Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20224
標題: 丙烯腈分解菌Mesorhizobium sp. F28醯胺酶基因突變株之建構及其腈水合酶基因之異源表現
Construction of amidase gene-defective mutant and heterologous expression of nitrile hydratase gene of acrylonitrile-utilizing bacterium-Mesorhizobium sp. F28
作者: 賀啟智
Ho, Kai-Chi
關鍵字: 丙烯腈丙烯醯胺;acrylonitrile;腈水合酶醯胺酶;acrylamide;nitrile hydratase;amidase
出版社: 生命科學系所
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摘要: 
腈化物 (nitrile) 泛指具有氰基 (-CN group) 的化合物,廣泛的存在於自然環境中,主要由植物產生,也可藉由化學的方式人工合成各式各樣的腈化合物。腈化物被廣泛使用於塑膠石化與農業化學工業中,因此這類工廠在運作的同時往往會產生大量含有腈化物的廢水,但對生物而言大部分的腈化物都有劇毒,可能導致基因突變與具致癌的潛在危險,故此若任由未經妥善處理之含腈化物廢水排放到環境中,會對自然環境及生物造成很大的危害。目前已發現許多自然環境中的微生物具有分解腈化物的能力,其中常見以水解 (hydrolysis) 方式來代謝腈化物能使其毒性降低,具有生物復育(bioremediation) 的意義。菌株Mesorhizobium sp. F28是本校環境工程學系李季眉老師的實驗室從國內某聚丙烯腈 (PAN) 人造纖維製造廠的廢水處理場曝氣槽之活性污泥中分離篩選得到的,經過研究分析後發現其具有可代謝腈化物的腈水合酶 (nitrile hydratase) 與醯胺酶 (amidase) 的酵素系統,在代謝丙烯腈的過程中可產生具高經濟價值的丙烯醯胺 (acrylamide) 中間產物,不過丙烯醯胺卻會因被進一步轉換成丙烯酸 (acrylic acid) 而不能累積。本實驗室先前已經選殖出F28的腈水合酶與醯胺酶的基因,並完成定序,因此本研究進一步採用數種商品化載體並藉由已知基因序列建構出重組質體,嘗試以熱休克轉形作用 (heat shock transformation) 、電穿孔法 (electroporation) 及凍融法 (freeze-thaw method) 等方法,將質體送到F28內,希望藉由同源重組 (homologus recombination) 或轉位作用 (transposition) 的方式進行F28醯胺酶基因突變株的建構,可惜經多次實驗後均未能成功。另一方面,將腈水合酶基因以替換成pET系列質體中之啟動子 (promoter) 或保留原有啟動子兩方式,建構出重組質體並送進各E. coli菌株中作異源表現,在測試重組菌株代謝丙烯腈的情形之後,發現保留原有啟動子的E. coli重組菌株JM110 (pBS-NH) ,丙烯醯胺最大累積濃度可以達到F28的43 %,而且不會有任何副產物丙醯酸的產生,也沒明顯被高濃度丙烯腈抑制的情形,比F28更具應用於代謝丙烯腈,累積丙烯醯胺的潛力。

Nitrile are compounds bearing a cyanide radical (-CN). These compounds, which exist widely in nature environment, are produced by plants in generally and synthesized by chemical technology. Nitriles are widely used in agricultural chemistry and petrochemical plastic industry, so the wastewater from related factories often contains a large amount of nitrile compounds. Because nitrile compounds are known to be mutagenic and carcinogenic, if the wastewater are released without any treatment, it will be harm to natural environment and organism. Many microbes are able to degrade nitrile compounds, they can play a meaningful role in bioremediation. Mesorhizobium sp. F28, a gram negative bacterium, was isolated from activated sludge of the wastewater treatment system of a polyacrylonitrile (PAN) fiber manufacture by Professor Chi Mei Lee’s laboratory (Department of Environmental Engineering, National Chung Hsing University, Taiwan). Dr. Lee’s studies indicated that Mesorhizobium sp. F28 possessed nitrile hydratase (NHase) and amidase, both of which are needed for degrading nitrile compounds. Nitrile hydratase catalyzes the degradation of acrylonitrile to useful economically important acrylamide, which is further converted into less useful acrylic acid by amidase. Our laboratory had cloned and sequenced the NHase and amidase genes of F28. One purpose of this study is to construct an amidase gene-defective mutant of F28 by the strategy of homologus recombination or transposition. So that this mutant can be utilized to produce acrylamide from acrylonitrile or wastewater containing acrylonitrile. Unfortunately, the recombinant plasmids constructed for homologous recombination and pUTmini-Tn5 Tc for transposition could not be transformed into F28 host cells by heat shock transformation, electroporation and freeze-thaw method. On the other hand, the heterologous expression of NHase gene in different E.coli strains were also tested acrylamide. When the NHase gene was cloned with pET expression vectors, the transformants showed very low or no NHase activity, due probably to formation of inclusion body. But when the NHase gene was cloned alony with its promoter, one transformant, JM110 (pBS-NH), showed high enzyme activity in terms of accumulated acrylamide production, about 43 % of what F28 produced. Furthermore, JM110 (pBS-NH) did not produced any acrylic acid as detected by HPLC and its NHase activity was not inhibited by high concentration of acrylonitrile. It is more potential than F28 in metabolizing acrylonitrile to produce acrylamide.
URI: http://hdl.handle.net/11455/20224
其他識別: U0005-1808201209342200
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