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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王愛玉 | |
| dc.contributor.author | Tai-Hung Chen | en |
| dc.contributor.author | 陳泰宏 | zh_TW |
| dc.date.accessioned | 2021-06-08T05:11:40Z | - |
| dc.date.copyright | 2006-07-28 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-21 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23846 | - |
| dc.description.abstract | 蔗糖轉化酶 (β-fructofuranosidase, EC 3.2.1.26) 是高等植物中參與蔗糖代謝的重要酵素之一，藉由蔗糖轉化酶的作用，蔗糖可被水解成葡萄糖與果糖，提供植物細胞利用。綠竹筍 (Bambusa oldhamii) 中至少存在三種蔗糖轉化酶異構酶，本研究以含有野生液泡型蔗糖轉化酶及其突變株cDNA的表現質體，轉形至酵母菌Pichia pastoris X-33進行重組蛋白質的表現。在1%甲醇誘導24小時後，以硫酸銨分劃及金屬螯合層析法等步驟純化液泡型蔗糖轉化酶及其突變株，進行酵素之蛋白質結構與功能關係的研究。
液泡型蔗糖轉化酶 (rIT3) 可被化學修試劑WRK、NBS及IAA抑制，蔗糖可保護酵素降低NBS的抑制作用。利用定位點突變配合酵素動力學實驗發現，Asp-127、Arg-251、Asp-252位置的突變會造成rIT3活性喪失，顯示這些胺基酸在酵素功能上扮演重要的角色。Asp-127、Glu-308為酵素催化主要的胺基酸，Cys-309以及Trp-151與穩定中間過渡狀態及酵素結構相關。rIT3與W151F、W151L、E308Q與C309A的最適反應pH值為5.0，在0~30℃的環境下較安定。突變株對蔗糖以及具有β-果糖苷基的棉仔糖親和力皆下降。在蔗糖轉化酶結構研究方面，成功利用同源模擬法 (Homology modeling)，以菊苣fructan 1-exohydrolase IIa為模版，得到綠竹筍液泡型蔗糖轉化酶可能的立體結構。此外，也成功得到rIT3可能的結晶條件，可作為後續微調最佳蛋白質結晶條件之參考。 | zh_TW |
| dc.description.abstract | Invertase (β-fructofuranosidase, EC 3.2.1.26) is one of the major enzymes in the sucrose metabolism of higher plants. The enzyme catalyzes the conversion of sucrose to glucose and fructose for plant cell utilization. There are at least three invertase isozymes in green bamboo (Bambusa oldhamii). To study the structure-function relationships of invertase, the expression plasmids containing wild-type vacuolar invertase or mutants cDNA were transformed into yeast Pichia pastoris X-33 for expression of recombinant proteins. After induction with 1% methanol for 24 hours, the recombinant wild-type and mutant proteins were purified by ammonium sulfate fractionation and affinity chromatography homogeneity.
The enzyme activity of recombinant vacuolar invertase (rIT3) could be inactivated to various extent by the chemical modifying reagents, N-ethyl-5- phenylisoxazolium-3’-sulfonate (WRK), N-bromosuccinimide (NBS) and iodoacetate (IAA). Sucrose could protect the enzyme against the inactivation by NBS. Mutations in Asp-127、Arg-251、Asp-252 abolished the rIT3 activity. These results suggested that they are important residues in invertase function. Asp-127 and Glu-308 play important roles in catalysis. Cys-309 and Trp-151 play important roles in transition state and structural stability. Wild-type rIT3、W151F、W151L、E308Q and C309A have a pH optimum of 5.0 and they are stable at 0~30℃. Mutants have lower affinity with sucrose and raffinose than wild-type rIT3. The predicted 3D structure of Bambusa oldhamii vacuolar invertase was setup by Homology modeling which was based on the model of Cichorium intybus fructan 1-exohydrolase IIa. The crystallization conditions of rIT3 were also tested successfully, which could be used to find out the best condition of rIT3 crystal. | en |
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| dc.description.tableofcontents | 目錄................................................. I
圖表目錄.............................................. V 縮寫表............................................... VII 摘要................................................. VIII Abstract.............................................. IX 第一章 緒論.......................................... 1 第一節 蔗糖轉化酶的生化性質......................... 1 1.1 酸性蔗糖轉化酶.............................. 2 1.2 中/鹼性可溶性細胞質蔗糖轉化酶.................. 2 第二節 蔗糖轉化酶的生理功能........................ 3 2.1 酸性可溶性液泡型蔗糖轉化酶.................... 3 2.2 酸性不溶性細胞壁型蔗糖轉化酶.................. 4 2.3 中/鹼性可溶性細胞質蔗糖轉化酶.................. 4 第三節 蔗糖轉化酶的調控............................. 5 3.1 荷爾蒙的調控................................ 5 3.2 基質和產物的調節............................ 5 3.3 抑制因子對蔗糖轉化酶的調控.................... 5 3.4 環境因子的調控.............................. 6 第四節 蔗糖轉化酶之分類地位......................... 6 第五節 Glycoside Hydrolases所催化的反應機制........ 6 5.1 保留機制................................... 7 5.2 反轉機制................................... 7 第六節 蔗糖轉化酶的立體結構......................... 8 6.1 蔗糖轉化酶的立體結構......................... 8 6.2 蔗糖轉化酶的酵素活性區....................... 9 6.3 蔗糖轉化酶 (GH32) 與 GH68、GH43家族酵素在結構上的相關性..................................... 