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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李平篤 | |
| dc.contributor.author | Yi-Lin Hsieh | en |
| dc.contributor.author | 謝毅霖 | zh_TW |
| dc.date.accessioned | 2021-06-13T03:37:09Z | - |
| dc.date.available | 2006-07-28 | |
| dc.date.copyright | 2006-07-28 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-27 | |
| dc.identifier.citation | 參考文獻
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32215 | - |
| dc.description.abstract | 苯丙胺酸脫氨裂解酶 (phenylalanine ammonia lyase, EC 4.3.1.5，簡稱 PAL) 是植物 phenylpropanoids 生合成之第一個酵素。其催化苯丙胺酸 (L-phenylalanine) 進行非氧化性脫氨反應，產生肉桂酸 (trans-cinnamic acid)，再經由許多酵素催化，可衍生出許多的二級代謝物，如黃酮素、木質素等。
本論文為探討在 PAL 的催化反應中，可能有哪些胺酸參與，故以點突變的方式，改變特定胺酸，並利用嗜甲醇酵母菌 (Pichia pastoris) 來表現突變後的蛋白質。再將突變之蛋白質與野生型做生化性質比較，便能得知該胺酸在 PAL 催化反應中可能之角色。 三種表現蛋白質 YPAL 2、YPAL 2-SA 及YPAL 2-FH 之C 端帶有 (His)6-tag，可經由鎳離子親和管柱進行純化。經 10% SDS-PAGE 得知，三種表現蛋白質之單元體分子量皆約為 80 kD。表現蛋白質 YPAL 2 與 YPAL 2-FH 對基質 L-Phe 之 Km 值分別為 0.28 mM 與 1.11 mM；Vmax 分別為 0.043 與0.014 nkat。YPAL 2 最適反應溫度為 60℃，最適反應 pH 為 8.0，反應活化能為 11.8 kcal/mol；YPAL 2-FH 由於活性過低及酵素用盡，僅能完成基質飽和曲線實驗；YPAL 2-SA 則由於完全沒有活性，故無法進行生化性質之測定。 將 YPAL 2 活性區的關鍵胺酸由 serine 改為 alanine (S200A) 使得 PAL的活性完全消失，原因可能為：在失去 serine 的情況下，將導致活性區的反應基團 MIO 無法形成。而將活性區附近的 phenylalanine 改為 histidine (F397H)可能會影響反應中間產物之形成，故 PAL 活性降的十分的低。 | zh_TW |
| dc.description.abstract | Abstract
Phenylalanine ammonia lyase (EC 4.3.1.5) is the first enzyme of the phenylpropanoid biosynthetic pathway. The enzyme catalyzes the non-oxidative deamination of L-phenylalanine to produce trans-cinnamic acid. This reaction leads to the biosynthesis of many phenylpropanoid derived compounds in plant such as flavonoids and lignin. In order to find out which amino acids play important roles in catalysis reaction of PAL, we use site-directed mutagenesis technique to change the specific amino acid residue of PAL and express it by the Pichia pastoris system. Three fusion protein YPAL 2、YPAL 2-SA and YPAL 2-FH containing C-terminal (His)6-tag was expressed then purified with Ni-column which retains proteins with polyhistidine tag. The subunit mass of the three expressed protein are about 80 kD. The Km values for phenylalanine are 0.28 mM for YPAL 2 and 1.11 mM for YPAL 2-FH. The Vmax values are 0.043 nkat for YPAL 2 and 0.014 nkat for YPAL 2-FH. The Kcat is 20.48 s-1 for YPAL 2 and 0.038 s-1 for YPAL 2-FH. The optimum temperature and pH for YPAL 2 are 60℃ and 8.0, respectively. The activation energy for YPAL 2 is 11.8 kcal/mol. Because of the amount and activity of YPAL 2-FH are very low we just can finish the substrate saturation experiment to deduce Km and Vmax values. In the other part, because the YPAL 2-SA totally loss its activity we can’t analyze its biochemical properties. By changing the amino acid in the active site of YPAL 2 from serine to alanine (S200A) could prevent the formation of MIO group which resulted in the losing of total activity. However, changing the amino acid near active site from phenylalanine to histidine (F397H) could affect the formation of intermediate and cause the decrease of PAL activity. