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研究生:汪玉婷
研究生(外文):Yu-Ting Wang
論文名稱:基因轉殖大腸桿菌吸附重金屬之精細結構分析
論文名稱(外文):Fine Structure Analyses of Recombinant Escherichia coli with Heavy Metal Adsorption
指導教授:林錕松
指導教授(外文):Kuen-Song Lin
學位類別:碩士
校院名稱:元智大學
系所名稱:化學工程學系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:191
中文關鍵詞:重金屬大腸細菌merP延伸細微結構X光吸收光譜X光吸收邊緣結構光譜光電子能譜譜儀場發射電子顯微鏡
外文關鍵詞:metalEscherichia colimerPESAFSXANESXPSFE-SEM
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本研究主要是探討大腸桿菌(BL21)、及源於抗汞革蘭氏陽性菌(GB)及陰性菌(GP)之汞鍵結蛋白質基因merP分別選殖至大腸桿菌宿主細胞,以IPTG誘導後,進行鉛、鎘、汞、鎳、鋅及銅等重金屬吸附實驗,藉由以下儀器分析探討菌體對重金屬之容忍度與重金屬離子對其影響。為了得知吸附重金屬後菌體裡內重金屬之含量,以感應耦合電漿放射光譜儀(ICP-AES)來測量其吸附量,由實驗結果發現,重金屬鉛、鎘、汞、鎳及銅之吸附量效果如下,G(B)>G(P)>BL21;而重金屬鋅之吸附量方面,則是G(P)>G(B)>BL21。並以場發射電子顯微鏡(FE-SEM),觀察細菌體吸附前後之菌體外形結構,細菌體經由重金屬吸附後,與原菌未吸附前的菌體形態相比,觀測菌體外形有無發生改變。在吸附鋅方面,菌體周圍產生出破碎物質,故菌體無法忍受;而在吸附鎳方面,菌體產生出黏稠物質包覆在細菌的周圍,藉此保護細菌不受重金屬的毒害;在吸附鉛、鎘、銅及汞方面,菌體吸附後並沒有很大的改變,還是呈現完整的菌體形態。
為了得知吸附重金屬後菌體裡內重金屬之含氧以及含硫化合物比例,藉由光電子能譜儀(XPS)分析則可發現三種菌體對於吸附不同重金屬有其不同化合物比例;在吸附銅與鎳的反應有相似的化合物比例模式,吸附量低時,Cu-S比例則與Cu-O比例相當,到較高吸附量時,則Cu-O比例則大於Cu-S。菌體對於吸附鋅與鎘也有相同化合物比例模式,不論濃度的高低,細菌喜好與硫鍵結,故菌體內的硫化物比氧化物多。在鉛的吸附反應中則恰恰好與鋅與鎘相反,不論吸附量的多寡,細菌體內的氧化物皆大於硫化物。另外,為了得知吸附重金屬後菌體裡內重金屬之細微結構,進一步以延伸細微結構X光吸收光譜(EXAFS)及X光吸收邊緣結構(XANES)光譜來分析。利用XANES則發現在吸附鉛、鎘、汞、鎳、鋅及銅重金屬後之價數為兩價的狀態;而在EXAFS光譜分析當中則發現吸附銅、鎳或鋅之反應配位數分別為4、6及6,而其Cu-S、Ni-S及Zn-S鍵長分別約為2.05、2.16及2.16 ± 0.05 Å。由化合物鍵結特性分析Cu-S、Ni-S及Zn-S之可能構造分別為四方平面晶系及六方晶系結構,但因生物體結構具有大量的C、O、N及S與其他微量金屬,其中金屬結構及可能因周圍配位之複雜性,造成CuS、NiS及ZnS晶體結構之扭曲或變形,以求得更穩定的鍵結型態。
Gram-positive (Bacillus cereus RC607) and Gram-negative (Pseudomonas sp. K-62) merP genes encoding metal-binding proteins cloned and over-expressed in Escherichia coli BL21 hosts were studied in the present work. The MerP protein possessing a highly conserved with two cysteine residues (Cys-X-X-Cys) for heavy bonding was over-expressed via IPTG induction and the resulting recombinant strains were used to adsorb heavy metals in aqueous solutions of Pb2+, Cd2+, Hg2+, Ni2+, Zn2+, and Cu2+. Experimentally, the adsorption capacities of these metals for the merP or merP-free cells were carefully measured by using ICP-AES. The experimental results indicate that the metal adsorption capacity of merP or merP-free host cells for Pb2+, Cd2+, Hg2+, Ni2+, and Cu2+ all decreased in the order of G(B)>G(P)>BL21 whereas the trend was G(P)>G(B)>BL21 for Zn2+.
