Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21903
標題: 丙烯腈分解菌Mesorhizobium sp. F28產生之丙烯腈水合酶的基因選殖、純化與特性分析
Nitrile hydratase from the Mesorhizobium sp.F28:Gene cloning and enzyme purification and characterization
作者: 李正文
CHENG-WEN, LI
關鍵字: nitrile hydratase;腈;nitrile compound;acrylamide;水合酵素;腈;化合物;丙烯醯胺
出版社: 生命科學系
摘要: 
腈化合物(nitrile compounds)被廣泛地用在農化、石化工業中,但所產生含有腈化合物的廢水卻對環境造成了嚴重的污染。有些微生物能攝取腈化物作為碳源,以腈水合酵素(nitrile hydratase)將之代謝降解成如丙烯醯胺(acrylamide)這類具經濟價值的醯胺產物。
丙烯腈分解菌F28是中興大學環工系李季眉教授篩選得到的一株出能以丙烯腈作為碳源並將之代謝產生丙烯醯胺的菌株,已完成此菌之各種特性及培養的實驗分析,本實驗研究採用16S rRNA分子定序法鑑定後發現此菌應歸為Mesorhizobium屬,因此將此菌命名為Mesorhizobium sp. F28。
本實驗研究的主要目的之一就是要選殖Mesorhizobium sp. F28的腈水合酵素基因。參考其它細菌之腈水合酵素基因的保留核甘酸序列設計與合成適當的引子,並使用南方墨點法、菌落雜交法(colony hybridization)、聚合酶鏈鎖反應(PCR)與inverse PCR等實驗技術與方法,本實驗研究選殖並完成全長2597 bp的核甘酸序列。與其他細菌的腈水合酵素經由核甘酸序列比對分析,結果顯示全長核甘酸序列上第1 nt~第485 nt的序列可以轉譯連續161個胺基酸的序列,相當於腈水合酵素β次單元的C端部分;第485 nt~第1103 nt的序列可以轉譯連續206個胺基酸的序列,其中包括可見於其它細菌的腈水合酵素α次單元內作為酵素活化區的C-T-L-C-S-C序列,因為此序列的第二個胺基酸是蘇胺酸,顯示F28的腈水合酵素之活性需要鈷離子作為輔助因子。第1102 nt ~ 第1531 nt的核甘酸序列可以連續轉譯143個胺基酸的序列,比對結果相當於腈水合酵素β次單元的同源蛋白(β-subunit homolog),此外在β次單元的同源蛋白基因下游第1547~第1573 nt的位置可見一個能形成stem- loop的inverted repeat sequence,應該就是腈水合酵素基因串的轉錄終點。
將腈水合酵素從Mesorhizobium sp. F28的細胞粗萃取液中純化出來之後,SDS-PAGE的分析結果預測顯示此酵素是由α和β兩個次單元所組成,其分子量分別為23 kDa與26 kDa,與從核甘酸序列分析結果所預測的大小符合。酵素活性分析顯示此酵素的最適活性溫度為37 ℃,最適pH值為8.0。

Acrylonitrile is wildly manufactured and extensively used by the chemical industry, but are generally toxin because of their cyano group. A nitrile-metabolizing microorganism, Mesorhizobium sp. F28, it utilizes nitrile hydratase( NHase) for conversion of acrylonitrile to acrylamide, followed by hydrolysis to acrylic acid and ammonia utilizing amidase.
In this report, we describe the cloning of a 2597-bp DNA fragment. Analysis of deduced amino acid sequences indicate that the nucleotide 1-485 (161 aa) belongs to incomplete NHase β-subunit (C-terminal portion), the nucleotide 485-1103 (206 aa) belongs to NHase α-subunit and the nucleotide 1102-1531 (143 aa) belongs to NHase β-subunit homolog. The NHase β- and α-subunits comprise a cobalt-containing member of the NHase family, which includes Pseudonocardia thermophila、 Rhodococcus sp. and Bacillus sp. BR449 NHases. A downstream linked gene, ORF-143, which showed a significant similarity with Rhodococcus rhodochrous J1 nnhG and nnlE in their amino acid sequences and gene location. Downstream of the ORF-143, there is a inverted repeat sequence composed of 26 nucleotides which formed a stem-loop structure and may serve as a transcriptional termination signal.
Nitrile hydratase was purified from the crude extract of F28. The enzyme was composed of two kinds of subunits, of which molrcular mass were 26 kDa and 23 kDa. The nitrile hydratase activity was examined at various temperature and pH value. Maximum activity was exhibited at 37℃, pH= 8.0.
URI: http://hdl.handle.net/11455/21903
Appears in Collections:生命科學系所

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