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研究生:曾詩喬
研究生(外文):Shih-Chiao Tseng
論文名稱:神經鞘磷脂水解酶的表現、純化及定性
論文名稱(外文):Expression, Purification and Characterization of Neutral Sphingomyelinase
指導教授:蕭世裕
指導教授(外文):Shyh-Yu Shaw
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
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:70
中文關鍵詞:枯草桿菌中性神經鞘磷脂水解脢
外文關鍵詞:Neutral sphingomyelinaseBacillus subtilis
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神經鞘磷脂代謝過程中扮演重要角色的磷脂水解酵素被稱之為神經鞘磷脂水解□(Sphingomyelinase),此酵素可誘發細胞凋亡、細胞分化、細胞增生與體內固醇平衡等不同的細胞調控。本實驗室已由枯草桿菌中鑑定出中性神經鞘磷脂水解□(Neutral Sphingomyelinase),此酵素分子量為34.3kDa,與已知之Bacillus cereus IAM1208 中之中性神經鞘磷脂水解□在胺基酸序列上有98.6%相同,以N-ω-trinitrophenylaminolauryl sphingomyelin (TNPAL-SM)為受質測定神經鞘磷脂水解□之酵素動力學及pH、離子對酵素活性的影響,得到酵素動力學常數Km 11.06 μM 及Vmax 10.23 nmol /min(10μg sphingomyelinase),最佳的反應環境以pH 7.4尤佳;酵素的活性被鈣離子所抑制並被鎂離子活化,為瞭解不同離子與中性神經鞘磷脂水解□的作用,以色胺酸螢光光譜分析不同濃度的鈣、鎂離子下的螢光強度及相對離子濃度活性,發現鈣離子色胺酸螢光光譜有一處明顯轉變,鎂離子色胺酸螢光光譜有二處明顯轉變,表示鈣離子在濃度10-6~10-8M有一處鍵結點、鎂離子在濃度10-7~10-8M與10-3~10-4M各有一處鍵結點,而在相對濃度的活性偵測可以發現鎂離子濃度低於10-4M時酵素活性消失,神經鞘磷脂代謝過程中扮演重要角色的磷脂水解酵素被稱之為神經鞘磷脂水解□(Sphingomyelinase),此酵素可誘發細胞凋亡、細胞分化、細胞增生與體內固醇平衡等不同的細胞調控。本實驗室已由枯草桿菌中鑑定出中性神經鞘磷脂水解□(Neutral Sphingomyelinase),此酵素分子量為34.3kDa,與已知之Bacillus cereus IAM1208 中之中性神經鞘磷脂水解□在胺基酸序列上有98.6%相同,以N-ω-trinitrophenylaminolauryl sphingomyelin (TNPAL-SM)為受質測定神經鞘磷脂水解□之酵素動力學及pH、離子對酵素活性的影響,得到酵素動力學常數Km 11.06 μM 及Vmax 10.23 nmol /min(10μg sphingomyelinase),最佳的反應環境以pH 7.4尤佳;酵素的活性被鈣離子所抑制並被鎂離子活化,為瞭解不同離子與中性神經鞘磷脂水解□的作用,以色胺酸螢光光譜分析不同濃度的鈣、鎂離子下的螢光強度及相對離子濃度活性,發現鈣離子色胺酸螢光光譜有一處明顯轉變,鎂離子色胺酸螢光光譜有二處明顯轉變,表示鈣離子在濃度10-6~10-8M有一處鍵結點、鎂離子在濃度10-7~10-8M與10-3~10-4M各有一處鍵結點,而在相對濃度的活性偵測可以發現鎂離子濃度低於10-4M時酵素活性消失,顯示低親和力鎂離子鍵結點與催化活性相關。
以往的研究顯示中性神經鞘磷脂水解□在大腸桿菌中表現之產率不佳,為了改善其在大腸桿菌中表現之產率,本研究利用培養皿誘導、低溫培養及低濃度誘導物IPTG等方式改良表現系統,研究結果發現帶有枯草桿菌訊息胜□之中性神經鞘磷脂水解□在此表現系統中可以在大腸桿菌BL21(DE3)中被大量表現並減少包涵體產生的現象,其產率高達10mg/L。
顯示低親和力鎂離子鍵結點與催化活性相關。
以往的研究顯示中性神經鞘磷脂水解□在大腸桿菌中表現之產率不佳,為了改善其在大腸桿菌中表現之產率,本研究利用培養皿誘導、低溫培養及低濃度誘導物IPTG等方式改良表現系統,研究結果發現帶有枯草桿菌訊息胜□之中性神經鞘磷脂水解□在此表現系統中可以在大腸桿菌BL21(DE3)中被大量表現並減少包涵體產生的現象,其產率高達10mg/L。
At the epicenter of the sphingomyelin-cell signaling pathway is a family of phospholipase called sphingomyelinase. These enzymes induce a variety of cell regulatory phenomenon such as programmed cell death (apoptosis), cell differentiation, cell proliferation, and sterol homeostasis. Sphingomyelinase may be useful for additional studies of apoptosis in experimental animals.
