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

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許永鈞(Yung-chun Hsu) 查詢紙本館藏

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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)

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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

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指導教授

黃雪莉(Shir-ly Huang)

審核日期

2014-8-21

推文