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研究生:劉惠娟
研究生(外文):Hui-Chuan Liu
論文名稱:BacillussubtilisATCC21332的改質與表面素生產相關蛋白質之研究
論文名稱(外文):Studies on the proteins involved in surfactin synthesis and improvement of Bacillus subtilis ATCC 21332 for surfactin production.
指導教授:康世旭
學位類別:碩士
校院名稱:元智大學
系所名稱:生物科技暨生物資訊研究所
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:2006
畢業學年度:94
語文別:中文
論文頁數:67
中文關鍵詞:枯草桿菌表面素
外文關鍵詞:Bacillus subtilissurfactin
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表面素是由Bacillus subtilis ATCC 21332以非核醣體胜肽合成機制所合成的脂胜肽生物界面活性劑,結構包含七個胺基酸所組成的環狀胜肽鏈,及一長碳鏈之脂肪酸支鏈。研究顯示有三個表面素合成酶,SrfA、SrfB、SrfC,參與胜肽鏈的胜肽合成。表面素是目前文獻上發現最有效的生物界面活性劑,但是在應用上,因侷限於產量低,生產成本高的瓶頸,因此尚無法達到商業化之目的。而造成表面素產量低的原因,可能是表面素合成酶啟動子僅在特定的條件下才會開始工作,以至於表面素合成酶表現量低,進而造成表面素產量低。所以我們嘗試以Gene targeting技術將表面素合成基因的啟動子置換為P43啟動子,但是到目前為止仍無法將質體送入。可能因為B. subtilis ATCC 21332難以進行遺傳改質工作,所以先嘗試建構一株易於進行遺傳改質的表面素生產菌株。目前已經成功得到一些能生產表面素的B. subtilis 168轉形株,其中以SWK569所生產的表面素最高,且遺傳穩定性高,同時這些B. subtilis 168轉形株的轉形效率亦略優於B. subtilis 168。針對這些轉形株以Gene targeting技術,置換表面素合成基因的啟動子為P43啟動子時,並不如預期般提升表面素產量,反而出現另一會造成血瓊脂培養基溶血的未知名物質,此物質的生成原因目前尚不清楚,需待進一步探討成因。雖然非核醣體胜肽合成機制研究已經超過數十年,但除了胜肽合成酶外,還需要哪些蛋白質參與或調控,目前均不清楚。本實驗室同時也利用二維電泳技術研究與表面素生產相關之蛋白質,分析在表面素合成時,哪些蛋白質表現量隨之變化。目前已找到二十個蛋白質,在合成表面素前後,表現量會隨之改變,將進一步探討其中十三個已確認之蛋白質與非核醣體胜肽合成機制的相關性。
Surfactin is a cyclic lipopeptidic biosurfactant produced by Bacillus subtilis ATCC 21332 with nonribosomal peptide synthesis mechanisn.Surfactin is composed of seven amino acids of lipopeptide and a long-chain fatty acid. The synthesis of peptide moiety of surfactin is catalyzed by three surfactin synthetases, SrfA , SrfB, and SrfC. Although surfactin is the most powerful biosurfactant, it’s application and commerializating is restricted because of low productivity and high production cost. The possible reason which cause low productivity of surfactin is the promoter of surfactin synthetases genes only work under the particular condition. Only few surfactin synthetases express to catalyze the surfactin synthesis. In order to upgrade the surfactin productivity, we tried to use the P43 promoter to replace the promoter of surfactin synthetas operon by gene targeting technology. Because of the poor transformation efficiency, no plasmid was transferred into B. subtilis ATCC 21332 to proceed further studies. it is necessary to get surfactin-producing strains with the ability to receive to proceed experiments for genetic engineering easily. Some B. subtilis 168 derive clones derived with highest surfactin productivity and genetic stability were gotten. The transformation frequencies of the B. subtilis 168 derive clones were better than B. subtilis 168 and 21332. P43 promoter replacement for promoter of surfactin synthetase operon was also performed, surfactin productivity was not improved and only some unknown products were synthesized to generate lysis zones on Blood agar. The nonribosomal peptide synthesis has been studied for more than fifty years. In addition to peptide synthetase, it is not clear which proteins regulate or participate in the nonribosomal peptide synthesis. We have carried out the preliminary proteomic analysis of B. subtilis ATCC 21332 proteins related to surfactin synthesis. Using two-dimensional electrophoretic separations followed by MALDI-TOF mass spectrometry, expression of 20 proteins alter during the surfactin synthesis and 13 protein were identified. Futuremore, functions of the 13 identified proteins in the surfactin synthesis will be studied.
中文摘要………………………………………………………..……..…...I
英文摘要…………………………………………………………………III
致謝………………………………………………………….....................V
目錄…………………………………………………………....................VI
表目錄………………………………………………………...................VII
圖目錄………………………………………………………………….VIII
前言……………………………………………………...…………….......1
材料與方法…………………………………....…………………………..8
結果…….…………….……………..…………………..……………......17
討論............................................................................................................26
結論……………………..……………………………………..…………32
參考文獻……………….………………………………..……………….55
附錄一………………….……………………………..……………….....60
附錄二………………….……………………………..………………….61
附錄三………………….……………………….….…………………….62
附錄四………………….………………………………….……………..63
附錄五………………….………………………….……………………..64
Arfin, S. M., and Umbarger, H. E. (1969). Purification and properties of the acetohydroxy acid isomeroreductase of Salmonella typhimurium. J Biol Chem 244, 1118-1127.

