臺灣博碩士論文加值系統

在金屬單加氧酵素，如何去進行在不同基質的C-H鍵氧化機制與酵素內部如何調控位置(區域)特異性或立體選擇性，一直都是讓人非常感興趣的議題。巨大芽胞桿菌（Bacillus megaterium）的細胞色素P450，使用大氣中的氧氣當做氧化劑，氧化C12－C20脂肪酸在ω-1，ω-2或ω-3 C-H鍵的位置。我們利用基因工程進行定點突變細胞色素P450 BM3 (Cytochrome P450 BM-3)的實驗，以結合理想化設計與導引式演化的定點突變法，對於P450 BM3的胺基酸序列進行定點突變，嘗試從靠近活性中心周圍不同的氨基酸位置，篩選出扮演關鍵性角色的位置。
本研究中，除了利用3mt (BM3 A74G F87V L188Q)蛋白來研究其催化中長度的烷烴化合物基質外，亦藉由實驗定點突變Ala328Phe （F328），進行不同碳數直鏈烷類篩選，發現位置選擇性會從2- 3- 和4-醇的催化，轉變為偏向2-號位置為主的選擇性。基質選擇性則由長碳鏈脂肪酸（C12-C22）變成具有丁烷催化能力。Leu188Pro（P450 BM3-FP188）可控制氣體分子進入的位置，能增加丁烷轉換成2-丁醇的催化活性12.5%；當我們引入Ala74Glu（P450 BM3-FPE74），可以大幅提升丁烷轉換成2-丁醇的催化活性提升至將近40％。
最後，一系列氘化和氟化的烷烴基質做為探針，並藉由酵素氧化這些烷烴基質的產物深入了解C-H鍵氧化控制機制。針對各式含氘丁烷與含氟辛烷的單加氧之反應性、羥基化的位置選擇性以及產物的掌性分布做歸納，找出其調控的動態基礎。

The control oxidation of aliphatic C-H bonds in regio- and/or stereo-selective manner by metalloenzymes is of great interest to scientists. Cytochrome P450 BM3 from Bacillus megaterium oxidizes C12-C20 fatty acids at the ω-1, ω-2, or ω-3 position of the C?{H bond, respectively. We employ the molecular manipulation techniques to overexpress the recombinant BM3 strain and carry out its site-directed mutagenesis study for generation of a variety of strains towards variable selective oxidation with different substrates.
In my study, strain 3mt, A74G F87V L188Q is exploited for the controlled oxidations of medium-chain length alkanes. Single mutation variant, BM3 mutant Ala328Phe (F328), was found with its capability of selective oxidation at the ω?{1 position of C4-C10 straight-chain alkanes. Interestingly, long-chain fatty acids (C12-C20) are no longer its substrates. It provides a great base to allow us engineering P450 BM3 protein from normal alkanes with medium-chain length such as n-octane gradually to smaller alkane such as n-butane for subsequent studies of controlled oxidation. The introduction of the second mutation, at Leu188Pro (P450 BM3-FP188) can improve the catalytic efficiency from butane to 2-butanol for 12.5%. In addtion, introducinge Ala74Glu (P450 BM3-FPE74) can shrink the substrate pocket down to half of the volume and significantly enhance the catalytic activity of butane to 2- butanol for nearly 40%.
Typically, enzymes invoke host–guest chemistry to sequester the substrates within the protein pockets, exploiting sizes, shapes and specific interactions such as hydrogen-bonding, electrostatic forces and/or van der Waals interactions to control the substrate specificity, regio-specificity and stereo-selectivity. To further investigate this issue, we also develope a series of deuterated and fluorinated aliphatic substrates as probes to gain insights into the controlled C-H oxidations of hydrocarbons facilitated by these enzymes. The effects resulting from the introduction of deuterated butane (isotopomers) and fluorinated octane (Bioisostere) substituents on the mechanistic insights for C-H oxidation and the controls for regio-specificity and stereo-selectivity of the C-H bond activation are discussed.

