Please use this identifier to cite or link to this item:
標題: 應用有機汞裂解酶及金屬硫蛋白質於清除汞化合物之探討
Application of MerB and MT proteins for the removal of mercurial compounds
作者: 江家成
Chiang, Chia-Cheng
關鍵字: bioaccumulation
organic mercury
organomercurial lyase(MerB)
Bacillus megaterium
scavenge free radical
金屬結合蛋白質(Metallothionein, MT)
Bacillus megaterium
出版社: 生命科學系所
引用: 李小龍. (2003). 金屬硫蛋白質鋅誘導合成及其在杜長大雜交豬和雜種野豬體內代謝規律的研究. 湖南農業大學. 碩士論文. 汪玉婷. (2004). 轉殖大腸桿菌吸附重金屬之精細結構分析. 元智大學化學工程學系. 碩士論文. 陳弘成. (1999). 汞污泥水池之魚體含汞量之研究. 台大漁推. 11, 15-19. 陳建仁. (2004). 烏腳病導因砷中毒. 科學人9月號. 葉顓銘, 陳少燕, 黃定鼎, 黃浩仁. (2004). 清理重金屬汙染的植物. 科學發展. 蔡懷楨. (2003). 動物基因轉殖技術與實驗. 教育部顧問室. 林國興. (2010). 應用重金屬抗性相關蛋白質於生物復育之研究. 博士論文. 吳文祥. (2008). 應用吳郭魚金屬硫蛋白質和汞離子結合蛋白質MerP於清除自由 基之探討. 碩士論文. Achard-Joris, M., Moreau, J.L., Lucas, M., Baudrimont, M., Mesmer-Dudons, N., Gonzalez, P., Boudou, A. and Bourdineaud, J.P.(2007). Role of metallothioneins in superoxide radical generation during copper redox cycling: Defining the fundamental function of metallothioneins. Biochimie, 89, 1474-1488. Acyna, E. G., Pacyna, J. M., Steenhuisen, F., Wilson, S. (2006). Global nthropogenic mercury emission inventory for 2000. Atmos Environ, 0, 4048-4063. Barkay, T., Miller, S. M., Summers, A. O. (2003). Bacterial mercury resistance from atoms to ecosystems. FEMS Microbiology Reviews, 27(2-3), 355-384. Begley, T. P., Walts, A. E., Walsh, C. T. (1986). Bacterial organomercurial lyase: overproduction, isolation, and characterization. Biochemistry, 25(22), 7186-7192. Belliveau, B. H., Tevirs, J. T. (1989). Mercury resistance and detoxification in bacteria. Appl Organometal Chem, 3, 283-294. Bontidean, I., Ahlqvist, J., Mulchandani, A., Chen, W., Bae, W., Mehra, R. K., Mortari, A., Csöregi, E. (2003). Novel synthetic phytochelatin-based capacitive biosensor for heavymetal ion detection. Biosensors and Bioelectronics, 18, 547-553. Brown, N. L., Camakaris, J., Lee, B. T., Williams, T., Morby, A. P., Parkhill, J., Rouch, D. A. (1991). Bacterial resistances to mercury and copper. J Cell Biochem, 46(2), 106-114. Chen, W. Y., John, J. A., Lin, C. H., Lin, H. F., Wu, S. C., Lin, C. H., Chang, C. Y. (2004). Expression of metallothionein gene during embryonic and early larval development in zebrafish. Aquatic Toxicology. , 69,215-217. Cherian, M. G. (1979). Metabolism of Orally Administered Cadmium-Metallothionein in Mice. Environmental Health Perspectives, 28, 127-130. Chung, R. S., Penkowa, M., Dittmann, J., King, C. E., Bartlett, C., Asmussen, J. W., Hidalgo, J., Carrasco, J., Leung, Y. K., Walker, A. K., Fung, S. J., Dunlop, S. A., Fitzgerald, M., Beazley, L. D., Chuah, M. I., Vickers, J. C., West, A. K. (2008). Redefining the role of metallothionein within the injured brain: extracellular metallothioneins play an important role in the astrocyte-neuron response to injury. J Biol Chem, 283(22), 15349-15358. Clarkson, T. W. (1997). The toxicology of mercury. Crit Rev Clin Lab Sci, 34, 369-403. Cousins, R. (1998). A role of zinc in the regulation of gene expression. Proc Nutr Soc, 57, 307-311. Duruibe, J. O., Ogwuegbu, M. O. C., Egwurugwu, J. N. (2007). Heavy metal pollution and human biotoxic effects. International Journal of Physical Sciences, 2 (5), 112-118. Falchuk, K. (1998). The molecular basis for the role of zinc in developmental biology. Mol Cell Biochem, 188, 41-48. Fu, Y., Wang, Y., Evans, S. (1998). Viral sequences enable efficient and tissue-specific expression of transgenes in Xenopus. Nat Biotech, 16, 253-257. Garbarino, J. R., Hayes, H., Roth, D., Antweider, R., Brinton, T. I., Taylor, H. (1995). Contaminants in the Mississippi River. Virginia, U.S.A. Goldwater, L. (1971). Mercury in the Environment. Sci Am, 224, 15-21. Goyer, R. A. (1996). Toxic Effects of Metals Casarett and Doull''s Toxicology, 23, 691-736. Hamdy, M. K., Noyes, O. R. (1975). Formation