淡江大學覺生紀念圖書館 (TKU Library)
進階搜尋


下載電子全文限經由淡江IP使用) 
系統識別號 U0002-2607201014204600
中文論文名稱 Serratia marcescens TKU011 生產 靈菌紅素之條件與定性
英文論文名稱 Production and characterization of prodigiosins from Serratia marcescens TKU011
校院名稱 淡江大學
系所名稱(中) 生命科學研究所碩士班
系所名稱(英) Graduate Institute of Life Sciences
學年度 98
學期 2
出版年 99
研究生中文姓名 王振宇
研究生英文姓名 Chen-Yu Wang
學號 697180262
學位類別 碩士
語文別 中文
口試日期 2010-07-09
論文頁數 50頁
口試委員 指導教授-王三郎
委員-陳佑汲
委員-梁慈雯
中文關鍵字 沙雷氏菌  烏賊軟骨粉末  靈菌紅素 
英文關鍵字 Serratia marcescens  squid pen powder  prodigiosin 
學科別分類 學科別醫學與生命科學生物學
中文摘要 Serratia marcescens TKU011 係一株以蝦頭殼粉末作為唯一碳/氮源
之蛋白酶及幾丁聚醣酶生產菌。本研究發現以烏賊軟骨粉末(SPP)作
為碳/氮源時具有可生產紅色色素-靈菌紅素(prodigiosin)的能力,菌株
醱酵產生之紅色色素經分離純化後,藉由1H-NMR 及ESI-MS 確認合成
之靈菌紅素屬於prodigiosin。TKU011 生產靈菌紅素之較適培養為含有
1.5%烏賊軟骨粉末、0.1% K2HPO4 及0.05% MgSO4〃7H2O 之50 mL 液
態培養基(pH 7~9)於30℃振盪培養一天後接著移到25℃繼續培養兩
天,靈菌紅素產量可達0.978 mg/mL。醱酵上清液經試驗推測亦含有脂
胜肽類生物界面活性劑沙雷濕潤素(serrawettin),表面張力值隨著靈菌
紅素的產量增加而降低。
英文摘要 A protease- and chitosanase-producing strain, Serratia marcescens TKU011, was cultivated by using shrimp-head powder(SHP)as the sole carbon/nitrogen source. In this study, a prodigiosin-like pigment was produced from TKU011 by using squid pen powder(SPP)as the sole carbon/nitrogen source. prodigiosin(PG)is one of prodigiosin-like pigments produced by S.marcescens TKU011 and was purified and characterized by1H-NMR and ESI-MS to determine its chemical structure and molecular weight. The optimized conditions for prodigiosin production were cells used to inoculate in 50mL of medium containing 1.5% SPP, 0.1% K2HPO4, 0.05% MgSO4.7H2O(pH7~9)shaken at 30℃ for one day and then cultivated at 25℃ for two days. The production of PG reached 0.978 mg/mL. Besides, TKU011 culture supernatant also contained the lipopeptide biosurfactant(serrawettin), and the production of serrawettin increased with PG production.
