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研究生:陳怡君
研究生(外文):Yi-June Chen
論文名稱:以蛋白質體學技術探討氧氣對產玻尿酸鏈球菌Streptococcuszooepidemicus蛋白質表現之影響
論文名稱(外文):Investigation of the impact of oxygen on the protein profiles of hyaluronan-producing bacterial Streptococcus zooepidemicus
指導教授:吳定峰吳定峰引用關係
指導教授(外文):Ting-Feng Wu
學位類別:碩士
校院名稱:南台科技大學
系所名稱:生物科技系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:74
中文關鍵詞:玻尿酸鏈球菌蛋白質體學氧氣
外文關鍵詞:HyaluronanStreptococcus zooepidemicus2-DEoxygen
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玻尿酸(Hyaluronan,HA),是一種不帶電荷,直鏈無分支的高分子多醣,由於具有充分之保水性及黏彈性,是一種目前在醫學及美容上常用的生醫材料;玻尿酸的來源有很多,目前用微生物發酵是最普遍的方式,用來發酵的菌種以鏈球菌屬為主,本研究是針對鏈球菌屬中的Streptococcus zooepidemicus,它是一株會產生玻尿酸莢膜之球菌,為一兼性厭氧菌,原本是一株感染馬科動物之病原菌,目前已馴化成為工業上做為量產玻尿酸的常用菌種,對於該菌種產玻尿酸之最適發酵條件幾乎已被研究。根據成功大學陳特良 老師實驗室的研究結果得知在通氧條件下該菌會產生較厚的玻尿酸莢膜保護菌體,若未通氣情況下玻尿酸的產生則會明顯降低。本實驗研究目的是以蛋白質體學技術探討這株產玻尿酸鏈球菌在通氣及未通氣兩種不同的培養條件下蛋白質表現之差異,以找出影響玻尿酸產生之關鍵蛋白質並推測其生理活性。以批次培養進行發酵,其培養基每升包含20g glucose、10g yeast extract、2.5g K HPO 、2g NaCl和1.5g MgSO ,在37 、pH 7.0下進行。在本研究中,利用二維電泳技術先將細菌細胞內所有蛋白質進行分離,以Image Master軟體進行膠片之分析比對,共找出了九個有差異的蛋白質點,再以串聯式質譜儀鑑Liquid Chromatography/Mass Spectrometry/Mass Spectrometry (LC-MS/MS)定出這些蛋白質身分,包括有UDP-N-acetyl-glucosamine pyrophosphorylase,putative dihydrolipoamide dehydrogenase,putative acetoin dehydrogenase,putative dihydrolipoamide S-acetyltransferase 及lactate dehydrogenase,經由這些蛋白質整合出在醣解路徑中影響玻尿酸產生之可能關鍵步驟,為驗證該結論,也在發酵槽進行部分實驗,初步得到了一致的結論。
Hyaluronan(HA) , a negatively charged , linear and unbranched polysaccharide , it possesses water retention capacity and viscoelastic and is a common biopolymer used in the medicine and cosmetics. HA can be produced from various source and more recently HA from microbial source is receiving attention, HA fermentation has been performed mostly by streptococci.In this study Streptococcus
zooepidemicus was used to investigate the protein profile under the influence of high oxygen concentration. S.zooepidemicus is a coccus of hyluranon capsule producing , aerotolerant anaerobes bacteria. It is a pathogen which has wide host range and can infect horses、pig、sheep..etc., Recently S. zooepidemicus has used in fermentation industry to produce hyaluronic acid, In the previous study conducted by Dr.Teh-Liang Chen laboratory, Department of Chemical Engineering, National Cheng-Kung University, higher amounts of hyaluronic acid were produced at the aeration(1 vvm, 600rpm) when compared to those at the anearation (600rpm). In order to explore the effect of oxygen on the synthesis of hyaluronic acid in S. zooepidemicus, the protein profiles of S. zooepidemicus at the aerations and anearation were compared using two-dimensional gel electrophoresis(2-DE) to search key proteins which might influence hyluronan biosynthesis. S.zooepidemicus is grown in batch culture with complex medium cotaining 20g glucose, 10g yeast extract, 2.5g K HPO , 2g NaCl and 1.5g MgSO per liter at 37 .In this study, The 2-DE gels were compared by Image Mastersoftware and nine differentially expressed protein spots were found. These proteins were identified by Liquid Chromatography/Mass Spectrometry/Mass Spectrometry (LC-MS/MS). The differentially expression proteins included UDP-N-acetyl-glucosamine pyrophosphorylase, putative dihydrolipoamide dehydrogenase, putative acetoin dehydrogenase, putative dihydrolipoamide S-acetyltransferase and lactate dehydrogenase. According to these proteins, the glycolytic pathway could be a key
point in the HA synthesis.
目 次

