導引式演化細胞色素P450 BM3做為辛烷次末端之羥基化酵素

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研究生：	盧昱穎 Yu-Ying Lu
論文名稱：	導引式演化細胞色素P450 BM3做為辛烷次末端之羥基化酵素 The Random Mutagenesis Studies of Cytochrome P450 BM3 for Regioselective C-2 Activation of Octane
指導教授：	蔡惠旭 Hui-Hsu Gavin Tsai 俞聖法 Steve, Sheng-Fa Yu
口試委員:
學位類別：	碩士 Master
系所名稱：	理學院 - 化學學系 Department of Chemistry
畢業學年度：	96
語文別：	中文
論文頁數：	252
中文關鍵詞：	細胞色素、羥基化酵素、辛烷、導引式演化
外文關鍵詞：	Octane, Regioselective, Cytochrome P450, Random Mutagenesis
相關次數：	點閱：8 下載：0
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由文獻可知Bacillus megaterium的細胞色素P450 BM3對其天然受質脂酸酸具有高度的專一性。我們成功的以大腸桿菌表現細胞色素P450 BM3，並且將random mutation P450序列以PCR複製載體或序列直接嵌入載體的方法，在不改變P450 reductase序列的情況下，探討三個定點突變(Ala74Gly, Phe87Val, Leu188Gln)與random mutation後P450蛋白質序列改變對辛烷催化反應的影響。
我們也利用iodoform reaction氧化在次末端具有羥基的2-octanol，並以GC或UV-Visible Spectroscopy分析氧化後的產物，建立可靠且快速檢測細菌氧化辛烷次末端碳氫鍵的轉化率的方法。
另一方面，我們也將Pseudomonas putida中參與camphor氧化的細胞色素P450cam (CamC)與電子傳遞的蛋白質Putidaredoxin (CamB)的序列，成功嵌入載體，並以E. coli表現出Putidaredoxin (CamB)蛋白質。

It was known that the Bacillus megaterium Cytochrome P450 BM3 exhibits high substrates specificity to catalyzing its congenital substrates, fatty acids. In this study, cytochrome P450 BM3 proteins were successfully heterologously expressed in Escherichia coli. A mutated Ala74Gly, Phe87Val, and Leu188Gln strain was also validate for octane oxidation. After employing random mutation studies of the P450BM3 encoded sequences, we obtained the other three variants to study their octane activation chemistry without any alteration of P450 reductase encoded sequences.
2-octanol exhibiting a hydroxyl group at the sub-terminal carbon could be further oxidized via the iodoform reaction. The oxidation products were analyzed by GC and UV-visible Spectroscopy. The conversion ratio of C-H bond on the 2nd carbon of octane by bacteria could be quantities facilely and reliably. We anticipate deploying this method for selecting appropriate strains to achieve the C-H activation of sub-terminal hydrocarbon in more specific manner.
In addition, the DNA of Pseudomonas putida Cytochrome P450cam (CamC) and Putidaredoxin (CamB) encoded sequences involved with the functions on camphor hydroxylation and catalytic redox suppliers, respectively, were constructed within the protein expression vectors pET 21a and 22b. Those constructed vectors were successfully transformed into the host, E. coli BL21 (DE3). The Putidaredoxin (CamB) protein could be expressed in E. coli heterlogously in the presence of IPTG.

