臺灣博碩士論文加值系統

木腐黴菌（Trichoderma koningii G-39）阿拉伯呋喃糖苷酵素(ABF)，屬於醣類水解酵素第54家族，其催化機制為保留機制（retention）且速率決定步驟為去醣基化（dearabinosylation）。其中E223和D299分別扮演親核基和一般酸/鹼基團之角色。在本研究中我們利用溶劑同位素效應（isotope solvent effect）再次強化D299在催化反應中之角色。然而，有趣的是突變酵素（E223G）仍保有明顯的活性。Aspergillus kawachii ABF是目前此家族中唯有三維立體結構，利用蛋白質序列相比對，發現兩者之相同度（identity）達72%，且其中活性區內之重要胺基酸均高度保留。因此我們利用其Aspergillus kawachii ABF之三維立體結構為模板，進行T. koningii ABF結構模擬。發現D191的位置可取代E223成為另一個重要胺基酸殘基，其與受質異位性碳原子（anomeric carbon）距離約4 Å，故當E223被突變成G223時，可能使E223G反應轉變為反轉機制（inverting），而本研究旨於探討此突變酵素之催化性質與機構。
本研究利用Pichia pastoris系統表現酵素，經由單菌落PCR確認基因轉殖結果，再由活性測試與蛋白質電泳方法，可以成功的篩選表現效率最佳之單菌落，其再進一步被誘導產生大量酵素。經80%飽和度之銨鹽沉澱與陽離子交換樹脂管柱層析，可以得到均質度達95%的酵素以利動力學研究使用。
根據酵素動力學的研究E223G/D191N與E223G/D191G活性值（kcat/ Km）降為E223G的0.69% ~ 4.6%，顯示D191在E223G中可能是重要胺基酸。另外，E223G/D299N活性值降為wild type的0.12%，E223G的2.8%，這很有可能說明D299不僅在wild type之中，同時也在E223G中是重要胺基酸。
E223G 和Wild type的pH activity profile顯示兩鐘形曲線分佈，這兩酵素催化過程都分別由兩重要胺基酸基團調控。對其他突變酵素之pH profile研究顯示，其中E223G/D191G之 pKa2=5.56，並且沒有pKa1。E223G/D299N之pKa1=2.23沒有pKa2。這些證據都顯示D191和D299分別是E223G的一般鹼基團與一般酸基團。
由分子模擬得知，D299和E223距離約為6 Å，是預期中保留機制兩重要胺基酸残基的距離。D299與D191距離為7.5 Å，則是預期可進行反轉機制的距離，因此我們認為E223G之催化反應為構型反轉之機構，而D191與D299是其重要胺基酸殘基，分別扮演一般酸與一般鹼之重要角色。

Convert a retaining ��-L-arabinofuranosidase to inverting enzyme
by single point mutation on E223


Student：Kai-Yu Chen Advisor：Dr. Yaw-Kuen Li

Department of Applied Chemistry
National Chiao-Tung University

ABSTRACT
The ��-L-arabinofuranosidase (ABF) from Trichoderma koningii G-39 is a retaining enzyme belonging to GH family 54. Our previous study showed that breakdown of arabinosyl-enzyme intermediate is the rate limiting step of the catalytic reaction. The essential groups are E223 (nucleophile) and D299 (general acid/base). In this study, the investigation of kinetic solvent isotope effect re-confirmed that the D299 functions as the general acid/base in the catalytic reaction.
Surprisingly, E223G mutant was found to remain significant activity, while E223Q was completely inactive. The structure of T. koningii ABF was obtained from the homology simulation by using the structure of Aspergillus kawachii ABF (family 54) as the template. The structure exhibited that the distance between D191 and anomeric carbon of substrate is about 4 Å and the space is suitable for accommodating a water molecule. D191 was, thus, proposed to be essential for the catalysis of E223G, and the mechanism of E223G might become an inverting process. For studying this hypothesis, a series of double mutants, such as E223G/D191N, E223G/D191G and E223G/D299N, were constructed and over-expressed in Pichia pastoris system. The colonies with high-level expression were selected through various steps of validation including colony PCR to confirm gene insertion and activity assay or protein electrophoresis (SDS-PAGE) to evaluate the protein expression level. After 80% ammonium sulfate precipitation following by a cation-exchanged chromatographic separation, enzymes can be purified to reach 95% homogeneity and used for further study.
Kinetic study revealed that the relative activity (kcat/Km) of E223G/D191N, E223G/D191G and E223G/D299N are 0.69%~4.6% of that of E223G. A bell-shaped pH-profile of E223G showed the catalytic activity of this enzyme is mediated by two pKas, 1.8 and 4.2. However, a sigmoidal pH-profile was observed for E223G/D191G (pKa2= 5.56) and for E223G/D299N (pKa2= 2.23), indicating that D191 and D299 are the general base and the general acid of E223G, respectively. These findings are consistent with the suggestions obtained from the simulated structure of T. koningii ABF.

