This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bühler, B. Articles by Schmid, A. Search for Related Content PubMed PubMed Citation Articles by Bühler, B. Articles by Schmid, A. Agricola Articles by Bühler, B. Articles by Schmid, A.

Characterization and Application of Xylene Monooxygenase for Multistep Biocatalysis

Institute of Biotechnology, Swiss Federal Institute of Technology Zurich, CH-8093 Zurich, Switzerland,¹ Research Fine Chemicals and Biotechnology, BASF Corporation, D-67056 Ludwigshafen, Germany²

Received 10 August 2001/ Accepted 5 November 2001

Xylene monooxygenase of Pseudomonas putida mt-2 catalyzes multistep oxidations of one methyl group of toluene and xylenes. Recombinant Escherichia coli expressing the monooxygenase genes xylM and xylA catalyzes the oxygenation of toluene, pseudocumene, the corresponding alcohols, and the corresponding aldehydes, all by a monooxygenation type of reaction (B. Bühler, A. Schmid, B. Hauer, and B. Witholt, J. Biol. Chem. 275:10085-10092, 2000). Using E. coli expressing xylMA, we investigated the kinetics of this one-enzyme three-step biotransformation. We found that unoxidized substrates like toluene and pseudocumene inhibit the second and third oxygenation steps and that the corresponding alcohols inhibit the third oxygenation step. These inhibitions might promote the energetically more favorable alcohol and aldehyde dehydrogenations in the wild type. Growth of E. coli was strongly affected by low concentrations of pseudocumene and its products. Toxicity and solubility problems were overcome by the use of a two-liquid-phase system with bis(2-ethylhexyl)phthalate as the carrier solvent, allowing high overall substrate and product concentrations. In a fed-batch-based two-liquid-phase process with pseudocumene as the substrate, we observed the consecutive accumulation of aldehyde, acid, and alcohol. Our results indicate that, depending on the reaction conditions, product formation could be directed to one specific product.

This article has been cited by other articles:

• Buhler, B., Park, J.-B., Blank, L. M., Schmid, A. (2008). NADH Availability Limits Asymmetric Biocatalytic Epoxidation in a Growing Recombinant Escherichia coli Strain. Appl. Environ. Microbiol. 74: 1436-1446 [Abstract] [Full Text]  
• Feingersch, R., Shainsky, J., Wood, T. K., Fishman, A. (2008). Protein Engineering of Toluene Monooxygenases for Synthesis of Chiral Sulfoxides. Appl. Environ. Microbiol. 74: 1555-1566 [Abstract] [Full Text]  
• Meyer, D., Witholt, B., Schmid, A. (2005). Suitability of Recombinant Escherichia coli and Pseudomonas putida Strains for Selective Biotransformation of m-Nitrotoluene by Xylene Monooxygenase. Appl. Environ. Microbiol. 71: 6624-6632 [Abstract] [Full Text]  
• Fishman, A., Tao, Y., Wood, T. K. (2004). Toluene 3-Monooxygenase of Ralstonia pickettii PKO1 Is a para-Hydroxylating Enzyme. J. Bacteriol. 186: 3117-3123 [Abstract] [Full Text]