臺灣博碩士論文加值系統

　　本研究選擇在細胞色素P450 BM3的328號位置進行變異，將Alanine 轉換為Phenyl Alanine，使得在反應性上，較原Wild-type 提升了2.5 到4倍左右。另一方面，以突變株A74G F87V L188Q進行改變位向選擇性的測試，結果此突變株，對於甲苯氧化的選擇性由原先WT 的100% sp2產物選擇性，改變為73%的sp2產物選擇性，並在更進一步優化的3mt　A328F變異株中達到了50%的sp3產物選擇性。

　　再者，本次實驗使用過氯酸亞鐵佐以過氧化氫，來模擬細胞色素P450 BM3活性中心鐵離子對於受質的氧化機制。使得能以調整過氯酸亞鐵濃度、過氧化氫添加速度以及反應溫度的方式，在sp3產物選擇性可達到最高96%以及sp2產物選擇性最高75%的程度。

　　最後以氘化苯與氘化甲苯輔助，評估細胞色素P450 BM3與過氯酸亞鐵動力學同位素效應，在氘化苯的氧化反應中，細胞色素P450 BM3與過氯酸亞鐵都呈現逆向二級動力學同位素效應，而在氘化甲苯的氧化反應中，則只有細胞色素P450 BM3維持逆向二級動力學同位素效應，過氯酸亞鐵在sp2產物中，則呈現正向二級動力學同位素效應。

According to our earlier study in cytochrome P450 BM3, for the oxidation of simple aromatics, we chose to mutate its amino acid residue at position 328 from alanine to
phenylalanine. The catalytic efficiency (turn over frequencies (TOF)) of toluene and benzene, mediated by this specific mutant, for the phenol and cresols production, can be increased 2.5 to 4 times higher than the wild-type strain. On the other hand, in the case of another cytochrome P450 BM3 variant, A74G F87V L188Q, except to the formation of o-cresol of toluene oxidation at sp2 position, one additional product of benzyl alcohol for sp3-selective product was observed and its ratio is 27%. Introducing one additional mutation site of A328F can enhance the sp3 product selectivity ratio up to 50%.
A biomimetic system of cytochrome P450 BM3 for the oxidation of benzene and toluene catalyzed by iron(II) perchlorate using H2O2(aq) in CH3CN was also undertaken in this study. The reactions can be facilely tuned and controlled to selectively yield either a single or double oxygenation of benzene as well as a sp3 or sp2 C–H bond oxidation of toluene.
In order to further delineate the reaction mechanisms of the oxy-functionalization at the sp3 and/or sp2 centers, for the comparison between cytochrome P450 BM3 and its
biomimetic system of Fe(ClO4)2 in an H2O2-H2O-CH3CN system, we conducted studies that included measurements of kinetic isotope effects (KIEs) by mixing C6D6 with C6H6 or C7D8 with C7H8, each in a 1:1 ratio. Our data reveal that both of the KIE data in benzene oxidations presented an inverse secondary KIE manner and, in the
case of toluene oxidation, the KIE for cytochrome P450 BM3 was also performed as Inverse secondary kinetic isotope effect (0.7-1.0). However, KIE for Fe(ClO4)2
biomimetics on the contrary was appeared as normal secondary kinetic isotope effect.

摘要 i
Abstract ii
誌謝. iii
一、導論1
芳香烴 1
　細胞色素P450（Cytochrome P450）2
　巨大芽孢桿菌（Bacillus megaterium，BM3） 4
　細胞色素P450 BM3 氧化機制 5
　細胞色素P450 BM3 位向選擇性 6
　定點變異的選擇 8
　動力學同位素效應（Kinetic isotope effect，KIE） 10
　研究目的 13
二、實驗藥品 14
三、儀器設備 17
四、實驗步驟與方法 19
　建立pET21a(DE3) Cytochrome P450 BM3 表現載體 19
　基因轉殖（Transformation） 20
　質體抽出（Plasmid extraction） 20
　定點突變（Site directed mutagenesis） 21
　蛋白質表達 27
　全細胞催化 27
　蛋白質純化28
　CBR 測量蛋白質濃度檢量線的建立 30
　以CBR 進行蛋白質濃度測量 30
　以一氧化碳對蛋白質濃度測定 31
　十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 31
　菸鹼醯胺腺嘌呤二核苷酸磷酸代謝測定 33
　過氯酸亞鐵（Iron(II) perchlorate，Fe(ClO4)2）催化 34
　氣相層析法濃度檢量線的建立 34
　同位素動力學效應的測定 36
五、結果與討論 37
　全細胞催化活性測試 37
　細胞色素P450BM3 直接催化活性分析 39
　以過氯酸亞鐵為基礎在乙腈-過氧化氫的仿生催化 41
　動力學同位素效應分析 46
六、結論 50
參考文獻 52

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