研究生: |
蔡景翔 Ching-hsiang Tsai |
---|---|
論文名稱: |
培養條件對基因重組大腸桿菌生產藍藻蛋白之探討 Effects of growth conditions on cyanophycin production by recombinant escherichia coli. |
指導教授: |
曾文祺
Wen-Chi Tseng |
口試委員: |
陳秀美
Hsiu-Mei Chen 方翠筠 Tsuei-Yun Fang |
學位類別: |
碩士 Master |
系所名稱: |
工程學院 - 化學工程系 Department of Chemical Engineering |
論文出版年: | 2010 |
畢業學年度: | 98 |
語文別: | 中文 |
論文頁數: | 91 |
中文關鍵詞: | 藍藻蛋白 、藍藻蛋白合成酶 、基因重組大腸桿菌 |
外文關鍵詞: | cyanophycin, cyanophycin synthetase, recombinant E. coli |
相關次數: | 點閱:253 下載:0 |
分享至: |
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報 |
藍藻蛋白又稱藻青素是一種非核醣體合成的聚合物,於1887年,被Borzi觀察藍綠藻菌所發現。藍藻蛋白是由藍綠藻菌體內產生的物質,但並非全部的藍綠藻都能產生藍藻蛋白。藍藻蛋白的組成為等莫耳比的精胺酸與天門冬胺酸,可藉由藍藻蛋白合成酶進行聚合反應,此聚合物的排列方式以天門冬胺酸為主要骨架,以精胺酸的α-胺基與天門冬胺酸的β-羧基連接。
本實驗使用含Synechocysis sp. PCC 6803的藍藻蛋白合成酶cphA,選殖入載體並送之大腸桿菌進行藍藻蛋白合成酶的表現,藉由培養大腸桿菌生產藍藻蛋白。由於本實驗使用pET表現系統,於培養過程中加入不同濃度的乳糖與IPTG進行藍藻蛋白生成的誘導,並改變不同溫度培養,探討藍藻蛋白合成酶之表現程度,以找出最佳培養溫度。微生物生產的藍藻蛋白,藉高效液相層析分析其胺基酸組成。
在實驗結果顯示,以0.01 mM IPTG有最佳的誘導效果。在溫度20 ℃下,藍藻蛋白合成酶有明顯表現,並有助於藍藻蛋白合成酶的生成。使用TB培養基進行微生物培養,能獲得較高產量。不可溶性藍藻蛋白的胺基酸組成為50±0.53 %天門冬胺酸、44.23±0.35 %精胺酸與5.77±0.39 %賴胺酸,而可溶性藍藻蛋白的胺基酸為50.24±0.36 %天門冬胺酸、38.27±0.31 %精胺酸與11.49±0.22 %賴胺酸。
綜合上述,將含有藍藻蛋白合成酶基因S. sp. PCC 6803(cphA)之E. coli BL21(DE3)CodonPlus,使用TB培養基在20 ℃、0.01 mM IPTG的培養條件下,能獲得較高藍藻蛋白之產量。
In 1887, cyanophycin is a non-ribosomal protein discovered by Borzi in cyanobacteria. Cyanophycin can be produced inside cyanobacteria. However, not all cyanobacteria are capable of producing cyanophycin. Cyanophycin is protein-like polymer which consists of equimolar amounts of aspartic acid and arginine arranged as a poly-aspartic acid backbone to which arginine residues are linked to the β-carboxyl group of each aspartate by its α-amino group.
This study reports the production of cyanophycin by recombinant Escherichia coli carrying the cphA gene from Synechocysis sp. PCC 6803. The pET expression system was employed to produce cyanophycin. Different concentrations of IPTG and lactose as well as different temperature were used to examine cyanophycin production.
The results showed that 0.01 mM IPTG performs inductive effects most efficiently. At 20 ℃, cyanophycin synthetase appears visibly on the electrophoresis gel. The use of TB medium is a better choice to produce cyanophycin by recombinant E. coli. Insoluble cyanophycin show a composition of aspartic acid, 50±0.53 %; arginine, 44.23±0.35 %; lysine, 5.77±0.39 %. Soluble cyanophycin showed a composition of aspartic acid, 50.24±0.36 %; arginine, 38.27±0.31 %; lysine, 11.49±0.22 %.
In conclusion, production of cyanophycin by recombinant Escherichia coli carrying the cphA gene from Synechocysis sp. PCC 6803 in TB medium at 20 ℃, and 0.01mM of IPTG , give higher production of cyanophycin.
1. Aboulmagd, E., F.B. Oppermann-Sanio, and A. Steinbüchel, Purification of Synechocystis sp. Strain PCC6308 Cyanophycin Synthetase and Its Characterization with Respect to Substrate and Primer Specificity. Applied and Environmental Microbiology, 2001. 67(5): p. 2176-2182.
