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研究生:林宏明
研究生(外文):Hung-Ming Lin
論文名稱:利用定點飽和突變研究抹香鯨肌紅蛋白中Ile-107位置對其過氧化能力之影響
論文名稱(外文):ite-Saturated Mutational Analysis of Isoleucine 107 from Sperm Whale Myoglobin on the Effect of Peroxidase Activity
指導教授:吳東昆
指導教授(外文):Tung-Kung Wu
學位類別:碩士
校院名稱:國立交通大學
系所名稱:生物科技系所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2006
畢業學年度:95
語文別:中文
論文頁數:96
中文關鍵詞:肌紅蛋白定點突變過氧化酶紫質
外文關鍵詞:myoglobinsite-directed mutagenesisperoxidaseprophyrin
相關次數:
  • 被引用被引用:1
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  • 下載下載:11
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肌紅蛋白(Myoglobin)為一種血基質蛋白(heme-protein),在脊椎動物體內具有儲存及攜帶氧氣的功能。本論文的研究主要是將不具酵素活性的肌紅蛋白突變成具有過氧化酵素(peroxidase)活性的功能。將mb基因殖入pET28a(+)表現質體中,利用定點突變及定點飽和突變技術改變His-64、Val-68、Ile-107這三個推測可改變活性的胺基酸。利用IPTG誘導蛋白質的大量表現以形成包涵體(inclusion body),接著以guanidine hydrochloride使包涵體變性再復性後,利用DEAE管柱純化,可以得到純度高達90%的脫輔基肌紅蛋白(apo-myoglobin),利用蛋白質輔基重組實驗可將血基質(heme)包入突變的脫輔基肌紅蛋白中。與過氧化氫和2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)反應後可以利用UV/VIS光譜儀偵測其一個電子傳遞的過氧化酵素活性。比較文獻的雙重突變H64D/V68L Mb以及利用定點飽和突變技術改變Ile-107的三重突變,在經由動力學參數測量後發現H64D/V68L/I107M Mb在與過氧化氫形成Compound I的效率可提高30%,與ABTS的一個電子傳遞效率則提高60%。另外也發現將Ile-107置換成體積較小的Ala及Val對形成Compound I的效率影響不大,但是卻可以提高一個電子傳遞效率47%及36%。因此認為Ile-107可能藉由立體效應去影響酵素與受質的一個電子傳遞效率。未來本實驗室會將各種不同金屬的紫質包入突變的脫輔基肌紅蛋白中,並研究其特性以期能應用並開發新型的染料敏化生物性太陽能電池(dye-sensitized biosolar cell)。
Myoglobin is a heme-protein, functioning as oxygen storage/carrier in vertebrates. In this study, the nonenzymatic myoglobin was functionally converted into a heme enzyme with peroxidase activity. Three amino acid residues, His-64/Val-68/Ile-107, located on the putative active site cavity, were subjected to site-directed mutagenesis and cloned into a pET-28a(+) expression vector. Following IPTG induction, the cell cultures were harvested and subjected to protein purification. After guanidine hydrochloride disruption and renaturation, the soluble apo-myoglobin was purified to homogeneity by DEAE chromatography. The heme molecule was reconstituted into these renatured apo-myoglobin mutants. A well established peroxidase activity with one-electron oxidation of 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was observed by the UV/VIS spectrophotometer from the myoglobin mutants, demonstrating its capability in electron transfer reaction. Among these mutations, as compared with the original MbH64D/V68L double mutant, MbH64D/V68L/I107M triple mutant exhibited a 30% activity increase in the compound I formation, whereas, a 60% activity increase in the one-electron oxidation was observed. Alternativily, the MbH64D/V68L/I107A or MbH64D/V68L/I107V showed the similar compound I formation rate, but with the one-electron oxidation rate of 47% or 36% increment respectively. These results indicated that the Ile-107 position may influence the one-electron oxidation of substrates via steric effect after the compound I formation. In the future, various metalloporphyrins with different metal ions will be reconstituted into the apo-myoglobin mutants to investigate their potentials as novel dye-sensitizers for biosolar cell application.
目錄 頁次
中文摘要…………………………………………………………………Ⅰ
英文摘要…………………………………………………………………Ⅲ
謝誌………………………………………………………………………V
目錄……………………………………………………………………VII
圖目錄……………………………………………………………………XI
表目錄…………………………………………………………………XVI


第一章 緒論 1
1-1 肌紅蛋白(myoglobin)的簡介 1
1-2 紫質(prophyrin)的簡介 4
1-3 血基質蛋白(heme protein) 5
1-4 過氧化酵素 6
1-5 肌紅蛋白經突變後具有過氧化酶酵素活性 9
1-6 染料敏化太陽能電池 (DSSC) 12
1-7 肌紅蛋白在人工光合作用反應中心的應用 14
1-8 研究目的 20
第二章 實驗材料及方法 22
2-1 實驗材料 22
2-1-1 化學藥品與材料 22
2-1-2 緩衝液及溶液配製 24
2-1-3 實驗儀器 28
2-1-4 菌株與載體 29
2-2 實驗方法 30
2-2-1 目標基因的建構 31
2-2-2 重組質體的建構 33
2-2-2-1 質體之轉化作用 34
2-2-3 定點突變與飽和定點突變實驗 35
2-2-4 脫輔基(apo-from)蛋白質的表現 38
2-2-5 包涵體(inclusion body)粗萃液之製備 39
2-2-6 包涵體(inclusion body)的變性與再摺疊 39
2-2-7 脫輔基(apo-form)蛋白質的純化 40
2-2-8 蛋白質分子量及純度分析 40
2-2-9 蛋白質輔基重組實驗 42
2-2-10 酵素最適化條件之研究 43
2-2-10-1 最適反應環境之酸鹼值測定 43
2-2-10-2溫度與活性之關係 44
2-2-11 酵素動力學參數測定 44
第三章 結果與討論 46
3-1 目標基因質體之建構 46
3-2 篩選目標 46
3-2-1 細胞粗萃液的活性篩選 49
3-3 脫輔基(apo-form)蛋白質表現及純化 51
3-4 蛋白質輔基重組 53
3-5 過氧化酵素活性最適反應條件 57
3-5-1最適反應之pH值 57
3-5-2溫度與活性之關係 59
3-6酵素動力學參數 60
第四章 結論與未來展望 82
第五章 參考文獻 84
附錄一 87
附錄1-1 建構不同金屬紫質的MbH64D/V68L/I107M三重突變肌紅蛋白 87
附錄二 89
附錄2-1 單株抗體的製備 89
附錄2-2 免疫動物 89
附錄2-3 骨髓癌細胞 (myeloma cell) 90
附錄2-4 細胞融合 91
附錄2-5 細胞增殖及取樣篩檢 92
附錄2-6 融合細胞之單株化 93
附錄2-7 單株抗體之產生 94
附錄2-8 利用西方墨點法 (Western blotting)確認單株抗體之專一性 94
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