目錄
中文摘要 1
英文摘要 3
致謝 5
目錄 7
圖目錄 12
表目錄 17
第一章 緒論 19
1-1巨大芽胞桿菌(Bacillus megaterium) 19
1-2 Cytochrome P450 的分類 20
1-2-1 Class I 21
1-2-2 Class II 21
1-2-3 Class III 22
1-2-4 Class IV 22
1-3 Cytochrome P450 BM3 (CYP101) 22
1-4 Cytochrome P450 BM3 的催化機制 23
1-5細胞色素P450的晶體結構 25
1-6 細胞色素P450 BM3的關鍵殘基所扮演的作用 27
1-6-1 Arg47 and Tyr51 27
1-6-2 Phe87 29
1-6-3 Thr268、Glu267 and Ala264 29
1-7設計酵素的方法 30
1-7-1理想化設計 (Rational Design) 30
1-7-2導引式演化 (Directed Evolution) 31
1-7-3結合理想化設計與導引式演化的定點突變法 32
1-8細胞色素P450的羥基化機制 34
1-8-1單加氧酶的羥基化反應之反應機構 34
1-9動力學同位素效應(Kinetic Isotope Effect，KIE) 36
1-9-1 一級同位素效應(Primary Kinetic Isotope Effect) 36
1-9-2 二級同位素效應(Secondary Kinetic Isotope Effect) 37
1-9-3 化學反應中的量子穿隧效應(Quantum Tunneling effect) 39
1-10 研究目的 40
第二章 實驗方法與步驟 43
2-1藥品試劑 43
2-2儀器設備 47
2-3實驗流程 52
2-4實驗步驟 53
2-4-1建立pET21a-p450 BM3的表現載體 53
2-4-2轉殖至表現勝任細胞(transformation) 55
2-4-3 抽取質體DNA 55
2-4-4確認pET21_BM3質體DNA的序列 57
2-4-5 定點突變 (Site-Directed Mutagenesis) 57
2-4-6 誘導表現蛋白質時間及溫度測試 63
2-4-7 超音波破菌(小量) 64
2-4-8 製作SDS-PAGE與蛋白質電泳分析 65
2-4-9 大規模養菌與表現蛋白質 68
2-4-10 高壓均質機 (大量) 69
2-4-11 純化蛋白質P450 BM3 70
2-4-12 利用CO-binding 對細胞色素P450 BM3蛋白質定量 71
2-4-13 利用BSA (Bovine serum albumin)對細胞色素P450 BM3蛋白質定量 72
2-4-14 進行3mt與3mt-F328突變株培養和全細胞（Whole Cell）之辛烷活性測試 74
2-4-15進行P450 BM3-F328、P450 BM3-FP188、P450 BM3-FPE74 突變株培養和全細胞（Whole Cell）之丁烷活性測試 75
2-4-16進行P450 BM3-F328、P450 BM3-FP188、P450 BM3-FPE74 純化後蛋白質與正丁烷的活性測試與產物分析 77
2-4-17衍生化的目的與功用 78
2-4-18衍生化P450 BM3-FP188、P450 BM3-FPE74細菌產物 (具有光學活性的含氘二丁醇) 79
第三章 結合理想化設計與導引式演化的定點突變法 81
3-1- 結合理想化設計與定點突變法之實驗結果 81
3-2 結合理想化設計與定點突變法之結果分析與討論 84
3-3 誘導溫度及時間對蛋白質P450 BM3 表現影響的分析 89
3-4 P450 BM3 蛋白質純化 91
3-5 蛋白質濃度分析 (protein assay) 94
3-6 進行P450 BM3-F328、P450 BM3-FP188、P450 BM3-FPE74 突變株培養和全細胞（Whole Cell）之丁烷活性測試實驗結果 95
3-7進行P450 BM3-F328、P450 BM3-FP188、P450 BM3-FPE74 純化後蛋白質與正丁烷的活性測試與產物分析 97
第四章 定點突變巨大芽孢桿菌細胞色素P450 BM3探討丁烷與含氘丁烷化合物的掌性機制 102
第五章 定點突變巨大芽孢桿菌細胞色素P450 BM3探討烷烴化合物和脂肪酸的次末端氧化機制 120
第六章 結論 135
參考文獻 138
附錄

圖目錄
【圖1-1】細胞色素P450 BM3之SEM圖 19
【圖1-2】細胞色素P450 家族的分類 21
【圖1-3】P450 domain and P450 reductase domain 23
【圖1-4】P450 BM3與脂肪酸的反應方式 23
【圖1-5】Cytochrome P450的催化機制 24
【圖1-6】 細胞色素P450 BM3鐵紫質區域與棕櫚油酸物晶體複合結構 25
【圖1-7】當基質進入BMP的通道，以透明的表面描繪。含鐵紫質輔基、N-palmitoylglycine和水分子都以CPK空間填充顯示表示。這些通道都可以進入大量的水分子存在指定的位置。但為了清楚起見，並不包括透明的表面。（A）不含基質的BMP晶體 （B）BMP/ N-palmitoylglycine複合晶體，基質用淺藍色表示 27
【圖1-8】NPG基質進入通道與結合在P450 BM3的疏水性口袋的活性中心 28
【圖1-9】細胞色素P450 BM3與基質相互作用的模型。（A）靜電作用力指引基質的羧酸基團朝向Arg47，羧酸與Tyr51的羥基之間形成氫鍵與基質更加穩定的結合一起。（B）基質的烷基鏈被吸入到活性中心通道，通道與基質的疏水性相互作用以確保有很強的連結性。（C）最後達到催化能力之方向性，以準備鐵紫質還原和催化循環的開始 28
