Burkholderia sp. SP4新穎酯解酵素之分離純化及性質分析

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。

關鍵字

鄰苯二甲酸二(2-乙基己基)酯 [di(2-ethylhexyl)phthalate,DEHP] ；伯克氏菌屬； DopA 及 DopB 酯解酵素；降解途徑；中間產物

並列摘要

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.

並列關鍵字

Di(2-ethylhexyl) phthalate (DEHP) ； Burkholderia sp. SP4 ； DopA and DopB esterases ； Degradation pathway ； Intermediates

參考文獻

林明慧。 2014 。環境流布濃度之鄰苯二甲酸二(2-乙基己基)酯對青鱂魚生殖毒性探討。臺灣大學農業化學研究所學位論文，1-100。

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Akita, K., Naitou, C., and Maruyama, K. (2001). Purification and characterization of an esterase from Micrococcus sp. YGJ1 hydrolyzing phthalate esters. Biosci Biotechnol Biochem, 65, 1680-1683.

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Chang, B.V., Yang, C.M., Cheng, C.H., and Yuan, S.Y. (2004). Biodegradation of phthalate esters by two bacteria strains. Chemosphere, 55, 533-538.

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Burkholderia sp. SP4新穎酯解酵素之分離純化及性質分析

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