論文目次 中文摘要 I
英文摘要 II
目錄 III
圖目錄 VII
表目錄 VIII
第一章 緒論.......................................1
第二章 文獻整理...................................2
2.1 Serratia marcescens 之分布與起源..............2
2.2 Serratia marcescens 之基本生化學特性..........2
2.3 Serratia marcescens 之應用....................3
2.3.1 幾丁質酶 (chitinase) 之生產.................3
2.3.2 蛋白酶之生產 ................................4
2.3.3 色素之生產..................................4
2.4 靈菌紅素之化學結構............................5
2.5 Prodigiosin 之一般特性........................7
2.6 Prodigiosin 在細胞中之位置....................7
2.7 Prodigiosin 之培養條件與生產研究..............7
2.7.1 培養條件對 prodigiosin 生成之影響...........7
2.7.2. 大量生產 prodigiosin 之研究...............10
2.8 Prodigiosin之應用............................11
2.8.1 Prodigiosin之抗癌效果......................11
2.8.2 抗生素.....................................11
2.8.3 免疫抑制劑.................................12
2.8.4 染料.......................................12
第三章 材料與方法................................13
3.1 實驗架構.....................................13
3.2 實驗菌株.....................................14
3.3 實驗材料.....................................14
3.4 實驗儀器.....................................15
3.5 碳氮源種類篩選...............................15
3.6 Prodigiosin 之定量..........................16
3.7 Prodigiosin 產量之計算方式..................16
3.8 Prodigiosin 之純化..........................17
3.9 較適培養條件探討.............................18
3.9.1 培養基酸鹼值...............................18
3.9.2 碳/氮源濃度的影響..........................18
3.9.3 培養體積...................................18
3.9.4 培養溫度...................................18
3.9.5 較適培養時間...............................19
3.10 微生物生長之分析............................19
3.11 表面張力測量................................19
第四章 結果與討論................................20
4.1 培養條件.....................................20
4.1.1 碳/氮源之選擇..............................20
4.1.2 各種水產廢棄物作為培養基對於靈菌紅素產量的影響...20
4.2 靈菌紅素之純化分離...........................22
4.3 以烏賊軟骨粉末作為培養基探討其他培養條件.....26
4.3.1 pH值之影響.................................26
4.3.2 碳氮源濃度之影響...........................28
4.3.3培養體積之影響..............................30
4.3.4 溫度之影響.................................31
4.4 靈菌紅素在不同pH值下的吸光值.................33
4.5 靈菌紅素在菌體內與上清液的比例比較...........34
4.6 照光與避光培養對於生產靈菌紅素的差異性.......35
4.7 靈菌紅素與表面張力值.........................37
4.8 其他不同碳氮源作為培養基的表面張力值.........38
4.9 其他不同碳氮源作為培養基的靈菌紅素生產量.....38
第五章 結論......................................42
參考文獻.........................................43

圖目錄
頁次
圖3.1. 實驗架構..................................13
圖4.1 TKU011於不同碳氮源培養基生產靈菌紅素之變化情形...21
圖4.2 prodigiosin之1H-NMR 的圖譜.................23
圖4.3 prodigiosin之13C-NMR 的圖譜................24
圖4.4 prodigiosin之ESI-MASS 圖譜.................25
圖4.5 TKU011不同pH值下與添加碳酸鈣後.............27
圖4.6 TKU011不同碳源濃度培養基與添加NPB緩衝液後..29
圖4.7 TKU011不同培養液體積生產靈菌紅素之變化情形.30
圖4.8 TKU011不同溫度培養基之
(A)菌體生長(B)生產靈菌紅素之變化情形.........32
圖4.9以OD535測量靈菌紅素在不同的pH值緩衝液之吸光值....33
圖4.10 上清液與沉澱物的靈菌紅素濃度比較.........34
圖4.11 照光與避光對於靈菌紅素生產的變化..........36
圖4.12 表面張力值與靈菌紅素生產濃度的比較........37
圖4.13 TKU011在不同培養基所得之
(A)表面張力值、(B)靈菌紅素濃度...............39

表目錄
頁次
表4.1 TKU011生產靈菌紅素之較適培養條件...........40
表4.2 不同文獻所載的prodigiosin類似物產量比較....41
參考文獻 林芊。2004。由Serratia marcescens 生產prodigiosin 之研究。國立台灣大學農業化學研究所碩士論文。
姚惠芳。2000。由 Serratia marcescens FC-R1 生產紅色色素之研究。國立台灣大學農業化學研究所碩士論文。
Anita, K., Mahnaz, M. A., and Fatemeh, A. F. 2006. Review of prodigiosin, pigmentation in Serratia marcescens. Journal of Biological Sciences, 6:1-13.
Alihosseini, F., Ju, K.S., Lango, J., Hammock , B.D., and Sun, G. 2008. Antibacterial colorants: characterization of prodiginines and their applications on textile materials. Biotechnology Progress, 24:742-747.
Anuradha, V.G., Nandini, A., Geetha, M., and Gautam, P. 2004. A novel medium for the enhanced cell growth and production of prodigiosin from Serratia marcescens isolated from soil. BMC Microbiology, 4:11.
Bunting, M. I. and Ingraham, L. J. 1942. The distribution of color variants in ageing broth cultures of Serratia marcescens #274, Journal of Bacteriology, 43:585-591.