【圖1-10】（A）I-螺旋的區域中，其中氫鍵網絡大致上都重新分配，這主要是由於Gly265，His266的轉動的重新定位的結果，與Wat202減少。Phe261側鏈的變動是受到Ile219和Phe158影響（B）圖中活性位點顯示（i）基環與其中所述近端的半胱氨酸Cys400配位體的位置，將導致在於鐵紫質平面的含鐵紫質輔基的鐵下沉（ⅱ）軸向水配位體（Wat23）的位移，因為Ala264剩餘氫鍵對'替代'結合位點（ⅲ）Wat202的減少，使它允許對I-螺旋扭結角度從131度收縮到51度（ⅳ）Phe87的扭轉和Leu437側鏈的退後相關聯 30
【圖1-11】細胞色素P450催化的氧化機制 34
【圖1-12】可偵測自由基回復反應機構的快速環丙烷探針 35
【圖1-13】一級同位素效應示意圖 37
【圖1-14】普通二級同位素效應示意圖 38
【圖1-15】逆向二級同位素效應示意圖 38
【圖2-1】Sticky end的方法示意圖 53
【圖2-2】pET21a(+)載體圖譜 54
【圖2-3】pET21a(+)載體中限制酶切點部分放大圖 54
【圖2-4】突變試劑PCR實驗流程示意圖 62
【圖2-5】P450的紫外光譜，氧化-P450還原-P450和CO-P450 71
【圖2-6】標準液OD595 對濃度的檢量線圖 73
【圖2-7】衍生化機制示意圖 79
【圖3-1】定點突變3mt瓊脂凝膠電泳圖 (1% Agar 0.5X TBE) 81
【圖3-2】定點突變3mt-F328瓊脂凝膠電泳圖 (1% Agar 0.5X TBE) 82
【圖3-3】定點突變P450 BM3-F328瓊脂凝膠電泳圖 (1% Agar 0.5X TBE) 82
【圖3-4】定點突變P450 BM3-FP188瓊脂凝膠電泳圖 (1% Agar 0.5X TBE) 83
【圖3-5】定點突變P450 BM3-FPE74瓊脂凝膠電泳圖 (1% Agar 0.5X TBE) 83
【圖3-6】30℃培養，IPTG 誘導蛋白質表現電泳圖 90
【圖3-7】37℃培養，IPTG 誘導蛋白質表現電泳圖 91
【圖3-8】P450 BM3-F328 蛋白質電泳分析圖 92
【圖3-9】P450 BM3-FP188 蛋白質電泳分析圖 93
【圖3-10】P450 BM3-FPE74 蛋白質電泳分析圖 94
【圖3-11】2-丁醇檢量線圖 98
【圖4-1】細胞色素P450 BM3疏水性口袋的基質 N-palmitoylglycine或palmic acid，分別從PDB：(A) 1JPZ and (B) 1FAG by Discovery Studio® 2.0 (Accelerys Inc.) 102
【圖4-2】在細胞色素突變株P450 BM3酵素的催化轉換下，以正丁烷為基質之模擬示意圖：(A)C-HS 朝向鐵紫質輔基的活性中心，產物為S- 形式的2-丁醇 (B) C-HR 朝向鐵紫質輔基的活性中心，產物為R- 形式的2-丁醇。使用軟體 Discovery Studio® 2.0 (Accelerys Inc.) 104
【圖4-3】模擬示意圖呈現了在正丁烷、（2R，3R）- [2-2H1,3-2H1]丁烷、（2R *，3S *）- [2-2H1,3-2H1]丁烷 與（2S，3S）- [2-2H1,3-2H1]丁烷其C-HR 或C-HS和C-DR或C-DS鍵 在P450 MB3活性位點口袋結合的立體化學的平衡之間的關係 109
【圖4-4】細胞色素P450 BM3突變株(P450 BM3-FP188) 其第二個碳原子C-H或C-D鍵的能量示意圖，分別為(A) （2R，3R） - [2 - 2H1,3-2H1] 丁烷和 (B)（2S，3S）- [2 - 2H1,3-2H1] 丁烷，經由活化氧原子通過高價數氧基鐵對於含氘丁烷基質進行羥基化反應。(A) G0HS 與 G0DR代表（2R，3R） - [2 - 2H1,3-2H1] 丁烷基質C-HS與C-DR朝向蛋白質結合活性位點的標準生成自由能的差異?v??w?孱0DS 與 ?孱0HR代表（2S，3S） - [2 - 2H1,3-2H1] 丁烷基質C-DS與C-HR朝向蛋白質結合活性位點的標準生成自由能的差異，?孱≠ROH 或 ?孱≠ROD 表示氧化d,l-[2-2H1,3-2H1]含氘丁烷基質的第二個碳原子C-H或C-D鍵的活化能，一般為??孱≠ = ?{RTln(kH/kD) 115
【圖4-5】 掌性含氘丁烷，（2R，3R）- [2-2H1,3-2H1]丁烷與（2S，3S）- [2-2H1,3-2H1] 丁烷，經細胞色素P450 BM3突變株酵素(FP188)作用後，呈現截然不同的對應物或光學活性過量數值 (ee)，分別為-93% (S)暨56% (R) 117
【圖5-1】 構型I和II的兩種構型為細胞色素P450 BM3 3mt突變株可以有效地將電子從NADPH提取轉移到三價鐵紫質，但是正辛烷氧化為2 -，3 - 和4 - 辛醇的產率相當少。從CE值小於10％來推測，蛋白質構型的轉換形成Cpd I過程太慢，使基質氧化速度也減慢。構型III中的正辛烷因錯位，無法接續著形成Cpd I而進一步被氧化 124
【圖5-2】 藉由細胞色素P450 BM3 3mt 酵素催化氟化辛烷1-4到其相對應的二號位置醇類 126
【圖5-3】從NADPH有效的電子轉移以及氧分子插入C－H鍵ω-1，2，3 或 4位置，可以得知以gem-difluorooctanes 做為基質，能縮緊細胞色素P450 BM3 3mt突變株的鐵紫質口袋 127
【圖5-4】氟化基質 129
【圖5-5】 (A)在細胞色素P450 BM 3的疏水口袋內的遠端水網絡輔助月桂酸（LA）的氧化作用之區域選擇性發生在ω-1，2或3位置。(B)氟化十二烷酸的CF3基團和細胞色素P450 BM3疏水口袋中的腔壁之間的獨特的凡得瓦力相互作用，大幅度促進區域選擇性氧化在ω-3位置 130

表目錄
【表2-1】LB 固態洋菜膠培養皿的成分 55
