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研究生:何威震
研究生(外文):WeiJen Ho
論文名稱:過氧化酵素(CoprinusCinereusperoxidase,CIP)之製備及酵素固定化
論文名稱(外文):Preparation and Immobilization of Coprinus Cinereus Peroxidase
指導教授:袁俊傑袁俊傑引用關係
指導教授(外文):Yuan, Chiun-Jye
學位類別:碩士
校院名稱:國立交通大學
系所名稱:生物科技研究所
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:2002
畢業學年度:90
語文別:中文
論文頁數:65
中文關鍵詞:過氧化酵素
外文關鍵詞:peroxidase
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Coprinus Cinereus 過氧化酵素(CIP)與山葵過氧化酵素(HRP)相似,擁有高度地催化活性及受質的特異性。先前的研究顯示CIP對於氧化胺基苯二醯井(luminol)產生化學冷光的反應上有更高的催化活性 (如常使用的受質ABTS)。這些特質使得過氧化酵素得以廣泛應用於檢測生物分子的檢測器、酵素免疫檢測、工業界防止染料洗脫的洗衣劑及各類生化與臨床檢驗等用途上。
本研究最主要的目標為建構出適合表現真菌屬的Coprinus cinereus 過氧化酵素的原核細胞表現系統。首先我們利用基因選殖與蛋白質工程技術的方式將全長的CIP基因選殖於pTrc99a的原核細胞表現載體中(此質體命名為pTrc-CIP),並以大腸桿菌JM105為宿主進行大量表現。我們發現所建構出的pTrc-CIP表現載體其最適化條件為在30℃下,菌液濃度為OD 0.8、添加0.8 mM IPTG並予以8小時的表現時間。至於CIP純化部份則是使用50%的硫銨進行沉澱,之後再通過陰離子管柱層析。此一以原核表現系統來大量表現具活性的真菌屬的過氧化酵素,目前已知為唯一成功的例子。另利用受質ABTS來計算酵素動力學參數,得知Km 及Vmax 值分別為0.082 mM 及2.1 μmole/ug/min。
在酵素固定化部份,我們已成功運用溶膠凝膠(sol-gel)方法建立一個酵素固定化之平臺,用此技術製成的基質具有優異的光學特性和熱力學、機械穩定性,且它形成的化學條件溫和,尤其適用於包埋生物大分子(酵素、蛋白質)且亦可提昇酵素使用效益。經由實驗數據可知,使用溶膠凝膠法來固定酵素,的確可以使酵素乾燥保存長達二個月以上活性不損而且亦可重覆使用十次。
The Coprinus cinereus peroxidase (CIP)shows a broad specificity for hydrogen donors and a high catalytic efficiency as does the well-known peroixdase from horseradish roots (HRP). Previous studies have shown that CIP exhibited a much higher catalytic activity to the common substrate, luminol as well as ABTS. This property permits a wide range of application for CIP, including high-sensitivity chemiluminescent determination of biological materials, construction of biosensor, enzyme immunoassay, and dye-transfer inhibition during laundering.
The major objective of the present study is to establish a bacterial expression system for CIP. We first constructed CIP expression vector by inserting full length CIP cDNA into the bacterial expression vector, pTrc99a, termed pTrc-CIP. The over expression of CIP was performed in E.coli strain JM105. We found that the pTrc-CIP could be expressed as a soluble form under the 30℃,in the presence of 0.8 mM IPTG for 8 h. The purification of recombinant CIP was carried out by 50% ammonium sulfate precipitation, followed by an anion-exchange chromatography. The study of CIP kinetic parameters was also conducted. The Km and Vmax values are 0.082 mM and 2.1 μmole/μg/min, respectively, by using ABTS as substrate. It is the first known so far example that the soluble, active fungal peroxidase can be successfully expressed in the bacterium.
In enzyme immobilization study, we successful established a platform technique for enzyme immobilization by using Sol-gel method. Sol-gel glass could be formed under low temperature and mild chemical condition.
The result shows that Sol-gel encapsulated HRP is reusable at least 10 times; its 80% activity can be maintained for around 55 days.
目錄
中文摘要………………………………………………………………..…i
英文摘要………………………………………………………………..…ii
誌謝…………………………………………………………………….…iii目錄………………………………………………………………….……iv
圖目錄……………………………………………………………….……v
表目錄……………………………………………………………….……vii縮寫表……………………………………………………………………viii
壹、背景介紹………………………………………………………….….1
貳、材料與方法……………………………………………………….…12
參、結果………………………………………………………………….21
肆、討論與結論………………………………………………………….35
伍、未來之研究方向…………………………………………………….37
陸、參考文獻…………………………………………………………….38
圖………………………………………………………………………….43
圖目錄
圖1、過氧化酵素作用機轉……………………………………………3
圖2、pTrc-CIP表現質體建構………………………………………13
圖3、pET22b(+)-CIP表現質體建構……………………………………13
圖4、pUT-CIP 表現質體建構………………………………………….14
圖5、pTrc-CIP,pET22(+)-CIP及pUT-CIP在JM105中表現結果...43
圖6、不同IPTG濃度對CIP在大腸桿菌JM105中表現之影響.……….44
圖7、不同菌液濃度對CIP在大腸桿菌JM105中表現之影響………….45
圖8、不同表現時間對CIP在大腸桿菌JM105中表現之影響………….46
圖9、添加Hemin對CIP 酵素在大腸桿菌JM105中表現之影響……47
圖10、不同濃度的hemin對CIP在細菌中表現之影響….............48
圖11、由細胞模間萃取之CIP酵素管柱純化剖面圖及電泳分析……49
圖12、從收集細菌培養液中純化CIP及電泳分析………………..…..50
圖13、由細胞質間抽取出之蛋白質之純化剖面圖及電泳分析……….51
圖14、不同H2O2濃度對CIP酵素之活性影響……………………….....52
圖15、CIP酵素在不同溫度中之活性圖……………………………….53
圖16、ABTS基質飽和曲線圖(受質抑制型) ………………………...54
圖17、CIP酵素之雙倒數圖…………………………………………….55
圖18、不同酸鹼值對溶膠(Sol)凝聚作用之影響…………………..56
圖19、溶膠凝膠(Sol-gel)縮聚後的老化(Aging)時間對固定酵素的作用影響...................................................57
圖20、包埋在溶膠凝膠(Sol-gel)中的酵素之活性測試……………59
表目錄
表1、CIP 與HRP的受質特異性……………………………………….4
表2 CIP、ARP與HRP的受質特異性………………………………..30
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