Bunting, M. I., Robinow, C. F., and Bunting, H. 1949. Factors affecting the elaboration of pigment and polysaccharide by Serratia marcescens. Journal of Bacteriology, 58:114-115.
Button, G. L., Miller, M. A., and Tsang, J. C. 1975. Antibiogram and lipid analysis of a pigmented strain of Serratia marcescens and its nonpigmented variants. Antimicrob. Agents Chemother, 7:219-222.
Castro, A. J., Deck, J. F., Hugo, M. T., Williams, L. R., and Zingg, M. R.
1958. Prodigiosin hydrochloride. Journal of Organic Chemistry ,23:1232-1233.
Campàs, C., Dalmau, M., Montaner, B., Barragán, M., Bellosillo, B., Colomer, D., Pons, G., Pérez-Tomás, R., and Gil, J. 2003. Prodigiosin induces apoptosis of Band T cells from B-cell chronic lymphocytic leukemia. Leukemia, 17:746-750.
Cho, L. K. N., Lowe, J. A., Maguire, R. B., and Tsang, J. C. 1987. Relationship of prodigiosin condensing enzyme activity to the biosynthesis of prodigiosin and its precursors in Serratia marcescens. Experientia, 43:397-399.
Clements-Jewery, S. 1976. The reversal of glucose repressed prodigiosin
production in Serratia marcescens by the cyclic 3'5'-adenosine
monophosphate inhibitor theophylline. Experientia, 32:421-533.
Deshpande, M.V. 1986. Enzymatic degradation of chitin & its biological applications. Journal of Scientific and Industrial Research,
45: 273-281.
Díaz-Ruis, C., montaner, B., and Pérez-Tomás, R. 2001. Prodigiosin
induces cell death and morphological changes indicative of apoptosis
in gastric cancer cell line HGT-1. Histology and Histopathology , 16: 415-421.
Ding. M. J. and Williams, R. P. 1983. Biosynthesis of prodigiosin by
white strains of Serratia marcescens isolated from patients. Journal of Clinical Microbiology, 17:476-480.
Eaves, G.N. and Jeffries, C.D. 1963. Isolation and properties of an
exocellular nuclease of Serratia marcescens. Journal of Bacteriology, 85: 273-278.
Füller, A. 2003. Chemistry and biology of reseophilin and the prodigiosin
alkaloids: a survery of the last 2500 years. Angewandte Chemie International Edition, 42:3582-3603.
Grimont, P. A. D. and Grimont, F. 1978. The genus Serratia. Annual Review of Microbiology, 32:221-248.
Grimont, P. A. D., and Grimont, F. 1984. Bergey’s Manual of Systematic
Bacteriology (N. R. Krieg. Ed.). Willams & wilkins, Baltimore, USA.,
1: 477-484.
Han, S. B., Kim, H. M., Kim, Y. H., Lee, C. W., Jang, E., and Son, K. H.
1998. T-cell specific immunosuppression by prodigiosin isolated
from Serratia marcescens. International Journal of Immunopharmacology, 20:1-13.
Hàse, C.C., and Finkelstein, R.A., 1993. Bacterial extracellular zinc-containing metalloproteases. Microbiology Review, 57:823-837.
Heinemann, B., Howard, A. J. and Palocz, H. J. 1970. Influence of dissolved oxygen levels on production of L-asparaginase and prodigiosin by Serratia marcescens. Applied Microbiology , 19:800-804.
Hearn, W. R., Medina-Castro, J. and Elson, M.K. 1968. Colour change of
prodigiosin. Nature, 220:170-171.
Hejazi, A. and Falkiner, F.R. 1997. Serratia marcescens. Journal of Medical Microbiology, 46:903-912.
Hubbard, R. and Rimington, C. 1950. The biosynthesis of prodigiosin, the tripyrrylmethene pigment from Bacillus prodigiosus (Serratia marcescens). Biochemistry Journal, 46:220-225.
Kobayashi, N. and Ichikawa, Y. 1983. Diversity of prodigiosin content in the stationary phase organisms of Serratia marcescens fractioned by Ficoll density gradient centrifugation. Microbiology and Immunology, 27:897-899.
