跳到主要內容

臺灣博碩士論文加值系統

(54.92.164.9) 您好!臺灣時間:2022/01/23 05:45
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:王思綺
研究生(外文):Szu-Chi Wang
論文名稱:以大腸桿菌生產肌酸酵素之研究:誘導劑IPTG之效應
論文名稱(外文):Production of creatinase by Escherichia coli:effect of inducer, IPTG
指導教授:陳特良
指導教授(外文):Teh-Liang Chen
學位類別:碩士
校院名稱:國立成功大學
系所名稱:化學工程學系碩博士班
學門:工程學門
學類:化學工程學類
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:47
中文關鍵詞:質體穩定性內醯胺酵素
外文關鍵詞:B-lactamaseplasmid stability
相關次數:
  • 被引用被引用:2
  • 點閱點閱:631
  • 評分評分:
  • 下載下載:85
  • 收藏至我的研究室書目清單書目收藏:0
本研究以基因重組菌Escherichia coli JM109/pSCR05醱酵生產肌酸酵素(Creatinase)。在批次醱酵中發現,於第2小時添加誘導劑0.5 mM IPTG進行誘導時,肌酸酵素的產量能夠於8小時達到約3.0 U/mL,菌體濃度達到約2.0 g/L。然而在每4小時置換60%培養液(內含IPTG)的Fill-and-Draw醱酵中發現,雖然菌體濃度約可回昇到1.5 g/L,但卻無法繼續生產肌酸酵素,其原因為質體已經脫落。研究發現,添加IPTG誘導細胞生產外來蛋白質時,質體會非常不穩定,且當外在選擇性壓力Ampicillin存在時,會導致細胞分泌出較多β-Lactamase以分解Ampicillin。增加Ampicillin的量,對質體穩定性並沒有幫助,只會造成細胞分泌出更多的β-Lactamase至胞外。額外添加Methicillin於培養液中以作為β-Lactamase之抑制劑,雖可短暫提昇質體穩定性,但卻會使細胞分泌更多的β-Lactamase。另外,若降低操作溫度以降低細胞的生長速率,則對質體穩定性的提升有很大的幫助。由此可見以大腸桿菌做為宿主細胞時,質體穩定性與細胞的生長速率有很大的關係。
A recombinant Escherichia coli, JM109/pSCR05, was used for creatinase production. In a batch culture, where 0.5 mM IPTG was added at 2 h, the creatinase yield was 3.0 U/mL at 8 h, and the cell density was 2.0 g/L. During fill-and-draw culture, where 60% of the medium (containing IPTG) was replaced every 4 hours, although the cells grew to 1.5 g/L, creatinase was no longer being produced because of plasmid instability. It was found that production of the foreign protein by IPTG induction resulted in serious plasmid instability, and the selective pressure, ampicillin, made the cells to excrete β-lactamase. Supplementing the medium with more ampicillin could not improve the plasmid stability, but resulted in more β-lactamase excreted. Extraneous addition of methicillin as an inhibitor of β-lactamase could temporarily improve the plasmid stability; nevertheless, it caused even more β-lactamase excreted. Furthermore, lowering culture temperature to reduce growth rate had a great improvement on plasmid stability. It therefore suggests that for E. coli as the host cell, the plasmid stability is highly dependent on the growth rate.