of Methyl Mercury by Bacteria. Appl Microbiol 30(3), 424-432. Hamer, D. H. (1986). Metallothionein. Annu Rev Biochem, 55, 913-951. Harada, M. (1995). Minamata disease: methylmercury poisoning in Japan caused by environmental pollution. Crit Rev Toxicol, 25,1-24. Hsiao, C. D., Hsieh, F. J., Tsai, H. J. (2001). Enhanced expression and stable transmission of transgenes flanked by inverted terminal repeats from adeno-associated virus in zebrafish. Dev Dyn, 220(4), 323-336. Huang, C. C., Narita, M., Yamagata, T., Endo, G. (1999). Identification of three merB genes and characterization of a board-spectrum mercury resistance module encoded by a class II transposon of Bacillus megaterium strain MB1. Gene, 239, 361-366. Hidalgo, J., Bernues, J., Thomas, D. G., Garvey, J. S. (1988). Effect of 2-mercaptoethanol on the electrophoretic behavior of rat and dogfish metallothionein and chromatographic evidence of a naturally occurring metallothionein polymerization. Comp Biochem Physiol C, 89(2), 191-196. Ibolya, B., Ahlqvist, J., Mulchandani, A., Chen, W., Bae, W., Mehra, R. K., Mortari A., Csoregi. E. 2003. Novel synthetic phytochelatin-based capacitive biosensor for heavymetal ion detection. Biosensors and Bioelectronics. 18, 547-553. Jeremias, H. R. (1991). Overview of metallothionein. Methods in enzymology. 205, 613-626. Ka¨gi, J. H. (1991). Overview of metallothionein. Methods Enzymol, 205, 613-626. Lazo, J. S., Kondo, Y., Dellapiazza, D., Michalska, A. E., Choo, K. H. Pitt, B. R. (1995). Enhanced sensitivity to oxidative stress in cultured embryoniccells from transgenic mice deficient in metallothionein I and II genes. J. Biol. Chem, 270, 5506-5510. Li, P., Feng, X. B., Qiu, G. L., Shang, L. H., Li, Z. G. (2009). Mercury pollution in Asia: A review of the contaminated sites. Journal of Hazardous Materials, 168(2-3), 591-601. Lin, K. H., Chien, M. F., Hsieh, J. L., Huang, C.-C. (2010). Mercury resistance and accumulation in Escherichia coli with cell surface expression of fish metallothionein. Appl Microbiol Biotechnol, 87, 561-569. Malten, M., Hollmann, R., Deckwer, W. D., Jahn, D. ( 2005). Production and secretion of recombinant Leuconostoc mesenteroides dextransucrase DsrS in Bacillus megaterium. Biotechnol Bioeng, 89(2), 206-218. Maret, W. (2008). Metallothionein redox biology in the cytoprotective and cytotoxic functions of zinc. Experimental Gerontology, 43, 363-369. Margoshes, M., Vallee, B. L. (1957). A cadmium protein from equinekidney cortex. J Am Chem Soc, 79, 4813-4814. McLean, E., Ash, R., Teskeredzic, Z., Teskeredzic, E. (1998). Intact protein absorption by the fish gut. 2. application potential and limitations. Ribarstvo, 3, 91-100. Miliauskas, G., Venskutonis, P. R., Beek, T. A. V. (2003). Screening of radical scavenging activity of some medicinal and aromatic plant extracts. Food Chemistry, 85. 231–237. Misra, T. K. (1992). Bacterial resistances to inorganic mercury salts and organomercurials. Plasmid, 27(1), 4-16. Morel, F., Kraepiel, A., Amyot M (1998). The Chemical Cycle and Bioaccumulation of Mercury. Annu Rev Ecol Syst, 29, 543-566. Nettesheim, D. G., Engeseth, H. R., Otvos, J. D. (1985). Products of metal exchange reactions of metallothionein. Biochemistry, 24(24), 6744-6751. Nordberg, G. F., Garvey., J. S., Chang, C. C. (1982). Metallothionein in plasma and urine of cadmium workers. Environmental Research, 28(1), 179-182. Pacyna, J. M., Pacynaa, E. G., Steenhuisenb, F., Wilson, S. (2003). Mapping 1995 global anthropogenic emissions of mercury. Atmos Environ Sci Technol, 37, 109-117. Penkowa, M., Tio, L., Giralt, M., Quintana, A., Molinero, A., Atrian, S., Vasak, M., Hidalgo, J. (2006). Specificity and divergence in the neurobiologic effects of different metallothioneins after brain injury. J Neurosci Res, 83(6), 974-984. Peplow, D. (1999). Environmental Impacts of Mining in Eastern Washington. Center for Water and Watershed Studies Fact Sheet. Petering, H. G., Tepper, L. B. (1976). Pharmacology and toxicologyof heavy metals: mercury. Pharmacol Ther 1, 131-151. Powlowski, J., Sahlman, L. (1999). Reactivity of the two essential cysteine residues of the periplasmic mercuric ion-binding protein, MerP. J Biol Chem, 274(47), 33320-33326. Priest, F. G. (1977). Extracellular enzyme synthesis in the genus Bacillus. Bacteriol Rev, 41, 711-753. