跳到主要內容

臺灣博碩士論文加值系統

(18.205.192.201) 您好!臺灣時間:2021/08/06 04:00
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

我願授權國圖
: 
twitterline
研究生:洪千喬
研究生(外文):Chian-Chiau Hung
論文名稱:利用大腸桿菌表達Cupriavidustaiwanensis184之phaCAB基因生產聚羥基烷酸
論文名稱(外文):Functional expressing of the phaCAB from Cupriavidus taiwanensis 184 in E.coli for polyhydroxybutyrate production.
指導教授:簡志青
學位類別:碩士
校院名稱:元智大學
系所名稱:生物科技與工程研究所
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:2009
畢業學年度:97
語文別:中文
論文頁數:70
中文關鍵詞:聚羥基烷酸Cupriavidus taiwanensisPHBPHAs
外文關鍵詞:PolyhydroxyalkanoatesCupriavidus taiwanensisPHBPHAs
相關次數:
  • 被引用被引用:0
  • 點閱點閱:134
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
聚羥基烷酸類(Polyhydroxyalkanoates, PHAs)為一種由微生物所合成之高分子聚合物,此類高分子與傳統石化塑膠具有相似的物化特性,並具生物相容性及生物可分解性,而Curpiavidus taiwanensis 184經研究證實可累積大量的PHB。本研究,將C. taiwanensis 184合成PHB相關基因轉殖進入Escherichia coli中,讓E. coli表現此段基因,藉以生產PHAs並且探討其產物的物理性質。
經由實驗結果挑選出菌株CCH30與CCH06,其PHB content約為66~70%,經醱酵槽測試後,在92小時菌體乾重達111.76 g/l,PHB的濃度達70.41 g/l,相當累積63%的PHB於菌體內,且CCH30所生產的PHB可以壓製成膜形。進一步的物理性質分析後,得知重組菌株CCH30所生產的PHB,玻璃轉移溫度(Tg)約為1 °C,融熔溫度(Tm)約為177 °C,結晶度(Xc)約為64%,熱裂解溫度(Td)約292°C。另外在不同營養源下,本研究之重組菌株所得到之PHB平均分子量約為12~29萬。本研究成功的轉殖表現C. taiwanensis 184的PHB相關合成基因,且顯示重組菌株所生產的PHB,可加工壓製成膜形,經由物性測試也得知其應用與加工的範圍,而由於不同的培養方式,也可做為產物性質上的調控。
Polyhydroxyalkanoates (PHAs) are polyesters synthesized by numerous microorganisms. These polyesters are biodegradable and have similar properties to those of conventional plastics. Cupriavidus taiwanensis strain 184 is phylogentically close related to the well known PHB producer Ralstonia eutropha (Cupriavidus necator) and is shown to be able to accumulate significant amount of polyhydroxybutyrate (PHB). In this study, we aim to clone the PHB syntheses genes (phaCAB) from C. taiwanensis 184 into an Escherichia coli to construct recombinant strains for biosynthesis of PHB.
Among the recombinant E. coli obtained from this study, we selected strains with plasmids CCH30 (DH5α/pTA30) and CCH06 (DH5α/pSR6) , which could produce higher contents of PHB compared to other clones examined. The contents of PHB of these two recombinant strains were about 66~70%. The final cell dry weight, PHB concertration, and PHB content of fermention results reached 111.76 g/l, 70.41 g/l, and 63% in 92 h, respectively.
The PHB product of strain CCH30 was further studied for its property by hot-press.The result showed the properies of this PHB are as follows: glass transition temperature (Tg) around 1 °C, melting temperature (Tm) around 177 °C, crystallinity (Xc%) around 64% and decomposition temperature (Td) around 292 °C. The molecular weight of PHB produced by this recombinant E. coli ranged between 120,000 and 290,000 when different
III
carbon source was provided.
We are currently investigating the optimization conditions for production of PHB by these recombinant E. coli and possible applications of the polymers produced.
