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    Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/6180


    Title: 烷烴氧化菌及氧化酵素之純化與功能性探討;Functional Studies of the Alkane Utilizing Bacteria Pseudomonas oleovorans and its ω- Hydroxylase
    Authors: 張鈞崴;Chun-Wei Chang
    Contributors: 化學研究所
    Keywords: 烷烴氧化酵素;alkane hydroxylase system
    Date: 2006-06-23
    Issue Date: 2009-09-22 10:14:56 (UTC+8)
    Publisher: 國立中央大學圖書館
    Abstract: 細胞膜非血基質二鐵烷類羥化酶 (alkane hydroxylase, alkB)來自Pseudomonas oleovorans可羥基化中等直鏈烷類,蛋白質序列比對和定點突變均顯示alkB和許多真核或原核的膜蛋白desaturase具有多個組胺酸可與活化中心的鐵離子配位。此外,還有兩個酵素rubredoxin-2 和 rubredoxin reductase在細胞質中,負責將電子從NADH傳遞至膜上的烷類羥化酶。 我們成功的從大腸桿菌中表現並且藉由金屬離子親和層析法(metal-affinity chromatography)純化具有His-tag的重組alkane hydroxylase和rubredoxin-2。經由光譜分析得知,所純化的His-tagged rubredoxin-2僅有一個三價鐵離子在C端,可被spinach ferredoxin reductase還原成二價鐵,而此還原過程還造成蛋白質二級結構的改變。在純化具有His-tag的重組alkane hydroxylase時,發現需要比一般His-tag的重組蛋白還要更高濃度的咪唑(imidazole)才可將其從Ni2+ column沖提出來。藉由Ni2+ column純化野生型alkane hydroxylase,我們得知在蛋白質表面與鐵催化中心配位的多個組胺酸是造成His-tag的重組alkB與Ni2+ column會有較高親和力的原因。加入FeSO4之後,野生型與His-tag重組的alkane hydroxylase均可將1,7-octadiene環氧化成1,2-epoxy-7-octene,當spinach ferredoxin reductase、NADPH和自行純化僅有一個鐵離子的His-tag重組rubredoxin-2均存在的活性測試中。這顯示了alkB中與鐵離子配位的多個組胺酸活化中心暴露在蛋白質表面,而藉由金屬離子親和層析法可以簡易的純化野生型膜蛋白alkane hydroxylase。 The integral membrane non-heme diiron alkane hydroxylase (alkB) from Pseudomonas oleovorans is able to hydroxylate medium-chain-length alkane. Primary protein sequence alignment and site-directed mutagenesis demonstrated that alkB and numbers of eukaryotic or prokaryotic integral-membrane desaturase comprise evolutionarily histidine-rich motifs probably coordinating the active site iron center. The other two components in the cytoplasm of the alkane hydroxylase complex, rubredoxin-2 and rubredoxin reductase, are responsible for reducing equivalent electron transfer from NADH to ω-hydroxylase. We successfully expressed and purified the recombinant His-tagged integral-membrane alkane hydroxylase and cytosolic rubredoxin-2 from E. coli lysates by a simple metal-affinity chromatography. On the basis of spectroscopic data, the isolated His-tagged rubredoxin-2 containing only one iron atom located at the C-terminal domain can be reduced by NADPH-dependent spinach ferredoxin reductase and subsequently results in conformational change of secondary structure. During purification, the recombinant His6-tagged ω-hydroxylase required more competing imidazole to elute out from Ni2+ column than the usual His6-tagged protein. After wild type alkane hydroxylase purification by Ni2+ column, we revealed that additional Ni2+ affinity for the recombinant His-tagged alkB come form surface poly-histidine residue motifs of ω-hydroxylase for catalytic iron coordination. In activity assay, both recombinant His-tagged and wild type alkane hydroxylase, after addition of FeSO4, are able to convert 1,7-octadiene to 1,2-epoxy-7-octene in the present of recombinant His-tagged 1Fe-rubredoxin-2, NADPH and spinach ferredoxin reductase. It supports the active site iron coordinating His-rich motifs of alkB are exposed to the protein surface, and simple purification of alkane hydroxylase was achieved by metal-affinity chromatography.
    Appears in Collections:[化學研究所] 博碩士論文

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