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T03:37:09Z (GMT). No. of bitstreams: 1 ntu-95-R93b47209-1.pdf: 1731299 bytes, checksum: 980e943f2ab556125ee4a0c5f381691e (MD5) Previous issue date: 2006 | en |
| dc.description.tableofcontents | 目錄
目錄…………………………………………………………………………………..Ⅰ 縮寫表……………………………………..……………………………….………IV 中文摘要…………………………...………………………….…………………….Ⅴ 英文摘要…...……..…………………………………………..……………………Ⅵ 第一章 緒論…………………………………………………………………………1 1.1 植物的二級代謝…..…………………………...……………………………1 1.2 類苯丙酸路徑 (Phenylpropanoid pathway)………………………………..3 1.3 苯丙胺酸脫氨裂解酶…………………………………………………….…5 1.4 苯丙胺酸脫氨裂解酶之催化機制……………………………………….....6 1.5 苯丙胺酸脫氨裂解酶之細胞定位……………………………………….....8 1.6 苯丙胺酸脫氨裂解酶之調控………………………………………...……9 1.6.1 生長環境因子對 PAL 的調控……………………………………...9 1.6.2 二級代謝產物的回饋抑制…………………………………………10 1.6.3 轉譯後磷酸化修飾…………………………………………………11 1.7 苯丙胺酸脫氨裂解酶在醫學上的應用…………………………………...12 1.8 嗜甲醇酵母菌 Pichia pastoris.....................................................................12 1.8.1 Pichia pastoris 對甲醇的代謝作用………………………………..13 1.8.2 Pichia pastoris 之表現載體………………………………………..15 1.8.3 表現質體嵌入染色體之作用方式…………………………………15 1.8.4 Pichia pastoris 之宿主酵母菌株 (host strains)…………………...17 1.9 實驗緣起…………………………………………………………………...17 第二章 材料與方法…………………………………………………………………18 第一節 實驗材料…………………………………………………………………18 1.1 菌種………………………………………………………………………...18 1.2 載體………………………………………………………………………...18 第二節 實驗藥品…………………………………………………………………18 2.1 一般化學試劑……………………………………………………………...18 2.2 限制酶及核酸修飾酵素…………………………...………………………18 2.3 培養基……………………………………………………………………...19 2.4 抗體…………………………………………………………………….......19 第三節 儀器設備…………………………………………………………………19 3.1 離心機……………………………………………………………………...19 3.2 分光光度計………………………………………………………………...19 3.3 核酸電泳設備……………………………………………………………...19 3.4 蛋白質電泳設備…………………………………………………………...19 3.5 其他………………………………………………………………………...19 第四節 一般實驗法………………………………………………………………20 4.1 質體 DNA 之小量抽取 (Spin-column method)….……………………...20 4.2 質體 DNA 之中量抽取 (Spin-column method)...……………………….20 4.3 限制酶切割分析…………………………………………………………...21 4.4 質體轉形法………………………………………………………………...21 4.5 質體快速檢定法…………………………………………………………...22 4.6 洋菜膠體電泳……………………………………………………………...23 4.7 蛋白質定量法……………………………………………………………...24 4.8 蛋白質電泳檢定法………………………………………………………...24 4.8.1 原態膠體電泳………………………………………………………24 4.8.2 SDS 膠體電泳……………………………………………………...27 4.8.3 膠片染色法…………………………………………………………28 4.8.4 膠片乾燥法…………………………………………………………29 4.9 蛋白質電泳轉印法………………………………………………………...29 4.10 免疫呈色法…..