目錄
中文摘要 ............................................... Ⅰ
英文摘要 ............................................... Ⅱ
目錄 ................................................... Ⅳ
圖索引 ................................................. Ⅵ
表索引 ................................................. Ⅶ
第一章 緒論 ............................................. 1
1-1 前言 ................................................ 1
1-2 研究動機與目的 ...................................... 2
第二章 文獻回顧.......................................... 4
2-1 生物可分解性塑膠簡介 ................................ 4
2-2 聚羥基烷酸介紹 ...................................... 4
2-3 臺灣貪銅菌(Cupriavidus taiwanensis)介紹 ............ 11
第三章 研究方法......................................... 12
3-1 論文研究架構 ....................................... 12
3-2 實驗材料 ........................................... 14
3-3 實驗儀器.............................................18
3-4 實驗方法 ........................................... 20
3-5 分析方法 ........................................... 31
第四章 結果與討論 ...................................... 38
4-1 利用聚合酵素連鎖反應取得phaCAB基因 ................. 38
4-2 序列比對 ........................................... 39
V
4-3 重組菌株建構(以pGEM-T為載體) ..................... 40
4-4 重組菌株建構(以pBluescript II KS+為載體) ......... 41
4-5 尼羅紅(Nile Red)測試 ............................... 44
4-6 重組菌株序列方向 .............................. 45 4-7 蛋白質電泳(SDS-PAGE)分析 ............................... 46
4-8 生長曲線(Growth curve)的測試 ....................... 48
4-9 產物偵測與鑑定 ..................................... 49
4-10 氣相層析儀(GC)分析 ................................ 50
4-11 利用DO-stat饋料批式醱酵策略進行發酵 ............... 55
4-12 穿透式電子顯微鏡(TEM)觀察 ......................... 56
4-13 聚羥基丁酸酯物理性質測試 .......................... 57
第五章 結論 ............................................ 66
參考文獻 ............................................... 68
附錄 .自述.......................................... 附錄21
圖索引
圖 1、PHAs結構 .......................................... 5
圖 2、Ralstonia eutropha穿透式電子顯微鏡圖 .............. 6
圖 3、PHAs合成路徑 ...................................... 7
圖 4、PHB相關合成基因 ................................... 8
圖 5、實驗流程圖-1 ......................................12
圖 6、實驗流程圖-2 ......................................13
圖 7、電泳圖-1 ..........................................38
圖 8、phaCAB基因圖示 ....................................39
圖 9、電泳圖-2 ..........................................40
圖 10、質體建構流程圖(以pGEM-T為載體) ...................42
圖 11、質體建構流程圖(以pBluescript II KS(+)為載體)......43
圖 12、尼羅紅測試結果 ...................................44
圖 13、質體方向示意圖 ...................................45
圖 14、SDS PAGE結果-1 ...................................46
圖 15、SDS PAGE結果-2 ...................................47
圖 16、菌株在M9培養液的生長曲線圖 .......................49
圖 17、氣相層析儀分析 ...................................50
圖 18、FTIR分析 .........................................51
圖 19、FTIR重疊圖 .......................................52
圖 20、培養之碳源補給條件測試 ...........................54
圖 21、利用DO-stat醱酵策略進行CCH30之醱酵 ...............55
圖 22、菌體內電子顯微鏡圖。 .............................57
V II
圖 23、重組菌株PHB萃取樣本 ..............................58
圖 24、研磨後之樣本 .....................................58
圖 25、不同來源之PHB分子量疊圖...........................59
圖 26、熱裂解測試圖 .....................................61
圖 27、PHB膜樣本 ........................................62
圖 28、DSC測試比較 ......................................65
表索引
表 1、目前國內生物可分解性塑膠廠商發產情形 ..............10
表 2、本研究所使用引子(Primer) ..........................26
表 3、PCR反應條件 .......................................27
表 4、DSC反應條件 .......................................35
表 5、蛋白質質譜鑑定結果 ................................48
表 6、重組菌株乾重與PHB累積情況 .........................52
表 7、IPTG誘導下,重組菌株乾重與PHAs累積比較 ............53
表 8、培養之碳源補給條件測試 ............................54
表 9、CCH30之醱酵實驗 ...................................56
表 10、不同來源之PHB分子量之比較.........................59
表 11、PHB熱裂解測試 ....................................61
表 12、各PHB之DSC測試比較 ...............................65
6 8
參考文獻
1. Amadou, C., G. Pascal, S. Mangenot, M. Glew, C. Bontemps, D. Capela, S. Carrere, S. Cruveiller, C. Dossat, A. Lajus, M. Marchetti, V. Poinsot, Z. Rouy, B. Servin, M. Saad, C. Schenowitz, V. Barbe, J. Batut, C. Medigue, and C. Masson-Boivin. 2008. Genome sequence of the beta-rhizobium Cupriavidus taiwanensis and comparative genomics of rhizobia. Genome Res 18:1472-83.
2. Barham, P. J., A. Keller, E. L. Otun, and P. A. Holmes. 1984. Crystallization and morphology of a bacterial thermoplastic: poly-3-hydroxybutyrate. Journal of Materials Science 19:2781-2794.
3. Chen, G. Q., and Q. Wu. 2005. The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials 26:6565-78.
4. Dewhirst, F. E., C. C. Chien, B. J. Paster, R. L. Ericson, R. P. Orcutt, D. B. Schauer, and J. G. Fox. 1999. Phylogeny of the defined murine microbiota: altered Schaedler flora. Appl Environ Microbiol 65:3287-92.
5. Doi, Y. 1990. Microbial Polyesters. Wiley-VCH, New York.
6. Holmes., P. A. 1988. Biologically produced (R)-3-hydroxyalkanoate polymers and copolymers. In: Bassett D.C. editor. Developments in crystalline polymers. Elsevier, London.
7. Howells, E. R. 1982. Opportunities in biotechnology for the chemical industry. Chem. Ind. 8:508-51.
8. Hyatt, J. W., and I. S. Hyatt. July 1870. U. S. 105338
9. Kahar, P., T. Tsuge, K. Taguchi, Y. Doi. 2004. High yield production of polyhydroxyalkanoates from soybean oil by Ralstonia eutropha and its recombinant strain. Polym. Degrad. Stab. 83:79-86.
