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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李平篤(Ping-Du Lee) | |
| dc.contributor.author | Guo-Jhang Ma | en |
| dc.contributor.author | 馬國彰 | zh_TW |
| dc.date.accessioned | 2021-06-15T04:52:25Z | - |
| dc.date.available | 2010-08-03 | |
| dc.date.copyright | 2010-08-03 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-07-31 | |
| 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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Biolog. 29, 535–550. 莊榮輝 (1985) 水稻蔗糖合成酶之生化及免疫學研究。博士論文，國立臺灣大學農 業化學研究所 謝陸盛 (2003) 綠竹葉與籜苯丙胺酸裂解酶之生化學研究。碩士論文，國立臺灣大學農業化學研究所 蘇玟珉 (2003) 綠竹筍苯丙胺酸裂解酶之生化學研究。碩士論文，國立臺灣大學農業化學研究所 潘宏記 (2004) 大腸桿菌表現綠竹苯丙胺酸脫氨裂解酶及功能鑑定。碩士論文，國立臺灣大學微生物與生化學研究所 鄭傑洋 (2005) 綠竹筍苯丙胺酸脫氨裂解酶在酵母菌中之表現與檢定。碩士論文，國立臺灣大學微生物與生化學研究所 謝毅霖 (2006) 經酵母菌表現之綠竹筍苯丙胺酸脫氨裂解酶的點突變。碩士論文，國立臺灣大學微生物與生化學研究所 謝陸盛 (2010) 綠竹苯丙胺酸脫氨裂解酶之生化學與分子生物學研究。博士論文， 國立臺灣大學微生物與生化學研究所 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/46051 | - |
| dc.description.abstract | 苯丙胺酸脫氨裂解酶 (Phenylalanine ammonia lyase; EC 4.3.1.5 簡稱 PAL) 為類苯丙酸生合成路徑中的第一個酵素。在植物體中，PAL常由多基因家族組成。其催化苯丙胺酸 (L-phenylalanine) 進行生物轉化反應，產生肉桂酸 (trans-cinnamic acid) 與氨 (ammonia)。在單子葉植物中，PAL可能具有催化酪胺酸脫氨裂解的能力。
BoPAL1 與 BoPAL2 的胺基酸序列相似度高達 96％，但兩者對於基質的專一性卻有不相同。將兩者於活性區中不同之胺基酸進行點突變互換，藉以觀察哪些胺基酸可能會對 PAL 基質專一性造成影響。經大腸桿菌表現系統之表現蛋白質 BoPAL1 F133H 與 BoPAL2 I198V、F134H、A149P、G389N，其對phenylalanine基質的親和力較原態差。對 tyrosine基質而言，表現蛋白質 BoPAL1 F133H 與 BoPAL2 A149P、 G389N 較原態有較高的酵素比活性。 BoPAL4 為綠竹 PAL 基因家族的一員。將表現載體 pTrcHisA/BoPAL4 轉形至大腸桿菌 Top10 中進行大量表現，所得到之表現蛋白 BoPAL4 於 N 端帶有 (His)6-tag，因此可透過鎳離子管柱層析進行純化。以膠體過濾層析法測得表現蛋白質 BoPAL4 原態分子量約為 290 kD，另外以 SDS-PAGE 測得表現蛋白質 BoPAL4 單元體分子量約為 70 kD，由上述結果推測表現蛋白質 BoPAL4 為同質四元體形式。表現蛋白質 BoPAL4 最適反應溫度為 50℃，最適反應 pH 為 9.0，且其活化能為 24.7 kcal/mol。表現蛋白質 BoPAL4 對 Phe 基質之 Km 值為 2072 μM 而 kcat/Km 值為 7.87。表現蛋白質 BoPAL4 對 tyrosine 基質之 Km 值為97 μM 而 kcat/Km 值為 5.56。與 BoPAL2 比較的結果，表現蛋白質 BoPAL4 對tyrosine基質具有較高的酵素活性。 | zh_TW |
| dc.description.abstract | Phenylalanine ammonia lyase (PAL; EC 4.3.1.5), the entry enzyme for phenylpropanoid biosynthesis, is often encoded by mutigene families in plant. It catalyzes the biotransformation of L-phenylalanine to trans-cinnamic acid and ammonia. In monocotyledon plants, PAL may exhibit the similar substrate specificities for tyrosine and phenylalanine and the protein-catalyst serves double duty.
