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

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, February 2002, p. 560-568, Vol. 68, No. 2
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.2.560-568.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Characterization and Application of Xylene Monooxygenase for Multistep Biocatalysis

Bruno Bühler,1 Bernard Witholt,1 Bernhard Hauer,2 and Andreas Schmid1*

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.

* Corresponding author. Mailing address: Institut für Biotechnologie, ETH Zürich, Hönggerberg HPT, CH-8093 Zürich, Switzerland. Phone: 41 1 633 36 91. Fax: 41 1 633 10 51. E-mail: andreas{at}biotech.biol.ethz.ch.

Applied and Environmental Microbiology, February 2002, p. 560-568, Vol. 68, No. 2
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.2.560-568.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Cunningham, D. S., Liu, Z., Domagalski, N., Koepsel, R. R., Ataai, M. M., Domach, M. M. (2009). Pyruvate Kinase-Deficient Escherichia coli Exhibits Increased Plasmid Copy Number and Cyclic AMP Levels. J. Bacteriol. 191: 3041-3049 [Abstract] [Full Text]  
  • 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]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»