中文摘要................................................................................................I
ABSTRACT...........................................................................................II
誌謝..................................................................................................III
目錄.................................................................................................IV
圖目錄.................................................................................................VI
第一章緒論.................................................................................1
1-1 基因重組工程....................................................................................1
1-1-1 基因重組技術.........................................................................1
1-1-2 基因重組菌醱酵工程.............................................................3
1-1-3 基因表現調節.........................................................................6
1-2 肌酸酵素及肌酸簡介及應用..........................................................10
1-3 研究目的..........................................................................................11
第二章材料與方法....................................................................12
2-1 實驗材料..........................................................................................12
2-1-1 菌株.......................................................................................12
2-1-2 藥品.......................................................................................12
2-1-3 實驗儀器與裝置...................................................................15
2-2 實驗方法..........................................................................................17
2-2-1 菌種保存...............................................................................17
2-2-2 醱酵培養...............................................................................17
2-2-3 溶氧電極之校正...................................................................18
2-3 分析方法..........................................................................................21
2-3-1 菌體濃度檢量線...................................................................21
2-3-2 菌體濃度分析.......................................................................21
2-3-3 酵素液之製備.......................................................................21
2-3-4 肌酸酵素活性分析...............................................................23
2-3-5 β-Lactamase活性分析..........................................................24
2-3-6 質體穩定性測定...................................................................26
第三章結果與討論....................................................................27
3-1半連續式醱酵:置換60 mL培養液內含IPTG............................27
3-2半連續式醱酵:置換60 mL培養液不含IPTG............................27
3-3 批次醱酵生產肌酸酵素:添加50 mg/L Ampicillin.....................30
3-4 批次醱酵生產肌酸酵素:添加100 mg/L Ampicillin...................30
3-5 批次醱酵生產肌酸酵素:添加200 mg/L Ampicillin...................33
3-6 批次醱酵生產肌酸酵素:添加50 mg/L Ampicillin及50 mg/L Methicillin........................................................................................37
3-7 批次醱酵生產肌酸酵素:添加50 mg/L Ampicillin,不添加IPTG..........................................................................................................37
3-8 批次醱酵生產肌酸酵素:改變操作溫度為30℃.........................40
第四章結論與未來展望.............................................................42
參考文獻.............................................................................................44
Bauer, S., and E. Ziv, “Dense growth of aerobic bacteria in a bench-scale fermentor,” Biotechnol. Bioeng., 18:81-94 (1976)
Bentley, W. E. and D. S. Kompala, “Stability in continuous culture in recombinant bacteria:a metabolic approach,” Biotechnol. Lett. 12:329-334 (1990)
Bentley, W. E., N. Mirjalili, D. C. Andersen, R. H. Davis, and D. S. Kompala, “Plasmid-encoded protein: the principle factor in the metabolic burden associated with recombinant bacteria,” Biotechnol. Bioeng., 35:668-681 (1990)
Chang, M. C., C. C. Chang, and J. C. Chang, “Cloning of a Creatinase Gene From Pseudomonas Putida in Escherichia coli by Using an Indicator Plate,” Appl. Envir. Microbiol., 58: 3437-3440 (1992)
Cheng, C., Y. L. Huang, and S. T. Yang, “A Novel feeding strategy for enhanced.plasmid stability and protein production in recombinant yeast fedbatch fermentation,” Biotechnol. Bioeng., 56:23-31 (1997)
Curless, C., J. Pope, and L. Tsai, “Effects of preinduction specific growth rate on recombinant alpha consensus interferon synthesis in Escherichia coli,” Biotechnol. Prog., 6:149-152 (1990)
Gleiser, I. E., and S. Bauer,”Growth of E. coli W to high cell concentration by oxygen linked control of carbon source concentration,” Biotechnol. Bioeng., 23:1015-1021 (1981)
Glick, B. R., and J. J. Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press Washington (1994)Bauer, S., and E. Ziv, “Dense growth of aerobic bacteria in a bench-scale fermentor,” Biotechnol. Bioeng., 18:81-94 (1976)
Bentley, W. E. and D. S. Kompala, “Stability in continuous culture in recombinant bacteria:a metabolic approach,” Biotechnol. Lett. 12:329-334 (1990)
Bentley, W. E., N. Mirjalili, D. C. Andersen, R. H. Davis, and D. S. Kompala, “Plasmid-encoded protein: the principle factor in the metabolic burden associated with recombinant bacteria,” Biotechnol. Bioeng., 35:668-681 (1990)