【表2-2】Plasmid Miniprep Purification Kit 內容物 56
【表2-3】 Quikchange® II XL Site-Directed Mutagenesis Kit 內容物 57
【表2-4】3mt (G74 V87 Q188) 突變引子與相關條件 58
【表2-5】3mt-F328 (G74 V87 Q188 F328) 突變引子與相關條件 59
【表2-6】P450 BM3-F328 (F328) 突變引子與相關條件 59
【表2-7】P450 BM3-F328-P188 (P188 F328) 突變引子與相關條件
59
【表2-8】P450 BM3-F328-P188-E74 (E74 P188 F328) 突變引子與相關條件 60
【表2-9】 突變試劑PCR實驗添加物 60
【表2-10】突變試劑PCR 實驗溫度循環條件設定 61
【表2-11】轉殖至突變試劑之勝任細胞添加物 63
【表2-12】誘導表現蛋白質時間及溫度測試 64
【表2-13】製作SDS-PAGE步驟與添加物 65
【表2-14】純化蛋白質P450 BM3 需要的緩衝溶液 70
【表3-1】兩種蛋白質濃度定量分析之比率差異 95
【表3-2】P450 BM3 WT和三株突變株進行與丁烷催化的全細胞活性測試 96
【表3-3】2-丁醇檢量線 98
【表3-4】純化後蛋白質與正丁烷的活性測試後產物生成量 99
【表3-5】P450 BM3-FPE74 蛋白質純化後與正丁烷基質反應後衍生化的數據分析整理 100
【表4-1】細胞色素P450 BM3突變株（A）P450 BM3-FP188與（B）P450 BM3-FPE74和正丁烷和各式含氘丁烷基質之羥基化產物，經由分析衍生化後具有(S)-形式與(R)-形式之2丁醇酯（(S)- 與(R)-butan-2-ol esters）產物，所推導出的平衡常數（KSS和KRR）和動力學同位素效應 (kH/kD) 110
【表5-1】藉由細胞色素P450 BM3酵素以月桂酸（LA），十五烷酸（PA）和正辛烷（OC）為基質，轉化氟化脂肪酸和辛烷1-10 (如【圖5-5】所示)。從NADPH消耗的周轉頻率（TOF）即(kcat,NADPH,app) 和產物的形成速率(kcat,ROH,app)以及從ES到ES≠狀態中含氟取代基上活化能影響(??孱≠NADPH,app and ??孱≠ROH,app) 去計算表觀速率常數(apparent kcat) 125

1.Gregersen, T.,“Rapid method for distinction of Gram-negative from Gram-positive bacteria”, European Journal of Applied Microbiology and Biotechnology, Vol. 5, No. 2, pp. 123-127 (1978)
2.Peters, M. W.; Meinhold, P.; Glieder, A.; Arnold, F. H.,“Regio-and enantioselective alkane hydroxylation with engineered cytochromes P450 BM-3”, Journal of the American Chemical Society, Vol. 125, No.44, pp. 13442-13450 (2003)
3.Noble, M.; Miles, C.; Chapman, S.; Lysek, D.; Mackay, A.; Reid, G.; Hanzlik, R.; Munro, A.,“Roles of key active-site residues in flavocytochrome P450 BM3”, Biochem. J, Vol. 339, pp. 371-379 (1999)
4.Urlacher, V. B.; Lutz-Wahl, S.; Schmid, R. D.,“Microbial P450 enzymes in biotechnology”, Applied microbiology and biotechnology, Vol. 64, No. 3, pp. 317-325 (2004)
5.Li, H.; Poulos, T. L.,“The structure of the cytochrome p450BM-3 haem domain complexed with the fatty acid substrate, palmitoleic acid”, Nature Structural & Molecular Biology, Vol. 4, No. 2, pp. 140-146 (1997)
6.Sevrioukova, I. F.; Li, H.; Zhang, H.; Peterson, J. A.; Poulos, T. L.,“Structure of a cytochrome P450–redox partner electron-transfer complex”, Proceedings of the National Academy of Sciences, Vol. 96, No. 5, pp. 1863-1868 (1999)
7.Ravichandran, K. G.; Boddupalli, S. S.; Hasermann, C.; Peterson, J. A.; Deisenhofer, J.,“Crystal structure of hemoprotein domain of P450BM-3, a prototype for microsomal P450's”Science, Vol.261, No. 5122, pp. 731-736 (1993)
