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研究生:劉玟燕
研究生(外文):Wen-Yan Liu
論文名稱:Uncultured Neocallimastigales β-1,4-D-木聚醣水解酶的動力學分析及結構模擬
論文名稱(外文):Kinetic studies and structural modeling for Uncultured Neocallimastigales β-1,4-D-xylanase
指導教授:蔡麗珠蔡麗珠引用關係
指導教授(外文):Li-Chu Tsai
口試委員:陳又嘉鄭貽生
口試委員(外文):Yo-Chia ChenYi-Sheng Cheng
口試日期:2012-07-24
學位類別:碩士
校院名稱:國立臺北科技大學
系所名稱:有機高分子研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:48
中文關鍵詞:14-β-D-木聚醣水解酶突變株模擬酵素動力學
外文關鍵詞:14-β-D-xylanasemutantmodelkinetic
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Uncultured Neocallimastigales 1,4-β-D-木聚醣水解酶(xynR8, EC 3.2.1.8)能催化水解木聚醣中β-1,4鍵結的醣苷鍵。我們觀察到此株xynR8與其突變株(N41D、N58D、N41D+N58D)皆有自然降解的現象,分子量從35kDa變成27kDa。酵素動力學結果顯示截短型的TxynR8和突變種(TN41D、TN58D、TN41D+TN58D)酵素對山毛櫸木中木聚醣的水解能力和效率都比原生種更佳。其中以TxynR8對山毛櫸木中木聚醣的活性最好,與受質間的比活性(specific activity)為11752 ± 324 units mg-1,而酵素的催化效率及專一性常數為kcat/Km為 1371 ml s-1 mg-1。我們也用Blast胺基酸序列比對找到TxynR8與晶體結構(PDB: 2VGD)有89%相似度,並用Modeler模擬TxynR8的結構,也將(PDB: 2VGD與1H4H)中類似受質分子放入模擬的TxynR8活性區域來觀察酵素活性區域的胺基酸與受質分子的鍵結關係,並得知突變位置Asn41 和 Asn58是扮演穩定結構和N端的loop,而Asn41也可能參與受質鍵結。而扮演催化水解反應的主要胺基酸是Glu144 和Glu232。

Uncultured Neocallimastigales 1,4-β-D-xylanase can hydrolyze and cleave β-1,4-glycosidic bonds in xylan. We found that 35 kDa xynR8 and its mutants (N41D、N58D、N41D+N58D) could degrade and the molecular weight became 27 kDa. The kinetic data showed that the truncated forms (TxynR8、TN41D、TN58D、 TN41D+TN58D) had more activity than that of wild type. TxynR8 had highest specific activity of 11752 ± 324 units mg-1 and the enzyme efficiency kcat/Km of 1371 ml s-1 mg-1 among all enzymes. In addition, we used Blast sequence alignment and Modeler to generate a TxynR8 structure based on the crystal structure (PDB:2VG9) which has 89% residue identity with TxynR8. The substrate-like (ferulic acid-1,5-arabinofuranose-α1,3-xylotriose) from PDB: 2VGD and (β-D-Xylopyranose) from PDB:1H4H were put into the active-site of xynR8 for enzyme-substrate interaction studied. Structural modeling suggests that residues Asn41 and Asn58 were required to stabilize the N-terminal loops and protein structure, and the Asn41 involved in substrate binding. The residues Glu144 and Glu232 play the important role in catalytic mechanism.

摘 要 i
Abstract ii
誌 謝 iii
目 錄 iv
表目錄 vi
縮寫表 ix
第一章 緒論 1
1.1 前言 1
1.2 醣苷鍵水解酶介紹 1
1.3 β-1,4-木聚醣水解酶 3
1.4 酵素動力學 9
1.5 結構模擬 11
1.6 研究動機 11
第二章 實驗方法與材料 12
2.1 蛋白質之大量表現與純化 12
2.2 酵素動力學 16
2.3 分子結構模擬 17
第三章 實驗結果 19
3.1 蛋白質純化與降解 19
3.2 MS質譜儀分析 21
3.3最適反應溫度與pH值 22
3.4 酵素動力學分析 23
3.5 分子結構模擬 26
3.6 不同家族β-1,4-Xylanase對受質水解效率 30
3.7 GH11嗜酸鹼性水解酶 32
第四章 討論 35
4.1 xynR8與突變株對xylan催化效率分析 35
4.2 TxynR8與截短突變株對xylan催化效率分析 35
4.3 xynR8之分子結構模擬 36
4.4 xynR8之養晶條件及數據收集 367
第五章 結論 40
參考文獻 41



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