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

盧昱穎(Yu-Ying Lu)

指導教授 ： 蔡惠旭 ； 俞聖法

國立中央大學/理學院/化學系/碩士(2007年)

摘要

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。

關鍵字

細胞色素 ； 羥基化酵素 ； 辛烷 ； 導引式演化

並列摘要

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.

並列關鍵字

Octane ； Regioselective ； Cytochrome P450 ； Random Mutagenesis

參考文獻

1. 洪惠瑩, Utilization of Cytochrome P450 BM3 as the Biocatalysts for Aliphatic Compounds Activation, in Department of Chemistry. 2007, National Cheng Kung University: Tainan.

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4. Peters, M.W., et al., Regio- and enantioselective alkane hydroxylation with engineered cytochromes P450 BM-3. J Am Chem Soc, 2003. 125(44): p. 13442-50.

5. Poulos, T.L., The past and present of P450cam structural biology. Biochem Biophys Res Commun, 2003. 312(1): p. 35-9.

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