臺灣博碩士論文加值系統

鄰苯二甲酸酯類 (Phthalates esters, PAEs) 常被用作塑膠產品中的塑化劑使用，可增加塑膠聚合物的柔軟性與延展性。已被發現具有内分泌干擾毒性，會隨著塑膠工業的發展而釋放累積在環境中，造成生物體及生態的危害。其中，以鄰苯二甲酸二(2-乙基己基)酯 [di(2-ethylhexyl) phthalate, DEHP] 為全世界使用量最廣泛的塑化劑。在前人的研究中分離到一株能降解 DEHP 的伯克氏菌 (Burkholderia sp. SP4)，但對於其降解基因和酵素方面仍所知甚少。本篇研究自 Burkholderia sp. SP4 基因庫中篩選出降解基因 dopA 及 dopB，將此二基因片段建構於載體pET21a上，轉型至E.coli BL21(DE3) 中表現並通過鎳離子親和性管柱純化出分子量為24 kDa的DopA 及35 kDa的 DopB 酯解酵素。DopA 最適反應溫度及 pH 值分別為 40°C 和 7，而 DopB 則為 50°C 和 7 ;在穩定性方面，兩種酯解酵素都在低於20°C 下酵素活性穩定且在 pH 值為 7-8 的範圍間具有良好的 pH 穩定性。DopA 和 DopB 對大部分金屬離子和介面活性劑有很好的耐受性，但 Zn2+、Mn2+ 金屬離子和 SDS、CTAB 等介面活性劑的存在明顯抑制酯解酵素活性。而在受質特異性分析表明相較於長鏈酯質，此酯解酵素偏好水解短鏈酯質 (p-NPC2)。由高效液相層析儀 (HPLC) 分析出在 96 小時內 dopA 之轉殖菌株對於 DEHP (200 mg/L) 分解率約 80 %，而 dopB 轉殖菌株則可達到將近 90 % 的降解率。利用薄層層析法 (TLC) 初步分析 Burkholderia sp. SP4 降解 DEHP 後可能會有鄰苯單甲酸(2-乙基己基)酯 [Mono(2-ethylhexyl) phthalate, MEHP] 和水楊酸 (Salicylic acid) 兩個中間產物的生成。再以氣相層析質譜法 (GC-MS) 確認分解 DEHP 後確實會產生 MEHP 之降解中間產物。本實驗分析 Burkholderia sp. SP4 初步的降解途徑並替酵素的開發和利用提供了參考依據，期望未來有助於清除環境中塑化劑汙染物 DEHP。

In order to increase the flexibility and ductility of plastic polymers, phthalates esters (PAEs) is a very commom group of ester plasticizer used in the plasitic product. PAEs has been identified as am endocrine disrupting chemical and environment accumulating toxicant. In this group of ester, di(2-ethylhexyl) phthalate (DEHP) is the most commonly used. In our previous study, we had isolated a microorganism, Burkholderia sp. SP4, which has the ability to degrade DEHP, but the mechanism of its degradation-related gene and enzyme are still unknown. In this study, dopA and dopB gene from the genomics library of Burkholderia sp. SP4, which are the esterase genes related to the degradation of DEHP were screened out. dopA and dopB were cloned into pET21a vector, and overexpressed in E.coli BL21(DE3). By using Ni-affinity column purification, DopA (24 kDa) and DopB (35 kDa) esterases were successfully purified. The optimum temperature and pH for DopA and DopB activity are 40°C, pH7 and 50°C, pH7 respectively. Both DopA and DopB are stable between pH values 7 and 8 and below 20°C. DopA and DopB have good tolerance against most metal ions and surfactants. But Zn2+, Mn2+, SDS and CTAB have significantly inhibited the esterase activity of DopA and DopB. The substrate specificity of DopA and DopB are specific for short-chain fatty acid such as p-NPC2. From the degradation rate analysis of DEHP by high performance liquid chromatography (HPLC), I have found 80 percent and 90 percent of DEHP (200 mg/L) were degraded by DopA and DopB, respectively, within 96 hours of incubation. Results of thin layer chromatography (TLC) have shown that the intermediates produced by the degradation of DEHP were mono(2-ethylhexyl) phthalate (MEHP) and salicylic acid. The presence of the intermediate MEHP was further comfirmed by gas chromatography–mass spectrometry (GC-MS). In this study, I have identified two esterases produced in Burkholderia sp. SP4. Expectedly, this study might be useful for degradation of environmental pollutant DEHP.

