Laboratory evolution of cytochrome p450 BM-3 monooxygenase for organic cosolvents |
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Authors: | Wong Tuck Seng Arnold Frances H Schwaneberg Ulrich |
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Institution: | Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, California 91125, USA. |
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Abstract: | Cytochrome p450 BM-3 (EC 1.14.14.1) catalyzes the hydroxylation and/or epoxidation of a broad range of substrates, including alkanes, alkenes, alcohols, fatty acids, amides, polyaromatic hydrocarbons, and heterocycles. For many of these notoriously water-insoluble compounds, p450 BM-3's K(m) values are in the millimolar range. Polar organic cosolvents are therefore added to increase substrate solubility and achieve high catalytic efficiency. Using p450 BM-3 as a catalyst for these important transformations requires that we improve its ability to tolerate the cosolvents. By directed evolution, we improved the activity of p450 BM-3 in the presence of dimethylsulfoxide (DMSO) and tetrahydrofuran (THF), achieving increases in specific activity up to 10-fold in 2% (v/v) THF and 6-fold in 25% (v/v) DMSO. The engineered p450 BM-3's are also significantly more resistant to acetone, acetonitrile, dimethylformamide, and ethanol as cosolvents in the reaction. |
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Keywords: | P450 BM‐3 CYP102 Bacillus megaterium organic solvent resistance random mutagenesis directed evolution |
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