臺灣博碩士論文加值系統

利用PCR (Polymerase Chain Reaction)方法取得Xenorhabdus luminescens脂肪基因，長約1.8kb，轉譯出約66kDa之蛋白質。將脂肪基因轉殖至pET20b(+)載體並於E.coli BL21(DE3)宿主中進行表現，在此系統中可大量表現但卻形成包函體(inclusion body)，以0.2% SDS溶解包函體可得到有活性的SDS-lipase。另將含有X. luminescens脂肪基因的pG3K質體送入BL21(DE3)宿主可得基礎表現之可溶性lipase。兩者皆以膠體電泳純化法(gel electrophoresis purified)純化。
生化性質分析方面，SDS-lipase與可溶性lipase最適作用pH值為8.0，最適作用溫度為40℃，可溶性lipase可至50℃。可溶性之lipase耐熱性比SDS-lipase高，置於100℃下處理一小時，soluble-lipase仍維持90%以上活性，而SDS-lipase於僅殘餘18%活性。金屬離子Fe2+提高了20%活性，而Co2+、Cu2+及Zn2+抑制了其活性。從基質(三酸甘油酯)特異性實驗中，對於鏈長度與酯鍵位置並無專一性，應屬於非特異性脂肪。SDS-lipase對短鏈酯類(四碳)有較高的kat/Km值，對長鏈酯類(12碳)Km值與soluble-lipase相近，但kcat值卻較低。以三酸甘油酯類為基質，X. luminescens脂肪活性比Candida rugosa脂肪高出9倍，比豬胰臟脂肪高出16倍，是一極具潛力的酵素。進行C端序列剪切實驗，將蛋白質C端剪切掉約一萬分子量時，仍保有活性，但超過則失活。

Xenorhabdu luminescens lipase gene encodes a 66kDa protein. The lipase gene was cloned with PCR (polymerase chain reaction) technique and overexpressed in E. coli BL21(DE3) using the pET20b(+) vector system, but the overexpressed protein formed inclusion body. Dissolving the inclusion body in 0.2% SDS can recover the lipase activity. On the other hand, the X. luminescens lipase gene expressed in BL21(DE3) using pG3K vector had basal expression of soluble lipase. Both enzymes were purified by gel electrophoresis.
The optimum pH of both lipases are pH 8. The optimum temperature for SDS-lipase and soluble lipase were 40℃ and 50℃, respectively. Soluble lipase is more stable than SDS-lipase. After 1 hour incubation in 100℃ water bath, soluble lipase and SDS-lipase retained 90% and 18% activity, respectively. Fe2+ enhanced 20% activity of lipase, but Co2+, Cu2+ and Zn2+ greatly decreased the activity. SDS lipase had higher catalytic efficiency (kcat/Km) toward p-nitrophenyl butyrate. When using p-nitrophenyl caprylate and p-nitrophenyl laurate as substrates, SDS lipase had similar Km but lower kcat than soluble lipase. The enzyme was a non-specific lipase, its activity was higher than Candida rugosa lipase and Porcine pancreatic lipase by 9 and 16 fold, respectively. The enzyme deleted 10 kDa from C terminal still retained lipase activity.

第一章 緒論
一、 脂肪的生化特性 1
二、 脂肪的結構特性 2
1. 一級胺基酸序列 2
2. 三級結構之特性…………………………………………..………2
3. 活化部位之催化三元體…………………………………………..3
4. 含氧陰離子洞 (oxyanion hole)……………………………………4
5. 蓋子 (The lid)……………………………………………………...4
三、 脂肪之催化反應機制 ..5
四、 脂肪的界面活化作用現象 ..5
五、 脂肪的應用性 7
六、 實驗緣起…………………………………………………..10
第二章 材料與方法
一、 材料、儀器與藥品 12
(一) 微生物材料 12
(二) 質體 12
(三) 實驗儀器 13
(四) 實驗藥品 15
二、 X. luminescens脂肪表現載體(expression vector)之構築 16
(一) 表現載體之構築設計 16
(二) 聚合連鎖反應 17
(三) 序列基因剪切 (serious deletion)………………………….....18
(四) 瓊脂醣膠電泳……………………………………….19
(五) DNA之回收…………………………………………19
(六) 限制之剪切作用…………………………………..20
(七) 接合反應……………………………………………..20
(八) E. coli通透性細胞之製備…………………………...20
(九) E. coli轉形作用(transformation)……………………..21
(十) 篩選(screening)……………………………………….21
(十一) 質體DNA 之抽取純化……………………………21
(十二) DNA序列分析……………………………………..23
三、 X. luminescens脂肪表現(expression)系統之建立 23
(一) E. coli培養方法 23
(二) 蛋白質誘發(induction)與樣品處理 23
四、 X. luminescens脂肪之純化 …………24
(一) E. coli之破碎 24
(二) 脂肪之純化 25
五、 X. luminescens脂肪之生化特性分析 26
(一) 電泳分析法 26
(二) 蛋白質濃度測定 29
(三) 脂肪活性測定 .29
(1) 脂肪活性測定…………………………………………....29
(2) SDS耐受性分析……………………………………….…...30
(3) 最適pH值…………………………………………………..30
(4) 最適作用溫度…………………………………………….…31
(5) 脂肪耐熱性分析…………………………...………….….31
(6) 金屬鹽類影響………………………………………….…….31
(7) 脂肪對不同受質練長度之活性分析………………….….32
(8) 脂肪酵素動力學分析………………………………….….32
第三章 結果與討論
一、 E. coli表現系統的選擇 33
二、 X. luminescens脂肪表現載體的構築 33
三、 重組蛋白質的表現 34
四、 重組蛋白質的純化 35
五、 重組蛋白質生化性質的分析 45
(一) 測定脂肪活性之波長選定………………………………...35
(二) 最適反應pH值(optimum pH) 36
(三) 最適反應溫度(optimum temperature)……………………….36
(四) 熱穩定性分析 37
(五) 金屬離子催化反應的影響 37
(六) 脂肪對不同受質鏈長度之活性分析……………………...38
六、 酵素動力學分析………………………………………….38
七、 脂肪活性比較………………………………………….39
八、 脂肪C端的序列剪切…………………………………40
第四章 結論與展望
一、 結論 42
二、 展望與後續實驗 43
參考文獻 44

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