8.Haines, D. C.; Tomchick, D. R.; Machius, M.; Peterson, J. A.,“Pivotal role of water in the mechanism of P450BM-3. Biochemistry”, Vol. 40 No. 45, pp. 13456-13465 (2001)
9.Whitehouse, C. J.; Bell, S. G.; Wong, L.-L., “P450BM3 (CYP102A1): connecting the dots”Chem. Soc. Rev., Vol. 41, No. 3, pp.1218-1260 (2012)
10.Ost, T. W.; Miles, C. S.; Murdoch, J.; Cheung, Y.-F.; Reid, G. A.; Chapman, S. K.; Munro, A. W.,“Rational re-design of the substrate binding site of flavocytochrome P450 BM3”,FEBS Letters, Vol. 486, No. 2, pp. 173-177 (2000)
11.Li, H.; Poulos, T.,“Conformational dynamics in cytochrome P450-substrate interactions”, Biochimi , Vol. 78, No. 8, pp. 695-699 (1996)
12.Hegde, A.; Haines, D. C.; Bondlela, M.; Chen, B.; Schaffer, N.; Tomchick, D. R.; Machius, M.; Nguyen, H.; Chowdhary, P. K.; Stewart, L.,“Interactions of substrates at the surface of P450s can greatly enhance substrate potency”, Biochemistry , Vol. 46, No. 49, pp. 14010-14017 (2007)
13.Haines, D. C.; Hegde, A.; Chen, B.; Zhao, W.; Bondlela, M.; Humphreys, J. M.; Mullin, D. A.; Tomchick, D. R.; Machius, M.; Peterson, J. A.,“A single active-site mutation of P450BM-3 dramatically enhances substrate binding and rate of product formation”, Biochemistry, Vol. 50, No. 39, pp. 8333-8341 (2011)
14.Wu, L. L.; Yang, C. L.; Lo, F. C.; Chiang, C. H.; Chang, C. W.; Ng, K. Y.; Chou, H. H.; Hung, H. Y.; Chan, S. I.; Yu, S. S. F.,“Tuning the Regio‐and Stereoselectivity of C-H Activation in n‐Octanes by Cytochrome P450 BM‐3 with Fluorine Substituents: Evidence for Interactions Between a C-F Bond and Aromatic π Systems”, Chemistry-A European Journal, Vol. 17, No. 17, pp. 4774-4787 (2011)
15.Oliver, C. F.; Modi, S.; Sutcliffe, M. J.; Primrose, W. U.; Lian, L.-Y.; Roberts, G. C.,“A single mutation in cytochrome P450 BM3 changes substrate orientation in a catalytic intermediate and the regiospecificity of hydroxylation”, Biochemistry, Vol. 36, No. 7, pp. 1567-1572 (1997)
16.Miura, Y. F., A.J. ,“omega-1, omega-2 and omega-3 hydroxylation of long-chain fatty acids, amides and alcohols by a soluble enzyme system from Bacillus megaterium”, Biochimica et Biophysica Acta, Vol. 388, pp. 305-317 (1975)
17.Shirane, N.; Sui, Z.; Peterson, J. A.; Ortiz de Montellano, P. R.,“Cytochrome P450BM-3 (CYP102): Regiospecificity of oxidation of. omega.-unsaturated fatty acids and mechanism-based inactivation”, Biochemistry , Vol. 32, No. 49, pp. 13732-13741 (1993)