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med, 26(9-10), 1231-1237. Rygus. T., Hillen, W. (1991). Inducible high-level expression of heterologous genes in Bacillus megaterium using the regulatory elements of the xylose-utilization operon. Appl. Microbiol. Biotechnol., 35, 594-599. Rabenstein, D. L. (1978). The Chemistry of Methylmercury Toxicology. Journal of Chemical Education, 55, 292-296. Rasmussen, P. (1994). Current methods f estimating atmospheric mercury fluxesin remote areas. Environ. Sci. Technol, 28, 2233-2241. Sambrook, J., Fritsch, E. F., Maniatis, T. 1989. Molecular cloning: A laboratory manual, 2nd edition. Cold Spring Laboratory. Cold Spring Harbor, New York. Sambrook, J., Fritsch, E. F., Maniatis, T. 2000. Molecular cloning: A laboratory manual, 3nd edition. Cold Spring Laboratory. Cold Spring Harbor, New York. Sato, M., Kondoh, M. (2002). Recent studies on metallothionein: protection against toxicity of heavy metals and oxygen free radicals. Tohoku J Exp Med, 196, 9-22. Shea, K. M., Perry, K. L., Shah, M. (2004). Health effects of methylmercury. Physi-cians for Social Responsibility publication. Shen, J. C., Liu, J., Zhuang, Z. X., Wang, X. R., Lee, F. S. (2006). Investigation of zinc binding metallothioneins'' polymerization in tris(hydroxymethyl)-aminomethane buffer by coupling of size exclusion chromatography with electrospray ionization mass spectrometry. Talanta, 69(4), 988-995. Stuart, G. W., McMurray, J. V., Westerfield, M. (1988). Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos. Development, 103, 403-412. Summers, A. O. (1986). Organization, Expression, and Evolution of Genes for Mercury Resistance. Annual Review of Microbiology, 40, 607-634 Summers, A. O. (1992). Untwist and shout: a heavy metal-responsive transcriptional regulator. J. Bacteriol., 174, 3097-3101. Sun, J., Wang, W., Hundertmark, C., Zeng, A . P., Jahn, D., Deckwer, W. D. (2006). A protein database constructed from low-coverage genomic sequence of Bacillus megaterium and its use for accelerated proteomic analysis. J Biotechnol, 124, 486-495. Suzuki, K. T., Yamamura, M. (1980). solation and characterization of metallothionein dimers. Biochem. Pharmacol, 29 689. Thomas, W., Clarkson, L. M. (2006). The Toxicology of Mercury and Its Chemical Compounds. Critical Reviews in Toxicology 36, 609-662. Tsubaki. (1977). Minimata disease. KodanshaLtd: Tokoyo. Vary, P. S. (1994). Prime time for Bacillus megaterium. Microbiology, 140(5), 1001-1013. VonBurg, R. (1995). Toxicology update. J Appl Toxicol 15, 483-493. Warkany, J., Hubbard D. M. (1951). Adverse mercurial reactions in the form of acrodynia and related conditions. Am J Dis Child 81, 335-373. Winship, K. A. (1986). Organic mercury compounds and their toxicity. Adv Drug React Ac Pois Rev, 3, 141-180. Zangger, K., Shen, G., Oz, G., Otvos, J. D., Armitage, I. M. (2001). Oxidative dimerization in metallothionein is a result of intermolecular disulphide bonds between cysteines in the alpha-domain. Biochem J, 359, 353-360. Zhang, B., Georgiev, O., Hagmann, M., Günes, C., Cramer, M., Faller, P., Vasák, M., Schaffner, W. (2003). Activity of metal-responsive transcription factor 1 by toxic heavy metals and H2O2 in vitro is modulated by metallothionein. Mol Cell Biol, 23(23), 8471-8485.
