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Research Collection Research Collection Doctoral Thesis Specific oxyfunctionalization of pseudocumene by multistep biocatalysis - Mechanism and scale up Author(s): Bühler, Bruno Albert Publication Date: 2003 Permanent Link: https://doi.org/10.3929/ethz-a-004630430 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library Diss. ETH No. 15206 SPECIFIC OXYFUNCTIONALIZATION OF PSEUDOCUMENE BY MULTISTEP BIOCATALYSIS – MECHANISM AND SCALE UP A dissertation submitted to the SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH for the degree of Doctor of Natural Sciences presented by BRUNO ALBERT BÜHLER Dipl. Nat. Sci., Swiss Federal Institute of Technology Zurich, Switzerland born December 26, 1973 Citizen of Homburg, TG (CH) Accepted on the recommendation of Prof. Dr. Bernard Witholt, examiner Dr. Andreas Schmid, co-examiner PD Dr. Bernhard Hauer, co-examiner Prof. Dr. Erick M. Carreira, co-examiner Zurich, 2003 II ACKNOWLEDGMENTS First of all, I would like to thank the promoters of my thesis: Prof. Dr. Bernard Witholt and Dr. Andreas Schmid, who enabled me to carry out this work at the IBT. Thanks a lot for always motivating me, for the helpful and interesting discussions, and for teaching me all the smaller and bigger “details” needed to make good science. Andreas, I thank you for always showing me the positive sides, when my critical attitude tended to end up in frustration, and for your excellent supervision. I am most grateful to PD Dr. Bernhard Hauer and Prof. Dr. Erick M. Carreira for being in the committee acting as coreferees. Furthermore, I would like to thank Bernhard Hauer, Thomas Zelinski, and Tilo Habicher from BASF for the good collaboration, their input in this project, and the good atmosphere during the project meetings including the nice dinners in the surroundings of Ludwigshafen. Adrie Straathof is gratefully acknowledged for his kind and competent collaboration and help during his sabbatical at the IBT. Thanks for excellent scientific input and helpful discussions also go to Birgit, Frank, Jan, Jin Byung, Kuno, Peter R., Sven, Wouter, and Zhi. For great technical assistance and teaching or helping me at various stages of the work, I am most thankful to Hans-Jürgen and Martina. Of course, I also thank the diploma students Irene and Pascal for their great contribution to this work. An especially big “thank you” I would like to address to Helena, who brilliantly mastered all administrative concerns. You always had an open door and a solution for all kind of organizational but also personal problems, not underestimating her impact on the social life creating a good atmosphere at the IBT. Thanks also to Petra and Rahel for perfectly managing the ordering stuff, to Helen and Monika for the clean up of glassware and coffee cups, to Collette for her excellent IT support, and to Peter Koller for repairing and tuning all kind of equipment. III My profound gratitude goes to my long term (all female) lab mates Barbara, Karin, Martina, and in former times Yvonne for being great colleagues and friends. I greatly appreciated the always funny atmosphere and your tolerance towards my chaotic behavior in terms of marking all the lab space with bottles and flasks and especially of creating a big mess on my desk and bench. I always will remember the nice evenings in the bistro, the parties and aperos in the aquarium, the skiing days, and other social events in connection with the IBT. Thanks to all the people who have contributed to the nice atmosphere at the Hönggerberg but also at other places in and around Zurich, especially to Barbara, Daniel, Georgios, Guy, Helena, Jochen, Karin, Martina, and Peter R.. Three people have become special friends and more during my PhD time at the IBT, Frank, Katja, and Kuno; I thank you very much not only for your scientific but especially for your personal assistance, the interesting and intensive discussions, and the nice and funny time, we had, have, and surely will have in the future. Furthermore, I would like to thank my friends from outside the institute, who always supported me personally or just by accompanying me for a beer. Finally and very greatly, I thank my Mum and Dad as well as my sister and her family for their moral support, helping me through all my ups and downs. This work was financially supported by the BASF corporation. IV CONTENTS Summary VI Zusammenfassung VII 1. Introduction 1 1.1. General Introduction 2 1.2. Biocatalysis in the Chemical Industry 3 1.2.1. Impact of Enzymes as Catalysts in the Chemical Industry 3 1.2.2. Bioprocesses and the Environment 4 1.2.3. Commercialized Applications of Enzymes in the Chemical Industry 4 1.2.4. Industrial Enzyme Applications in the Exploratory Phase 