| Summary: | 博士 === 國立臺灣科技大學 === 應用科技研究所 === 104 === Cytochrome P450 BM3 is an iron heme protein in Bacillus megaterium；it is capable of binding with oxygen and proceeding oxidation of the natural substrate-fatty acids C12-C20 in length to form hydroxyl group at ω-1, ω-2 or ω-3 position. The corresponding gene of P450BM3 was replicated and inserted in a DNA vector via molecular bioengineering technology. The constructed DNA was transformed and cloned in Escherichia coli host to achieve the protein expression and reveal its function by the catalytic oxidation of hydrocarbons.
Crystal structure from Protein Data Bank（PDB：1FAG）allows us to understand the environment around the active site. On the basis of these insights, we can redesign cytochrome P450BM3 to switch its substrates from natural substrate-fatty acids to simple aromatics by directed evolution including site-directed mutagenesis study. According to our rational design, in order to achieve our goal, the amino acid residues 328 or 330 have to be substituted with phenylalanine by site-directed mutagenesis techniques. The variants with single site, double, triple as well as four sites have been deliberated.
From the catalysis that using benzene or toluene as substrates, single-site variants （Phe328 or Phe330）, double-sites variant（Phe328／Met401）, triple-sites variants （3mt, Ile47／Phe51／Phe328 and Phe47／Ser51／Phe328）and four-sites variant 3mt Phe328 are able to show the activities. Phe328 is a major site to present the activities towards benzene and toluene. The distribution of main product was changed to benzyl alcohol when 3mt combined with Phe328 in toluene catalysis. By comparison with other type oxygenase, i.e. non-heme iron monooxygenase, the variants of cytochrome P450BM3 can generate o-cresol but the membrane bound xylene monooxygenase can only achieve sp3 oxidation for benzyl alcohol formation when toluene used as the substrate.
From the studies of stable carbon isotope fractionation, the three variants of cytochrome P450BM3 shifted the δ13C（‰） of phenol down to ca. -38 when using benzene (-31) as a substrate. For the catalysis that using toluene (-24) as a substrate, the product δ13C（‰）values of the benzyl alcohol are -24.4~-27.4 and those of o-cresol and p-cresol are at the range of -27.4~-36.5 and -25.7~-29.0, respectively. The derived 12C/13C kinetic isotope effects (1.03~1.09) for benzene oxidation (~1.04) are relatively bigger indicated the transition state of oxygenation related to the bond breakage of C-H bond and bond formation of C-O is mechanistically significant (central transition state). However, most of the 12C/13C kinetic isotope effects obtained for the formation of benzyl alcohol, o-cresol and p-cresol ranged from 1.00~1.03 suggest the processes of both sp2 and sp3 C-H bond breaking as well as C-O bond formation encounter either in a very early or late transition state expect to the case of 3mt A328F variant corresponding to the formation of o-cresol (KIE (12C/13C) ~ 1.07).
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