18.Ost, T. W.; Munro, A. W.; Mowat, C. G.; Taylor, P. R.; Pesseguiero, A.; Fulco, A. J.; Cho, A. K.; Cheesman, M. A.; Walkinshaw, M. D.; Chapman, S. K., “Structural and spectroscopic analysis of the F393H mutant of flavocytochrome P450 BM”, Biochemistry, Vol. 40, No. 45, pp. 13430-13438 (2011)
19.Cedrone, F.; Ménez, A.; Quéméneur, E.,“Tailoring new enzyme functions by rational redesign”, Current opinion in structural biology, Vol. 10, No. 4, pp. 405-410 (2000)
20.Hult, K.; Berglund, P.,“Enzyme promiscuity: mechanism and applications”TRENDS in Biotechnology, Vol. 25, No. 5, pp. 231-238 (2007)
21.Tracewell, C. A.; Arnold, F. H.,“Directed enzyme evolution: climbing fitness peaks one amino acid at a time”, Current opinion in chemical biology, Vol. 13, No 1, pp. 3-9 (2009)
22.Farinas, E. T.; Schwaneberg, U.; Glieder, A.; Arnold, F. H.,“Directed evolution of a cytochrome P450 monooxygenase for alkane oxidation”, Advanced Synthesis & Catalysis, Vol. 343, No. 6-7, pp. 601-606 (2001)
23.Meinhold, P.; Peters, M. W.; Hartwick, A.; Hernandez, A. R.; Arnold, F. H., “Engineering cytochrome P450 BM3 for terminal alkane hydroxylation”, Advanced Synthesis & Catalysis, Vol. 348, No. 6, pp. 763-772 (2006)
24.Shilov, A. E.; Shul'pin, G. B.,“Activation of C-H bonds by metal complexes”, Chemical reviews, Vol. 97, No. 8, pp. 2879-2932 (1997)
25.陳耀甥、張鈞崴、楊宗霖、吳麗嵐、江志祥、拉姆、羅文一、陳長謙、俞聖法， 「金屬單加氧酵素內烷烴基質其位置與立體選擇性的活化控制」，化學，第七十一卷，第一期，第1-15頁 (2013)
26.王永梅、王业柳、田间，「同位素效应简介」，大学化学，第十九卷，第三期，第32-39頁 (2004)
27.Yu, S. S.-F.; Wu, L.-Y.; Chen, K. H.-C.; Luo, W.-I.; Huang, D.-S.; Chan, S. I., “The stereospecific hydroxylation of [2, 2-2H2] butane and chiral dideuteriobutanes by the particulate methane monooxygenase from Methylococcus capsulatus (Bath)”, Journal of Biological Chemistry, Vol. 278, No. 42, pp. 40658-40669 (2003)
28.洪惠瑩，「Cytochrome P450 BM-3 作為烷烴化合物的催化試劑」，碩士論文，國立成功大學，台南 (2007)
29.Omura, T.; Sanders, E.; Estabrook, R.; Cooper, D.; Rosenthal, O.,“Isolation from adrenal cortex of a nonheme iron protein and a flavoprotein functional as a reduced triphosphopyridine nucleotide-cytochrome P-450 reductase”, Archives of Biochemistry and Biophysics, Vol. 117, No. 3, pp. 660-673 (1966)
30.Seifert, A.; Vomund, S.; Grohmann, K.; Kriening, S.; Urlacher, V. B.; Laschat, S.; Pleiss, J.,“Rational Design of a Minimal and Highly Enriched CYP102A1 Mutant Library with Improved Regio‐, Stereo‐and Chemoselectivity”, Chembiochem, Vol. 10, No. 5, pp. 853-861 (2009)
31.Appel, D.; Lutz-Wahl, S.; Fischer, P.; Schwaneberg, U.; Schmid, R. D.,“A P450 BM-3 mutant hydroxylates alkanes, cycloalkanes, arenes and heteroarenes”, Journal of biotechnology, Vol. 88, No. 2, pp. 167-171 (2001)