We have identified a neutral sphingomyelinase from Bacillus subtilis IAM1208. The enzyme, with a molecular mass of 34.3 kDa, has 98.6% amino acid sequence identity with sphingomyelinase from Bacillus cereus IAM 1208. When N-ω-trinitrophenylaminolauryl sphingomyelin (TNPAL-SM) was used as a substrate, the purified neutral-sphingomyelinase exhibited a Km of 11.34μM and a Vmax of 10.225 nmol /min/10μg of protein at 37℃, pH 7.4. The pH and ion effect on N-SMase activity was examined. The optimal pH of N-SMase was at pH 7.4 .The enzyme activity was inhibited by Ca2+ but was activated by Mg2+. The effect of bivalent cations on the activity and Tryptophan fluorescence spectra of N-SMase were also examined. In the Ca2+-dependence curve of tryptophan fluorescence intensity, one distinct transition was observed between 10-6 to 10-8M, In the Mg2+-dependence curve of fluorescence intensity, two distinct transition was observed between10-7 to 10-8 and 10-3 to 10-4M and the enzyme activity was disappeared in the presence of Mg2+ below 10-4 M, suggesting that the binding of to the low-affinity site is essential for the catalysis.
In literature, the expression of sphingomyelinase of Bacillus cereus was not efficient in E. coli. To improve the yield sphingomyelinase of Bacillus cereus, we have modified the expression and induction system by changing the plating method, lowered temperature, and lowered concentration of IPTG. In this report, we have expressed sphingomyelinase from Bacillus subtilis in E. coli and decreased the amount of sphingomyelinase in inclusion bodies with the yield of 10mg protein in 1L of Bacteria culture.
中文摘要 I
英文摘要 II
目錄 IIV
圖目錄 VII
表目錄 X

壹、前言 1
貳、文獻探討 3
一、神經鞘磷脂生物功能與疾病 3
1. 神經鞘磷脂 3
2. 尼曼-匹克氏病 4
二、中性神經鞘磷脂水解□ 5
1. 動物中性神經鞘磷脂水解□生理功能 5
1.1神經鞘磷脂循環與細胞凋亡 5
1.2膽固醇調節與心血管疾病 6
2. 微生物之神經鞘磷脂水解□ 7
2.1溶血現象 7
2.2致病能力 9
三、Bacillus cereus之中性神經鞘磷脂水解□ 9
1. B. cereus神經鞘磷脂水解□性質 9
2. B. cereus神經鞘磷脂水解□純化與表現 11
四、Bacillus subtilis之中性神經鞘磷脂水解□ 12
參、材料與方法 13
一、材料 13
二、儀器 13
三、枯草桿菌神經鞘磷脂水解□之純化 14
1. 枯草桿菌培養 15
2. 快速蛋白質液相層析儀(FPLC)純化 15
3. 蛋白質電泳分析 16
四、大腸桿菌之轉殖、表現與蛋白質純化 17
1.勝任細胞的製備 17
2.Transformation轉型作用 17
3.轉殖大腸桿菌之誘導作用 18
4.轉殖大腸桿菌之神經鞘磷脂水解□純化 18
5.蛋白質定量 19
6.TCA蛋白質沈澱法 19
7.質譜儀分析與N端胺基酸定序 20
五、神經鞘磷脂水解□之性質研究 20
1. 活性測試 20
2. 動力學測定 20
3. 最適反應環境測定 21
4. 色胺酸螢光光譜分析 23
六、無訊息胜□中性神經鞘磷脂水解□選殖 24
肆、結果 27
一、枯草桿菌神經鞘磷脂水解□之純化 27
二、大腸桿菌的表現與純化 27
三、酵素活性測試 28
伍、討論 31
一、枯草桿菌中性神經鞘磷脂水解□純化 31
二、大腸桿菌之培養皿誘導法 31
三、中性神經鞘磷脂水解□性質探討 34
陸、結論 37
柒、參考文獻 38
圖 42
表 69
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