研究生: |
陳信中 SHEN-JHONG CHEN |
---|---|
論文名稱: |
利用大腸桿菌生產藍藻蛋白之評估 assessment of cyanophycin production from Escherichia coli |
指導教授: |
曾文祺
Wen-Chi Tseng |
口試委員: |
廖本瑞
Ben-Ruey Liaw 方翠筠 Tsuei-Yun Fang 鄭如忠 Ru-Jong Jeng |
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: | 2009 |
畢業學年度: | 97 |
語文別: | 中文 |
論文頁數: | 92 |
中文關鍵詞: | 藍藻蛋白 、大腸桿菌 |
外文關鍵詞: | Escherichia coli, cyanophycin |
相關次數: | 點閱:396 下載:0 |
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藍藻蛋白(cyanophycin),是一存在於藍藻菌中,作為能量,碳和氮來源的包涵體(inclusion body)。本實驗除了生產藍藻蛋白並分析組成,也對藍藻蛋白的物性做測定並嘗試將藍藻蛋白製成薄膜,並分析其成膜後的特性。
在使用基因重組的大腸桿菌生產藍藻蛋白的過程中,可藉由外加胺基酸來提高產率,產率會由1.3%~1.4% (w/w)提升至3.5%。再使用酸萃取法,溶出堆積在菌體中的藍藻蛋白。最後使用逆向高壓層析法分析其胺基酸組成。
經分析後,藍藻蛋白的胺基酸組成為精胺酸(arginine):天冬門胺酸(aspartic acid)約為1:1,並含有極少量的離胺酸(lysine)。另外,藉由蛋白質電泳,可略估分子量約為24.4kDa~30kDa。
物性測定項目有微差掃瞄熱卡計分析(Differential Scanning Calorimeter)、傅立葉轉換紅外線光譜(Fourier Transform Infrared Spectrometer)分析以及熱重分析(Thermogravimetry Analysis),經測定後可知藍藻蛋白的熔點約為194°C,且裂解溫度為246.9°C。且外加胺基酸並不影響藍藻蛋白的物性。
對於其成膜的物性,測量部份包含接觸角、減弱全反射兩部份。
發現以0.1N HCl作為溶劑的接觸角約為63°,而以0.1N HCl :甲醇=1:1做為溶劑的接觸角為66°,且成膜後官能基不變。
另外,也試著使用外加不同莫爾比率的胺基酸來改變藍藻蛋白的組成,不過藉由DSC和蛋白質電泳分析來看,對藍藻蛋白的組成並沒有影響。
Cyanophycin is a water-insoluble reserve polymer of cyanobacteria, and is a product of nonribosomal peptide synthesis. We used recombinant Escherichia coli to produce cyanophycin, and extracted cyanophycin from the cells by the acid extraction method, and then analyzed its amino acids composition by high performance liquid chromatography.
In order to increase the cyanophycin yield, we supplemented additional amino acids in the medium when induction. After the addition of amino acids, the yield increased from 1.3~1.4% (w/w) to nearly 3.5% (w/w).
The results of high performance liquid chromatography analysis showed that the composition of amino acids is arginine : aspartic acid approximately 1 : 1, and the content of lysine is very low, almost undetectable.
Another aim of this study is to measure the physical properties of cyanophycin. We used differential scanning calorimeter, fourier transform infrared spectrometer and thermogravimetry analysis. We found the melting point of cyanophycin is approximately 196°C, and pyrolysis temperature is 246°C.
We also used cyanophycin to prepare membranes, and used fourier transform infrared spectrometer to analyze its function groups. We found that the spectra of the cyanophycin powder and membrane are almost the same. The contact angle of cyanophycin membrane prepared with 0.1N HCl is 63°, and the contact angle of cyanophycin membrane prepared with 0.1N HCl / methanol is 66°.
We also added amino acids in different ratios. The analysis of differential scanning calorimeter and sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that different ratios had no effect on the structure of chyanophycin.
