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研究生:謝儒樑
研究生(外文):Ju-Liang Hsieh
論文名稱:應用汞離子結合蛋白MerP於重金屬生物修復上之研究
論文名稱(外文):Application of MerP proteins in bioremediation of heavy metals
指導教授:黃介辰
指導教授(外文):Chieh-Chen Huang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:45
中文關鍵詞:生物修復merP重金屬去毒Bacillus cereus RC607Pseudomonas aerugenosa K-62汞抗性基因組
外文關鍵詞:bioremediationmercurymerPheavy metaldetoxificationBacillus cereus RC607Pseudomonas aerugenosa K-62mer operon
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結合分生科技的生物修復技術已逐漸成為熱門的研究話題。在重金屬的生物修復技術方面,可利用基因工程的方式將微生物的重金屬抗性蛋白的基因選殖至配合實際需求的目標生物中,提升該生物對重金屬的抗性或累積性,甚至選擇性地累積某種特定的重金屬,這類的轉殖生物所建構的處理系統被認為是深具潛力的重金屬處理技術。
本研究是利用具汞抗性的革蘭氏陽性菌Bacillus cereus RC607,以及陰性菌Pseudomonas aerugenosa K-62汞抗性基因組中的汞離子結合蛋白MerP,分別在大腸菌E. coli BL21 (DE3) pLysS中表現。對汞及其他重金屬進行平板抗性測試的結果顯示,對汞及鋅較具明顯的抗性提升效果。至於對含汞的溶液,其抗性可由濃度5 μΜ提升至30 μΜ以上。在乾菌重2 mg及汞離子濃度10 μΜ的條件下,溶液中的汞離子幾乎可完全吸附;而在25 μΜ下,陽性菌的汞離子吸附效果比陰性菌的好。
The molecular biotechnology based bioremediation technology has been attracting the interests in controlling heavy metal pollution. By using genetic engineering, we can use bacterial heavy-metal detoxification proteins to promote the heavy metal resistance or metal-accumulation ability in a proper host.
In this research, the mercuric ion binding proteins, MerP, from the mer operon in a Gram-positive bacterial strain Bacillus cereus RC607 and a Gram-negative bacterial strain Pseudomonas aerugenosa K-62 were over expressed in E. coli BL21 (DE3) pLysS, respectively. In liquid media amended with mercury ions, the resistances were promoted from 5 μM to more than 30 μM in both recombinant bacteria. Two grams of recombinant cells were able to completely adsorb 10μM of Hg2+ . While in 25 μM Hg2+ solution, the recombinant strain expressing Gram-positive merP had better metal adsorption capacity than the strain expressing Gram-negative merP.
目錄
頁次
目錄……………………………………………………………………… I
表目錄…………………………...…………………….....……………..III
圖目錄………………………………………………………………..…IV
中文摘要…………………………………………………………...……V
Abstract………………………………… ..…………………………….VI
第一章 緒論……………………………………………………………..1
1-1 研究背景…………………………………………………………….1
1-2 研究動機與目的…………………………………………………….3
第二章 研究背景及文獻回顧…………………………………………..5
2-1汞之物理和化學性質………………………………….…………….5
2-2汞之毒性……………………………………………………………..5
2-3 汞之用途和汞污染來源…………………………………………….6
2-4 自然界的汞循環………………………………………………….…8
2-5 細菌的汞抗性機制………………………………………………….8
2-6 細菌的汞抗性基因的利用………………………………….………9
2-7 MerP的介紹………………………………………………………..10
2-8 其他重金屬的生物修復…………………………………………...10
第三章 材料與方法……………………………………………………12
3-1 材料…………………………………………………………….…..12
3-1-1 菌種及質體……………………………………………………...12
3-1-2培養液……………………………………………………………12
3-2 實驗方法…………………………………………………………..13
3-2-1 細菌的培養與保存……………………………………………...13
3-2-2 大腸菌質體的精製……………………………………………...13
3-2-3 聚合酶鏈反應(Polymerase chain reaction, PCR)……………….14
3-2-4 DNA片段回收及純化…………………………………………...14
3-2-5 DNA的黏合反應………………………………………………...15
3-2-6大腸菌的質體轉形(transformation) …………………………….15
3-2-7 核酸膠體電泳分析與紀錄……………………………………...16
3-2-8 pETBmerP及pETPmerP質體的構築…………………………..17
3-2-9具merP基因大腸菌的重金屬抗性平板測試…………………..19
3-2-10 具merP基因大腸菌的汞抗性液態測試………………….…..19
