臺灣博碩士論文加值系統

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

Chatterjee, S. (1994). “Neutral sphingomyelinase action stimulates signal transduction of tumor necrosis factor-alpha in the synthesis of cholesteryl esters in human fibroblasts.” J Biol Chem 269(2) 879-82.Chatterjee, S. (1999). “Neutral sphingomyelinase: past, present and future.” Chem Phys Lipids 102(1-2) 79-96.Choi, J. H., K. J. Jeong, et al. (2000). “Efficient secretory production of alkaline phosphatase by high cell density culture of recombinant Escherichia coli using the Bacillus sp. endoxylanase signal sequence.” Appl Microbiol Biotechnol 53(6) 640-5.Drobniewski, F. A. (1993). “Bacillus cereus and related species.” Clin Microbiol Rev 6(4) 324-38.Gatt, S. (1966). “Enzymatic hydrolysis of sphingolipids. I. Hydrolysis and synthesis of ceramides by an enzyme from rat brain.” J Biol Chem 241(16) 3724-30.Granum, P. E. and H. Nissen (1993). “Sphingomyelinase is part of the ''enterotoxin complex'' produced by Bacillus cereus.” FEMS Microbiol Lett 110(1) 97-100.Gupta, A. K. and H. Rudney (1991). “Plasma membrane sphingomyelin and the regulation of HMG-CoA reductase activity and cholesterol biosynthesis in cell cultures.” J Lipid Res 32(1) 125-36.Hamanaka, R., K. Kohno, et al. (1992). “Induction of low density lipoprotein receptor and a transcription factor SP-1 by tumor necrosis factor in human microvascular endothelial cells.” J Biol Chem 267(19) 13160-5.Ikezawa, H., M. Matsushita, et al. (1986). “Effects of metal ions on sphingomyelinase activity of Bacillus cereus.” Arch Biochem Biophys 249(2) 588-95.Ikezawa, H., M. Mori, et al. (1978). “Studies on sphingomyelinase of Bacillus cereus. I. Purification and properties.” Biochim Biophys Acta 528(2) 247-56.Ikezawa, H., M. Mori, et al. (1980). “Studies on sphingomyelinase of Bacillus cereus: hydrolytic and hemolytic actions on erythrocyte membranes.” Arch Biochem Biophys 199(2) 572-8.Jan, J. T., S. Chatterjee, et al. (2000). “Sindbis virus entry into cells triggers apoptosis by activating sphingomyelinase, leading to the release of ceramide.” J Virol 74(14) 6425-32.Jarvis, W. D., R. N. Kolesnick, et al. (1994). “Induction of apoptotic DNA damage and cell death by activation of the sphingomyelin pathway.” Proc Natl Acad Sci U S A 91(1) 73-7.Kim, M. Y., C. Linardic, et al. (1991). “Identification of sphingomyelin turnover as an effector mechanism for the action of tumor necrosis factor alpha and gamma-interferon. Specific role in cell differentiation.” J Biol Chem 266(1) 484-9.Lawler, J. F., Jr., M. Yin, et al. (1998). “Tumor necrosis factor-alpha stimulates the maturation of sterol regulatory element binding protein-1 in human hepatocytes through the action of neutral sphingomyelinase.” J Biol Chem 273(9) 5053-9.Masamune, A., T. Shimosegawa, et al. (1999). “Helicobacter pylori-dependent ceramide production may mediate increased interleukin 8 expression in human gastric cancer cell lines.” Gastroenterology 116(6) 1330-41.Matsuo, Y., A. Yamada, et al. (1996). “A distant evolutionary relationship between bacterial sphingomyelinase and mammalian DNase I.” Protein Sci 5(12) 2459-67.Miranda, S. R., S. Erlich, et al. (1997). “Bone marrow transplantation in acid sphingomyelinase-deficient mice: engraftment and cell migration into the brain as a function of radiation, age, and phenotype.” Blood 90(1) 444-52.Nakamura, M. and W. R. Cross (1968). “The lecithinase (alpha toxin) activity of strains of Clostridium perfringens.” Proc Soc Exp Biol Med 127(3) 719-22.Obeid, L. M., C. M. Linardic, et al. (1993). “Programmed cell death induced by ceramide.” Science 259(5102) 1769-71.Okazaki, T., R. M. Bell, et al. (1989). “Sphingomyelin turnover induced by vitamin D3 in HL-60 cells. Role in cell differentiation.” J Biol Chem 264(32) 19076-80.Pinkney, M., E. Beachey, et al. (1989). “The thiol-activated toxin streptolysin O does not require a thiol group for cytolytic activity.” Infect Immun 57(8) 2553-8.Rodrigues-Lima, F., A. C. Fensome, et al. (2000). “Structural requirements for catalysis and membrane targeting of mammalian enzymes with neutral sphingomyelinase and lysophospholipid phospholipase C activities. Analysis by chemical modification and site- directed mutagenesis.” J Biol Chem 275(36) 28316-25.Rogolsky, M. (1979). “Nonenteric toxins of Staphylococcus aureus.” Microbiol Rev 43(3) 320-60.Schneider, P. B. and E. P. Kennedy (1967). “Sphingomyelinase in normal human spleens and in spleens from subjects with Niemann-Pick disease.” J Lipid Res 8(3) 202-9.Slotte, J. P. and E. L. Bierman (1988). “Depletion of plasma-membrane sphingomyelin rapidly alters the distribution of cholesterol between plasma membranes and intracellular cholesterol pools in cultured fibroblasts.” Biochem J 250(3) 653-8.Suchi, M., T. Dinur, et al. (1992). “Retroviral-mediated transfer of the human acid sphingomyelinase cDNA: correction of the metabolic defect in cultured Niemann-Pick disease cells.” Proc Natl Acad Sci U S A 89(8) 3227-31.Suter-Crazzolara, C. and K. Unsicker (1995). “Improved expression of toxic proteins in E. coli.” Biotechniques 19(2) 202-4.Tamura, H., K. Tameishi, et al. (1992). “Mass production of sphingomyelinase of Bacillus cereus by a protein- hyperproducing strain, Bacillus brevis 47, and its purification.” J Biochem (Tokyo) 112(4) 488-91.Tomita, M., R. Taguchi, et al. (1982). “Molecular properties and kinetic studies on sphingomyelinase of Bacillus cereus.” Biochim Biophys Acta 704(1) 90-9.Tomita, M., J. Togami, et al. (1992). “Effect of 22R-hydroxycholesterol on the action of sphingomyelinase from Bacillus cereus toward bovine erythrocytes.” Toxicon 30(8) 801-13.Tomita, M., Y. Ueda, et al. (1993). “The role of acidic amino-acid residues in catalytic and adsorptive sites of Bacillus cereus sphingomyelinase.” Biochim Biophys Acta 1203(1) 85-92.Yamada, A., N. Tsukagoshi, et al. (1988). “Nucleotide sequence and expression in Escherichia coli of the gene coding for sphingomyelinase of Bacillus cereus.” Eur J Biochem 175(2) 213-20.Zhang, H., N. N. Desai, et al. (1991). “Sphingosine-1-phosphate, a novel lipid, involved in cellular proliferation.” J Cell Biol 114(1) 155-67.