Kobayashi, N. and Ichikawa, Y. 1985. Decrease in respiration activity related to prodigiosin synthesis in Serratia marcescens. Microbiology and Immunology, 29:301-308.
Kamata, K., S. Okamoto, S. Oka, H. Kamata, Agisawa, H.Y., and Hirata, H. 2000. Cycloprodigiosin hydrochloride suppresses tumor necrosis factor (TNF) -induced transcriptional activation by NF-κB. FEBS Letters, 507:74–80.
Kawauchi, K., Shibutani, K., Agisawa, H.Y., Kamata, H., Nakatsuji, S., and Anzai, H. 1997. A possible immunosuppressant, cycloprodigiosin hydrochloride,obtained from Pseudoalteromonas denitrificans. Biochemical and Biophysical Research Communications, 237:543–547.
Matsuyama, T., Murakami, T., Fujita, M., Fujita, S., and Yano, I. 1986.
Extracellular vesicle formation and biosurfactant production by Serratia marcescens. Journal of General Microbiology, 132:865-875.
Matsuyama, T. and Nakagawa,Y. 1996. Bacterial wetting agents working in colonization of bacteria on surface environments. Colloids and Surfaces B: Biointerfaces, 7:207-214.
Minke, R. and Blackwell, J. 1987. The structure of α-chitin. Journal of Molecular Biology, 120:167-181.
Monreal, J. and Reese, E. T. 1969. The chitinase of Serratia marcescens. Canadian Journal of Microbiology, 15:689-696.
Montaner, B. S., Navarro, M. PiquéM. Vilaseca, M. Martinell, E. Giralt, J.Gil, and Pérez-Tomás, R. 2000. Prodigiosin from the supernatant of
Serratia marcescens induces apoptosis in haenatopoietic cancer cell line. British Journal of Pharmacology, 131: 585-593.
Montaner, B. and Pérez-Tomás, R. 2001. Prodigiosin-induced apoptosis in human colon cancer cells. Life Sciences, 68: 2025-2036.
Morita, Y., Kondoh, K., Hasan, Q., Sakaguchi, T., Murakami, Y., Yokoyama, K., Tamiya, E., 1997. Purification and characterization of a cold-active protease from psychrotrophic Serratia marcescens AP3801. Journal of the American Oil Chemists' Society, 74:1377-1383.
O’Rear, J., Alberti, L., and Harshey,R. M. 1992. Mutations that impair
swarming motility in Serratia marcescens 274 include but not limited
to those affecting chemotaxis or flagellar function. Journal of Bacteriology, 174:6125-6137.
Ohnishi, Y., Horinouchi, S. 1996. Extracellular production of a Serratia
marcescens serine protease in Escherichia coli. Bioscience Biotechnology and Biochemistry, 60:1551-1558.
Ohnishi, Y., Beppu, T., Horinouchi, S. 1997. Two genes encoding serine protease homologues in Serratia marcescens and characterization of their products in Escherichia coli. Journal of Biochemistry, 121:902-913.
Patricia, S.M., Nelson, D., and Marcela, H. 2000. Cytotoxicity of prodigiosin and benznidazole on V79 cells. Toxicology Letters, 116: 237–242
Paruchuri, D. K. and Harshey, R. M. 1987. Flagellar variation in Serratia
marcescens in associated with color variation. Journal of Bacteriology, 169:61-65.
Qadri, S. M. H., and Willaims, R. P. 1972. Induction of prodigiosin
biosynthesis after shift-down in temperature of nonproliferating cells
of Serratia marcescens, Applied Microbiology, 23:704-709.
Reid, J. D., and Ogrydziak, D. M. 1981. Chitinase-overproducing mutant
of Serratia marcescens. Applied and Environmental Microbiology
, 41:664-669.
Ricardo, P. T., Beatriz, M., Esther, L., and Vanessa, S. C. 2003. The
prodigiosin, proapoptotic drugs with anticancer properties. Biochemical Pharmacology,66:1447-1452.
Roberts, D.P., McKenna, L.F., Lakshman, D.K., Meyer, S.L.F., Kong, H.,
Souza, J.T., Lydon, J., Baker, C.J., Buyer, J.S., and Chung, S. 2007. Suppression of damping-off of cucumber caused by Pythium ultimum with live cells and extracts of Serratia marcescens N4-5. Soil Biology and Biochemistry, 39:2275–2288.