3-2-11 具merP基因大腸菌的汞吸附測試……………………………20
3-2-12 其他重金屬對具merP基因大腸菌的汞吸附干擾測試………21
3-2-13 汞濃度之測定………………………………………………….22
第四章 結果……………………………………………………………23
4-1 具merP基因的轉殖株之重金屬抗性平板測試………………...23
4-2 merP基因轉殖株於液態培養基之抗性測試…………………..24
4-3 merP基因轉殖株的汞吸附測試…………………………………..24
4-4 其他重金屬對具merP菌的汞吸附干擾測試…………………….25
第五章 討論……………………………………………………………26
5-1 在平板抗性測試中,抑制圈的探討………………..………..……26
5-2 具有merP的轉形株,能增加汞抗性的探討………….…..……..27
5-3 BL21/pETBmerP與BL21/pETPmerP對汞的吸附與抗性不同的探討…………………………………………………………………..28
5-4 BL21/pETBmerP與BL21/pETPmerP在鋅、銅、鉛、鎘及鎳等多種重金屬干擾對汞的吸附能力差異測試的探討………………...29
5-5 具merP菌在生物修復的探討……………………………………29
5-6 MerP在生復修復潛力的探討……………………………………..30
參考文獻………………………………………………………………..31
附錄一…………………………………………………………………..44
附錄二…………………………………………………………………..45
表目錄
Table 1.…………………………………………………….…….…….. 37
Table 2…………………………………………………………………..38
Table 3…………………………………………………………………..39
圖目錄
Fig. 1…………………………………………………………………..40
Fig. 2…………………………………………………………………..41
Fig. 3…………………………………………………………………..42
Fig. 4…………………………………………………………………..43
Barrineau, P., P. Gilbert, W. J. Jackson, C. S. Jones, A. O. Summers, and S. Wisdom. 1984. The DNA sequence of the mercury resistance operon of the IncFII plasmid NR1. J. Mol. Appl. Genet. 2:601-619.
Begley, T. P., A. E. Walts, and C. T. Walsh. 1986. Mechanistic studies of a protonolytic organomercurial cleaving enzyme: bacterial organomercurial lyase. Biochemistry. 25:7192-7200.
Belliveau, B. H. and J. T. Tevirs. 1989. Mercury resistance and detoxification in bacteria. Appl. Organometal. Chem. 3:283-294.
Bizily, S. P., C. L. Rugh, R. B. Meagher. 2000. Phytodetoxification of hazardous organomercurials by genetically engineered plants. Nat Biotechnol. 18:213-7.
Bizily, S. P., C. L. Rugh, A. O. Summers, and R. B. Meagher. 1999. Phytoremediation of methylmercury pollution: merB expression in Arabidopsis thaliana confers resistance to organomercurials. Proc Natl Acad Sci U S A. 96:6808-13.
Bogdanova E. S., I. A. Bass, L. S. Minakhin, M. A. Petrova, S. Z. Mindlin, A. A. Volodin, E. S. Kalyaeva, J. M. Tiedje, J. L. Hobman, N. L. Brown, and V. G. Nikiforov. 1998. Horizontal spread of mer operons among gram-positive bacteria in natural environments. Microbiology.144:609-20.
Brown, N. L., J. Camakaris, B. T. O. Lee, T. Williams, A. P. Morby, J. Parkhill, and D. A. Rouch. 1991. Bacterial resistances to mercury and copper. J. Cell. Biochem. 46:106-114.
Brown, N. L., S. J. Ford, R. D. Pridmore, and D. C. Fritzinger. 1983. Nucleotide Sequence of a gene from the Pseudomonas transposon Tn501. Biochemistry. 22:4089-95.
Diels, L., S. Van Roy, M. Mergeay, W. Doyen, S. Taghavi, and R. Leysen. 1993. Immobilization of bacteria in composite membranes and development of tubular membrane reactors for heavy metal recuperation, p. 275-293. In R. Peterson (ed.), Effective membrane processes: new perspectives. Kluwer Academic Publishers, Dordrecht, The Netherlands.
Goldwater, L. 1971. Mercury in the Environment. Sci Am. 224:15-21.
Grill, E. 1987. Phytochelatins, the heavy metal binding peptides of plants: characterization and sequence determination. Experientia Suppl. 52:317-22.