摘要 iv
英文摘要 v
致謝 vii
目次 viii
圖目錄 x
表目錄 xii
第一章 前言 1
第二章 文獻探討 4
2.1 玻尿酸的介紹 4
2.1.1 玻尿酸的結構 4
2.1.2 玻尿酸的來源 5
2.1.3 玻尿酸的生合成路徑 6
2.1.4 玻尿酸的應用 7
2.2 Streptococcu介紹 7
2.2.1 Streptococcus zooepidemicus分類 8
2.2.2 Streptococcus zooepidemicus特性 9
2.3 蛋白質體學介紹 10
2.3.1 二維電泳介紹 11
第三章 研究材料與方法 13
3.1 研究材料 13
3.1.1 Streptococcus zooepidemicus 菌體來源 13
3.1.2 藥品 14
3.1.3 實驗器材 16
3.2 研究方法 17
3.2.1 Streptococcus zooepidemicus 菌體蛋白質萃取 17
3.2.2 蛋白質等電點聚焦電泳法 (IEF) 17
3.2.3 聚丙烯膠電泳(SDS-PAGE) 17
3.2.4 銀染 18
3.2.5 二維電泳膠片軟體影像分析及比對 18
3.2.6 蛋白質身分鑑(LC/MS/MS) 19
第四章 結果 20
4.1 Streptococcus zooepidemicus二維電泳條件的探討 20
4.2 氧氣對Streptococcus zooepidemicus之蛋白質圖譜的條件下影響 21
4.3 具差異性蛋白質點身分確認 21
4.4 Streptococcus zooepidemicus發酵槽驗證 22
第五章 討論 24
第六章 參考文獻 30



圖目錄


圖一 通氣培養之Streptococcus zooepidemicus,0.1g 菌體加500ul
lysis buffer ,11cm ,pH4-7之二維電泳圖 34
圖二 通氣培養之Streptococcus zooepidemicus,0.2g 菌體加1000ul lysis buffer,18cm ,pH4-7之二維電泳圖 35
圖三 通氣培養之Streptococcus zooepidemicus ,0.1g 菌體加2000ul lysis buffer,18cm ,pH4-7之二維電泳圖 36
圖四 通氣培養之Streptococcus zooepidemicus ,0.1g 菌體加2000ul lysis buffer及0.3% SDS,18cm,pH4-7之二維電泳圖 37
圖五 (A)未通氣培養之Streptococcus zooepidemicus 0.1g 菌體2000ul
lysis buffer及0.3% SDS,18cm,pH4-7之二維電泳圖 38
圖五 (B)通氣培養之Streptococcus zooepidemicus 0.1g 菌 體加2000ul
lysis buffer及0.3% SDS,18cm,pH4-7之二維電泳圖 39
圖六 通氣培養及未通氣培養下表現具差異的九個蛋白質點的放大圖 40
圖七 玻尿酸及相關酵素推測之生合成路徑 41
附圖一 玻尿酸一級結構 42
附圖二 玻尿酸二級結構 42
附圖三 玻尿酸之三級結構 43
附圖四 玻尿酸之生合成路徑 44
附圖五 Streptococcus zooepidemicus 型態 45
附圖六 Streptococcus zooepidemicus 發酵槽厭氧批次發酵結果 46
附圖七 Streptococcus zooepidemicus第4小時加入 4g/L acetoin發酵槽厭氧批次發酵結果 47
附圖八 Streptococcus zooepidemicus第6.5個小時加入0.5g/L acetoin 發酵槽厭氧批次發酵結果 48

附圖九Streptococcus zooepidemicus 發酵槽好氧批次發酵結果 49
附圖十Streptococcus zooepidemicus 第6.5個小時加入0.5g/L acetoin 發酵槽好氧批次發酵結果 50


表目錄


表一 通氣培養與未通氣培養下兩者間具差異蛋白質點之統計結果 51
表二 具差異之蛋白質點經LC/MS/MS所鑑定出之蛋白質相關資料 52
表三 LC/MS/MS 軟體與蛋白質資料庫比對的相關參數定義 53
表四 表現具差異之蛋白質點的LC/MS/MS比對結果 54
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