中文摘要	i
Abstract	ii
謝誌	iv
一、序論	1
1. Bacillus megaterium (巨大芽胞桿菌)	1
1-1. Cytochrome P450 的分類	1
1-1-1. Class I CYPs:	2
1-1-2. Class II:	3
1-1-3. Class III:	3
1-1-4. Class IV	4
1-2. Cytochrome P450 BM3 (CYP101)	6
1-3. Cytochrome P450 BM3的催化機制	7
2. Pseudomonas putida	8
2-1. 樟腦代謝路徑(Camphor metabolism pathway)	10
3. Molecualar cloning原理與方法	10
3-1. 原核細菌之染色體與質體基因抽取之方法	10
3-1-1. 破菌	10
3-1-2. 去除蛋白質	11
3-1-3. 分離沈澱DNA	11
3-2. 基因蛋白質之交互作用(DNA and protein interaction)	11
3-2-1. 基因連接酶(Ligase)	11
3-2-2. 磷酸酶(kinase)	11
3-2-3. 去磷酸酶(phophatase)	12
3-2-4. 限制酶(restriction enzyme)	12
3-2-5. 星號活性(Star activity)	12
3-2-6. DNA聚合酶(DNA Polymerase)	12
3-2-7. 聚合酶鏈鎖反應(Polymerase Chain Reaction，PCR)	13
3-2-8. 引子(Primers)	13
3-2-9. 蛋白質降解酶(Protease, proteinase)	13
3-2-10. 去氧核醣核酸與核醣核酸降解酶(Deoxyribonuclease, DNase and Ribonuclease RNase)	13
3-3. 細菌的生長曲線(Bacteria growth curve)	13
3-3-1. 遲緩期（Lag phase）	14
3-3-2. 對數期(Logarithmic phase)	14
3-3-3. 穩定期（Stationary phase）	14
3-3-4. 衰亡期（Decline phase or death phase）	14
3-4. IPTG誘導大腸桿菌大量表現蛋白質的原理	15
3-4-1. 轉錄調控因數啟動區(Open Reading Frame, ORF)	15
3-4-2. 以IPTG誘導大腸桿菌進行蛋白質大量表現(overexpression)	15
3-5. 重組質體基因的技術	16
3-5-1. P450嵌入pET21a載體切位的設計	16
3-5-2. Putidaredoxin (CamB)嵌入pET21a載體切位的設計	18
3-5-3. P450cam (CamC)嵌入pET22b載體切位的設計	20
3-6. 使用套件(Kit)進行隨機突變與定點突變	21
3-6-1. 使用StratageneTM GeneMorph IITM EZClone Kit進行Random mutation	21
3-6-2. 使用StratageneTM QuickChange IITM XL Site-Directed Mutagenesis Kit進行site-directed mutagenesis	25
3-7. 使用sticky end primers之PCR產物嵌入表現載體之原理	28
3-8. P450酵素進行導引式演化的策略	28
3-9. 降低Inclusion bodies的形成以達全細胞合成的目的	30
4. Idodoform reaction定量次末端碳原子具有羥基(-OH group)之化合物的原理與方法[15, 16]	32
5. 研究目的	35
二、實驗	37
1. 藥品試劑和儀器設備	37
1-1. 藥品試劑	37
1-2. 儀器設備	41
2. 實驗步驟	52
2-1. 第一種隨機突變(Random mutation)之方法	53
2-1-1. Bacillus megaterium (巨大芽胞桿菌)菌種培養及選種	53
2-1-2. 建立表現載體pET21a_P450BM3WT	54
2-1-3. 於E. coli strain BL21(DE3)中大量表現(over expression)P450蛋白質	54
2-1-4. pET21a_P450BM3WT 表現載體之P450基因A74G F87V L188Q定點突變(Site-directed Mutagenesis)	55
2-1-5. E. coli strain pET21a_P450BM3WT DH5alpha菌種培養	55
2-1-6. 錐形瓶放大培養	56
2-1-7. 離心(Centrifugation)收菌	57
2-1-8. 抽取質體基因(Plasmid DNA Extraction)	57
2-1-9. pET21a_P450BM3WT Random Mutation Using GeneMorph IITM EZClone Kit	58
2-1-9-1. 以T7 primer與AseI primer隨機突變P450序列	58
2-1-9-2. 以PCR random mutation P450序列	59
2-1-9-3. T7 primer與AseI primer進行PCR之反應物與反應條件	60
2-1-9-4. 以PCR產物作為mega primers以PCR複製P450外之質體序列成為新的載體pET21a_P450BM3mutant	61
2-1-10. Transformation至宿主細胞E. coli strain DH5alpha	62