2. Ziegler, K., Diener, A., Herpin, C., Richter, R., Deutzmann, R., Lockau, W., Molecular characterization of cyanophycin synthetase, the enzyme catalyzing the biosynthesis of the cyanobacterial reserve material multi-L- arginyl-poly-L-aspartate (cyanophycin). European Journal of Biochemistry, 1998. 254(1): p. 154-159.
3. Borzi, A., Le comunicazioni intracellulari delle Nostochinee. Malpighia, 1887. 1: p. 174-203.
4. Simon, R., Inclusion bodies in the cyanobacteria: cyanophycin, polyphosphate, polyhedral bodies. In: Fay P, Baalen C van(eds). The Cyanobacteria,1987: p. 199-225.
5. Simon, R.D. and P. Weathers, Determination of the structure of the novel polypeptide containing aspartic acid and arginine which is found in cyanobacteria. Biochimica et Biophysica Acta, 1976. 420(1): p. 165-176.
6. Simon, R.D.,Weathers, P., The biosynthesis of multi - L - arginyl - poly (L-aspartic acid) in the filamentous cyanobacterium Anabaena cylindrica. Biochimica et Biophysica Acta, 1976. 422(2): p. 407-418.
7. Lawry, N.H. and Simon R.D., The normal and induced occurrence of cyanophycin inclusion bodies in several blue-green algae. Journal of Phycology, 1982. 18: p. 391-399.
8. Allen, M.M., Cyanobacterial cell inclusions. Annual Review of Microbiology, 1984. 38: p. 1-25.
9. Simon, R.D., Measurement of the cyanophycin granule polypeptide contained in the blue green alga Anabaena cylindrica. Journal of Bacteriology, 1973. 114(3): p. 1213-1216.
10. Mackerras, A.H., N.M. De Chazal, and G.D. Smith, Transient accumulations of cyanophycin in Anabaena cylindrica and Synechocystis 6308. Journal of General Microbiology, 1990. 136(10): p. 2057-2065.
11. Dembinska ME, A.M., Cyanophycin granule size variation in Aphanocapsa. Journal of General Microbiology, 1988. 143: p. 295-298.
12. Lang, N.J., The fine structure of blue-green algae. Annual Review of Microbiology, 1968. 22: p. 15-46.
13. Allen, M., Inclusions: cyanophycin. Methods Enzymol, 1988. 167: p. 207-213.
14. Golecki, J.R. and U.R. Heinrich, Ultrastructural and electron spectroscopic analyses of cyanobacteria and bacteria. Journal of Microscopy, 1991. 162(1): p. 147-154.
15. Aboulmagd, E., F.B. Oppermann-Sanio, and A. Steinbüchel, Molecular characterization of the cyanophycin synthetase from Synechocystis sp. strain PCC6308. Archives of Microbiology, 2000. 174(5): p. 297-306.
16. Berg, H., Ziegler, K., Piotukh, K., Baier, K., Lockau, W, Volkmer-Engert, R., Biosynthesis of the cyanobacterial reserve polymer multi - L - arginyl- poly - L - aspartic acid (cyanophycin): mechanism of the cyanophycin synthetase reaction studied with synthetic primers. European Journal of Biochemistry, 2000. 267(17): p. 5561-5570.
17. Hai, T., F.B. Oppermann-Sanio, and A. Steinbuchel, Purification and characterization of cyanophycin and cyanophycin synthetase from the thermophilic Synechococcus sp. MA19. FEMS Microbiology Letters, 1999. 181(2): p. 229-236.
18. Aboulmagd, E., Voss, I., Oppermann-Sanio, F. B., Steinbuchel, A., Heterologous expression of cyanophycin synthetase and cyanophycin synthesis in the industrial relevant bacteria Corynebacterium glutamicum and Ralstonia eutropha and in Pseudomonas putida. Biomacromolecules, 2001. 2(4): p. 1338-1342.
19. Simon, R.D., Cyanophycin Granules from the Blue-Green Alga Anabaena cylindrica: A Reserve Material Consisting of Copolymers of Aspartic Acid and Arginine. Proceedings of the National Academy of Sciences of the United States of America, 1971. 68(2): p. 265-267.
20. Mooibroek, H., Oosterhuis, N., Giuseppin, M., Toonen, M., Franssen, H., Scott, E., Sanders, J., Steinbuchel, A., Assessment of technological options and economical feasibility for cyanophycin biopolymer and high-value amino acid production. Applied Microbiology and Biotechnology, 2007. 77(2): p. 257-267.
21. Howarth, R.W. and J.J. Cole, Molybdenum availability, nitrogen limitation, and phytoplankton growth in natural waters. Science, 1985. 229(4714): p. 653-655.
22. Rodriguez-Lopez, M., M.L. Muñoz Calvo, and J. Gomez-Acebo, The effect of rifamycins in the ultrastructure of Anacystis montana. Journal of Ultrasructure Research, 1971. 36(5-6): p. 595-602.
