分離五株雌激素與口服避孕藥降解菌及其轉化產物之初探

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：103

、訪客IP：18.223.172.180

姓名

許永鈞(Yung-chun Hsu) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

分離五株雌激素與口服避孕藥降解菌及其轉化產物之初探
(Isolation and preliminary characterization of five bacterial isolates on the transformation of natural estrogens and an oral contraceptive drug)

相關論文

★ 陰離子界面活性劑sodium dodecylbenzene sulfonate分解菌篩選與脫磺酸酵素研究	★ 鄰苯二酚加氧酵素的熱穩定性提昇研究
★ Triton X-100 分解菌之分離和分解酵素之特性研究	★ Triton X-100加氧酵素之純化與定性
★ Lactobacillus reuteri於酸性與膽鹽環境中之蛋白質體研究	★ 蕃茄根部受銅逆境之基因調控
★ Pseudomonas nitroreducens TX1 異化辛基苯酚聚氧乙基醇之功能性蛋白質體學：以二維電泳法分析等電點4-8之蛋白質表現	★ Pseudomonas nitroreducens TX1之具耗氧活性之麩胺酸合成酶之單離
★ 人類細胞株生產含多種亞型的干擾素-a之蛋白質體學研究	★ 辛基苯酚之分解:分解菌和生物復育之菌相研究
★ 分解辛基苯酚聚氧乙基醇之耗氧酵素（二氫硫辛醯胺脫氫酶）的純化與定性	★ AtNPR1轉殖番茄之性狀分析及抗病機制研究
★ Pseudomonas putida TX2分解辛基苯酚聚氧乙基醇及其具雌激素活性代謝物之研究	★ 以功能性蛋白質體學研究Pseudomonas nitroreducens TX1生長於辛基苯酚聚氧乙基醇之代謝與逆境反應
★ 以功能性蛋白質體學研究Pseudomonas putida TX2生長於辛基苯酚聚氧乙基醇與辛基苯酚之代謝與逆境反應	★ 以功能性基因體學研究細菌異化辛基苯酚聚氧乙基醇及抗逆境之基因

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

至系統瀏覽論文 ( 永不開放)

摘要(中)

環境中的內分泌干擾物自 1990 年代起，已成為一個重要的環境議題。本研究所使用的內分泌干擾物中，雌酮、雌二醇為人類與動物自然產生的激素，乙炔基雌二醇為一種使用在口服避孕藥的合成雌激素。這些內分泌干擾物隨著人類和動物的排泄物直接或間接排出至自然環境中。本研究目的為自豬糞（採樣自桃園養豬場）與活性汙泥（採樣自台北迪化汙水處理廠）中分離出可降解雌激素的降解菌，本研究中，挑選五株降解菌進一步探討其降低雌激素的機制。透過核醣體的測定，此五株降解菌分別為菌株 SH11,SH28, SH101, SH102, SH104。上述分離菌株皆可生長在濃度為 0.003% 與 0.0003% 的乙炔基雌二醇中；除了雌激素外，菌株 SH28 亦能以其他環境荷爾蒙做為唯一碳源生長，如辛基酚與壬基酚。經過高液效層析儀的分析，所有分離菌都將雌二醇轉換成雌酮，菌株SH11、SH28、SH102在濃度10 ppm下，五天可降解70%以上的雌二醇，其中菌株SH11降解率高達90.8%；而菌株SH28 (濃度10 ppm，10天)、SH102 (濃度5 ppm，5天)、SH104 (濃度10 ppm5天)可降解50%的乙炔基雌二醇，其中菌株SH102可降解64.6%的乙炔基雌二醇。在降解雌激素過程中，尚有其他十個未知化合物利用其滯留時間不同而被鑑定出，當中六個化合物比原本更具疏水性。此外，利用聚合酶鏈鎖反應增幅具潛力的環加氧酶與切環酵素基因，結果顯示SH11, SH28具有catecho 1,2-dioxygenase與multiple component phenol hydroxylase 基因。

摘要(英)