2-1-11. P450 random mutation sequence嵌入pET21a_P450BM3WT鑑定	62
2-1-11-1. 以EcoRI確認細菌之質體大小	63
2-1-11-2. 以T7 primer與AseI primer確認質體之P450序列大小	63
2-1-12. 比對定序結果	64
2-2. 第二種隨機突變(Random mutation)之方法	64
2-2-1. 使用GeneMorph IITM EZClone Random Mutagenesis Kit以sticky end primers random mutation P450序列	65
2-2-1-1. 設計PCR所需的二組EcoRI與AseI sticky end primers	65
2-2-1-2. 二組EcoRI與AseI sticky end primers進行PCR之條件	66
2-2-2. 磷酸化PCR產物(Phosphorylation)	68
2-2-3. DNA載體的準備(Vector Double Digests)	68
2-2-3-1. 純化以AseI切一刀pET21a_P450BM3WT之DNA	68
2-2-3-2. 確認純化後載體之大小	71
2-2-3-3. 使用EcoRI切一刀純化後之產物	71
2-2-4. P450mutant嵌入pET21a_P450BM3WT載體	72
2-2-5. Transformation至宿主細胞E. coli strain DH5alpha	73
2-2-6. 抽取細菌Plasmid DNA	74
2-2-7.重組後質體的確認	74
2-2-7-1. 以EcoRI確認P450mutant嵌入pET21a_P450BM3WT後之質體大小	74
2-2-7-2. 以PCR確認嵌入後載體上P450mutant大小	75
2-3. Iodoform reaction顯色測試活性	76
2-3-1. 辛烷及2-辛醇之校正曲線	76
2-3-2. 碘仿反應及其校正曲線	77
2-3-3. 含P450 BM3定點突變之E. coli的辛烷反應及碘仿反應	77
2-4. Pseudomonas putida ATCC 17453 Cytochrome P450cam(CamC) and Putidaredoxin(CamB) overexpression in E. coli	79
2-4-1. Pseudomonas putida ATCC 17453菌種培養及選種	80
2-4-2. Pseudomonas putida ATCC 17453錐形瓶放大培養及偵測菌種growth curve	81
2-4-3. 離心(Centrifugation)收菌	82
2-4-4. 抽取Plasmid DNA	82
2-4-5. 複製P450cam (CamC)與Putidaredoxin (CamB)序列	84
2-3-5-1. Putidaredoxin (CamB)進行PCR的二組EcoRI與XhoI sticky end primers (用於嵌入pET21a載體)	84
2-4-5-2. Putidaredoxin (CamB)二組EcoRI與XhoI sticky end primers PCR之條件	85
2-3-5-3. 設計P450cam (CamC) PCR的一組blunt end primers(用於嵌入TA載體)	87
2-3-5-4. P450cam (CamC)一組blunt end primers進行PCR之條件	88
2-4-5-5. P450cam (CamC) PCR所需的二組NdeI與XhoI sticky end primers(用於嵌入pET22b載體)	89
2-3-5-6. P450cam (CamC)二組NdeI與XhoI sticky end primers PCR之條件	89
2-3-6. 純化PCR產物	91
2-4-7. Phosphorylation DNA序列	91
2-4-7-1. Phosphorylation Putidaredoxin (CamB)序列	91
2-4-7-2. phosphorylation P450cam (CamC)序列	92
2-4-8. pET21a載體的準備(Vector Double Digests)	92
2-4-8-1.  pET22b載體的準備(Vector Double Digests)	93
2-4-9.嵌入(ligation)載體	93
2-4-9-1. Putidaredoxin (CamB)序列嵌入pET21a載體	93
2-4-9-2. P450cam (CamC)序列嵌入pET22b載體	94
2-4-10. Transformation至宿主細胞E. coli strain DH5alpha	94
2-4-10-1. 抽取Putidaredoxin (CamB)嵌入pET21a載體宿主細胞之Plasmid DNA	95
2-4-10-2. 抽取P450cam (CamC)嵌入TA載體宿主細胞之Plasmid DNA	96
2-4-10-3. 抽取P450cam (CamC)嵌入pET22b載體宿主細胞之Plasmid DNA	96
2-4-11. 重組後質體的確認	97
2-4-11-1以XhoI確認pET21a_Putidaredoxin (CamB)之大小	97
2-4-11-2. 以第一組EcoRI與XhoI primer PCR確認pET21a_Putidaredoxin (CamB)上Putidaredoxin (CamB)大小	98
