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Cost-Effective Whole-Cell Assay for Laboratory Evolution of Hydroxylases in Escherichia coliDivision of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA Cytochrome P450 BM-3 from Bacillus megaterium catalyzes the subterminal hydroxylation of medium- and long-chain fatty acids at the to-1, w-2, and c-3 positions. A continuous spectrophotometric assay for P450 BM-3 based on the conversion of p-nitrophenoxycarboxylic acids (pNCA) to co-oxycarboxylic acids and the chromophore p-nitrophenolate was reported recently. However, this pNCA assay procedure contained steps that limited its application in high throughput screening, including expression of P450 BM-3 variant F87A in 4-ml cultures, centrifugation, resuspension of the cell pellet, and cell lysis. We have shown that permeabilization of the outer membrane of Escherichia coli DH5a with polymyxin B sulfate, EDTA, polyethylenimine, or sodium hexametaphosphate results in rapid conversion of 12-pNCA. A NADPH-generating system consisting of NADP+, D/L-isocitric acid, and the D/L-isocitrate dehydrogenase of E. coli DH5a reduced the cofactor expense more than 10-fold. By avoiding cell lysis, re-suspension, and centrifugation, the high throughput protocol allows screening of thousands of samples per day.
Journal of Biomolecular Screening, Vol. 6, No. 2,
111-117 (2001) This article has been cited by other articles:
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