臺灣博碩士論文加值系統

本實驗室從Variovorax paradoxus Iso1中選殖到含N-醯基-D-胺基酸醯基水解酶的基因片段，全長11337 bp，經定序及電腦分析的結果，共定義出10個開架讀取區（Open reading frame），其中有2個比對不到功能外，其餘分別為intergrase、D-胺基酸氧化酶、N-醯基-D-胺基酸醯基水解酶、2個Regulators及一組ABC transporter。D-胺基酸氧化酶，其轉譯出的蛋白質共416個胺基酸，預測分子量約44,981 Da，經基因選殖至表現載體pET21a及pQE30表現後，其在pET系統中皆為inclusion body而測不到表現活性，在pQE系統中雖然表現量低，但仍可測到較佳的活性，因此可由此確定其功能。在轉錄層次的分析上，從北方式雜交的結果中，確定D-胺基酸氧化酶和N-醯基-D-胺基酸醯基水解酶轉錄時會位在同一條RNA上，且其長度約為2.8 knt，剛好約為兩個基因長度的總合；而此RNA的訊號，在無誘導物或誘導物為D-Met、N-acetyl-L-Met的情形下幾乎偵測不到表現，但若加入N-acetyl-D-Met時，表現量會上升。在轉譯層次上，我們以在基本培養基、或LB中添加不同誘導物的方式，測定此兩個基因可能的誘導情形及相互關係：從西方式雜交分析N-醯基-D-胺基酸醯基水解酶及活性電泳分析D-胺基酸氧化酶表現的結果，證明此兩個基因皆在相同的條件下有相似的誘導表現。因此，由轉譯及轉錄結果的分析，證明此兩個基因應為同一操縱子。在調控子部份，選殖除了操縱子外還含不同調控子片段的選殖株，分析結果發現這兩個調控子應都和此操縱子的調控有關。代謝定額感應因子方面，經由生物感測法確定V. paradoxus Iso1的酵素液能將acyl-homoserine lactone（AHL）轉變成去活化態，但N-醯基-D-胺基酸醯基水解酶的表現株卻不行，因此推翻我們之前預期此酵素可代謝AHL的假設。高效能液相層析法的分析中，在以Iso1的粗酵素液反應N-octanoyl-Homoserine lactone時，其產物的滯留時間和標準品比對後，發現應有一個八個碳脂肪酸的產物，且此產物會隨時間有增加的趨勢；同時利用OPT-thiol衍生法分析Homoserine lactone（HSL）的生成，發現HSL亦有隨時間增加的現象，因此，應存有一個AHL-acylase於V. paradoxus中，用來代謝AHL。

We sequenced a gene fragment in pBluescriptII KS (+), our lab cloned before, containing N-acyl-D-amino acid amidohydrolase (N-D-AAase) from Variovorax paradoxus Iso1. Sequence analysis revealed ten open reading frames, except N-acyl-D-amino acid amidohydrolase, including intergrase, D-amino acid dehydrogenase, two regulators and three ABC transporters. The putative D-amino acid dehydrogenase (DAAO), encodes 416 amino acids, was through gene cloning, expression, and measure activity to confirm its function. We choused two expression systems-pQE30 and pET21a-to express the DAAO, and its activity is very low in pET21a and higher in pQE30. It seemed to have no expression pattern on SDS PAGE, but we could make sure its function through measuring activity. DAAO and N-D-AAase could be induced by N-acetyl-D-amino acids on transcription level and expressed in the same 2.8knt RNA, it is the sum of these two genes length. Through the data, Western blot of N-D-AAase and zymography of DAAO, they also have similar expression pattern when add different kinds of inducers in minimal medium or LB. These data reveals that the two enzymes may constitute an operon to cooperate metabolize similar substrate and be regulated by the same promoter. There are two regulators localized separately at upstream and downstream of this operon. When cloning the different truncated regulators fragment in pUC119 and measure their effect on the activity of N-D-AAase, we found they maybe play a role on the regulation of this operon. The degradation of quorum sensing factors (QSFs) experiment denied our assumption that N-D-AAase maybe active on them. The crude enzyme of N-D-AAase expression strain- pKKdam3 -couldn’t degrade QSFs but the total lysat of wild type could degrade QSFs at 37℃ very soon. When using N-octanoyl-homoserine lactone as substrate, we found a caprylic acid product increase thorough time goes by from the HPLC data. And we analyzed the homoserine lactone derived the OPT-thiol method under the same condition; HSL increased through time, too. From these results, we make propose that there should be an acyl-homoserine lactone（AHL）acylase in V. paradoxus Iso1 to degrade AHL.