Solé, M., Rius, N., Francia, A., and Lorén, J. G. 1994. The effect of pH on prodigiosin production by non-proliferation cells of Serratia marcescens. Letters in Applied Microbiology, 19: 341-344.
Solé, M., A. Francia, N. Rius, and Lorén, J. G. 1997. The role of pH in the ‘glucose effect’ on prodigiosin production by non-proliferating cells of Serratia marcescens. Letters in Applied Microbiology, 25: 81-84.
Solé, M., Rius, N., and Lorén, J. G. 2000. Rapid ectracellular acidification induced by glucose metabolism in non-proliferating cells of Serratia marcescens. International Microbiology, 3: 39-43.
Song, M.J., Bae, J., Lee, D.S., Kim, C.H., Kim, J.S., Kim, S.W., and Hong, S.I. 2006. Purification and characterization of prodigiosin produced by integrated bioreactor from Serratia sp. KH-95. Journal of Bioscience and Bioengineering, 101:157-161.
Songia, S., Mortellaro, A., Taverna, S., Fornasiero, C., Scheiber, E. A., and Erba, E. 1997. Characterization of the new immunosuppressive drug undecylprodigiosin in human lymphocytes. Journal of Immunology, 158:3987-3995.
Wang, S.L., Peng, J.H., Liang, T.W., and Liu, K.C. 2008. Purification and characterization of a chitosanase from Serratia marcescens TKU011. Carbohydrate Research, 343:1316-1323.
Wang, X., Tao, J., Wei, D., Shen, Y., and Tong, W. 2004. Development of an adsorption process for the direct separation and purification of prodigiosin from culture broth. Biotechnology and Applied Biochemistry, 40:277-280
Wei, Y.H., Yu, W.J., and Chen, W.C. 2005. Enhanced undecylprodigiosin production from Serratia marcescens SS-1 by medium formulation and amino-acid supplementation. Journal of Bioscience and Bioengineering, 100: 466-471.
Wei, Y.H., and Chen,W.C. 2005. Enhanced production of prodigiosin-like pigment from Serratia marcescens SMΔR by medium improvement and oil-supplementation strategies. Journal of Bioscience and Bioengineering, 99:616-622.
Williams, R. P., Gott, C. L., Qadri, S. M. H. and Scott, R. H.. 1971. Influence of temperature of incubation and type of growth medium on pigmentation in Serratia marcescens. Journal of Bacteriology, 106:438-443.
Williams, R. P. 1973. Biosynthesis of prodigiosin, a secondary metabolite of Serratia marcescens. Applied Microbiology, 25:396-402.
Williamson N. R., Simonsen, H. T., Ahmed, R. A. A., Goldet, G., Slater, H., Woodley, L., Leeper, F. J., and Salmond, G. P. C. 2005. Biosynthesis of the red antibiotic, prodigiosin, in Serratia : identification of a novel 2-methyl-3-n-amylpyrrole (MAP) assembly pathway, definition of the terminal condensing enzyme, and implications for undecylprodigiosin biosynthesis in Streptomyces. Molecular Microbiology, 56: 971–989.
Witney, F. R., Failla, M. L. and Weinberg, E. D. 1977. Phosphate inhibition of secondary metabolism in Serratia marcescens. Applied and Environmental Microbiology, 33:1042-1046.
Yamamoto, D., Kiyozuca, Y., Uemura, Y., Yamamoto, C., Takemoto, H.,and Hirata, H. 2000. Cycloprodigiosin hydrochloride, a H+/Cl- symporter, induces apoptosis in human breast cancer cell lines. Journal of Cancer Research and Clinical Oncology, 126:191–197.
Yu, V. L., 1979. Serratia marcescens. Historical perspective and clinical review. New England Journal of Medicine, 300:887-893.
論文使用權限
  • 同意紙本無償授權給館內讀者為學術之目的重製使用,於2015-07-29公開。
  • 同意授權瀏覽/列印電子全文服務,於2015-07-29起公開。


  • 若您有任何疑問,請與我們聯絡!
    圖書館: 請來電 (02)2621-5656 轉 2281 或 來信