烷烴氧化菌及氧化酵素之純化與功能性探討

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張鈞崴(Chun-Wei Chang) 查詢紙本館藏

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論文名稱

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)

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摘要(中)

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。

摘要(英)

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.

關鍵字(中)

★ 烷烴氧化酵素

關鍵字(英)

★ alkane hydroxylase system

論文目次

Abstract.......................................................................................................................III
中文摘要.....................................................................................................................IV
Chapter 1: Introduction ..............................................................................................1
1.0 Preface.............................................................................................................1
1.1 Alkane Oxygenase and its Related Enzymes ...............................................3
1.1.1 Rubredoxin and rubredoxin reductase ..................................................3
1.1.2 ω-hydroxylase and related integral-membrane proteins .......................7
1.1.3 Other non-heme soluble diiron metalloproteins .................................12
1.1.3.1 Hemerythrin .............................................................................12
1.1.3.2 Soluble methane monooxygenase............................................13
1.1.3.3 Ribonucleotide reductase .........................................................13
1.1.3.4 Stearoyl-ACPΔ9-desaturase .....................................................14
1.1.4 Models of the non-heme diiron alkane monooxygenase ....................15
1.2 The Reaction Mechanism of Cytochrome P-450.......................................16
1.3 Implication of the Hydroxylation Mechanism for the Particulate
Methane Monooxygenases from Methylococcus capsulatus (Bath) .........18
1.4 Design of the Carbon-Position-Specific Fluorine Enriched Hydrocarbons
as Substrates of the ω-Hydroxylase............................................................19
Chapter 2: Experimental Methods...........................................................................21
2.1 Materials and Instrumentation...................................................................21
2.1.1 Materials .............................................................................................21
2.1.2 Instrumentation ...................................................................................22
2.2 Culturing and Growth of P. oleovorans......................................................23
2.3 Construction of Expression Vectors ...........................................................26
2.3.1 Chromosomal DNA extraction ...........................................................26
2.3.2 Polymerase chain reaction (PCR) .......................................................26
2.3.3 Purification of PCR product................................................................27
2.3.4 Phosphorylation ..................................................................................28
2.3.5 Plasmid extraction...............................................................................28
2.3.6 Vector preparation...............................................................................28
2.3.7 Ligation ...............................................................................................29
2.3.8 Transformation....................................................................................29
2.3.9 Screening of recombinants..................................................................29
2.3.10 Small–scale protein induction...........................................................30
2.4 Overexpression and Purification of Recombinant Rubredoxin-2 ...........30
2.5 Expression and Purification of Recombinant ω-Hydroxylase.................31
2.6 Membrane Preparations from P. oleovorans .............................................32
2.7 Purification of Wild type ω-Hydroxylase ..................................................33
2.7.1 Method 1 .............................................................................................33
2.7.2 Method 2 .............................................................................................33
2.8 In-gel Digestion and Peptide Mass Fingerprinting (PMF).......................33
2.9 Assay of Alkane Hydroxylase Activity........................................................34
2.10 The Synthesis of 2,2-Difluorooctane and 3,3-Difluorooctane ................34
Chatpter 3: Results and Discussion..........................................................................36
3.1 Construction of Expression Vectors ...........................................................36
3.1.1 Chromosomal DNA extraction ...........................................................36
3.1.2 Polymerase chain reaction ..................................................................36
3.1.3 Cloning and protein induction ............................................................38
3.2 Expression of Recombinant Rubredoxin Reductase.................................38
3.3 Expression and Purification of Recombinant Rubredoxin-2...................40
3.4 Spectral Properties of Recombinant His-tagged Rubredoxin-2 ..............44
3.4.1 UV/visible Spectra ..............................................................................44
3.4.2 CD Spectra ..........................................................................................45
3.4.3 EPR spectra.........................................................................................47
3.5 Expression and Purification of Recombinant ω-Hydroxylase.................48
3.6 Purification of Wild Type ω-Hydroxylase from P. oleovorans ................51
3.7 Assay of Alkane Hydroxylase Activity........................................................55
3.8 The Preparation of 2,2-Difluorooctane and 3,3-Difluorooctane as
Substrates of ω-Hydroxylase. ....................................................................57
Chapter 4: Conclusions .............................................................................................60
References:..................................................................................................................62
Appendix.....................................................................................................................66

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指導教授

俞聖法、蔡惠旭
(Steve Sheng-Fa Ya、Hui-Hsu Gavin Tsai)

審核日期

2006-7-21

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