32.Fasan, R.; Meharenna, Y. T.; Snow, C. D.; Poulos, T. L.; Arnold, F. H., “Evolutionary history of a specialized P450 propane monooxygenase”Journal of molecular biology, Vol. 383, No. 5, pp. 1069-1080 (2008)
33.Chiang, C. H.; Ramu, R.; Tu, Y. J.; Yang, C. L.; Ng, K. Y.; Luo, W. I.; Chen, C. H.; Lu, Y. Y.; Liu, C. L.; Yu, S. S. F.,“Regioselective Hydroxylation of C12–C15 Fatty Acids with Fluorinated Substituents by Cytochrome P450 BM3”, Chemistry-A European Journal, Vol. 19, No. 41, pp. 13680-13691 (2013)
34.Cryle, M. J.; Matovic, N. J.; De Voss, J. J.,“Weak Hydrogen Bridges: A Systematic Theoretical Study on the Nature and Strength of C-H…F-C Interactions”, Tetrahedron Letters, Vol. 48, No. 1, pp. 133-136 (2007)
35.Cryle, M. J.; De Voss, J. J.,“Facile determination of the absolute stereochemistry of hydroxy fatty acids by GC: application to the analysis of fatty acid oxidation by a P450BM3 mutant”, Tetrahedron: Asymmetry, Vol. 18, No. 4, pp. 547-551 (2007)
36.Truan, G.; Komandla, M. R.; Falck, J. R.; Peterson, J. A.,“P450BM-3: absolute configuration of the primary metabolites of palmitic acid”, Archives of biochemistry and biophysics, Vol. 366, No. 2, pp. 192-198 (1999)
37.Chen, Y.-S.; Luo, W.-I.; Yang, C.-L.; Tu, Y.-J.; Chang, C.-W.; Chiang, C.-H.; Chang, C.-Y.; Chan, S. I.; Yu, S. S.-F.,“Controlled oxidation of aliphatic C-H bonds in metallo-monooxygenases: Mechanistic insights derived from studies on deuterated and fluorinated hydrocarbons”, Journal of inorganic biochemistry, Vol. 134, pp. 118-133 (2014)
38.Alonso, H.; Roujeinikova, A.,“Characterization and two-dimensional crystallization of membrane component AlkB of the medium-chain alkane hydroxylase system from Pseudomonas putida GPo1”, Applied and environmental microbiology, Vol. 78, No. 22, pp. 7946-7953 (2012)
39.Chen, P. P.-Y.; Chan, S. I.,“Theoretical modeling of the hydroxylation of methane as mediated by the particulate methane monooxygenase”, Journal of inorganic biochemistry, Vol. 100, No. 4, pp. 801-809 (2006)
40.Li, H.; Poulos, T. L., “Fatty acid metabolism, conformational change, and electron transfer in cytochrome P-450 BM-3”, Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids, Vol. 1441, No. 2, pp. 141-149 (1999)
41.O'Hagan, D., “Understanding organofluorine chemistry. An introduction to the C–F bond”, Chemical Society Reviews, Vol. 37, No. 2, pp. 308-319 (2008)
42.Hyla‐Kryspin, I.; Haufe, G.; Grimme, S., “Weak Hydrogen Bridges: A Systematic Theoretical Study on the Nature and Strength of C-H⋅⋅⋅ F-C Interactions”, Chemistry-A European Journal, Vol. 10, No. 14, pp. 3411-3422 (2004)