BoPAL1 showed 96％ sequence identity with BoPAL2, but had very different situation in substrate specificity. By exchanging the different amino acid nearby active site to figure out which amino acid was important for substrate specificity, the fusion proteins BoPAL1 F133H and BoPAL2 I198V, F134H, A149P, G389N showed the poor substrate affinity to phenylalanine than those of wild-type PALs. To tyrosine substrate,fusion protein BoPAL1 F133H and BoPAL2 A149P, G389N increase enzyme specific activities. BoPAL4 is one of the Bamboo PAL gene families. Expression constructs pTrcHisA/BoPAL4 was transformed into E.coli Top10. Fusion protein BoPAL4 contains (histidine)6-tag in N-terminus and can be purified by Ni2+-column chromatography. By using gel filtration chromatography, the molecular mass of expressed BoPAL4 was estimated to be 290 kD, and monomer mass was determined to be 70 kD by SDS-PAGE. As the result, BoPAL4 was estimated to be homotetramer. The optimum temperature and pH for PAL activity were 50℃ and 9.0, respectively. The activation energy was 24.7 kcal/mol for BoPAL4. The Km value for phenylalanine was 2072 μM, and kcat/Km was 7.87. For tyrosine substrate, Km value was 97 μM, and kcat/Km was 5.56. Expressed BoPAL4 has highly tyrosine ammonia lyase activity than that of BoPAL2. | en |
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| dc.description.tableofcontents | 目錄…………………………………………………………………………………..a
縮寫表……………………………………..……………………………….………I 中文摘要…………………………...………………………….…………………….II Abstract……...……..…………………………………………..……………………III 第一章 緒論…………………………………………………...………………………1 1.1 竹……………………………………………………………………………....1 1.2 植物二次代謝....………………………………………………………………2 1.3 Phenylpropanoid pathway……….………………………………………….…5 1.4 苯丙胺酸脫氨裂解酶..………………….………………………………….....7 1.5 苯丙胺酸脫氨裂解酶之分子生物學研究…………………………...…….....9 1.6 苯丙胺酸脫氨裂解酶之細胞定位………….…………………………….....10 1.7 苯丙胺酸脫氨裂解酶之蛋白質結構與催化機制.…………………….........11 1.8 禾本科植物之 PAL 具有 TAL 活性...........................................................15 1.9 苯丙胺酸脫氨裂解酶之調控..........................................................................16 1.9.1 光照………………………………….………………………………..17 1.9.2 植物逆境…………………….………………………………………..17 1.9.3 植物生長調節劑.......................................................…………………18 1.9.4 二次代謝產物的回饋抑制………...………..……...………………...18 1.9.5 轉譯後磷酸化修飾……...……………………………………………18 1.10 苯丙胺酸脫氨裂解酶之應用………………………………………...……...19 1.11 實驗緣起……………………………………………………………………..20 第二章 材料與方法……………………………………………...…………………..22 2.1 實驗材料………………...…………………………………………………...22 2.1.1 質體 DNA…………………………………….……………………...22 2.1.2 菌種…………………………………………………………………...23 2.2 實驗藥品………………………………...…………………...………………23 2.2.1 一般化學藥品….…….…………………………………………….…23 2.2.2 抗體…………………………………………………………………...23 2.2.3 培養基……………………………………………………………...…23 2.3 實驗儀器…………………...………………………………………………...24 2.3.1 離心機…………………………………………………………...……24 2.3.2 分光光度計…………………………………………………………...24 2.3.3 核酸電泳設備………………………………………………………...24 2.3.4 蛋白質電泳設備…………………………………………………...…24 2.3.5 其他…………………………………………………………………...24 2.4 質體 DNA 之小量抽取..…………………………………………………...25 2.5 定點突變…………………...…………………………………………..