摘要: 近年來,重金屬汙染已普遍成為河川、漁業養殖用水及其他水域極待解決之問題,其中因為有機汞會破壞中樞神經系統因而最具毒性。自然界存在著穩定的汞循環,但隨著工業發展,任意排放汞之故,導致過量汞釋放於環境中,伴隨著生物累積性,許多魚類的高等獵食者,體內之重金屬含量較易超過標準,當人們攝食這些魚類,便會遭受重金屬毒害,最有名例子為日本水俁病。因此如何讓有機汞不累積於魚體內,是現階段極待研發之目標;如何有效清除汞,可以仿效自然界,微生物可透過有機汞裂解酶MerB將有機汞轉化為較不具毒性之無機汞,甚至還能將無機汞轉化成汞蒸氣,釋放於胞外。而其他生物雖然不具這套機制,但其具有金屬結合蛋白質(Metallothionein, MT),能透過螯合汞之方式,降低汞之毒性,並將之排除於體外;且金屬結合蛋白質其結構上富含半胱胺酸,近年來被廣泛證實具有清除自由基之能力,因此可以清除因汞所造成之自由基。本研究利用這兩種蛋白質的能力,將MerB基因專一表現於斑馬魚肌肉及神經細胞上,期望能達到將有機汞轉化成較不具毒性之無機汞並不累積於體內及不破壞神經細胞之目標,且透過餵食MT之方式,經由腸胃道吸收進入血液循環,螯合住游離態的汞,並將之帶往腎臟排除於體外;本研究更利用Bacillus megaterium將MT產製於胞外,便於純化。結果顯示,本研究已成功轉殖出於肌肉表現MerB之轉殖基因斑馬魚,更驗證其能有效提高對汞之半致死濃度(LC50)。而MT於清除自由基之能力上,以ABTS為例,BM-25-TMT清除自由基之能力比控制組高出19%,以DPPH為例,比控制組高出9%;且於平板抗性實驗驗證,於汞濃度300 ppm、400 ppm、500 ppm下,BM-25-TMT能提升對汞之抗性。進一步透過餵食MT之方式,期望提高斑馬魚對汞之耐受性,結果顯示以濃度100 nM之PMA進行攻毒測試18小時後,餵食MT之斑馬魚其存活率比控制組高出15%;於300 nM之MMC攻毒中,餵食MT之斑馬魚其存活率比控制組高出10%。
In recent years, there are heavy metal pollution problem about river, aquaculture water and other aquatic environments need to resolve. Among heavy metal, because organic mercury which would damage the central nervous system so the most toxic. The nature has the stable mercury circulation system, but with the industrial development, high level mercury are emitted into environment. Because of bioaccumulation, large predatory fish bioaccumulate high concentrations of mercury.When people eat these fish, will be subjected to heavy metal poisoning. The most famous example is Japan Minamata disease. Therefore, how to let the organic mercury not accumulate in fish is goal of the present need research and development. How to effectively remove mercury, could follow the example of nature. Bacterial organomercurial lyase(MerB) conducts protolytic cleavage of the Hg-C bond, this can transform methylmercury to less toxic inorganic mercury, while another enzyme, mercuric ion reductase(MerA), reduces Hg(II) to elemental mercury, Hg(0),and release. Metallothionein can chelate mercury, then Metallothionein-metal complex can transported via the blood to the kidneys and release heavy metal. Metallothioneins are cysteine-rich metal-binding proteins, report indicate they have ability to scavenge free radical. so it can scavenge free radical from mercury-induced. In this study, MerB gene specific express at zebrafish muscle cell and nerve cell, in order to transform methylmercury to less toxic inorganic mercury, do not accumulate in fish and damage nerve cell. Through oral administration of MT then through gastrointestinal gut absorb into blood circulation, binding free mercury and sent it to kidneys release. Here use Bacillus megaterium as a host, it has ability to release MT to medium and easy to purify. Result indicate, MerB can specific express at muscle, and can transfer MerB to F1 generation. And LC50 values of mercury to transgenic zebrafish is higher than wild type. Furthermore , the supernatant medium from BM-25-TMT performed free radical scavenging ability was 19% higher than control in ABTS, and 9% higher than control in DPPH. And disc resistant assay show that in 300ppm, 400ppm, 500ppm mercury standar stress, BM-25-TMT had better mercury resistant. Finally, zebrafish through oral administration of MT indicate, in 100 nM PMA stress, the survival rates of administration of MT was 15% higher than control, in 300 nM MMC stress was 10% higher than control.
其他識別: U0005-1808201015022700
Appears in Collections:生命科學系所



Show full item record
TAIR Related Article

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.