目錄
頁次
中文摘要
英文摘要
目錄…………………………………………………………………….…I
表次……………………………………………………………….……VII
圖次……………………………………………………………….…...VIII
附錄表次…………………………………………………………………X
附錄表次………………………………………………………………..XI
壹、 前言
一、Variovorax paradoxus的簡介…………………………………...1
二、D-胺基酸的重要性及生產……………………………….….…. 3
三、N-醯基-D-胺基酸醯基水解酶的相關介紹……………………...7
四、定額感應因子( quorum-sensing factor)的相關研究
1. 定額感應因子的簡介…………………………..…...…….…8
2. 定額感應因子的去活化機制………………………...…….11
3. 定量定額感應因子的原理……………………………...….14
五、研究緣起及目的…………………………………………… .. 15
貳、材料與方法
I、 材料
一、 實驗菌株與質體……………………………………………...17
二、 培養基…………………………………………….…………..17
三、 藥品與試劑…………………………………………………...17
四、 實驗中使用的套組（kit）…………………………………...18
五、 各種溶液及緩衝溶液
1.DNA瓊脂膠體電泳試劑…………………………...…….. 19
2.RNA電泳試劑……………………………………………. 19
3.蛋白質電泳膠體試劑……………………………………... 20
4.西方氏雜交試劑………………………………………….. 22
5.蛋白質定量試劑及其標準品……………………...……... 22
6.N-醯基-D-胺基酸醯基水解酶酵素活性測試相關溶液…..23
7.D胺基酸氧化酶酵素活性測試相關溶液…….…………. 23
六、 儀器………………………………………………………….. 24
七、 引子…………………………………………….……………. 26
八、 其他…………………………………………….……………. 26
II、實驗方法
一、 本實驗所用的一般DNA技術
1. DNA瓊脂膠體電泳………………………………………..…… 28
2.質體的製備……………………………………………..………... 28
3. DNA片段純化…………………………………………..……… 29
4. 接合作用………………………………………………………... 29
5. 勝任細胞之製備…………………………………………………29
6. 轉形作用………………………………………………………... 30
二、V. paradoxus Iso1 N-醯基-D-胺基酸醯基水解酶基因選殖株的定序及序列分析……………………………………………………..….. 31
三、N-醯基-D-胺基酸醯基水解酶北方氏分析
1. RNA的萃取…………………………………………….……….. 31
2. DIG-labeling 探針製備…………………………………..……... 33
3. 甲醛洋菜膠體電泳 (1 %)………………………………..…….. 33
4. 北方氏漬片製備………………………….…………………….. 34
5. 北方式雜交……………………………….…………………….. 34
6. DIG-核酸探針之免疫雜合反應………………………………... 34
四、V. paradoxus Iso1 D胺基酸氧化酶的選殖與E.coli的表現
1.不同表現載體之建構………………………………...…………...35
2.不同表現載體中D胺基酸氧化酶的表現及其粗酵素液製備… 36
3. D胺基酸氧化酶酵素活性之測定…………………………….…36
五、N-醯基-D-胺基酸醯基水解酶及D胺基酸氧化酶轉譯層次表現情形分析
1. 誘導物測試……………………………………….…...……. 37
2. N-醯基-D-胺基酸醯基水解酶之酵素活性分析…………... 37
3. SDS-PAGE分析……………….………..…...………….….. 38
4. 西方免疫雜交分析……………………...………….………. 38
5. D胺基酸氧化酶酵素活性電泳……………………………. 39
六、調控子於E.coli系統中的研究
1. 含不同調控子片段的建構………………………………… 40
2. 含不同調控子片段對N-醯基-D-胺基酸醯基水解酶酵素活性的影響…………………….……………………………… 40
七、N-醯基-D-胺基酸醯基水解酶代謝acyl-HSL的情形
1. acyl-HSL溶液的配置………………………………..……...41
2. 粗酵素液的製備…………………..………………………... 41
3. Reporter strain的最適測定條件分析……………………… 42
4. 以生物方法（bioassay）定量定額感應因子
（quorum sensing factor）的降解情形…………………...…43
5. 以高效能液相層析方式分析定額感應因子
（quorum sensing factor）的降解途徑………………….…. 43
參、結果
一、 N-醯基-D-胺基酸醯基水解酶上下游基因的選殖
及功能性探討……………………………..….…...…………45
二、N-醯基-D-胺基酸醯基水解酶操縱子調控之研究
﹙一﹚轉錄層次
1. RNA的製備……………………………...……….....………... 48
2. N-醯基-D-胺基酸醯基水解酶操縱子轉錄的情形………...…49
3.不同誘導物對N-醯基-D-胺基酸醯基水解酶mRNA之 表現情形……………………………………..……………….. 49
4.可能之啟動子﹙promoter﹚及終止子﹙terminator﹚電腦分析…..49
﹙二﹚轉譯層次
1. 基本培養基……………………………..……...…...………... 50
2. 營養培養基﹙LB﹚……………………………..….…...…… 51
三、D型胺基酸氧化酶的選殖與E.coli的表現
1. 基因選殖……………………………………....….…...……... 52