10 第七節 研究緣起與實驗目的........................... 11 第二章 材料與方法..................................... 13 第一節 實驗材料................................... 13 1.1 菌種...................................... 13 1.2 質體...................................... 13 第二節 實驗藥品................................... 13 2.1 一般化學藥品................................ 13 2.2 分生實驗用藥品與限制酶....................... 13 2.3 培養基..................................... 13 第三節 實驗儀器................................... 14 3.1 離心機..................................... 14 3.2 核酸電泳系統................................ 14 3.3 蛋白質電泳系統.............................. 14 3.4 震盪培養箱................................. 14 3.5 其它...................................... 14 第四節 實驗方法................................... 15 4.1 DNA之抽取、檢定與分析........................ 15 4.1.1 質體的抽取............................. 15 4.1.2 DNA瓊脂糖膠體電泳...................... 16 4.2 以定位點突變法 (site-directed mutagenesis) 構築液泡型蔗糖轉化酶突變株之表現質體...................... 16 4.2.1 引子對的設計........................... 16 4.2.2 聚合酶鏈鎖反應 (polymerase chain reaction, PCR) 合成全長質體DNA......................... 17 4.2.3 限制酶Dpn I切割......................... 18 4.2.4 質體轉形 (transformation) .................. 18 4.2.5 菌種保存.............................. 19 4.3 於酵母菌 (Pichia pastoris) 中表現重組蛋白質......... 19 4.3.1 酵母菌勝任細胞 (competent cells) 的製備....... 19 4.3.2 電穿孔轉形............................. 19 4.3.3 轉形酵母菌株的鑑定...................... 20 4.3.3.1 MM-MD培養基鑑定.................. 20 4.3.3.2 插入基因之確認..................... 20 4.3.4 最佳表現菌株與最佳表現培養條件............ 21 4.3.5 重組蛋白質之表現與純化................... 21 4.3.5.1 重組蛋白質之表現................... 21 4.3.5.2 重組蛋白質之純化................... 22 4.4 重組蛋白質之檢定............................ 22 4.4.1 蛋白質定量法........................... 22 4.4.2 蔗糖轉化酶活性測定—還原糖定量法.......... 23 4.4.3 聚丙醯胺膠體電泳........................ 23 4.4.4 西方轉印法............................. 24 4.4.5 免疫染色法............................. 24 4.5 重組蔗糖轉化酶及其突變株之生化性質分析.......... 25 4.5.1 突變株活性分析......................... 25 4.5.2 酵素動力學研究......................... 25 4.5.3 最適反應pH............................ 26 4.5.4 熱安定性.............................. 26 4.5.5 基質特異性............................. 26 4.6 化學修飾劑對重組野生型蔗糖轉化酶活性之影響....... 26 4.7 重組蔗糖轉化酶的結晶條件...................... 27 第三章 結果與討論..................................... 29 第一節 綠竹液泡型蔗糖轉化酶與活性相關之保守性胺基酸區域探討........................................ 29 第二節 綠竹液泡型蔗糖轉化酶可能的立體結構.............. 29 第三節 於酵母菌 (Pichia pastoris) 中表現重組蛋白質......... 30 3.1 以定位點突變法構築液泡型蔗糖轉化酶突變株之表現質體 30 3.2 轉形酵母菌株之鑑定........................... 30 3.3 最佳表現條件探討............................ 31 3.4 重組野生型蔗糖轉化酶及其突變株降解情形探討....... 31 3.5 重組野生型蔗糖轉化酶及其突變株在外泌液的活性測定.. 33 3.6 重組野生型蔗糖轉化酶及其突變株的純化............ 33 第四節 重組野生型蔗糖轉化酶及其突變株之生化性質分析..... 33 4.1 純化之突變株活性分析......................... 33 4.2 酵素動力學研究.............................. 35 4.3 最適反應pH................................. 36 4.4 熱安定性................................... 37 4.5 基質特異性................................. 38 第五節 化學修飾劑對重組野生型蔗糖轉化酶活性之影響....... 38 5.1 重組蔗糖轉化酶經WRK修飾之研究................ 38 5.2 重組蔗糖轉化酶經NBS修飾之研究................ 39 5.3 重組蔗糖轉化酶經IAA修飾之研究................. 39 5.4 討論...................................... 39 第六節 重組液泡型蔗糖轉化酶的結晶條件................. 40 第四章 總結........................................... 41 第一節 本論文主要的研究成果......................... 41 第二節 未來展望................................... 42 2.1 進一步對NDPNG motif (sucrose-binding box) 作點突變分析........................................ 42 2.2 生物資訊軟體的應用........................... 42 2.3 rIT3蛋白質結晶結構........................... 42 參考文獻............................................. 44 圖與表............................................... 50 附錄................................................. 82 | |
| dc.language.iso | zh-TW | |
| dc.title | 以定位點突變法探討綠竹液泡型蔗糖轉化酶活性區之結構與功能 | zh_TW |
| dc.title | Studies on the catalytic site of the Bambusa oldhamii
vacuolar invertase | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 宋賢一 | |
| dc.contributor.oralexamcommittee | 楊健志,楊啟伸,蔣啟玲,張珍田 | |
| dc.subject.keyword | 液泡型蔗糖轉化酶, | zh_TW |
| dc.subject.keyword | vacuolar invertase, | en |
| dc.relation.page | 82 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2006-07-23 | |
| 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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