bacterial strain able to degrade branched nonylphenol. Appl. Environ. Microbiol. 65:746-751.
Trevors, J. T. 1995. Extraction and amplification of 16S rRNA genes from deep marine sediments and seawater to assess bacterial community diversity. pp.220-222. In Nucleic acid in the environment:Methods and applications. J. D. van Elsas (Eds.) Springer, D. C.
Worsey, M. J., F. C. H. Frankliu and P. A. Williams. 1978. Regulation of the degradative pathway enzymes coded for by the TOL plasmid (pWWO) from Pseudomonas putida mt-2. J. Bacteriol. 134:757-764.
Whyte, L. G., C. W. Greer and W. E. Inniss. 1996. Assessment of the biodegradation potential of psychrotrophic microorganisms. 42:99-106.
Yates, J. R. and F. J. Mondello. 1989. Sequence similarities in the genes encoding polychlorinated biphenyl degradation by Pseudomonas strain LB400 and Alcaligenes eutrophus H850. J. Bacteriol. 171:1733-1735.
Yen, K. M. and I. C. Gunsalus. 1985. Regulation of naphthalene catabolic genes of plasmid NAH7. J. Bacteriol. 162:1008-1013.
Yen, K. M., M. R. Karl, L. M. Blatt, M. J. Simon, R. B. Winter, P. R. Fausset, H. S. Lu, A. A. Harcourt and K. K. Chen. 1991. Cloning and characterization of a Pseudomonas mendocina KR1 gene cluster encoding toluene-4-monooxygenase. J. Bacteriol. 173:5315-5327.
Yen, K. M. and M. R. Karl. 1992. Identification of a new gene, tmo F, in the Pseudomonas mendocina KR1 gene cluster encoding toluene-4- monooxygenase. J. Bacteriol. 174:7253-7261.
Yeh, W. K., D. T. Gibson and T.-N. Liu. 1977. Toluene dioxygenase: A multicomponent enzyme system. Biochem. Biophy. Res. Commun. 78:401-410.
Zylstra, G. J. 1994. Molecular analysis of aromatic hydrocarbon degradation. pp.83-115. In Molecular environmental biology. Seymour J. Gerte(ed.).
Zylstra, G. J. and D. T. Gibson. 1989. Toluene degradation by Pseudomonas putida F1. J. Biol. Chem. 264:14940-14946.

指導教授

黃雪莉(Shir-Ly Huang)

審核日期

2000-7-17

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