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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李平篤 | |
| dc.contributor.author | Wei-Hsueh Lai | en |
| dc.contributor.author | 賴韋學 | zh_TW |
| dc.date.accessioned | 2021-06-15T01:35:05Z | - |
| dc.date.available | 2009-07-24 | |
| dc.date.copyright | 2009-07-24 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-07-17 | |
| dc.identifier.citation | Abell, C.W., and Shen, R.S. (1987). Phenylalanine ammonia-lyase from the yeast Rhodotorula glutinis. Methods Enzymol. 142, 242– 8.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43062 | - |
| dc.description.abstract | 苯丙胺酸裂解酶 (phenylalanine ammonia lyase, PAL EC 4.3.1.5) 為植物體內 phenylpropanoid 代謝路徑中的第一個反應酵素。可催化 L-phenylalanine 行脫氨反應成 trans-cinnamic acid 和 氨，以促使植物二級代謝產物及大量 phenylpropanoids 衍生物生合成，包括木質素 (lignin)、黃酮素 (flavonoid) 等。本論文以白蘆筍嫩莖為材料，經緩衝液粗抽、硫酸銨分劃、膠體過濾法 (Sepharacyl S-300)、疏水性作用層析法 (Phenyl-Sepharose) 及快速蛋白質液相層析法 (FPLC) 後，最後得到純化倍率為 65 倍，比活性為 41 nkat/mg protein。以 FPLC Superose 12 測得 PAL 原態分子量約 288 kD，次單元體分子量由 SDS-PAGE 測得為約 75 kD，因此推測綠竹 PAL 可能為四元體結構。利用綠竹免疫染色法之專一性特性，顯示純化所得結果確實為 PAL。
白蘆筍 PAL 之生化性質，最適反應溫度為 55℃，Km 值為 982 μM，活化能為 22.5 kcal/mol，最適反應 pH 為 8.5。酵素活性會受到鎂、鈣離子的活化；錳、鈷、汞離子的抑制。二級代謝產物，如 trans-cinnamic acid, p-amino-benzonic acid, caffeic acid 及 tannic acid 對 PAL 有回饋抑制現象。PAL 對基質 L-phenylalanine 有專一性。酵素催化活化區域具有 serine、tyrosine、histidine 胺酸。 | zh_TW |
| dc.description.abstract | Phenylalanine ammonia lyase (PAL, EC 4.3.1.5) is the first enzyme of the phenylpropanoid metabolic pathway. The enzyme catalyzes the deamination of L-phenylalanine to form trans-cinnamic acid and ammonia. This reaction leads to biosynthesis of many phenylpropanoid-derived secondary metabolic products in plants, such as flavonoids and lignin. In this study, PAL was purified from white asparagus spears successively by buffer extraction, ammonium sulfate precipitation, gel filtration (Sephacryl S-300), hydrophobic interaction chromatography (Phenyl-Sepharose), and fast protein liquid chromatography (FPLC). The purified PAL with 65-fold, and specific activity of 41 nkat/mg protein. Using Superose 12 column (FPLC), the molecular weight of asparagus PAL of native form was estimated to be 288 kD, the subunit weight was determined to be about 75 kD by SDS-PAGE, using immunoblot by bamboo PAL antibody, and the PAL might be a tetrameric form.
Biochemical properties of asparagus PAL：The optimum temperature of PAL activity was 55.℃ The Km value for L-phenylalanine was 982 μM. The activation energy was 22.5 kcal/mol. The optimum pH of PAL activity was 8.5. Asparagus PAL activity is activated by Ca2+, Mg2+ and inhibited by Mn2+, Co2+, Hg2+. Secondary metabolites e.g. trans-cinnamic acid, p-amino-benzonic acid, caffeic acid and tannic acid with phenomena of feedback inhibition on asparagus PAL. PAL had specificity to L-phenylalanine. Enzyme catalytic site presented serine, tyrosine and histidine residues. | en |
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| dc.description.tableofcontents | 目錄……………………………………………………………………………………a