The morphology or microstructures of cells markedly changed after metal adsorption and exhibited signals of merP host cell lysis by FE-SEM microphotos. The degree of transformation of the cells adsorbing metal cations analyzed by FE-SEM was Zn>Ni>Pb>Cd>Cu. The element analysis, oxidation states, fine structures of metals in merP or merP-free cells were also investigated by using XPS, EA, EDS, XANES or EXAFS spectroscopies. The XPS analysis shows that the of metal oxides excluding Pb species was higher than that of metal sulfides in higher metal adsorption capacity of in merP cells. From XANES data, valencies of all metal cations were 2. It showed that the metal cations might preferably attack SH groups and combined into M-S bonding. The EXAFS spectra indicating that the Cu-S, Ni-S, and Zn-S species in merP host cells had bond distances of 2.05, 2.16, and 1.97Å, respectively. Coordination numbers of the Cu, Ni, and Zn elements in merP host cells were 4, 6, and 6, respectively. These results might offer a further explanation of the transformation for more stable metal-S structures in cells postulated with plane or hexagonal bonding under complex environment.
中文摘要 i
ABSTRACT ii
誌謝 iii
目錄圖目錄 iv
圖目錄 vii
表目錄 xx
第一章 前言 1
第二章 文獻回顧與原理 4
2-1 重金屬之簡介 4
2-1-1 重金屬之定義 4
2-1-2 重金屬污染之來源 4
2-2 重金屬之特性及危害 5
2-2-1 鉛 5
2-2-2 鎘 6
2-2-3 銅 7
2-2-4 鎳 8
2-2-5 鋅 8
2-2-6 汞 9
2-3 微生物對重金屬的吸附與回收 12
2-3-1 微生物處理重金屬的機制 12
2-3-1-1 生物轉換 13
2-3-1-2 菌體外的累積與沉澱 13
2-3-1-3 菌體表面之積聚 13
2-3-1-4 細胞內之累積 14
2-3-2 生物對重金屬之去除原理 15
2-4 以基因改質微生物進行生物復育 15
2-5 微生物的抗汞機制 17
2-6 MerP簡介 17
第三章 實驗設備與研究方法 18
3-1 實驗藥品 18
3-2 實驗設備 19
3-3 菌種簡介及培養 20
3-3-1 菌種簡介 20
3-3-2 培養基製備 21
3-3-2-1 固態培養基 21
3-3-2-2 液態培養基 21
3-3-3 菌種培養與誘導 21
3-3-4 細胞之光學密度測量 22
3-3-5 濃縮菌液的製備 22
3-4 重金屬溶液之配製與測量 23
3-4-1 重金屬測量 23
3-5 重金屬吸附實驗 23
3-6 菌種的乾燥與保存 24
第四章 結果與討論 26
4-1 菌體吸附重金屬前後之硫及金屬含量分析 26
4-1-1 吸附效果測定 26
4-1-2 元素分析 27
4-2 菌體吸附重金屬前後之表面分析 28
4-2-1 場發掃描式電子顯微鏡之分析 28
4-2-1-1 E. coil BL21 (DE3)/pLysS菌種之分析 28
4-2-1-2 E. coli BL21 (DE3)/pLysS/pET-21b G+P菌種之分析 36
4-2-1-3 E. coli BL21 (DE3)/pLysS/pET-21b G-P菌種之分析 43
4-2-2 X光光電子能譜儀分析 51
4-2-2-1 E. coil BL21 (DE3)/pLysS菌種之分析 54
4-2-2-2 E. coli BL21 (DE3)/pLysS/pET-21b G+P菌種之分析 61
4-2-2-3 E. coli BL21 (DE3)/pLysS/pET-21b G-P菌種之分析 68
4-3 菌體與重金屬之結構分析 76
4-3-1 X光吸收邊緣結構光譜 76
4-3-1-1 E. coil BL21 (DE3)/pLysS菌種之分析 76
4-3-1-2 E. coli BL21 (DE3)/pLysS/pET-21b G+P菌種之分析 84
4-3-1-3 E. coli BL21 (DE3)/pLysS/pET-21b G-P菌種之分析 89
4-3-2 延伸細微結構X光吸收光譜分析 94
4-3-2-1 E. coil BL21 (DE3)/pLysS菌種之分析 94
4-3-2-2 E. coli BL21 (DE3)/pLysS/pET-21b G+P菌種之分析 103
4-3-2-3 E. coli BL21 (DE3)/pLysS/pET-21b G-P菌種之分析 109
第五章 結論與未來研究方向 118
5-1 細菌體菌相之表面分析 118
5-2 細菌體中金屬化合物之分析 118
5-3 細菌體中結構之分析 119
參考文獻 124
附錄A BL21吸附不同金屬之FE-SEM分析 130
附錄B G(B)吸附不同金屬之FE-SEM分析 132
附錄C G(P)吸附不同金屬之FE-SEM分析 133
附錄D BL21吸附不同重金屬之XPS全譜圖 135
附錄E G(B)吸附不同重金屬之XPS全譜圖 145
附錄F G(P)吸附不同重金屬之XPS全譜圖 155
附錄G BL21吸附不同重金屬之同步輻射光譜 165
附錄H G(B)吸附不同重金屬之同步輻射光譜 174
附錄I G(P)吸附不同重金屬之同步輻射光譜 183
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