Since the early 1990s, the presence of endocrine-disrupting compounds (EDCs) in the environment displaying estrogen-like activities has become a major issue in environmental research and policy. Both natural estrogen, estrone (E1), 17β-estradiol (E2) and synthetic estrogen, 17α-ethinylestraiol (EE2), are excreted in urine and feces of humans and livestocks reach the natural environment through discharge from sewage treatment plants and manure disposal units. The aim of this study is to isolate estrogen-degrading bacteria from pig manure and sewage treatment plant and study their mechanism in the reduced estrogenic activities. Five bacteria were isolated, they are two Gram-positive bacteria,strain SH11 and SH102, three Gram-negative bacteria strain SH28, SH101 and SH104. All isolates can grow in the media containing 0.0003% or 0.003% 17α-ethinylestradiol. In addition to estrogens the strain SH28 could be cultivated on other EDCs, such as octylphenol and nonylphenol. High-performance liquid chromatography analysis of E2 degradation metabolites revealed that all isolates were able to convert C17-OH to C17-keto and produce estrone. Degradation of E2 up to 70% for strain SH11, strain SH28, strain SH102 and EE2 up to 50% for strain SH28, SH102, SH104. The best degradation rate was 90.8% of E2 was degraded by strain11 in 10 ppm at 5 days, 64.6% of EE2 was degraded by strain SH102 in 5 ppm at 5 days. Besides E1, there were ten unknown compounds which they distinguish from retention time during those isolates degradation E1, E2, EE2. Six unknown compounds are more hydrophobic than the original carbon source. The presence of potential degradation genes were preliminarily screened by polymerase chain reaction using the primer sets to amplify specific fragments. The result showed that strain SH11 and strain SH28 have both gene sequences of multiple component phenol hydroxylase and catechol 1, 2- dioxygenase were present in their genomes.

關鍵字(中)

★ 內分泌干擾物
★ 雌酮
★ 雌二醇
★ 乙炔基雌二醇

關鍵字(英)

★ endocrine-disrupting compounds
★ estrone
★ 17β-estradiol
★ 17α-ethinylestraiol

論文目次

Introduction
1. Endocrine disrupting chemicals 1 1.1 Classification of EDCs 2
1.1 Current status of Taiwan 2
2. Estrogen 3
2.1 Estrone 4
2.2 17β-estradiol 4
2.3 Estraiol 5
2.4 17α-ethinylestraiol 5
2.5 Catabolism of estrogens 5
3. Behavior of estrogens in environment
3.1 Sources 6
3.2 Estrogen activity 7
3.3 Exposure 8
4. Degradation of estrogens
4.1 Photodegradation 9
4.2 Ozonation 9
4.3 Adsorption by activated carbon 10
4.4 Fungi and algal 11
4.5 Bacteria 11
5. Research aims 13
6 Study outline 14
Materials and methods
1. Chemicals 15
2. Bacterial isolation, culture conditions and characteristic
2.1 Isolation 15
2.2 Culture media 15
3. Enrichment 16
4. Screening condition and cultivation 17
5. Identification 17
5.1 16S rRNA gene sequencing and phylogenetic analysis 17
5.2 Observation of bacteria 18
5.3 Bacterial growth curve 18
6. Identification of metabolites from the biodegradation of estrogens
6.1 Extraction 19
6.2 HPLC determination 19
6.3 Calibration curve 20
6.4 The recovery of estrogens 20
6.5 Quantification 20
7. Degradation gene analysis
7.1 The primer for general detection 21
7.2 DNA extraction and PCR amplification 21
8. Yeast estrogenic screening (YES) assay 22

Result
1. The bacteria were isolated from estrogens and 4-tert-octylphenol 23
2. The growth of isolates from pig manure and sewage treatment plant 23
3. The growth curve of bacteria 23
4. Identification and characteristic of bacteria
4.1 Identification of 16S rRNA gene sequencing 24
4.2 Gram stain and morphology 24
4.3 The growth curve of the selected bacteria 25
5. Analysis of degradation metabolites by isolates
5.1 The separated of estrogen by using HPLC 26
5.2 Standard curve, quantification and recovery 27
6. Preliminarily analysis of degradation of estrogens by isolates 27
7. Detection of various catabolic 28
8. Yeast estrogenic screening (YES) assay 28

Discussion 29
Conclusions 37
Reference 38
Figure 44
Table 68
Appendix 83

參考文獻

1. 陳信豪、黃守潔、陳玉盆、闕麗卿、施養志， 2012 利用高效液相層析法分析化粧品中Estradiol、Ethinyl estradiol及Estrone等成分。食品藥物研究年報3，第252-256頁。臺北，臺灣。