縮寫表………………………………………………………………………………….i 中文摘要………………………………………………………………………………ii 英文摘要……………………………………………………………………………...iii 第一章 緒論…………………………………………………………………………..1 1.1 蘆筍……………………………………..….………………………1 1.2 植物二級代謝與 Phenylpropanoid pathway…..……………………..……..2 1.3 苯丙胺酸裂解酶………………………….………………………………….4 1.4 PAL之細胞定位......…..….…...….…..……..……………..5 1.5 PAL之調控……..……………..…..….…………..…………...6 1.5.1 生長環境因子對 PAL 轉錄方面的調控………………….………6 1.5.2 二次代謝產物的回饋抑制……………………………………….…...8 1.5.2 轉譯後磷酸化修飾………………………………………………...….9 1.6 禾本科植物 PAL 可能具有 TAL 活性…………………………………9 1.7 PAL 之催化機制...……………….…………….……….…….…………10 1.8 PAL在醫學上的應用..…………..…..………….……..………………..13 1.9 實驗緣起…………………………………………………………………..14 第二章 材料與方法….……..…………………………………………………...…..16 2.1 材料、藥品與儀器..…………..…..………….……..………..……………..16 2.1.1 材料..…………..…………………….……..………………...……..16 2.1.2 藥品與儀器..…………..……….……..………..…..…………….....16 2.2 蛋白質定量法..…………..…..………….…….………..……………….....17 2.3 酵素活性分析法..…………..…..………….….………..……………….....18 2.3.1 PAL 活性..………………….….……………..……..………….....18 2.4 電泳檢定系統..………………….….……………..……..……..……….....19 2.4.1 原態膠體電泳 (native-PAGE) …………..……..…..…..……….....20 2.4.2 SDS-膠體電泳 (SDS-PAGE) …………...……..…..…..……..….....23 2.4.3 膠體染色法………...…….….……………..……..……..……….....26 2.4.3.1 CBR蛋白質染色法.….……………..……..……..……….....26 2.4.3.2 膠片乾燥法.….……………..……..……………..……….....27 2.5 蛋白質電泳轉印與免疫染色……………..……..……………..……….....27 2.5.1 蛋白質電泳轉印法 …………..……..…..……………..……….....28 2.5.2 免疫染色法…………...……..…..…..………………………..….....29 2.6 PAL 的純化…..…..…..…………………………………….………..….....31 2.6.1 粗抽及硫酸銨分劃………………………………….………..….....31 2.6.2 膠體過濾層析……………………………………….………..….....33 2.6.3 疏水性作用管柱層析……………………………….………..….....35 2.6.4 快速蛋白質液相管柱層析（FPLC）……………….………..….....38 2.7 分子量測定…..…..…..…………………………………….………..….....40 2.7.1 Superose 6 和 Superose 12 (FPLC) 膠體過濾層析………..….....40 2.7.2 SDS-PAGE..…..…………………………………….…….…..….....42 2.8 酵素生化性質..…..…………………………………….………..….....42 2.8.1 酵素反應最適 pH 值………………………….….………...….....42 2.8.2 最適反應溫度…………………………………………..…......43 2.8.3 活化能 (Activation energy, Ea) ……………..………..….....43 2.8.4 熱安性性………....………....………....……......………..….....43 2.8.5 Km 值………....………....………....……………..............…..….....43 2.8.6 金屬離子對活性影響………. …....……….....………..….....44 2.8.7 二級代謝物對活性影響……….... …....…..….………….....44 第三章 結果與討論…………………………………………………………………46 3.1 白蘆筍嫩莖 PAL 純化策略………………………………...……………46 3.1.1 純化之流程……………………………………………....…………46 3.1.2 活性測定…………………………………………….……………47 3.1.3 硫酸銨分劃………………………………………….……………47 3.1.4 Gel filtration (Sephacryl S-300) ……………………….……………48 3.1.5 Hydrophoic interaction chromatography (Phenyl-Sepharose) …….49 3.1.6 FPLC (Mono Q HR5/5) ……………………………….……………50 3.1.7 各純化步驟電泳分析比較………………………….……………50 3.1.8 純化表……………………………………………….……………50 3.2分子量測定………………………………………………….……………50 3.2.1 原態分子量測定………………………….……………………51 3.2.2 次單元體分子量測定………………………….……….………51 3.3 白蘆筍嫩莖 PAL 生化性質分析………………….………….….………51 3.3.1 Time course………………………….……………………..……51 3.3.2 最適反應溫度與活化能………………………….………………52 3.3.3 熱安定性………………………….………………………………52 3.3.4 最適反應 pH………………………….……………….…………53 3.3.5 Km 值……………...............…………….……………….…………53 3.3.6 金屬離子影響………………………….……………….…………53 3.3.7 二級代謝物對 PAL 活性的影響………………………….……54 3.3.8 基質專一性………………………….…………………..…………54 3.3.9 化學修飾劑對活性影響………………………….………………55 第四章 結論與展望…………………………………………………………………56 4.1 白蘆筍嫩莖苯丙胺酸裂解酶純化…………….…………………..………56 4.2 苯丙胺酸裂解酶生化性質比較…………….…………………..…………56 4.3 未來展望…………….…………………..…………………………………58 參考文獻……………………………..………………………………………………87 | |
| dc.language.iso | zh-TW | |
| dc.subject | 筍 | zh_TW |
| dc.subject | 苯丙胺酸裂 | zh_TW |
| dc.subject | 解酶 | zh_TW |
| dc.subject | Asparagus | en |
| dc.subject | Phenylalanine Ammonia Lyase | en |
| dc.title | 白蘆筍苯丙胺酸裂解酶之生化學研究 | zh_TW |
| dc.title | Biochemical Studies of Phenylalanine Ammonia Lyase from White Asparagus Spears (Asparagus officinalis) | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林棋財,楊健志 | |
| dc.subject.keyword | 苯丙胺酸裂,解酶,蘆,筍, | zh_TW |
| dc.subject.keyword | Phenylalanine Ammonia Lyase,Asparagus, | en |
| dc.relation.page | 93 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2009-07-17 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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