陰離子界面活性劑sodium dodecylbenzene sulfonate分解菌篩選與脫磺酸酵素研究;Isolation of a bacterium degrading sodium dodecylbenzene sulfonate and characterization of desulfonation enzyme

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Title:	陰離子界面活性劑sodium dodecylbenzene sulfonate分解菌篩選與脫磺酸酵素研究;Isolation of a bacterium degrading sodium dodecylbenzene sulfonate and characterization of desulfonation enzyme
Authors:	林奕成;Yi-Chen Lin
Contributors:	生命科學研究所
Keywords:	界面活性劑;生物分解;菌種鑑定;脫磺酸;surfactant;biodegradation;bacterium identifica
Date:	2001-07-20
Issue Date:	2009-09-22 10:16:28 (UTC+8)
Publisher:	國立中央大學圖書館
Abstract:	Sodium dodecylbenzene sulfonate (DBS) 屬於linear alkylbenzene sulfonate (LAS) 類之陰離子性界面活性劑，被廣泛使用於工業、農業和家庭清潔用品中，易於堆積在環境中並對生物體造成毒性。本研究以定量DBS添加入台中典型土樣，置於30°C恆溫並保持溼度，一個月後取出0.5 g土壤於含有0.3 % succinate的sulfate-limited minimal salts培養液中連續繼代培養，經分離後篩選出一株革蘭氏陰性桿菌能以DBS為唯一硫源生長。在鑑定此菌時採用BioLog方法但BioLog革蘭氏陰性菌之資料庫中無相同之菌株；而以其16S rDNA變異區及全長序列皆比對出數株相同度一樣高的菌種，繼而利用與其他菌株之16S rDNA序列分析在演化上親源性最高的菌株，Enterobacter agglomerans（AF157688）為關係最近之一株菌，經1,000次運算之bootstrap值為92，乃命名為Enterobacter sp. SH3。液相層析/質譜儀 (LC/MS) 的定量分析結果顯示此菌在含0.05 % DBS的sulfate-limited minimal salts培養液中，DBS的量隨SH3菌株生長下降至穩定期時已使用了80 %，但沒有分析到代謝產物。此菌可同時利用多種直鏈烷基磺酸鹽及苯環類磺酸鹽為唯一硫源生長，為其一大特殊之處。針對其脫磺酸能力本研究利用5,5’-dithiobis(2-nitrobenzoic acid)和sulfite的呈色反應偵測脫磺酸酵素脫去DBS所生成sulfite之活性。利用此菌細胞粗萃液於10 mM Tris-HCl緩衝液中酵素催化的最適pH值為9.0，並發現外加500 mM NADPH和3 mM FMN於酵素活性反應中時可增加活性1.6倍；目前已知上述之初步生化特性和一個由Escherichia coli EC1250中分離出會對直鏈烷基磺酸鹽進行脫磺酸反應的FMNH2-dependent alkanesulfonate monooxygenase十分類似。經由chromatofocusing和gel filtration管柱層析得知此酵素活性區落在pI值4.12 ~ 5.18，而可能的分子量在60 ~ 150 kDa之間。最終得到的部分純化蛋白質（240 mg），因量少不易測得活性乃利用二維電泳將之分離仍可見到約十多個蛋白質點，這些蛋白質點將以介質輔助雷射揮離離子化飛行時間質譜儀（MALDI-TOF MS）進一步鑑定之，希望未來能分離此在微生物生理和污染物分解應用皆具潛力之酵素。 Linear alkylbenzene sulfonates (LAS) are the most widely used anionic surfactants today. These compounds accumulate in the environment and show toxic effects on organisms in polluted water. This strain utilized sodium dodecylbenzene sulfonate (DBS) as sole source of sulfur in a minimal salts medium containing 0.3 % succinate. The bacterium can’t be identified by BioLog method in Gram-negative database. Further identification by 16S rDNA sequence was performed and showed 98 % identity to many Enterobacter species. Phylogenetic tree was constructed from an alignment of 1,193 nucleotides of 16S rDNA sequences showing the highest phylogenic relationship with Enterobacter agglomerans. And the bacterium was designated Enterobacter sp. SH3. The growth of strain was demonstrated as a function in the decreasing of DBS, which was analyzed by liquid chromatography-mass spectroscopy (LC-MS). It’s specific that Enterobacter. sp. SH3 can utilize several alkanesulfonates and aromatic sulfonates as sole sulfur source to grow. The action of the release of sulfite from DBS was measured using Ellman’s reagent followed by the absorbance increased at 430 nm as an enzyme assay. The highest enzyme activity was observed in the addition of 500 mM NADPH and 3 mM FMN in 0.3 ml 10 mM Tris-HCl, pH 9.0. The preliminary biochemical feature of the enzyme acting in Enterobacter sp. SH3 was showed to be similar to an FMNH2-dependent alkanesulfonate monooxygenase from Escherichia coli EC1250. A series of column chromatography was applied to purify the desulfonation enzyme. The active fraction from chromatofocusing chromatography was at pI of 5.18－4.12. The native molecular weight was at the range of 60－150 kDa by Sephacryl S-200 gel filtration chromatography. This highly purified desulfonation enzyme pool (240 mg) was further separated by 2D-gel electrophoresis and identified by MALDI-TOF
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