臺灣博碩士論文加值系統

本研究利用PCR的方式，次選殖Ralstonia solanacearum GMI1000中的aac基因，利用四種不同的表現載體來建構Aac蛋白的表現菌株，純化此酵素並分析其酵素特性。經由胺基酸序列比對分析，發現Aac與Ralstonia sp. XJ12B的AHL acylase以及Ralstonia metallidurans CH34的penicillin amidase分別有89 %與88 %的相似性，屬於N-terminal nucleophillic (Ntn) family。由軟體預測Aac蛋白為一分子量大小85 kD，pI 8.0，且N端帶有signal peptide的蛋白質。利用四種表現載體，並選擇三種不同的大腸桿菌作為表現菌株，其中以pET43.1a(+) Factor Xa-GMI1000aac重組質體，在表現菌株Rosetta (DE3)2中誘導表現蛋白質，於30 ℃下能夠獲得較多量的可溶性Aac蛋白。而其他兩種重組質體体pET32a(+) Factor Xa-GMI1000aac與pET41a- GMI1000aac，在25 ℃下亦能得到可溶性的Aac蛋白。比較此三種表現系統所誘導表現蛋白質之比活性，其中以pET43.1a(+) Factor Xa-GMI1000 aac系統表現的蛋白質有最高的活性，以C8HSL為基質活性可達59 U/μg。重組Aac蛋白質經純化並去除序列上之融合蛋白後，其最適反應溫度為30 ℃，最適酸鹼值為7.5，在4℃下保存的酵素穩定度，保存至第五天時，活性僅為原來的55 ％。Aac對於中長鏈的AHL (C7HSL、C8HSL、C10HSL、C12HSL)具有活性，其中對於C8HSL有最高的比活性，而對於短鏈以及含有氧取代基的AHL (C4HSL、C6HSL、3OC6HSL)則不具分解能力，但對於3OC8HSL則有些微的活性 (2.73 U/μg)。以質譜儀 (Q-Tof Ultima)分析經Aac作用的AHL水解產物，證實產物中有HSL的存在，Aac在AHL分解酵素中屬於AHL acylase可得到直接的証明。

目錄

口試委員審定書...........................................i
誌謝....................................................ii
中文摘要................................................iv
英文摘要................................................vi
目錄..................................................viii
表次...................................................xiv

圖次....................................................xv
附錄圖次..............................................xvii
符號縮寫表...........................................xviii

壹、 前言
一、 群體密度調控系統(Quorum sensing)
1.Quorum sensing之發現..............................1
2.Quorum sensing在微生物中之功能....................2
3.Quorum sensing作用機制............................2
二、 群體密度調控系統清除機制(Quorum-quenching)
1.Quorum-quenching之發現...........................4
2.Quorum-quenching之機制...........................5
3.Quorum-quenching之應用...........................6
三、 Quorum sensing中訊息小分子簡介
1.各種訊息小分子....................................6
2.N-acyl-homoserine lactone (AHL)的生合成與分解.......7
四、 N-醯化高絲胺酸內酯分解酵素(AHL-degrading enzyme)
1.N-醯化高絲胺酸內酯醯化酶(AHL acylase)..............8
2.N-醯化高絲胺酸內酯酶(AHL lactonase)................9
3.AHL-degrading enzyme在菌體中的角色...............10
五、 青枯菌(Ralstonia solanacearum)之介紹...................11
六、 研究緣起與目的......................................12

貳、 實驗方法及步驟
I. 實驗材料
一、 實驗菌株及質體....................................14
二、 培養基............................................14
三、 藥品與試劑........................................14
四、 實驗中使用之套組(kit) ............................16
五、 儀器..............................................17
六、 引子..............................................18
II. 實驗方法
一、 Ralstonia solanacearum GMI1000的aac基因
的次選殖及分析
1. 帶有aac基因之質體DNA的製備...................19
2. 引子之設計.....................................20
3. 利用PCR次選殖R.solanacearum GMI1000的
aac基因........................................20
4. 接合反應 ......................................20
5. 製備勝任細胞...................................21
6. 電衝法轉型作用.................................21
7. 建構表現載體...................................21
8. R.solanacearum GMI1000的aac基因定序與序
列分析.........................................22
二、 Ralstonia solanacearum GMI1000的Aac蛋白質
的表現與純化
1. 誘導表現Aac蛋白質.............................22
2. Aac蛋白質的收集與純化..........................23
3. 蛋白質定量.....................................24
4. 蛋白質膠體電泳.................................24
5. 抗體製備.......................................24
6. 西氏轉印 ......................................25