摘要 i
Abstract ii
目錄 iv
表目錄 ix
圖目錄 x
附錄目錄 xii
1.緒論 1
1.1.研究緣起 1
1.2.研究方向和目的 2
2.文獻回顧 3
2.1.環境荷爾蒙 3
2.2.鄰苯二甲酸酯類 (Phthalate esters, PAEs) 5
2.2.1.鄰苯二甲酸酯類特性 5
2.2.2.鄰苯二甲酸酯類之應用 8
2.2.3.鄰苯二甲酸酯類於台灣汙染分佈情形 10
2.3.鄰苯二甲酸二(2-乙基己基)酯 [Di-(2-ethylhexyl) phthalate, DEHP] 12
2.3.1.DEHP 的物化性質 12
2.3.2.人體曝露於 DEHP 的途徑與潛在的健康危害 14
2.4. DEHP 的分解與降解途徑 17
2.5.降解鄰苯二甲酸酯類化合物 (PAEs) 相關的酵素 26
3.材料與方法 27
3.1.實驗器材 28
3.1.1.藥品、酵素及相關套組 28
3.1.2.實驗設備 28
3.1.3.分析及製圖軟體 29
3.2.培養基 30
3.3.分解 DEHP 之菌種來源及篩選降解基因建構轉殖菌株 32
3.3.1.菌株來源及菌種鑑定 32
3.3.2.篩選及分析 Burkholderia sp. SP4 菌株降解 DEHP 之基因 32
3.3.3.抽取染色體 DNA 32
3.3.4.聚合酶連鎖反應 (Polymerase chain reaction, PCR) 33
3.3.5. PCR 產物純化 34
3.3.6. DNA 瓊脂膠體 (Agarose gel) 電泳 34
3.3.7. PCR 產物電泳膠體回收與純化 34
3.3.8.質體 DNA 之製備 34
3.3.9.限制酶截切作用 35
3.3.10.接合反應 (Ligation) 36
3.3.11.轉形作用(Transformation) 36
3.4.DEHP 之降解率分析 (High-performance liquid chromatography, HPLC) 37
3.4.1.樣品配置 37
3.4.2.樣品萃取及前處理 37
3.4.3.高效液相層析儀測定 DEHP 的參數與條件 37
3.4.4.檢量線之建立 38
3.4.5.降解率之計算 38
3.5.蛋白質分析 39
3.5.1.重組蛋白質的誘導表現 39
3.5.2.蛋白質之純化 39
3.5.3.十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (Sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) 40
3.5.4.蛋白質透析 41
3.6.酯解酵素的酶學性質測定 42
3.6.1.酯解酵素活性測定 42
3.6.2.測定溫度對酵素活性及熱穩定性的影響 42
3.6.3.測定 pH 值對酵素活性及穩定性的影響 43
3.6.4.不同金屬離子對酵素活性之影響 43
3.6.5.不同介面活性劑對酵素活性之影響 44
3.6.6.酯解酵素受質特異性測試 44
3.7.以薄層層析法 (Thin-layer chromatography, TLC) 初步分析降解中間產物的生成 45
3.8.以氣相層析質譜法 (Gas chromatography–mass spectrometry, GC-MS) 檢測Burkholderia sp. SP4 產生之 DEHP 降解中間產物 45
4.結果與討論 46
4.1.Burkholderia sp. SP4 降解 DEHP 基因的篩選及分析 46
4.1.1.篩選 DEHP 降解基因以及序列分析 46
4.1.2.降解基因蛋白質序列以及分析 46
4.2.建構並篩選具有降解基因的轉殖菌株及菌株降解 DEHP 之能力測試 47
4.3.轉殖菌株降解 DEHP 分析 47
4.3.1利用高效液相層析法 (High-performance liquid chromatography, HPLC) 分析 dopA 及 dopB 轉殖菌株對 DEHP 之降解情形 47
4.3.2. dopA 轉殖菌株及 dopB 轉殖菌株對於 DEHP 降解曲線及效率之分析 48
4.4.蛋白質分析 49
4.4.1.以蛋白質變性膠體電泳 (SDS-PAGE) 分析酯解酵素誘導表現及純化情形 49
4.4.2.酯解酵素之酶學性質分析 49
4.4.2.1.酯解酵素之受質特異性 50
4.4.2.2.酯解酵素於不同溫度之活性與穩定性測定 51
4.4.2.3.酯解酵素於不同 pH 值之活性與穩定性測定 51
4.4.2.4.不同介面活性劑對酯解酵素活性的影響 52
4.4.2.5.不同金屬離子對酯解酵素活性的影響 53
4.5.分析 Burkholderia sp. SP4 降解 DEHP 之中間產物及降解途徑 54
4.5.1.以薄層層析法 (Thin-layer chromatography, TLC) 初步分析 Burkholderia sp. SP4 降解 DEHP 之產物 54
4.5.2.以氣相層析質譜法 (Gas chromatography–mass spectrometry, GC-MS) 確認 Burkholderia sp. SP4 生成之 DEHP 降解中間產物及分析降解途徑 54
5.綜合討論 57
6.結論 61
7.參考文獻 62
8.圖表集 69

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