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研究生:張為超
論文名稱:厭氧菌Achromobactercycloclaste亞硝酸鹽還原酵素的基因選殖蛋白質表現及蛋白質工程之研究
論文名稱(外文):The cloning, expression and protein engineering studies on nitrite reductase of Achromobacter cycloclastes
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:機械工程學研究所
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:1998
畢業學年度:87
語文別:中文
中文關鍵詞:
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純化格蘭氏陰性兼性厭氧菌Achromobacter cycloclastes的染色體DNA,並構築其染色體基因庫。利用已知的含銅亞硝酸鹽還原酵素( copper nitrite reductase, CuNIR )蛋白質序列設計引子,以聚合連鎖反應( PCR )方法合成探針,用以選殖染色體基因庫。經定序得到完全對應於CuNIR基酸的基因序列,經序列分析得知在成熟型的基因產物前具有一段長38個基酸的訊號( signal peptide ),用以導引成熟型的CuNIR進入它的生理活性區域 - periplasmic space。在CuNIR基因上游具有控制蛋白質於厭氧狀態下表現的FNR box;及典型的SD sequence,與起始密碼子ATG共同形成核醣體結合區域。將包含CuNIR基因之表現質體轉殖於大腸桿菌系統,可誘導其表現重組蛋白rNIR,rNIR具有正常的氧化吸收光譜,可被抗NIR血清所辨識,經蛋白酵素enterokinase切割後具有完全的酵素活性。一般的分子篩層析測出NIR分子量約7萬,無法反映正確NIR三原體分子量,本實驗室以Tris-tricine SDS-PAGE分析NIR,成功地區分出單元體、雙元體與三元體的存在,提供了一項簡單而迅速的方式來分析三元體結構。以螯合劑剝奪銅離子,顯示銅的鍵結並非維繫NIR三元體的結構的必要條件;三元體結構對熱不穩定,隨著溫度增加,三元體結構也逐漸瓦解;此外,並構築及表現C端不同缺損程度的重組NIR,以Tris-tricine SDS-PAGE電泳分析觀察到:隨著C端缺損程度的增加,三元體的比例逐漸減少,而單元體比例增加;顯示C端結構對於三元體的穩定極為重要。三元體的構造與酵素活性相關,由C端多8個基酸的突變種雖仍維持三元體結構,但卻失去酵素活性的結果得知:三元體結構為表現酵素活性的必要、而非充份條件。對於一型銅與二型銅間的電子傳遞方式,本實驗室提出Solvent Channel假說,認為NIR分子內的電子傳遞是藉由鍵結於分子間的水分子為橋樑。以定點突變的方法改變影響此通路的基酸,發現當I257位置突變為E257之後,雖然一型銅位置維持完整,但可能因電子傳遞受阻而使其完全失去酵素活性。

The nitrite reductase (NIR) gene has been cloned and characterized from Achromobacter cycloclastes. NIR gene encodes a protein of 378 amino acid residues including a putative signal peptide of 38 residues. The DNA-derived amino acid sequence of NIR is in complete agreement with that from Edman degradation. NIR gene contains its own FNR box in the 5’ upstream region and a TA-rich region which could be the transcription start site. NIR is expressed in E. coli. The recombinant NIR could be recognized by rabbit antisera and demonstrated full enzyme activity.The tris-tricine SDS-PAGE has been used to analyze the quaternary structure of NIR protein. NIR migrates as trimer and monomer in this system while it behaves as dimer in HPLC gel-filtration or tris-glycine SDS-PAGE. The dissociation of trimer to monomer is effected by heating, suggesting that the molecular interaction between monomer is heat-labile. We have constructed and expressed a series of mutants with deletion of a pentapeptide, an undecapeptide, or a heptadecapeptide from the C-terminus of NIR designated as ENIRC-5, ENIRC-11 and ENIRC-17, respectively. A C-terminally extended mutated protein, ENIRC+8, is also produced, which contains an extra octapeptide attached to the C-terminus of the wide-type NIR. ENIRC-5 maintains as trimer and retained 72% of the wile-type NIR. However, ENIRC-11 and ENIRC-17 behave as monomers in the tris-tricine SDS-PAGE and lose all their enzyme activity. Although ENIRC+8 maintains its trimer stucture it is enzymatially inactive. These results clearly indicate that the C-terminal undecapeptide is essential for maintaining the quaternary structure as well as the full enzymatic activity.
We propose a “solvent channel” model for intramolecular electron transfer between type I and type II copper sites. Water molecule bound in NIR protein may be involved in electron transfer. A NIR mutant, I257E, loses its enzyme activity, indicating that the I257 is important in the electron transfer pathway.



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