2. 胡菽蕓，2011。Methylobacterium radiotolerans SH771 生長於辛基苯酚的特性與代謝物分析。國立中央大學生命科學研究所碩士論文。桃園，臺灣。

3. 李美慧，1999。常見環境荷爾蒙物質及其影響，環境荷爾蒙研討會論文集，臺北生技中心，第4-13 頁。臺北，臺灣。

4. 林怡雯，2009。辛基苯酚聚氧乙基醇分解菌之多樣性及 Pseudomonas sp.
TX1 代謝物分析。桃園，臺灣。

5. Bila, D., A. F. Montalvao, A. Azevedo Dde, and M. Dezotti. "Estrogenic Activity Removal of 17beta-Estradiol by Ozonation and Identification of by-Products." Chemosphere 69, no. 5 (2007): 736-46.

Blanquez, P., and B. Guieysse. "Continuous Biodegradation of 17beta-Estradiol and 17alpha-Ethynylestradiol by Trametes Versicolor." J Hazard Mater 150, no. 2 (2008): 459-62.

6. Chang, H. S., K. H. Choo, B. Lee, and S. J. Choi. "The Methods of Identification, Analysis, and Removal of Endocrine Disrupting Compounds (Edcs) in Water." J Hazard Mater 172, no. 1 (2009): 1-12.

7. Colucci, M. S., and E. Topp. "Persistence of Estrogenic Hormones in Agricultural Soils: Ii. 17alpha-Ethynylestradiol." J Environ Qual 30, no. 6 (2001): 2077-80.

8. Combalbert, S., and G. Hernandez-Raquet. "Occurrence, Fate, and Biodegradation of Estrogens in Sewage and Manure." Appl Microbiol Biotechnol 86, no. 6 (2010): 1671-92.

9. Della Greca, M., G. Pinto, P. Pistillo, A. Pollio, L. Previtera, and F. Temussi. "Biotransformation of Ethinylestradiol by Microalgae." Chemosphere 70, no. 11 (2008): 2047-53.

10. Diamanti-Kandarakis, E., J. P. Bourguignon, L. C. Giudice, R. Hauser, G. S. Prins, A. M. Soto, R. T. Zoeller, and A. C. Gore. "Endocrine-Disrupting Chemicals: An Endocrine Society Scientific Statement." Endocr Rev 30, no. 4 (2009): 293-342.

11. DiDiego, M. L., J. A. Eggert, R. H. Pruitt, and L. L. Larcom. "Unmasking the Truth Behind Endocrine Disruptors." Nurse Pract 30, no. 10 (2005): 54-9.

12. Dray, J., T. F. Dray, and A. Ullmann. "[Hydrolysis of Urinary Metabolites of Different Steroid Hormones by -Glucuronidase from Escherichia Coli]." Ann Inst Pasteur (Paris) 123, no. 6 (1972): 853-7.

13. Drzyzga, O., L. Fernandez de las Heras, V. Morales, J. M. Navarro Llorens, and J. Perera. "Cholesterol Degradation by Gordonia Cholesterolivorans." Appl Environ Microbiol 77, no. 14 (2011): 4802-10.

14. Haiyan, R., J. Shulan, N. ud din Ahmad, W. Dao, and C. Chengwu. "Degradation Characteristics and Metabolic Pathway of 17alpha-Ethynylestradiol by Sphingobacterium Sp. Jcr5." Chemosphere 66, no. 2 (2007): 340-6.

15. Ifelebuegu, A. O., J. N. Lester, J. Churchley, and E. Cartmell. "Removal of an Endocrine Disrupting Chemical (17alpha-Ethinyloestradiol) from Wastewater Effluent by Activated Carbon Adsorption: Effects of Activated Carbon Type and Competitive Adsorption." Environ Technol 27, no. 12 (2006): 1343-9.

16. Ke, J., W. Zhuang, K. Y. Gin, M. Reinhard, L. T. Hoon, and J. H. Tay. "Characterization of Estrogen-Degrading Bacteria Isolated from an Artificial Sandy Aquifer with Ultrafiltered Secondary Effluent as the Medium." Appl Microbiol Biotechnol 75, no. 5 (2007): 1163-71.