…….25 2.6 E.coli competent cell 製作…………………………………………………..27 2.7 質體之轉形…………………………………………………………………..27 2.8 一般分子生物學操作………………………………………………………..28 2.8.1 DNA 洋菜膠體電泳法………………………………………………28 2.8.2 聚合酶鏈鎖反應……………………………………………………...29 2.9 重組蛋白之誘導條件討論…………………………………………………..30 2.9.1 表現載體來源………………………………………………………...30 2.9.2 最適 IPTG 濃度與溫度探討………………………………………..30 2.9.3 最適時間探討………………………………………………………...30 2.10 重組蛋白質之純化…………………………………………………………..31 2.10.1 表現菌株大量培養…………………………………………………...31 2.10.2 粗抽液之取得………………………………………………………...31 2.10.3 鎳離子親和管柱層析………………………………………………...32 2.11 活性分析法…………………………………………………………………..32 2.11.1 苯丙胺酸脫氨裂解酶活性分析法…………………………………...32 2.11.2 酪胺酸脫氨裂解酶活性分析法……………………………………...33 2.12 蛋白質定量法………………………………………………………………..34 2.13 電泳檢定系統………………………………………………………………..34 2.13.1 SDS-膠體電泳………………………………………………………...34 2.14 膠體染色法…………………………………………………………………..37 2.15 蛋白質電泳轉印與免疫染色法……………………………………………..37 2.15.1 蛋白質電泳轉印……………………………………………………...38 2.15.2 免疫染色法…………………………………………………………...38 2.16 重組蛋白質之生化性質分析………………………………………………..39 2.16.1 分子量測定…………………………………………………………...39 2.16.2 最適反應溫度………………………………………………………...40 2.16.3 最適反應時間………………………………………………………...41 2.16.4 最適反應 pH值……………………………………………………...41 2.16.5 活化能………………………………………………………………...41 2.16.6 酵素動力學…………………………………………………………...41 2.17 蛋白質立體結構模擬………………………………………………………..41 第三章 結果與討論…………………...……………………………………………..42 3.1 大腸桿菌 BoPALs 表現載體之點突變系統建立...……………………… .42 3.1.1 BoPALs立體結構模擬…………………………………………….…42 3.1.2 點突變位置選擇.......……………………….………………………...42 3.2 大腸桿菌 BoPALs 表現系統最適反應條件.……………………….……...43 3.2.1 最適誘導溫度與 IPTG 濃度......…………….……………………...43 3.2.2 最適誘導時間...........…………………………………………………44 3.3 大腸桿菌 BoPALs 重組蛋白質純化流程......……...………………………44 3.4 大腸桿菌 BoPALs 重組蛋白質生化性質探討………….………………...45 3.4.1 分子量測定...…………………………………………………………46 3.4.2 重組蛋白質反應時間與吸光值關係……………………...…………46 3.4.3 PAL 產物與吸光值關係…………………………..…………………47 3.4.4 最適反應溫度……………………………...…………………………47 3.4.5 活化能…………………...………………………………………...….47 3.4.6 最適反應 pH 值..……………………………………………………49 3.5 大腸桿菌 BoPALs 與突變株重組蛋白質酵素動力學…….………….…..49 3.5.1 對苯丙胺酸催化之酵素動力學……………………………………...49 3.5.2 對酪胺酸催化之酵素動力學………………………………………...51 3.6 總結...……………………...…………………………………………………52 3.6.1 BoPAL1、BoPAL2 與突變株基質專一性探討…….…...………….52 3.6.2 BoPAL4 表現系統建立與生化性質分析.......………………………55 第四章 未來展望……………...………………………………………………….….56 4.1 點突變之應用...............……………………………………………………...56 4.2 蛋白質立體結構研究...........………………………………………………...56 4.3 同質與異質四元體相關研究…………...…………………………………...56 結果圖表集………………………………...…………………………………………..57 參考文獻………………………………………….…………………………………....98 附錄…………………………………………………………………………..……….104 | |
| dc.language.iso | zh-TW | |
| dc.subject | 點突變 | zh_TW |
| dc.subject | 基質專一性 | zh_TW |
| dc.subject | 綠竹 | zh_TW |
| dc.subject | 苯丙胺酸脫氨裂解酶 | zh_TW |
| dc.subject | Bambusa oldhamii | en |
| dc.subject | Substrate Specificity | en |
| dc.subject | Phenylalanine Ammonia-lyase | en |
| dc.subject | Site-Directed Mutagenesis | en |
| dc.title | 利用點突變方式探討綠竹苯丙胺酸脫氨裂解酶中
胺基酸序列與基質專一性的相互關係 | zh_TW |
| dc.title | Identification of Essential Amino Acids for Tyrosine Substrate Specificity in Bamboo Phenylalanine Ammonia-lyase by Site-Directed Mutagenesis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 楊健志(Chien-Chih Yang),林棋財,王恆隆 | |
| dc.subject.keyword | 苯丙胺酸脫氨裂解酶,基質專一性,點突變,綠竹, | zh_TW |
| dc.subject.keyword | Phenylalanine Ammonia-lyase,Substrate Specificity,Site-Directed Mutagenesis,Bambusa oldhamii, | en |
| dc.relation.page | 110 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-08-02 | |
| 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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