23. Allen, M.M. and M.A. Hawley, Protein degradation and synthesis of cyanophycin granule polypeptide in Aphanocapsa sp. Journal of Bacteriology, 1983. 154(3): p. 1480-1484.
24. Allen, M.M. and P.J. Weathers, Structure and composition of cyanophycin granules in the cyanobacterium Aphanocapsa 6308. Journal of Bacteriology, 1980. 141(2): p. 959-962.
25. Liotenberg, S., Campbell, D., Rippka, R., Houmard, J., de Marsac, N. T., Effect of the nitrogen source on phycobiliprotein synthesis and cell reserves in a chromatically adapting filamentous cyanobacterium. Microbiology, 1996. 142 (3): p. 611-622.
26. Hannig, G. and S.C. Makrides, Strategies for optimizing heterologous protein expression in Escherichia coli. Trends in Biotechnology, 1998. 16(2): p. 54-60.
27. Frey, K.M., Oppermann-Sanio, F. B., Schmidt, H., Steinbüchel, A., Technical-scale production of cyanophycin with recombinant strains of Escherichia coli. Applied and Environmental Microbiology, 2002. 68(7): p. 3377-3384.
28. Schwamborn, M., Chemical synthesis of polyaspartates: A biodegradable alternative to currently used polycarboxylate homo- and copolymers. Polymer Degradation and Stability, 1998. 59(1-3): p. 39-45.
29. Voss, I. and A. Steinbuchel, Application of a KDPG-aldolase gene-dependent addiction system for enhanced production of cyanophycin in Ralstonia eutropha strain H16. Metabolic Engineering, 2006. 8(1): p. 66-78.
30. Diniz, S.C., I. Voss, and A. Steinbuchel, Optimization of cyanophycin production in recombinant strains of Pseudomonas putida and Ralstonia eutropha employing elementary mode analysis and statistical experimental design. Biotechnology and Bioengineering, 2006. 93(4): p. 698-717.
31. Poirier, Y., Polyhydroxyalknoate synthesis in plants as a tool for biotechnology and basic studies of lipid metabolism. Progress in Lipid Research, 2002. 41(2): p. 131-155.
32. Romano, A., De, Roo, G., Vreugdenhil, D., Jamar, D., Van, der, Plas, L., Witholt, B., Eggink, G., Mooibroek, H., Evidence of medium - chain - length - polyhydroxyalkanoate accumulation in transgenic potatolines expressing the P. oleovorans Pha-C1 polymerase in the cytoplasm. Biochemical Engineering Journal, 2003. 16: p. 135-143.
33. Mooibroek, H., Oosterhuis, N., Giuseppin, M., Toonen, M., Franssen, H., Scott, E., Sanders, J., Steinbuchel, A., Assessment of technological options and economical feasibility for cyanophycin biopolymer and high-value amino acid production. Applied Microbiology and Biotechnology, 2007. 77(2): p. 257-267.
34. Stanford. Moore, D. H. Spackman, , W. H. Stein, Chromatography of Amino Acids on Sulfonated Polystyrene Resins. An Improved System. Analytical Chemistry, 1958. 30: p. 1185-1190.
35. Hamilton, P.B., Ion exchange chromatography of amino acids: A single column, high resolving, fully automatic procedure. Analytical Chemistry, 1963. 35(13): p. 2055-2064.
36. Weigele, M., DeBernardo, S. L., Tengi, J. P., Leimgruber, W., A novel reagent for the fluorometric assay of primary amines. Journal of the American Chemical Society, 1972. 94(16): p. 5927-5928.
37. Hill, D.W., Walters, F. H., Wilson, T. D., Stuart, J. D., High performance liquid chromatographic determination of amino acids in the picomole range. Analytical Chemistry, 1979. 51(8): p. 1338-1341.
38. Roth, M., Fluorescence reaction for amino acids. Analytical Chemistry, 1971. 43: p. 880-882.
39. Fernstrom, M.H. and J.D. Fernstrom, Rapid measurement of free amino acids in serum and CSF using high-performance liquid chromatography. Life Sciences, 1981. 29(20): p. 2119-2130.
40. Koop, D.R., Morgan, E. T., Tarr, G. E., Coon, M. J., Purification and characterization of a unique isozyme of cytochrome P-450 from liver microsomes of ethanol-treated rabbits. Journal of Biological Chemistry, 1982. 257(14): p. 8472-8480.
41. Bidlingmeyer, B.A., S.A. Cohen, and T.L. Tarvin, Rapid analysis of amino acids using pre-column derivatization. Journal of Chromatography - Biomedical Applications, 1984. 336(1): p. 93-104.
42. Steinle, A., K. Bergander, and A. Steinbuchel, Metabolic engineering of Saccharomyces cerevisiae for production of novel cyanophycins with an extended range of constituent amino acids. Applied and Environmental Microbiology, 2009. 75(11): p. 3437-3446.