Laboratory evolution of cytochrome P450 BM‐3 monooxygenase for organic cosolvents
- 31 December 2003
- journal article
- research article
- Published by Wiley in Biotechnology & Bioengineering
- Vol. 85 (3) , 351-358
- https://doi.org/10.1002/bit.10896
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 Km 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.Keywords
This publication has 26 references indexed in Scilit:
- Use of Kinetic Isotope Effects to Delineate the Role of Phenylalanine 87 in P450BM-3Bioorganic Chemistry, 2002
- Modification of the fatty acid specificity of cytochrome P450 BM-3 from Bacillus megaterium by directed evolution: a validated assayJournal of Molecular Catalysis B: Enzymatic, 2001
- P450 monooxygenase in biotechnology: I. Single-step, large-scale purification method for cytochrome P450 BM-3 by anion-exchange chromatographyJournal of Chromatography A, 1999
- A Continuous Spectrophotometric Assay for P450 BM-3, a Fatty Acid Hydroxylating Enzyme, and Its Mutant F87AAnalytical Biochemistry, 1999
- Functional Interactions in Cytochrome P450BM3: Flavin Semiquinone Intermediates, Role of NADP(H), and Mechanism of Electron Transfer by the Flavoprotein DomainBiochemistry, 1997
- An Active Site Substitution, F87V, Converts Cytochrome P450 BM-3 into a Regio- and Stereoselective (14S,15R)-Arachidonic Acid EpoxygenaseJournal of Biological Chemistry, 1997
- The Highly Stereoselective Oxidation of Polyunsaturated Fatty Acids by Cytochrome P450BM-3Journal of Biological Chemistry, 1996
- Role of the Linker Region Connecting the Reductase and Heme Domains in Cytochrome P450BM-3Biochemistry, 1995
- A fully modular vector system for the optimization of gene expression in Escherichia coliPlasmid, 1991
- ω-1, ω-2 and ω-3 Hydroxylation of long-chain fatty acids, amides and alcohols by a soluble enzyme system from Bacillus megatyeriumBiochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1975