Hamlett, N. V., E. C. Landale, B. H. Davis and A. O. Summers. 1992. Roles of the Tn21 merT, merP, and merC gene products in mercury resistance and mercury binding.174:6377-6385.
Hamer, D. H. 1986. Metallothionein. Annu. Rev. Biochem. 55, 913—951
Hamdy, M. K., and O. R. Noyes. 1975. Formation of methylmercury by bacteria. Appl. Microbiol. 30:424-432.
Harada, M. 1995. Minamata disease: methylmercury poisoning in Japan caused by environmental pollution. Crit. Rev. Toxicol. 25:1-24.
Huang, C. C., M. Narita, T. Yamagata and G. Endo. 1999b. Identification of three merB genes and characterization of a broad-spectrum mercury resistance module encoded by a class Ⅱ transposon of Bacillus megaterium strain MB1. Gene. 239:361-366.
Huang, C. C., M. Narita, T. Yamagata and Y. Itoh. 1999a. Structure analysis of class Ⅱ transposon encoding the mercury resistance of the Gram-positive bacterium Bacillus megaterium MB1, a strain isolated from Minamata Bay, Japan. Gene. 234:361-369.
Jackson, W. J., and A. O. Summers. 1982. Biochemical characterization of HgCl2-inducible polypeptides encoded by the mer operon of plasmid R100. J. Bacteriol. 151:962-970.
Jernelov, A. and H. Lann. 1971. Mercury accumulation in food chains. Okkos. 22:403-406.
Kägi, J. H. R., and A. Schäffer. 1988. Biochemistry of metallothionein. Biochemistry. 27: 8509—8515.
Kägi, J. H. R. 1991. Overview of metallothioneins. Methods Enzymol. 205: 613—626
Kamps, L. R., R. Carr, and H. Miller. 1972. Bull. Environ. Contam. Toxicol. 8:273-279.
Kiyono, M., and H. Pan-Hou. 1999. DNA sequence and expression of a defective mer operon from Pseudomonas K-62 plasmid pMR26. Biol Pharm Bull. 22:910-4.
Kotrba, P., L. Doleckova, V. de Lorenzo, T. Ruml. 1999. Enhanced bioaccumulation of heavy metal ions by bacterial cells due to surface display of short metal binding peptides. Appl. Environ. Microbiol. 65:1092—98.
Kotrba, P., P. Pospisil, V. de Lorenzo, and T. Ruml. 1999. Enhanced metallosorption of Escherichia coli cells due to surface display of beta- and alpha-domains of mammalian metallothionein as a fusion to LamB protein. J. Receptor Signal Transduct. Res. 19:703—715.
Lund, P. A. and N. L. Brown. 1987. Role of the merT and merP gene products of transposon Tn501 in the induction and expression of resistance to mercuric ions. Gene. 52:207-214.
Macaskie, L. E., and A. C. R. Dean. 1990. Metal sequestering biochemicals, p. 200-248. In B. Volesky (ed.), Biosorption of heavy metals. CRC Press, Boca Raton, Fla.
Macaskie, L. E., A. C. R. Dean, A. K. Cheetham, R. J. B. Jakeman, and A. J. Skarnulis. 1987. Cadmium accumulation by Citrobacter sp.: the chemical nature of the accumulated metal precipitate and its location on the bacterial cells. J. Gen. Microbiol. 133:539-544.
Mejare, M. and L. Bulow. 2001. Metal-binding proteins and peptides in bioremediation and phytoremediation of heavy metals. Trends biotechnol.19:67-73.
Mindlin, S., G. Kholodii, Z. Gorlenko, S. Minakhina, L. Minakhin, E. Kalyaeva, A. Kopteva, M. Petrova, O. Yurieva, and V. Nikiforov. 2001. Mercury resistance transposons of gram-negative environmental bacteria and their classification. Res. Microbiol. 152: 811-822.
Misra, T. K. 1992. Bacterial resistance to inorganic mercury salts and organomercurials. Plasmid. 27:4-16.
Morby, A. P., J. L. Hobman, and N. L. Brown. 1995. The role of cysteine residues in the transport of mercuric ions by the Tn501 MerT and MerP mercury-resistance proteins. Mol. Microbiol. 17:25-35.
Nakahara, H., S. Silver, T. Miki, and R. H. Rownd. 1979. Hypersensitivity to Hg2+ and hyperbinding activity associated with cloned fragments of the mercurial resistance operon of the plasmid NR1. J. Bacteriol. 140:161-166.