2-4-11-3. 以PCR確認TA_P450cam (CamC)上P450cam (CamC)大小	99
2-4-11-4.以第一組NdeI與XhoI primers PCR確認pET22b_P450cam (CamC)上P450cam (CamC)之大小	99
2-4-12. Transformation至表現宿主細胞E. coli strain BL21(DE3)	99
2-4-13. 異丙基-beta-D-1-硫代半乳糖苷(Isopropyl beta-D-1-thiogalactopyranoside, IPTG) induction表現蛋白質時間、溫度與IPTG濃度測試	100
2-4-13-1. 第一組IPTG誘導Putidaredoxin (CamB)大量表現之條件	100
2-4-13-2. 第二組IPTG誘導Putidaredoxin (CamB)大量表現之條件	102
2-4-14. 破菌(Cell lysis)	103
2-4-15. 不同誘導條件Putidaredoxin (CamB)蛋白質之表現情形 (Protein Assay)	104
2-4-16. 蛋白質電泳(SDS-PAGE)	105
2-4-17. 蛋白質大量表現	107
2-4-18. 大量破菌(Cell lysis)	108
2-4-19. 純化蛋白質Putidaredoxin (CamB)	109
2-4-20. 蛋白質定性	112
三、結果與討論	114
1. 第一種Random mutation之方法	114
【圖3-1】第一種Random mutation方法之流程圖 1-1.  P450 BM3 Random Mutation Using GeneMorph IITM EZClone Kit	114
1-1.  P450 BM3 Random Mutation Using GeneMorph IITM EZClone Kit	115
1-1-1. 以PCR random mutation P450序列	115
1-1-2. 以上一步驟之P450mutant作為mega primers以PCR合成新的pET21a_P450mutant載體	116
1-2.  DNA嵌入表現載體鑑定	117
1-2-1. 以EcoRI確認細菌質體之大小	117
1-2-2. T7與AseI primers PCR確認pET21a_P450mutant載體上P450mutant之大小	118
1-2-3. 比對定序結果	119
1-2-4. Iodoform Reaction顯色測試活性	119
2-1. 二組EcoRI與AseI sticky end primers進行PCR之結果	124
2-1-1. 第一組sticky end primers	125
2-1-2. 第二組sticky end primers	125
2-2. DNA載體的準備(Vector Double Digests)	126
2-2-1. AseI切割pET21a_P450BM3WT並切下純化8.5kbp的band	126
2-2-2. 確認純化後載體大小正確	128
2-2-3. 使用EcoRI將上一步驟之8.5kbp的band再切一刀後純化7kbp的band	129
2-3. 序列嵌入載體之確認	130
2-3-1. 以EcoRI確認細菌之質體大小	130
2-3-2. 第一組EcoRI與AseI primers PCR確認pET21a_P450mutant載體上P450mutant之大小	131
2-4. 定序結果	133
2-5. Pseudomonas putida ATCC 17453 Cytochrome P450cam(CamC) and Putidaredoxin(CamB) overexpression in E. coli	136
2-5-1. 抽取Plasmid DNA	139
2-5-2-1. Putidaredoxin (CamB)序列以嵌入pET21a載體	140
2-5-2-2. 複製P450cam (CamC)序列用以嵌入TA載體	142
2-5-3. Putidaredoxin (CamB)序列嵌入pET21a載體鑑定	144
2-5-3-1. 以XhoI確認pET21a_Putidaredoxin (CamB)之大小	144
2-5-3-2. 第一組EcoRI與XhoI primers PCR確認pET21a_Putidaredoxin (CamB)上Putidaredoxin (CamB)之大小	146
2-5-3-3. PCR確認TA_P450cam (CamC)上P450cam (CamC)之大小	146
2-5-3-4. 以PCR確認pET22b_P450cam (CamC)上P450cam (CamC)之大小	147
2-5-4. 蛋白質分析(Protein assay)	148
2-5-5. Induction溫度及時間對蛋白質Putidaredoxin (CamB)表現影響的分析	149
2-5-5-1. 第一組IPTG誘導Putidaredoxin (CamB)大量表現之條件	151
2-5-5-2. 第二組IPTG誘導Putidaredoxin (CamB)大量表現之條件	155
2-5-6. 純化大量Putidaredoxin (CamB)蛋白質	157
2-5-7. 蛋白質Putidaredoxin (CamB)定性	160
四、結論	163
五、參考文獻	165
六、附錄	167

                                

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