17. Kuch, H. M., and K. Ballschmiter. "Determination of Endogenous and Exogenous Estrogens in Effluents from Sewage Treatment Plants at the Ng/L-Level." Fresenius J Anal Chem 366, no. 4 (2000): 392-5.

18. Kuch, H. M., and K. Ballschmiter. "Determination of Endocrine-Disrupting Phenolic Compounds and Estrogens in Surface and Drinking Water by Hrgc-(Nci)-Ms in the Picogram Per Liter Range." Environ Sci Technol 35, no. 15 (2001): 3201-6.

19. Kurisu, F., M. Ogura, S. Saitoh, A. Yamazoe, and O. Yagi. "Degradation of Natural Estrogen and Identification of the Metabolites Produced by Soil Isolates of Rhodococcus Sp. And Sphingomonas Sp." J Biosci Bioeng 109, no. 6 (2010): 576-82.

20. Lai, K. M., M. D. Scrimshaw, and J. N. Lester. "Biotransformation and Bioconcentration of Steroid Estrogens by Chlorella Vulgaris." Appl Environ Microbiol 68, no. 2 (2002): 859-64.

21. Lamb, D. C., D. E. Kelly, S. Masaphy, G. L. Jones, and S. L. Kelly. "Engineering of Heterologous Cytochrome P450 in Acinetobacter Sp.: Application for Pollutant Degradation." Biochem Biophys Res Commun 276, no. 2 (2000): 797-802.

22. Liu, Z. H., Y. Kanjo, and S. Mizutani. "Removal Mechanisms for Endocrine Disrupting Compounds (Edcs) in Wastewater Treatment - Physical Means, Biodegradation, and Chemical Advanced Oxidation: A Review." Sci Total Environ 407, no. 2 (2009): 731-48.

23. MacNaughton, S. J., J. R. Stephen, A. D. Venosa, G. A. Davis, Y. J. Chang, and D. C. White. "Microbial Population Changes During Bioremediation of an Experimental Oil Spill." Appl Environ Microbiol 65, no. 8 (1999): 3566-74.

24. Maniero, M. G., D. M. Bila, and M. Dezotti. "Degradation and Estrogenic Activity Removal of 17beta-Estradiol and 17alpha-Ethinylestradiol by Ozonation and O3/H2o2." Sci Total Environ 407, no. 1 (2008): 105-15.

25. Ohko, Y., K. Iuchi, C. Niwa, T. Tatsuma, T. Nakashima, T. Iguchi, Y. Kubota, and A. Fujishima. "17 Beta-Estradiol Degradation by Tio2 Photocatalysis as a Means of Reducing Estrogenic Activity." Environ Sci Technol 36, no. 19 (2002): 4175-81.

26. Pauwels, B., K. Wille, H. Noppe, H. De Brabander, T. Van de Wiele, W. Verstraete, and N. Boon. "17alpha-Ethinylestradiol Cometabolism by Bacteria Degrading Estrone, 17beta-Estradiol and Estriol." Biodegradation 19, no. 5 (2008): 683-93.

27. Pessione, E., F. Bosco, V. Specchia, and C. Giunta. "Acinetobacter Radioresistens Metabolizing Aromatic Compounds. 1. Optimization of the Operative Conditions for Phenol Degradation." Microbios 88, no. 357 (1996): 213-21.

28. Pillon, A., N. Servant, F. Vignon, P. Balaguer, and J. C. Nicolas. "In Vivo Bioluminescence Imaging to Evaluate Estrogenic Activities of Endocrine Disrupters." Anal Biochem 340, no. 2 (2005): 295-302.

29. Rudder, Jd, T. V. Wiele, W. Dhooge, F. Comhaire, and W. Verstraete. "Advanced Water Treatment with Manganese Oxide for the Removal of 17alpha-Ethynylestradiol (Ee2)." Water Res 38, no. 1 (2004): 184-92.

30. Sabirova, J. S., L. F. Cloetens, L. Vanhaecke, I. Forrez, W. Verstraete, and N. Boon. "Manganese-Oxidizing Bacteria Mediate the Degradation of 17alpha-Ethinylestradiol." Microb Biotechnol 1, no. 6 (2008): 507-12.