Arima, K., Kakinuma, A., and Tamura, G. (1968). Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation. Biochem Biophys Res Commun 31, 488-494.

Banerjee, S., and Hansen, J. N. (1988). Structure and expression of a gene encoding the presursor of subtilin, a small protein antibiotic. J Biol Chem 263, 9508-9514.

Biswas, I., Gruss, A., Ehrlich, S. D., and Maguin, E. (1993). High-efficiency gene inactivation and replacement system for gram-positive bacteria. J Bacteriol 175, 3628-3635.

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254.

Chen, C. L., Chang, L. K., Chang, Y. S., Liu, S. T., and Tschen, J. S. (1995). Transposon mutagenesis and cloning of the genes encoding the enzymes of fengycin biosynthesis in Bacillus subtilis. Mol Gen Genet 248, 121-125.

Cohen, S. N., Chang, A. C., and Hsu, L. (1972). Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A 69, 2110-2114.

Desai, J. D., and Desai, A. J. (1993). Surfactant science series, In Production of biosurfactants, N. Kosaric, ed. (Basel and Hong Kong), pp. 65-97

Dubnau, D., and Davidoff-Abelson, R. (1971). Fate of transforming DNA following uptake by competent Bacillus subtilis. I. Formation and properties of the donor-recipient complex. J Mol Biol 56, 209-221.

Fuma, S., Fujishima, Y., Corbell, N., D''Souza, C., Nakano, M. M., Zuber, P., and Yamane, K. (1993). Nucleotide sequence of 5'' portion of srfA that contains the region required for competence establishment in Bacillus subtilus. Nucleic Acids Res 21, 93-97.

Hemila, H., Palva, A., Paulin, L., Arvidson, S., and Palva, I. (1990). Secretory S complex of Bacillus subtilis: sequence analysis and identity to pyruvate dehydrogenase. J Bacteriol 172, 5052-5063.

Holmes, D. S., and Quigley, M. (1981). A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114, 193-197.

Imanaka, T., Fujii, M., Aramori, I., and Aiba, S. (1982). Transformation of Bacillus stearothermophilus with plasmid DNA and characterization of shuttle vector plasmids between Bacillus stearothermophilus and Bacillus subtilis. J Bacteriol 149, 824-830.

Jung, G. (1991). Lantibiotics-ribosomally Synthetisized biologylly active Polypeptids containing sulfide bridges and α, β-didehydroamino acid. Angew Chemical International Ed England 30, 1051-1068.

Kakinuma, A., Hori, M., Isono, M., Tamura, G., and Arima, K. (1969). Determination of amino acid sequence in furfactin, a crystalline peptidolipid surfactant produced by Bacillus subtilis. Agric Biol Chem 33, 971-997.

Kleinkauf, H., and Von Dohren, H. (1996). A nonribosomal system of peptide biosynthesis. Eur J Biochem 236, 335-351.

Kleinkauf, H., and von Dohren, H. (1997). Enzymatic generation of complex peptides. Prog Drug Res 48, 27-53.

Kratzschmar, J., Krause, M., and Marahiel, M. A. (1989). Gramicidin S biosynthesis operon containing the structural genes grsA and grsB has an open reading frame encoding a protein homologous to fatty acid thioesterases. J Bacteriol 171, 5422-5429.

Kunst, F., Ogasawara, N., Moszer, I., Albertini, A. M., Alloni, G., Azevedo, V., Bertero, M. G., Bessieres, P., Bolotin, A., Borchert, S., et al. (1997). The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390, 249-256.

Lai, E. M., Phadke, N. D., Kachman, M. T., Giorno, R., Vazquez, S., Vazquez, J. A., Maddock, J. R., and Driks, A. (2003). Proteomic analysis of the spore coats of Bacillus subtilis and Bacillus anthracis. J Bacteriol 185, 1443-1454.