Chang, M. C., C. C. Chang, and J. C. Chang, “Cloning of a Creatinase Gene From Pseudomonas Putida in Escherichia coli by Using an Indicator Plate,” Appl. Envir. Microbiol., 58: 3437-3440 (1992)
Cheng, C., Y. L. Huang, and S. T. Yang, “A Novel feeding strategy for enhanced.plasmid stability and protein production in recombinant yeast fedbatch fermentation,” Biotechnol. Bioeng., 56:23-31 (1997)
Curless, C., J. Pope, and L. Tsai, “Effects of preinduction specific growth rate on recombinant alpha consensus interferon synthesis in Escherichia coli,” Biotechnol. Prog., 6:149-152 (1990)
Gleiser, I. E., and S. Bauer,”Growth of E. coli W to high cell concentration by oxygen linked control of carbon source concentration,” Biotechnol. Bioeng., 23:1015-1021 (1981)
Glick, B. R., and J. J. Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press Washington (1994)
Gupta, R., P. Sharma, and V. V. Vyas, “Effect of growth environment on the stability of recombinant shuttle plasmid, pCppS-31, in Escherichia coli,” J. Biotechnol., 41:29-37 (1995)
Heiengard, D., and G. Tidenstrom, “Determination of serum creatine by a direct colorimertic method,” Clin. Chim. Asta. , 43:305-311 (1973)
Hoeffken, H. W., S. H. Knof, P. A. Bartlettt, R. Huber, H. Moellering, and G. Schumacher, ”Crystal structure determination, refinement and molecular model of creatine amidinolydrolase from Pseudomonas putida,” J. Mol. Biol., 204:417-433 (1988)
Kaplan, A., and D. Naugler, “Creatinine hydrolase and creatine amidinohydrolase Ι. presence in cell-free extracts of Arthrobacter ureafaciens,” Mol. Cell. Biochem., 3:9-15 (1974)
Kim, C. H., J. Y. Lee, M. G. Kim, K. B. Song, J. W. Seo, B. H. Chung, S. J. Chang, and S. K. Rhee, “Fermentation strategy to enhance plasmid stability during the cultivation of Escherichia coli for the production of recombinant levansucrase,” J. Ferment. Bioeng., 86:391-394 (1998)
Koyama, Y., S. Kitao, H. Yamamoto-Otake, M. Susuki, and E. Nakano, “Cloning and expression of the creatinase gene from Flavobaterium sp. U-188 in Escherichia coli,” Agric. Biol. Chem., 54:1453-1457 (1990)
Matsuda, Y., N. Wakamatsu, Y. Inouye, S. Uede, Y. Hashimoto, K. Asano, and S. Nakamura, “Purification and characterization of creatine amidinohydrolase of Alcaligenes Origin,” Chem. Pharm. Bull., 34:2155-2160 (1986)
Majewski, R. A., and M. M. Domach, “Simple constrained optimization view of acetate overflow in E. coli,” Biotechnol. Bioeng., 35:732-738 (1990)
Nancib, N., and J. Boudrant, “Effect of growth rate on stability and gene expression of a recombinant plasmid during continuous culture of E. coli in a non-selective medium,” Biotechnol. Lett., 14:643-648 (1992)
O’Connor, G. M., F. Sanchez-Rierra, and C. L. Cooney, “Design and evaluation of control strategies for high cell density fermentations,” Biotechnol. Bioeng., 39:293-304 (1992)
Parker, C., and D. Dibiasio, “Effect of growth rate and expression level on plasmid stability in Saccharomyces cerevisiae,” Biotechnol. Bioeng., 29:215-221 (1987)
Prescott, L., J. P. Harley, and D. A. Klein, Microbiology, McGraw Hill, New York (1999)
Rinas, U., “Synthesis rates of cellular proteins involved in translation and protein folding are strongly altered in response to overexpression of basic fibroblast growth factor by recombinant Escherichia coli,” Biotechnol. Prog., 12:196-200 (1996)
Ryan, W., and S. J. Parulekar, “Recombinant protein synthesis and plasmid instability in continuous cultures of Escherichia coli JM103 harboring a high copy number plasmid,” Biotechnol. Bioeng., 37:415-429 (1991)
Sambrook, J., E. F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, second ed. Cold Spring Habor Laboratory Press, Cold Spring Habor, NY. (1989)
Sargent, M. G., “Rapid Fixed-Time Assay for Penicillinase.,” J. Bacteriology, 95:1493-1494 (1968)
Seo, J. H., and J. E. Bailey, “Effects of recombinant plasmid content on growth properties and cloned gene product formation in Escherichia coli,” Biotechnol. Bioeng.,27:1668-1674 (1985)
Shoham, Y., and A. L. Demain, “Effect of medium composition on the maintenance of a recombinant plasmid in B. subtilis,” Enzyme Microb. Technol, 12:330 (1990)
Tamarin, R., and R. W. Leavitt, Principles of Genetics 3rd ed Wm. C. Brown Publishers. Iowa (1991)
Togna, A. P., M. L. Shuler, and D. B. Wilson, “Effect of plasmid copy number and runway plasmid replication on over production and excretion of β-lactamase from Escherichia coli,” Biotechnol. Prog., 9:31-39 (1993)
Vind, J., M. A. Soerensen, M. D. Rasmussen, and S. Pedersen, “Synthesis of proteins in Escherichia coli is limited by the concentration of free ribosomes: expression from reporter genes does not always reflect functional mRNA level,” J. Mol. Biol 231:678-688 (1993)
Yee, L., and H. W. Blanch, “Recombinant trypsin production in high cell density fed-batch cultures in Escherichia coli,” Biotechnol. Bioeng., 41:781-790 (1992)
Zabriskie, D. W., and E. J. Arcuri, “Factor influencing productivity of fermentations employing recombinant microorganisms,” Enzyme. Microb. Technol., 8:706-711 (1986)
連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top