31. Shi, J. H., Y. Suzuki, B. D. Lee, S. Nakai, and M. Hosomi. "Isolation and Characterization of the Ethynylestradiol-Biodegrading Microorganism Fusarium Proliferatum Strain Hns-1." Water Sci Technol 45, no. 12 (2002): 175-9.

32. Sun, W., S. Li, J. Mai, and J. Ni. "Initial Photocatalytic Degradation Intermediates/Pathways of 17alpha-Ethynylestradiol: Effect of Ph and Methanol." Chemosphere 81, no. 1 (2010): 92-9.

33. Takeo, M., M. Nishimura, H. Takahashi, C. Kitamura, D. Kato, and S. Negoro. "Purification and Characterization of Alkylcatechol 2,3-Dioxygenase from Butylphenol Degradation Pathway of Pseudomonas Putida Mt4." J Biosci Bioeng 104, no. 4 (2007): 309-14.

34. Tanenbaum, D. M., Y. Wang, S. P. Williams, and P. B. Sigler. "Crystallographic Comparison of the Estrogen and Progesterone Receptor′s Ligand Binding Domains." Proc Natl Acad Sci U S A 95, no. 11 (1998): 5998-6003.

35. Ternes, T. A., J. Stuber, N. Herrmann, D. McDowell, A. Ried, M. Kampmann, and B. Teiser. "Ozonation: A Tool for Removal of Pharmaceuticals, Contrast Media and Musk Fragrances from Wastewater?" Water Res 37, no. 8 (2003): 1976-82.

36. Tuan, N. N., H. C. Hsieh, Y. W. Lin, and S. L. Huang. "Analysis of Bacterial Degradation Pathways for Long-Chain Alkylphenols Involving Phenol Hydroxylase, Alkylphenol Monooxygenase and Catechol Dioxygenase Genes." Bioresour Technol 102, no. 5 (2011): 4232-40.

37. Tuan, N. N., Y. W. Lin, and S. L. Huang. "Catabolism of 4-Alkylphenols by Acinetobacter Sp. Op5: Genetic Organization of the Oph Gene Cluster and Characterization of Alkylcatechol 2, 3-Dioxygenase." Bioresour Technol 131 (2013): 420-8.

38. Velicu, M., and R. Suri. "Presence of Steroid Hormones and Antibiotics in Surface Water of Agricultural, Suburban and Mixed-Use Areas." Environ Monit Assess 154, no. 1-4 (2009): 349-59.

39. Watanabe, K. "Microorganisms Relevant to Bioremediation." Curr Opin Biotechnol 12, no. 3 (2001): 237-41.

40. White, R., S. Jobling, S. A. Hoare, J. P. Sumpter, and M. G. Parker. "Environmentally Persistent Alkylphenolic Compounds Are Estrogenic." Endocrinology 135, no. 1 (1994): 175-82.

41. Wise, A., K. O′Brien, and T. Woodruff. "Are Oral Contraceptives a Significant Contributor to the Estrogenicity of Drinking Water?" Environ Sci Technol 45, no. 1 (2011): 51-60.

42. Yu, C. P., R. A. Deeb, and K. H. Chu. "Microbial Degradation of Steroidal Estrogens." Chemosphere 91, no. 9 (2013): 1225-35.

43. Yu, C. P., H. Roh, and K. H. Chu. "17beta-Estradiol-Degrading Bacteria Isolated from Activated Sludge." Environ Sci Technol 41, no. 2 (2007): 486-92.

44. Zhang, H., D. Cui, B. Wang, Y. H. Han, P. Balimane, Z. Yang, M. Sinz, and A. D. Rodrigues. "Pharmacokinetic Drug Interactions Involving 17alpha-Ethinylestradiol: A New Look at an Old Drug." Clin Pharmacokinet 46, no. 2 (2007): 133-57.

45. Zuo, Y., K. Zhang, and S. Zhou. "Determination of Estrogenic Steroids and Microbial and Photochemical Degradation of 17alpha-Ethinylestradiol (Ee2) in Lake Surface Water, a Case Study." Environ Sci Process Impacts 15, no. 8 (2013): 1529-35.

指導教授

黃雪莉(Shir-ly Huang)

審核日期

2014-8-21

推文