2. 在E.coli系統的表現情形……………………………….……52
四、N-醯基-D-胺基酸醯基水解酶調控子研究
1. 含不同調控子片段的建構………….………………………..53
2. 含不同調控子片段對N-醯基-D-胺基酸醯基水解酶酵素活性的影響………………………………………………….…….. 53
五、V. paradoxus Iso1代謝數量感應因子情形的分析
1. 測定利用生物分析法（Bioassay）定量定額感應因子的最佳條件…………………………………………………...….…… 54
2. 定額感應因子被不同酵素代謝情形的分析……………….. 54
3. 以高效能液相層析方式分析數量感應因子在V. paradoxus Iso1中可能的代謝機制
（1）N-ocatanoyl-HSL的降解情形………………………….. 55
（2）N-ocatanoyl-HSL的降解產物中HSL的分析結果…...…56
肆、討論
一、N-醯基-D-胺基酸醯基水解酶上下游基因的功能性探討……57
二、N-醯基-D-胺基酸醯基水解酶操縱子轉錄層次的研究………58
三、N-醯基-D-胺基酸醯基水解酶操縱子轉譯層次的研究………58
四、D胺基酸氧化酶基因的選殖與表現………………………….60
五、N-醯基-D-胺基酸醯基水解酶調控子研究………………….…61
六、V. paradoxus Iso1代謝定額感應因子情形的分析
1. 以生物分析法（bioassay）分析定額感應因子降解
的情形……………………...………………………...…… 61
2. 高效能液相層析方式分析定額感應因子在
V. paradoxus Iso1中的代謝機制………………………... 63
伍、結論………………....……………………………………………..…65
陸、參考文獻……………….……………………………………….……67
表次
表一、本研究所使用的菌株…………………………………………… 78
表二、本研究所使用的質體…………………………………………… 79
表三、定序pBK-damD4所使用的引子及其序列……………………. 80
表四、本研究所使用的非定序用引子………………………………… 81
表五、含N-醯基-D-胺基酸醯基水解酶上下游基因載體：pBKD4
經定序後電腦分析的結果………...………………..………….. 82
表六、在培養基中加入不同誘導物後對N-醯基-D-胺基酸醯基水解
酶表現的影響……………………………………………….…. 84
表七、含不同調控子的片段對N-醯基-D-胺基酸醯基水解酶表現
的影響………………………………………………………..….. 85
圖次
圖一、pBK-damD4定序所使用的引子及其相對位置……………….. 86
圖二、ABC transporter保守性區塊及疏水性區域分析…………….....87
圖三、V. paradoxus 之可能ATP鍵結蛋白（ORF9）與其他功能 相似蛋白質胺基酸序列比對………………………………..….. 88
圖四、北方氏雜交結果……………………………………………….....89
圖五、N-醯基-D-胺基酸醯基水解酶操縱子之可能啟動子及終止子 的分析………………………………………………………...… 90
圖六、在基本培養基中不同誘導物對N-醯基-D-胺基酸醯基水解酶及D-胺基酸氧化酶表現的影響…………………………...…….... 91
圖七、在LB中不同誘導物對N-醯基-D-胺基酸醯基水解酶及D-胺基酸氧化酶表現的影響…………………………………………..….. 92
圖八、daao基因的選殖……………………………………………….....93
圖九、D型胺基酸氧化酶在28度下的誘導表現情形………………....94
圖十、D型胺基酸氧化酶在10度下的誘導表現情形……………….....95
圖十一、V. paradoxus Iso1 D-胺基酸氧化酶酵素基因於表現載體pQE-30的活性表現情形……………………..…………….....96
圖十二、含不同調控子片段的建構圖……………………………….....97
圖十三、E.coli pSB 536（C4-HSL反應菌株）質體圖譜及最適測定條件的分析……………………………………………………..….. 98
圖十四、E.coli pSB401（C8-HSL反應菌株）質體圖譜及最適測定條件的分析………………………………………………………....99
圖十五、E.coli pSB1075（C12-HSL反應菌株）最適測定條件的分析及質體圖譜……………………………………………………..100
圖十六、C4-HSL被不同蛋白質的降解情形……………...…………..101
圖十七、C8-HSL被不同蛋白質的降解情形……………...…………...102
圖十八、C12-HSL被不同蛋白質的降解情形…………………………103
圖十九、AHL（C8-HSL）的代謝途徑級產物分子量…………………104
圖二十、V. paradoxus Iso1代謝N-octanoyl-HSL 後可能代謝產物標準品的高能液相層析檢測圖………...…………………….…..105
圖二十一、V. paradoxus Iso1的粗酵素液對N-octanoyl-HSL 作用後的液相層析檢測圖………………………...………………...…106
圖二十二、以OPT衍生法分析HSL標準品的高效能液相層析檢測圖
………………..……………...……………….……………107
圖二十三、V. paradoxus Iso1的粗酵素液對N-octanoyl-HSL作用後以OPT衍生法分析HSL的液相層析檢測圖………………108
附錄表次
附錄表一、本實驗所使用之培養基成分………………………..……..109
附錄圖次
附錄圖一、利用生物感測方式定量數量感應因子的原理：
V. fischeri luciferase基因的調控…….…………..……...111
附錄圖二、N-醯基-D-胺基酸醯基水解酶的作用機制及
活性呈色原理…….…………..…….……………………112
附錄圖三、D胺基酸氧化酶的作用機制及活性呈色原理…………113
附錄圖四、D胺基酸氧化酶的作用機制及活性電泳呈色原理....…114

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