Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23073
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
有機汞
organic mercury
organomercurial lyase(MerB)
Metallothionein
Bacillus megaterium
LC50
scavenge free radical
有機汞裂解酶MerB
金屬結合蛋白質(Metallothionein, MT)
Bacillus megaterium
半致死濃度(LC50)
清除自由基
出版社: 生命科學系所
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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.
URI: http://hdl.handle.net/11455/23073
其他識別: U0005-1808201015022700
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1808201015022700
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