1. Voss I., Steinbuchel A., Application of a KDPG-aldolase gene-dependent addiction system for enhanced production of cyanophycin in Ralstonia eutropha strain H16.Metablic engineering, 2006. 8(1): 66-78.
2. Mooibroek H., et al., Assessment of technological options and economical feasibility for cyanophycin biopolymer and high-value amino acid production. Applied Microbiology and Biotechnology, 2007. 77(2): 257-267.
3. Garret R.H., Grisham C.M., Biochemistry, 2000. Saunders College Publishers.
4. Moffatt B.A., Studier F.W., Use of Bacteriophage T-7 RNA Polymerase to Direct Selective High-Level Expression of Cloned Genes. Journal of Molecular Biology, 1986. 189(1): 113-130.
5. Aboulmagd E., Sanio F.B.O., Steinbuchel A., 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): 2176-2182.
6. Aboulmagd E., Sanio F.B.O., Steinbuchel A., Molecular characterization of the cyanophycin synthetase from Synechocystis sp strain PCC6308. Archives of Microbiology, 2000. 174(5): 297-306.
7. Hai T., Frey K.M., Steinbuchel A., Engineered cyanophycin synthetase (CphA) from Nostoc ellipsosporum confers enhanced CphA activity and cyanophycin accumulation to Escherichia coli. Applied and environmental microbiology, 2006. 72(12): 7652-7660.
8. Frey K.M., et al., Technical-scale production of cyanophycin with recombinant strains of Escherichia coli. Applied and environmental microbiology, 2002. 68(7): 3377-3384.
9. Ziegler K., et al., 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): 154-159.
10. Conrad U., Polymers from plants to develop biodegradable plastics. Trends In Plant Science, 2005. 10(11): 511-512.
11. Simon R.D., Weathers P., 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): 165-176.
12. Bosch L., Alegria A., Farre R., Application of the 6-aminoquinolyl-N-hydroxysccinimidyl carbamate (AQC) reagent to the RP-HPLC determination of amino acids in infant foods. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 2006. 831(1-2): 176-183.
13. Ziegler F., et al., Plasma amino-acid determinations by reversed-phase HPLC: Improvement of the orthophthalaldehyde method and comparison with ion exchange chromatography. The Journal of automatic chemistry, 1992. 14(4): 145-149.
14. van Eijk H.M., et al., Fully automated liquid-chromatographic determination of amino acids. Clinical chemistry, 1988. 34(12): 2510-2513.
15. Catherine Cooper, Nicolle Packer, and Keith Williams,.Amino Acid Analysis Protocols. 2001.
16. Woo, K.L., Hwang Q.C., Kim H.S., Determination of amino acids in the foods by reversed-phase high-performance liquid chromatography with a new precolumn derivative, butylthiocarbamyl amino acid, compared to the conventional phenylthiocarbamyl derivatives and ion-exchange chromatography. Journal of chromatography. A, 1996. 740(1): 31-40.
17. Bank R.A., et al., Amino acid analysis by reverse-phase high-performance liquid chromatography: improved derivatization and detection conditions with 9-fluorenylmethyl chloroformate. Analytical biochemistry, 1996. 240(2): 167-176.
18. Marquez F.J., et al., Determination of 27 dansyl amino acid derivatives in biological fluids by reversed-phase high-performance liquid chromatography. Journal of chromatography, 1986. 380(2): 275-283.
19. Giuffrida A., et al., The 3-amino-derivative of gamma-cyclodextrin as chiral selector of Dns-amino acids in electrokinetic chromatography." Journal of chromatography. A, 2009. 1216(17): 3678-3686.
20. Le Boucher J., et al., Amino acid determination in biological fluids by automated ion-exchange chromatography: performance of Hitachi L-8500A. Clinical chemistry, 1997. 43(8 Pt 1): 1421-1428.
21. Nakano K., Rischke M., Mrkl H., Influence of acetic acid on the growth of Escherichia coliEscherichia coli K12 during high-cell-density cultivation in a dialysis reactor. Applied Microbiology and Biotechnology . 1997. 48: 597-601