8 1.2.5. Potential of Biocatalysis and Future Perspectives 11 1.3. Oxyfuctionalization of Hydrocarbons 13 1.3.1. Bacterial Hydrocarbon Degradation; Potential for Applications in Organic Synthesis 13 1.3.2. Oxyfunctionalization of sp3-Hybridized Carbons 16 1.3.2.1. Heme Dependent Oxygenases 19 1.3.2.2. Nonheme Iron Oxygenases 21 1.4. Application of Oxygenases 28 1.4.1. Biocatalyst Selection 28 1.4.2. Intrinsic Oxygenase Properties 29 1.4.2.1. Specific Activity 29 1.4.2.2. Uncoupling 31 1.4.2.3. Multiple Oxidation 31 1.4.3. Physiological Aspects – Biocatalyst Stability 33 1.4.3.1. Product Degradation 33 1.4.3.2. Recombinant Biocatalysis 33 1.4.3.3. Cofactor Recycling in Whole cells 34 1.4.3.4. Toxicity of Substrates and Products 35 1.4.4. Bioprocess Engineering Aspects 37 1.4.4.1. In Situ Product Removal 37 1.4.4.2. Solid Phase Extraction 37 V 1.4.4.3. Two-Liquid Phase Concept 38 1.4.4.4. Oxygen Transfer and Explosion Hazard 45 1.4.5. Perspectives of Biocatalytic Oxyfuctionalization 46 1.5. Scope of this Thesis 47 2. Xylene Monooxygenase Catalyzes the Multistep Oxygenation of Toluene and Pseudocumene to Corresponding Alcohols, Aldehydes and Acids in Escherichia coli JM101 49 3. Characterization and Application of Xylene Monooxygenase for Multistep Biocatalysis 75 4. Use of the Two-Liquid Phase Concept to Exploit Kinetically Controlled Multistep Biocatalysis 101 5. Chemical Biotechnology for the Specific Oxyfunctionalization of Hydrocarbons on a Technical Scale 131 6. Modeling and Simulation of a Whole-Cell Multistep Biooxidation Process in a Two-Liquid Phase System 151 7. Conclusions and Outlook 197 8. References 205 Curriculum Vitae 229 VI SUMMARY Oxygenases catalyze, among other interesting reactions, highly selective hydrocarbon oxyfunctionalizations, which are important in industrial organic synthesis but difficult to achieve by chemical means. Many enzymatic oxygenations have been described, but few of these have been scaled up to a commercial level, due to the complexity of oxygenase-based biocatalysts and demanding process implementation. The topic of this thesis was the specific oxyfunctionalization of xylenes by the use of xylene monooxygenase (XMO) from Pseudomonas putida mt-2. Recombinant Escherichia coli expressing the XMO genes xylM and xylA were found to catalyze the multistep oxidation of xylenes to corresponding alcohols, aldehydes, and acids. For all steps, the incorporation of molecular oxygen was verified. Kinetic analyzes to characterize this multistep oxygenation allowed to determine the kinetic parameters for the individual oxygenation steps. Furthermore, we found that the substrates toluene and pseudocumene and the corresponding alcohols inhibit aldehyde oxidation and that elevated toluene and pseudocumene concentrations also weakly inhibit alcohol oxidation. As an example for a technically demanding biooxidation, we exploited this complex multistep reaction for the production of 3,4-dimethylbenzaldehyde from pseudocumene. For this purpose, we combined recombinant whole-cell catalysis in an aqueous-organic two-liquid phase system with fed-batch cultivation in an optimized medium. A system with bis(2- ethylhexyl)phthalate as organic carrier solvent allowed the production of 3,4-dimethylbenz- aldehyde as the predominant product. Process optimization, scale up, and suitable downstream processing enabled the production of 484 ml 97% pure 3,4-dimethylbenz- aldehyde on a 30-L scale. A productivity of 31 g L-1 day-1 and a product concentration of 37 g L-1 in the organic phase were achieved. The higher biocatalyst activity at the expense of cell growth as a consequence of a pH increase pointed to a pH-influenced competition for NADH between XMO and the respiratory chain. A mathematical model including a pH-dependent metabolic inhibition of the NADH-consuming bioconversions allowed consistent simulation of experimental biotransformations performed at varying conditions. Moreover, bioconver- sion kinetics and process simulation based on this model indicated efficient substrate-cell transfer and the occurrence of direct substrate uptake from organic phase droplets. This work describes the characterization of a complex oxygenase-based biocatalyst and its implementation into a process on a technical scale, thus illustrating the general feasibility of industrial biocatalytic oxyfunctionalization. VII ZUSAMMENFASSUNG Oxygenasen katalysieren eine Vielzahl interessanter Reaktionen, darunter hoch spezifische Oxyfunktionalisierungen von Kohlenwasserstoffen. Solche, für die chemische Industrie relevante Reaktionen sind jedoch mit organisch chemischen Methoden schwierig durchführbar. Viele enzymatische Oxygenierungen wurden beschrieben, aber wenige
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