來自Bacillus megaterium的細胞色素P450 BM3對受質是非常有專一性的，使用大氣中的氧氣當作氧化劑，除天然受質：脂肪酸外幾乎不接受其他的化合物。我們成功的從大腸桿菌中表現P450 BM3並且藉由金屬離子親和層析管柱純化具有His-tag的重組蛋白質。在大腸桿菌內大量表現的P450 BM3依然具有活性，且輔因子也一同被表現。這個蛋白質在加入NADPH即可催化脂肪酸。從UV/visible光譜分析中, 此His-tagged P450 BM3 內依然保有鐵的存在，且從GC/MS分析得知，此His-tagged P450 BM3也具有催化的能力。
接著，我們利用定點突變的方法改變P450 BM3的三個胺基酸序列(Leu188Gln, Phe87Val, Ala74Gly)，這個突變過後的蛋白質內依然含鐵。
辛烷並不會被原生型的P450 BM3所氧化，但是此P450 BM3 mutant可以將辛烷氧化成2-辛醇, 3-辛醇, 和4-辛醇。P450 BM3 mutant也可以催化含氟的化合物，實驗結果提供我們有用的資訊在於設計新的含氟探針，以便測試P450系統對甲烷催化的能力。

Cytochrome P450 BM3 from Bacillus megaterium display a rather narrow substrate specificity. It can only hydroxylates fatty acid by using atmospheric dioxygen as an oxidant. We successfully expressed and purified the recombinant His-tagged protein (P450 BM3) from E. coli. by a simple metal-affinity chromatography. The hydroxylate activity is retained in E. coli., where the engineered P450 BM3 can be expressed at high levels and the cofactor is supplied endogeneously. This protein can retain its hydroxylate function to catalyze the fatty acid-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH). From UV/visible, the His-tagged P450 BM3 obtains heme. From GC/MS, the His-tagged P450 BM3 is characterized enzymeologically.
And then we engineer P450 BM3 using site-directed mutagensis. A triple mutant P450 BM3 (Leu188Gln, Phe87Val, Ala74Gly) also obtains heme. Octane is not oxidized by the wild-type enzyme, but this mutant hydroxylates octane to 2-octanol, 3-octanol, and 4-octanol. P450 BM3 mutant also has the capability to hydroxylate fluorinated compound. The outcome supplied us some imperative information to design new fluorinated probes towards direct evolving the P450 system for methane activation utilities.

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