Ni’Bhriain, N. N., S. Silver, and T. J. Foster. 1983. Tn5 insertion mutations in the mercuric ion resistance genes derived from plasmid R100. J. Bacteriol. 155:690-703.
Nogawa, K. and T. Kido. 1993. Biological monitoring of cadmium exposure in itai-itai disease epidemiology. Int. Arch. Occup. Environ. Health.65:43-46.
Okkeri, J., and T. Haltia. 1999. Expression and mutagenesis of ZntA, a zinc-transporting P-type ATPase from Escherichia coli. Biochemistry. 38:14109-16
Pazirandeh, M., B. M. Wells, R. L. Ryan. 1998. Development of bacterium-based heavy metal biosorbents: Enhanced uptake of cadmium and mercury by Escherichia coli expressing a metal binding motif. Appl. Environ. Microbiol. 64:4068—72.
Powlowski, J., and L. Sahlman. 1999. Reactivity of the two essential cysteine residues of the periplasmic mercuric ion-binding protein, MerP. J Biol Chem. 274:33320-26.
Qian, H., L. Sahlman, P. Eriksson, C. Hambraeus, U. Edlund and I. Sethson. 1998. NMR Solution structure of the oxidized form of MerP, a mercury ion binding protein involved in bacterial mercuric ion resistance. Biochemistry. 37:9316-22.
Rensing, C., B. Fan, R. Sharma, B. Mitra, and B. P. Rosen. 2000. CopA: An Escherichia coli Cu(I)-translocating P-type ATPase. Proc. Natl. Acad. Sci. U S A. 97:652-6
Robinson, J. B. and O. H. Tuovinen. 1984. Mechanisms of microbial resistance and detoxification of mercury and organomercury compounds: Physiological, Bio-chemical, and genetic analysis. Microbial. Rev. 48:95-124.
Robinson, N. J., A. M. Tommey, C. Kuske, and P. J. Jackson. 1993. Plant metallothioneins. J. Biochem. 295:1-10.
Rugh, C. L., H. D. Wilde, N. M. Stack, D. M. Thompson, A. O. Summers, and R. B. Meagher. 1996. Mercuric ion reduction and resistance in transgenic Arabidopsis thaliana plants expressing a modified bacterial merA gene. Proc Natl Acad Sci U S A.93:3182-7.
Rugh, C. L., J. F. Senecoff, R. B. Meagher, and S. A. Merkle. 1998.Development of transgenic yellow poplar for mercury phytoremediation. Nat Biotechnol. 16:925-8.
Sahlman, L., and B.-H. Jonsson. 1992. Purification and properties of the mercuric-ion-binding protein MerP. Eur. J. Biochem. 205:375-381.
Sahlman, L., and E. G. Skärfstad. 1993. Mercuric ion binding abilities of MerP variants containing only one cysteine.Biochem. Biophys. Res. Commun. 196:583-588.
Sahlman, L., Wong, W., and Powlowski, J. 1997. A mercuric ion uptake role for the integral inner membrane protein, MerC, involved in bacterial mercuric ion resistance. J. Biol. Chem. 272:29518-26
Saldano, B. A., P. Bien, and P. Kwan. 1975. Air-borne organomercury and elemental mercury emissions with emphasis on central sewage facilities. Atmos. Environ. 9:941-944.
Silver, S. and L. T. Phung. 1996. Bacterial heavy metal resistance: new surprises. Annu. Rev. Microbiol. 50:753-789.
Sousa, C., A. Cebolla, and V. de Lorenzo. 1996. Enhanced metalloadsorption of bacterial cells displaying poly-His peptides. Bio/Technology. 14:1017—20.
Sousa, C., P. Kotrba, T. Ruml, A. Cebolla, and V. de Lorenzo. 1998. Metalloadsorption by Escherichia coli cells displaying yeast and mammalian metallothioneins anchored to the outer membrane protein LamB. J. Bacteriol. 180:2280—84.
Summers, A. O. 1986. Organization, expression, and evolution of genes for mercury resistance. Annu. Rev. Microbiol. 40:607-634.
Summers, A. O. 1992. Untwist and shout: a heavy metal responsive transcriptional regulator. J. Bacteriol. 174:3097-3101.
Wang, Y., M . Moore, H. S. Levinson, S. Silver, C. Walsh and I. Mahler. 1989. Nucleotide sequence of a chromosomal mercury resistance determinant from a Bacillus sp. with broad-spectrum mercury resistance. J. Bacteriol. 171:83-92.
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