43.Rybalova, T.; Bagryanskaya, I. Y., “C-F... π, F... H, and F... F intermolecular interactions and F-aggregation: Role in crystal engineering of fluoroorganic compounds”, Journal of Structural Chemistry, Vol. 50, No.4, pp. 741-753 (2009)
44.Müller, K.; Faeh, C.; Diederich, F.,“Fluorine in pharmaceuticals: looking beyond intuition”, Science, Vol. 317 No. 5846, pp. 1881-1886 (2007)
45.Mecinovic, J.; Snyder, P. W.; Mirica, K. A.; Bai, S.; Mack, E. T.; Kwant, R. L.; Moustakas, D. T.; Héroux, A.; Whitesides, G. M.,“Fluoroalkyl and alkyl chains have similar hydrophobicities in binding to the “hydrophobic wall” of carbonic anhydrase”, Journal of the American Chemical Society, Vol. 133, NO. 35, pp. 14017-14026 (2011)
46.Dalvi, V. H.; Rossky, P. J.,“Molecular origins of fluorocarbon hydrophobicity”, Proceedings of the National Academy of Sciences, Vol. 107, No. 31, pp. 13603-13607 (2010)
47.Smart, B. E.,“Fluorine substituent effects (on bioactivity)”, Journal of Fluorine Chemistry, Vol. 109, No. 1, pp. 3-11 (2011)
48.Pongdee, R.; Liu, H.-w.,“Elucidation of enzyme mechanisms using fluorinated substrate analogues”, Bioorganic chemistry, Vol. 32, No. 5, pp. 393-437 (2004)
49.Ng, K. Y.; Tu, L. C.; Wang, Y. S.; Chan, S. I.; Yu, S. S. F.,“Probing the hydrophobic pocket of the active site in the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) by variable stereoselective alkane hydroxylation and olefin epoxidation”, ChemBioChem, Vol. 9, No. 7, pp. 1116-1123 (2008)
50.Purser, S.; Moore, P. R.; Swallow, S.; Gouverneur, V.,“Fluorine in medicinal chemistry”, Chemical Society Reviews, Vol. 37, No. 2, pp. 320-330 (2008)
51.Böhm, H. J.; Banner, D.; Bendels, S.; Kansy, M.; Kuhn, B.; Müller, K.; Obst‐Sander, U.; Stahl, M.,“Fluorine in medicinal chemistry”, ChemBioChem, Vol. 5, No. 5, pp. 637-643 (2004)
52.Li, Q. S.; Schwaneberg, U.; Fischer, P.; Schmid, R. D.,“Directed Evolution of the Fatty‐Acid Hydroxylase P450 BM‐3 into an Indole‐Hydroxylating Catalyst”, Chemistry-A European Journal, Vol. 6, No. 9, pp. 1531-1536 (2000)
53.Zilly, F. E.; Acevedo, J. P.; Augustyniak, W.; Deege, A.; Häusig, U. W.; Reetz, M. T.,“Tuning a P450 enzyme for methane oxidation”, Angewandte Chemie, Vol. 123 No. 12, pp. 2772-2776 (2011)
54.Li, C.; Shaik, S.,“How do perfluorinated alkanoic acids elicit cytochrome P450 to catalyze methane hydroxylation? An MD and QM/MM study”, RSC advances, Vol. 3, No. 9, pp. 2995-3005 (2013)