10. Lee, S. Y. 1996. Bacterial polyhydroxyalkanoates. Biotechnol Bioeng 49:1-14.
11. Lee, S. Y. 1997. E. coli moves into the plastic age. Nat Biotechnol 15:17-8.
6 9
12. Luengo, J. M., B. Garcia, A. Sandoval, G. Naharro, and E. R. Olivera. 2003. Bioplastics from microorganisms. Curr Opin Microbiol 6:251-60.
13. Maehara, A., Y. Doi, T. Nishiyama, Y. Takagi, S. Ueda, H. Nakano, and T. Yamane. 2001. PhaR, a protein of unknown function conserved among short-chain-length polyhydroxyalkanoic acids producing bacteria, is a DNA-binding protein and represses Paracoccus denitrificans phaP expression in vitro. FEMS Microbiol Lett 200:9-15.
14. Nobes, G. A. R., D. Maysinger, and R. H. Marchessault. 1998. Polyhydroxyalkanoates: Materials for Delivery Systems. Drug Delivery 5:167 - 177.
15. Oeding, V., and H. G. Schlegel. 1973. Beta-ketothiolase from Hydrogenomonas eutropha H16 and its significance in the regulation of poly-beta-hydroxybutyrate metabolism. Biochem J 134:239-48.
16. Potter, M., M. H. Madkour, F. Mayer, and A. Steinbuchel. 2002. Regulation of phasin expression and polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha H16. Microbiology 148:2413-26.
17. Salehizadeh, H., and M. C. Van Loosdrecht. 2004. Production of polyhydroxyalkanoates by mixed culture: recent trends and biotechnological importance. Biotechnol Adv 22:261-79.
18. Slater, S., K. L. Houmiel, M. Tran, T. A. Mitsky, N. B. Taylor, S. R. Padgette, and K. J. Gruys. 1998. Multiple beta-ketothiolases mediate poly(beta-hydroxyalkanoate) copolymer synthesis in Ralstonia eutropha. J Bacteriol 180:1979-87.
19. Spiekermann, P., B. H. Rehm, R. Kalscheuer, D. Baumeister, and A. Steinbuchel. 1999. A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Arch Microbiol 171:73-80.
20. Steinbuchel, A., and Y. Doi. 2005. Biotechnology of Biopolymers: From Synthesis to Patents, vol. 2. WILEY-VCH, Weinheim.
21. Stubbe, J., and J. Tian. 2003. Polyhydroxyalkanoate (PHA) hemeostasis: the role of PHA synthase. Nat Prod Rep 20:445-57.
7 0
22. Timothy R. McDermott, S. M. G. C. P. V. P. H. G. 1989. Carbon metabolism in Bradyrhizobium japonicum bacteroids. FEMS Microbiology Letters 63:327-340.
23. Valentin, H. E., D. L. Broyles, L. A. Casagrande, S. M. Colburn, W. L. Creely, P. A. DeLaquil, H. M. Felton, K. A. Gonzalez, K. L. Houmiel, K. Lutke, D. A. Mahadeo, T. A. Mitsky, S. R. Padgette, S. E. Reiser, S. Slater, D. M. Stark, R. T. Stock, D. A. Stone, N. B. Taylor, G. M. Thorne, M. Tran, and K. J. Gruys. 1999. PHA production, from bacteria to plants. Int J Biol Macromol 25:303-6.
24. Vaneechoutte, M., P. Kampfer, T. De Baere, E. Falsen, and G. Verschraegen. 2004. Wautersia gen. nov., a novel genus accommodating the phylogenetic lineage including Ralstonia eutropha and related species, and proposal of Ralstonia [Pseudomonas] syzygii (Roberts et al. 1990) comb. nov. Int J Syst Evol Microbiol 54:317-27.
25. Wieczorek, R., A. Pries, A. Steinbuchel, and F. Mayer. 1995. Analysis of a 24-kilodalton protein associated with the polyhydroxyalkanoic acid granules in Alcaligenes eutrophus. J Bacteriol 177:2425-35.
26. Williams, S. F., D. P. Martin, D. M. Horowitz, and O. P. Peoples. 1999. PHA applications: addressing the price performance issue: I. Tissue engineering. Int J Biol Macromol 25:111-21.
27. Wu, Q., Y. Wang, and G. Q. Chen. 2009. Medical application of microbial biopolyesters polyhydroxyalkanoates. Artif Cells Blood Substit Immobil Biotechnol 37:1-12.
28. York, G. M., J. Stubbe, and A. J. Sinskey. 2002. The Ralstonia eutropha PhaR protein couples synthesis of the PhaP phasin to the presence of polyhydroxybutyrate in cells and promotes polyhydroxybutyrate production. J Bacteriol 184:59-66.
29. 黃晉貴. 2004. 台灣本土根瘤菌Wautersia taiwanensis 184生產聚羥基丁酸醱酵策略之探討. 元智大學.
30. 鍾淑芳. 2004. 以台灣本土根瘤菌生產聚羥基烷酸之研究. 元智大學.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top