Lambalot, R. H., Gehring, A. M., Flugel, R. S., Zuber, P., LaCelle, M., Marahiel, M. A., Reid, R., Khosla, C., and Walsh, C. T. (1996). A new enzyme superfamily - the phosphopantetheinyl transferases. Chem Biol 3, 923-936.

Leyva-Vazquez, M. A., and Setlow, P. (1994). Cloning and nucleotide sequences of the genes encoding triose phosphate isomerase, phosphoglycerate mutase, and enolase from Bacillus subtilis. J Bacteriol 176, 3903-3910.

Massey, V. (1963). Lipoyl dehydrogenase, In The Enzymes, P. D. Boyer, Lardy, H. and Myrbäck, K. , ed., pp. 275-306.

Massey, V., Gibson, Q. H., and Veeger, C. (1960). Intermediates in the catalytic action of lipoyl dehydrogenase (diaphorase). Biochem J 77, 341-351.

Meyer-Arendt, E., Beisenherz, G., and Bücher, T. (1953). Triosephosphate isomerase. Naturwissenschaften 40, 59.

Meyerhof, O., and Beck, L. V. (1944). Triosephosphate isomerase. J Biol Chem 156, 109-120.

Mootz, H. D., and Marahiel, M. A. (1997). The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains. J Bacteriol 179, 6843-6850.

Nakano, M. M., Marahiel, M. A., and Zuber, P. (1988). Identification of a genetic locus required for biosynthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis. J Bacteriol 170, 5662-5668.

Omura, S., Iwai, Y., Masuma, R., Hayashi, M., Furusato, T., and Takagaki, T. (1980). A new peptide antibiotic, alboleutin. J Antibiot (Tokyo) 33, 758-759.

Peypoux, F., Bonmatin, J. M., and Wallach, J. (1999). Recent trends in the biochemistry of surfactin. Appl Microbiol Biotechnol 51, 553-563.

Pfeifer, E., Pavela-Vrancic, M., von Dohren, H., and Kleinkauf, H. (1995). Characterization of tyrocidine synthetase 1 (TY1): requirement of posttranslational modification for peptide biosynthesis. Biochemistry 34, 7450-7459.

Sahl, H. G. (1991). Pore formation in bacterial membrane by cationic lantibioticd, In Nisin and Novel Lantibiotics., pp. 347-358.

Schuermann, M., and Sprenger, G. A. (2001). Fructose-6-phosphate aldolase is a novel class I aldolase from Escherichia coli and is related to a novel group of bacterial transaldolases. J Biol Chem 276, 11055-11061.

Siezen, R. J., Kuipers, O. P., and de Vos, W. M. (1996). Comparison of lantibiotic gene clusters and encoded proteins. Antonie Van Leeuwenhoek 69, 171-184.

Singh, P., and Cameotra, S. S. (2004). Potential applications of microbial surfactants in biomedical sciences. Trends Biotechnol 22, 142-146.

Stachelhaus, T., Schneider, A., and Marahiel, M. A. (1996). Engineered biosynthesis of peptide antibiotics. Biochem Pharmacol 52, 177-186.

Stein, T., Vater, J., et al., (1996). The multiple carrier model of nonribosomal peptide biosynthesis at modular multienzymatic templates. J Biol Chem 271, 15428-15435.

Trach, K., Chapman, J. W., Piggot, P., LeCoq, D., and Hoch, J. A. (1988). Complete sequence and transcriptional analysis of the spo0F region of the Bacillus subtilis chromosome. J Bacteriol 170, 4194-4208.

Wei Y. H., Wang, L. F., Chang, J. S. and Kung, S. S. (2003). Identification of induced acidification in iron-enriched cultures of Bacillus subtilis during biosurfactant fermentation. J. Biosci. Bioeng 96, 174-178.


Wang, P. Z., and Doi, R. H. (1984). Overlapping promoters transcribed by bacillus subtilis σ55 and σ37 RNA polymerase holoenzymes during growth and stationary phases. J Biol Chem 259, 8619-8625.

Zuber, P., Nakano, M. M., and Marahiel, M. A. (1993). peptide antibiotics, In Bacillus subtilis and other gram-positive bacteria, A. L. Sonenshein, J. A. Hoch, and e. R. Losick, eds. (Washington, D C.: American Society for Microbiogy), pp. 897-916.

陳奇良 (1995). 枯草桿菌(B. subtilis)F29-3中豐原素合成基因的分析. 國立中興大學植物學研究所博士論文.

魏毓宏 (2001). 二價金屬無機鹽類之添加對生物界面活性劑表面素生產之影響. 化工 48, 1-8.
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