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研究生:李秀雄
研究生(外文):Hsiu-Hsiung Li
論文名稱:1.利用Thioredoxin改善CandidarugosaLIP4之熱穩定性.2.龜山島嗜高溫熱溫泉菌纖維素?基因之選殖與表現.
論文名稱(外文):Fusion of Thioredoxin to improve thermostability of Candida rugosa LIP4. Cloning and overexpression of the Thermoanaerobacterium sp. NTOU2 cellulase gene
指導教授:唐世杰
指導教授(外文):Shye-Jye Tang
學位類別:碩士
校院名稱:國立臺灣海洋大學
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:2008
畢業學年度:96
語文別:中文
論文頁數:95
中文關鍵詞:Candida rugosa脂肪?Thioredoxin
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Candida rugosa 所產的五種菌體外脂肪?,皆含有534個胺基酸,其序列上具有高度的相似性。實驗室先前在進行LIP1基因工程時,所挑到ㄧ個mutant 脂肪?,命名為LIPF。LIPF與LIP1僅差異20個胺基酸,相同度(identity)為96 %,其催化三元體、雙硫鍵、鹽橋、含氧離子洞、糖基化位置等位置與LIP1都相同,但LIPF卻不具有脂肪?活性。
本實驗利用定位突變法(Site-directed mutagenesis),對LIPF的二十個胺基酸進行突變,期望能找到影響活性的胺基酸,探討CRL序列與結構及活性的相關性。目前已突變的十個mutant LIPF,各帶有1至6個突變點,皆未發現有活性產生。
實驗室先前利用硫氧化蛋白(Thioredoxin)融合在NLJF314N的N端,發現其熱穩定性較原NLJF314N好。為提高熱LIP4穩定性,本論文亦將Thioredoxin融合在LIP4的N端,發現其耐熱性較原LIP4效果提升了1.3倍。

由國立台灣海洋大學海生所實驗室於龜山島海底熱泉中分離出一株新菌種,命名為Thermoanaerobacterium sp. NTOU2,經該實驗室發現此菌能利用多種醣類包括 glucose, xylan, cellulose, mannose等促進其生長。將Thermoanaerobacterium sp. NTOU2基因定序解碼後,經由資料庫比對發現,該株菌含多種分解醣類的酵素基因,包括木糖?、纖維?等。
本論文將Thermoanaerobacterium sp. NTOU2的纖維分解酵素選殖出來,利用E. coli BL21(DE3)作為宿主,表現出纖維分解酵素,經由活性測試後發現,於37℃、50℃和70℃三種反應溫度下,由實驗結果可知此酵素具有熱穩定性,該酵素的蛋白質粗萃取物活性於70℃具有最高活性,於37℃時活性最低,70℃下酵素活性為37℃的兩倍。
The yeast Candida rugosa produces five extracellular lipases (LIP1 - LIP5), all containing 534 amino acids and displaying high homology in protein sequences. A mutant form lipase, LIPF, was found and it contains twenty amino acids differing form LIP1, and showing 95% identity with LIP1. However, LIPF has no has no lipase activity.
To understand the relationship of lipase activity between LIPF and LIP1. We had done ten mutant LIPF, we performed site-directed mutagenesis to replace LIPF sequence into LIP1. Ten mutants, containing 1 to 6 corrected amino acids, were show no lipase activities. The future works may keep on performing.
LIP4 shows low thermal stability. To improve thermal stability of lipase, Thioredoxin was fused with LIP4 to generate Trx-LIP4. The fusion lipase were exhibit more thermal stability than LIP4 and T1/2 of Trx-LIP4 was increased 1.3 folds. Our results demonstrated that fusion a thermal stable protein may improve lipase thermal stability.

A new thermophilic bacterium, named Thermoanaerobacterium sp.NTOU2 had isolated from acidic hydrothermal fluids off Gueishan Island in Taiwna by Institute of Marine Biology NTOU. And it can capable of growth using glucose, xylan, cellulose, mannose, etc. After whole genome sequencing of Thermoanaerobacterium sp. NTOU2, 1200 genes were found in Thermoanaerobacterium sp. NTOU2 and NTOU2 contains cellulase and xylanase gene.
In this study, cellulase gene was cloned and overexpressed in E. coli.
This enzyme was analyzed the activity in 37℃、50℃and 70℃. In our finding, the cellulase was thermal stable.
目錄
目錄………………………………………………………………………1
中文摘要…………………………………………………………………2
英文摘要…………………………………………………………………3
縮寫表……………………………………………………………………4
序論………………………………………………………………………5
一、脂肪?的應用………………………………………………………5
二、脂肪?的生化特性…………………………………………………6
三、脂肪?的結構特性…………………………………………………8
四、Candida rugosa脂肪?…………………………………………10
五、Candida rugosa脂肪?結構……………………………………11
六、研究方向……………………………………………………………15
材料與方法 ………………………………………………………………17
一、材料 …………………………………………………………………17
二、方法 …………………………………………………………………23
實驗結果 …………………………………………………………………35
討論 ………………………………………………………………………38
參考文獻 …………………………………………………………………41
圖 …………………………………………………………………………44
附錄 ………………………………………………………………………51


目錄
目錄………………………………………………………………………1
中文摘要…………………………………………………………………2
英文摘要…………………………………………………………………3
序論………………………………………………………………………4
一、龜山島海底熱泉嗜高溫菌…………………………………………4
二、嗜高溫菌酵素………………………………………………………5
三、纖維素………………………………………………………………6
四、纖維分解酵素………………………………………………………6
五、龜山島海底熱泉嗜高溫菌纖維分解酵素…………………………9
六、研究目的……………………………………………………………11
材料與方法 ……………………………………………………………12
一、材料……………………………………………………………… 12
二、方法 ………………………………………………………………14
實驗結果 ………………………………………………………………20
討論 ……………………………………………………………………22
參考文獻 ………………………………………………………………25
圖 ………………………………………………………………………27
附錄 ……………………………………………………………………31
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