…………………………………………………………….30 第五節 Pichia pastoris 表現系統之建立………………………………………..31 5.1 表現載體之點突變………………………………………………………...31 5.2 Pichia pastoris competent cell 之製備…………………………………..32 5.3 表現載體之電穿孔轉形…………………………………………………...33 5.4 ZeocinTM 抗生素篩選具高套數之轉形株................................................34 5.5 酵母菌染色體 DNA 之抽取………..……………………………………34 5.6 Mut+ 表現型酵母菌之 PCR 鑑定………….…………………………..35 5.7 Pichia pastoris 轉形株 Mut 表現型之鑑定………………....…………35 5.8 最佳誘導時間探討………………………………………………………...36 5.9 苯丙胺酸脫氨裂解酶活性分析法…..…………………………………...37 5.10 重組蛋白質之大量表現…………..……………………………………….38 5.11 重組蛋白質之純化………………………………………………………...38 第六節 重組蛋白質之生化活性分析……………………………………………40 6.1 分子量測定………………………………………………………………...40 6.2 酵素動力學研究…………………………………………………………...40 6.3 最適反應溫度……………………………………………………………...40 6.4 活化能 (Activation energy, Ea)……………………………………………41 6.5 熱安定性…………………………………………………………………...41 6.6 酵素反應最適 pH 值……………………………………………………..41 6.7 金屬離子對活性之影響…………………………………………………...41 第三章 實驗結果……………………………………………………………………42 第一節 pPBoPAL 2表現載體之點突變…………………………………………42 1.1 點突變的選擇……………………………………………………………...42 1.2 pPBoPAL 2表現載體之點突變……..…………………………………...42 1.3 pPBoPAL 2表現載體之酵母菌轉形與篩選………………...…………..43 第二節 綠竹筍苯丙胺酸脫氨裂解酶之大量表現與純化………………………44 2.1 最適誘導時間探討………………………………………………………...44 2.2 苯丙胺酸脫氨裂解酶之純化與鑑定……………………………………...45 第三節 綠竹筍苯丙胺酸脫氨裂解酶的生化性質探討…………………………46 3.1 分子量……………………………………………………………………...46 3.2 產物與吸光值關係………………………………………………………...46 3.3 表現蛋白質反應時間與吸光值關係……………………………………...47 3.4 酵素動力學研究…………………………………………………………...47 3.5 最適反應溫度……………………………………………………………...47 3.6 活化能……………………………………………………………………...47 3.7 熱安定性…………………………………………………………………...49 3.8 最適反應 pH 值……..…….……………………………………………...49 3.9 金屬離子影響……………………………………………………………...49 第四章 結論與展望…………………………………………………………………50 4.1 點突變與酵母菌表現系統之建立………………………………………...50 4.2 酵母菌表現蛋白質之純化………………………………………………...50 4.3 酵母菌表現蛋白質之分子形式…………………………………………...51 4.4 酵母菌表現蛋白質之生化性質…………………………………………...51 4.5 點突變對表現蛋白質之影響……………………………………………...52 4.6 未來展望…………………………………………………………………...53 4.6.1 點突變之應用………………………………………………………53 4.6.2 蛋白質結構研究……………………………………………………53 4.6.3 TAL 活性探討……………………………………………………...53 4.6.4 綠竹筍懸浮細胞之誘導……………………………………………53 結果圖表集…………………………………………………………………………..54 參考文獻……………………………………………………………………………..81 | |
| dc.language.iso | zh-TW | |
| dc.subject | 苯丙胺酸脫氨裂解酶 | zh_TW |
| dc.subject | phenylalanine ammonia lyase | en |
| dc.title | 經酵母菌表現之綠竹筍苯丙胺酸脫氨裂解酶 | zh_TW |
| dc.title | Site-Directed Mutagenesis of Bamboo Phenylalanine
Ammonia Lyase Expressed in Yeast (Pichia pastoris) | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 楊健志,林棋財 | |
| dc.subject.keyword | 苯丙胺酸脫氨裂解酶, | zh_TW |
| dc.subject.keyword | phenylalanine ammonia lyase, | en |
| dc.relation.page | 89 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2006-07-27 | |
| 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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