三、 Ralstonia solanacearum GMI1000的Aac蛋白質
的酵素特性分析
1. 酵素基質試劑的配製.............................26
2. Whole cell bio-assay...........................26
3. 酵素活性測定法.................................27
4. 利用MS分析水解產物............................27
參、 實驗結果
一、 Ralstonia solanacearum GMI1000的aac基因序列比
對與分析...........................................28
二、 次選殖aac基因於不同表現載體與表現菌株............28
三、 帶有aac基因的重組質體之最適蛋白質表
現情形............................................29
四、 Ralstonia solanacearum GMI1000的Aac蛋白質的
酵素特性分析
1. 最適反應條件....................................30
2. 酵素安定性 .....................................30
3. 酵素基質專一性分析..............................30
4. 水解產物分析....................................30

肆、 討論
一、利用不同表現載體與不同表現菌株來誘導表現
Ralstonia solanacearum GMI1000中的Aac蛋白..........33
二、Aac蛋白之酵素特性分析..............................34

伍、 結論...............................................36

陸、參考文獻............................................37

表次

表一、本研究所使用之菌株與質體..........................44
表二、本研究所使用之引子................................46
表三、Ralstonia solanacearum GMI1000中的Aac蛋白質
與其他相似蛋白質之序列比較........................47
表四、本研究中不同表現載體中融合蛋白之比較..............48
表五、E. coli中的少見密碼子在Ralstonia solanacearum
GMI1000的Aac蛋白質分布情形.....................49
表六、不同表現系統中Aac酵素活性之比較..................50
表七、不同溫度與pH下Aac酵素活性之比較.................51
表八、Aac蛋白對於不同AHL的基質專一性...................52
表九、Aac蛋白在4℃下的酵素穩定性.......................53


圖次

圖一、Ralstonia solanacearum GMI1000中的Aac之核酸
與蛋白序列........................................54
圖二、Aac之多序列比對..................................61
圖三、Aac蛋白親疏水性之預測............................66
圖四、本研究中所使用的四種表現載體......................67
圖五、PCR Ralstonia solanacearum GMI1000的aac DNA
片段之電泳圖......................................68
圖六、表現載體與建構質體經限制酶截切後之電泳圖..........69
圖七、不同溫度、不同IPTG濃度下在不同表現系統
中Aac蛋白表現之情形..............................70
圖八、Aac蛋白在30℃下、Rosetta(DE3)2中的表現情形.......72
圖九、Aac蛋白之SDS-PAGE電泳及免疫染色圖..............73
圖十、利用whole cell bio-assay來鑑定不同表現系統中
Aac的酵素活性....................................74
圖十一、Aac在不同溫度與酸鹼度酵素活性之比較............75
圖十二、Aac於4℃下之酵素穩定性.........................76
圖十三、利用質譜分析來鑑定Aac分解AHL之水解產物.......77


附錄圖次

附錄圖一、Vibrio fischeri的quorum sensing中的
LuxI/LuxR系統................................80
附錄圖二、 Pseudomonas aeruginosa的quorum sensing
中的LasI/LasR-RhlI/RhlR系統..................81
附錄圖三、格蘭氏陽性菌中以peptide調控quorum sensing
的一般模式....................................82
附錄圖四、Vibrio harveyi中的混合型quorum sensing機制.....83
附錄圖五、AHL的生合成途徑.............................84
附錄圖六、AHL的結構與經酵素分解之產物.................85

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