目錄
中文摘要......................................................................................i
英文摘要......................................................................................ii
謝誌..............................................................................................v
目錄..............................................................................................vi
表目錄..........................................................................................viii
圖目錄..........................................................................................viii


第一章 緒論................................................................................. 1
1-1 半纖維水解酵素群................................................................. 1
1-2 阿拉伯呋喃糖苷酵素（��-L-Arabinofuranosidase）................6
1-3 阿拉伯呋喃糖苷酵素反應機制的研究………………………….9
1-4 研究目的...............................................................................14

第二章 實驗方法.....................................................................15
2-1 定點突變..............................................................................15
2-2 利用電送法將質體送入表現之宿主細胞GS115（His-）......16
2-2-1 一般敘述.......................................................................16
2-2-2 P. pastoris之轉型（transformation）............................16
2-2-2-1 勝任細胞（Competent cell）之製備..........................16
2-2-2-2 利用電送法（electroporation）轉型...........................17
2-2-3 利用聚合酶連鎖（PCR）反應分析DNA........................18
2-3 阿拉伯呋喃糖苷酵素在P. pastoris系統之誘導纯化條件…....19
2-3-1 一般敘述.........................................................................19
2-3-2 阿拉伯呋喃糖苷酵素的誘導條件.....................................19
2-3-3 阿拉伯呋喃糖苷酵素的純化............................................20
2-3-4 決定蛋白質分子量與純度................................................20
2-3-5 蛋白質濃度的確定...........................................................21
2-4 阿拉伯呋喃糖苷酵素反應機制之研究......................................22
2-4-1 一般敘述............................................................................22
2-4-2 阿拉伯呋喃糖苷酵素野生株與各突變株Km、kcat之測定....22
2-4-3 阿拉伯呋喃糖苷酵素野生株與各突變株和pH之關係.........22
2-4-4 由CD（circular dichroism）光譜觀測野生株和突變株酵素之二級結構.................................................................................23

第三章 結果與討論......................................................................24
3-1 酵素之表現與純化..................................................................24
3-1-1 single colony PCR確認電送法轉型.................................25
3-1-2 篩選最佳菌落.................... .............................................26
3-1-3 阿拉伯呋喃糖苷酵素的表現.............................................26
3-1-4 酵素的純化.................... ...... ..........................................28
3-2 再確認D299在催化反應的角色.............................................30
3-3 Wild type與E223G之pH-profile的比較................................31
3-4 E223G之轉糖研究..................................................................33
3-5 突變酵素之活性分析...............................................................35
3-6 E223G/D191G和E223G/D299N之pH-profile研究...............37
3-7 CD光譜研究............................................................................39
3-8 阿拉伯呋喃糖苷酵素反應機制的探討......................................41

第四章 結論................................................................................42

第五章 參考文獻.........................................................................43
附錄..............................................................................................45

附錄一 不同來源之阿拉伯呋喃糖苷酵素性質比較
附錄二 各突變株所使用之寡核甘酸引子
附錄三 蛋白質濃度的測定
附錄四 Aspergillus kawachii IFO4308與Trichoderma koningii G-39 ��-L-arabinofuranosidase蛋白質序列之比對
表目錄

表 1-1 參與水解木聚醣的酵素列表................................................2
表 1-2 第二類ABF的次分類（subclass）......................................8
表 3-1 wild type及D299G之同位素效應結果.............................30
表 3-2 wild type和各突變株酵素活性之比較...............................36
表 3-3 CD程式分析二級結構......................................................40

圖目錄

圖 1-1 ο-acetyl-4-ο-methylglucuronoxylan (hardwood xylan)的組成......................................................................................3
圖 1-2 arabino-4-ο-methylglucuronoxylan (softwood xylan)的組成
..........................................................................................4
圖 1-3 木聚醣水解酵素群.............................................................5
圖 1-4 水解木聚寡糖的木糖苷酵素...............................................5
圖 1-5 阿拉伯聚醣（L-arabinan）主要結構..................................6
圖 1-6 E223G酵素推測之反應機制.............................................11
圖 1-7 ��-L-arabinofuranosidase野生株的水解反應機制..............12
圖 1-8 家族54之ABF部分蛋白質序列比對.......................... .......13
圖 1-9 ��-L-arabinofuranosidase from Aspergillus kawachii IFO4308之重要胺基酸......................................................................14
圖 3-1 single colony PCR電泳圖................................................25
圖 3-2 酵素蛋白質電泳圖............................................................27
圖 3-3 wild type及各突變株酵素之SP管柱層析圖....................28
圖3-4 α-L-arabinofuranosidase經管柱層析纯化後之蛋白質電泳分析.......................................................................................29
圖 3-5 wild type酵素在各pH值中的活性變化............................31
圖 3-6 E223G酵素在各pH值中的活性變化...............................32
圖 3-7 ��-L-arabinofuranosidase分子模擬...................................34
圖 3-8 以雙倒數做圖法求E223G酵素對pNPAF之Km、
kcat...................................................................................36
圖 3-9 E223G /D191G酵素在各pH值中的活性變化..................37
圖 3-10 E223G /D299N酵素在各pH值中的活性變化...................38
圖 3-11 wild type與D191G/E223G、E223G/D299N和E223G之CD光譜..................................................................................39

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