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Phenylacetylcarbinol(PAC) Synthesis in Aqueous/Organic Two-Phase System

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Phenylacetylcarbinol(PAC) Synthesis in Aqueous/Organic Two-Phase System Bioprocess Development for (R)-phenylacetylcarbinol (PAC) Synthesis in Aqueous/Organic Two-Phase System Cindy Gunawan, B.E. A thesis submitted in fulfillment of the requirements for the Degree of Doctor of Philosophy School of Biotechnology and Biomolecular Sciences University of New South Wales Sydney, Australia March 2006 Declaration I hereby declare that this submission is my own work and to the best of my knowledge it contains no material previously published or written by another person, nor material which to a substantial extent has been accepted for the award of any degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in this thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowledged. __________________________ Cindy Gunawan for papi and mami Acknowledgements i ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my supervisor Professor Peter L. Rogers and co-supervisor Dr Bettina Rosche for their endless guidance, support, and patience throughout my project and in the preparation of this thesis. I am very thankful to the Australian Government for awarding me the Endeavour International Postgraduate Research Scholarship, which has given me priceless opportunities and invaluable experience in research. I would also like to thank BASF Ludwigshafen for their sponsorhip of the project. I am very grateful to Dr. Martin Zarka, Dr. Russel Cail, and Malcolm Noble for their technical support and to Gerry Ferhard, Peter, and John of the Faculty of Science Workshop for constructing the Lewis cell. A warm thank you to all my colleagues Lia, Allen, Noppol, André, and Richard for their opinions, support, and those happy times. I would also like to thank all my friends Eny, Ronachai, Onn, Yin, Nico, Adrian and all the staff in the School of Biotechnology and Biomolecular Sciences (BABS) for their friendship. My greatest gratitude to my parents, sisters and brothers; Lingling, Mona, Cangcang, and Penpen for their kindness and warm love. Finally, I wish to thank my husband Frans for his beautiful heart and endless encouragement. Abstract ii ABSTRACT (R)-phenylacetylcarbinol or R-PAC is a chiral precursor for the synthesis of pharmaceuticals ephedrine and pseudoephedrine. PAC is produced through biotransformation of pyruvate and benzaldehyde catalyzed by pyruvate decarboxylase (PDC) enzyme. The present research project aims at characterizing a two-phase aqueous/organic process for enzymatic PAC production. In a comparative study of several selected yeast PDCs, the highest PAC formation was achieved in systems with relatively high benzaldehyde concentrations when using C. utilis PDC. C. tropicalis PDC was associated with the lowest by-product acetoin formation although it also produced lower PAC concentrations. C. utilis PDC was therefore selected as the biocatalyst for the development of the two-phase PAC production. From an enzyme stability study it was established that PDC deactivation rates in the two- phase aqueous/octanol-benzaldehyde system were affected by: (1) soluble octanol and benzaldehyde in the aqueous phase, (2) agitation rate, (3) aqueous/organic interfacial area, and (4) initial enzyme concentration. PDC deactivation was less severe in the slowly stirred phase-separated system (low interfacial area) compared to the rapidly stirred emulsion system (high interfacial area), however the latter system was presumably associated with a faster rate of organic-aqueous benzaldehyde transfer. To find a balance between maintaining enzyme stability while enhancing PAC productivity, a two-phase system was designed to reduce the interfacial contact by decreasing the organic to aqueous phase volume ratio. Lowering the ratio from 1:1 to 0.43:1 resulted in increased overall PAC production at 4°C and 20°C (2.5 M MOPS, partially purified PDC) with a higher concentration at the higher temperature. The PAC was highly concentrated in the organic phase with 212 g/L at 0.43:1 in comparison to 111 g/L at 1:1 ratio at 20°C. The potential of further two-phase process simplification was evaluated by reducing the expensive MOPS concentration to 20 mM (pH controlled at 7.0) and employment of Abstract iii whole cell PDC. It was found that 20°C was the optimum temperature for PAC production in such a system, however under these conditions lowering the phase ratio resulted in decreased overall PAC production. Two-phase PAC production was relatively low in 20 mM MOPS compared to biotransformations in 2.5 M MOPS. Addition of 2.5 M dipropylene glycol (DPG) into the aqueous phase with 20 mM MOPS at 0.25:1 ratio and 20°C improved the production with organic phase containing 95 g/L PAC. Although the productivity was lower, the system may have the benefit of a reduction in production cost. Publications iv PUBLICATIONS Published Paper C. Gunawan, G. Satianegara, A.K. Chen, M. Breuer, B..Hauer, P.L. Rogers, B. Rosche. (2006). Yeast Pyruvate Decarboxylases: Variation in Biocatalytic Characteristics for (R)- phenylacetylcarbinol Production. FEMS Yeast Research: doi:10.1111/j.1567- 1364.2006.00138.x (with pending volume, issue and page numbers). Paper in preparation C. Gunawan, M. Breuer, B..Hauer, P.L. Rogers, B. Rosche. (2006). Key Factors Influencing Enzyme Stability and Biotransformation in Two-Phase Aqueous/Organic System for (R)-phenylacetylcarbinol Production. In preparation for submission to Biotechnology and Bioengineering Journal. Poster and oral presentations C. Gunawan, M. Breuer, B. Hauer, P.L. Rogers, B. Rosche. (2003). Impact of Process Parameters on R-phenylacetylcarbinol (PAC) Production in Aqueous/Organic Two-Phase Biotransformation, Fermentation and Bioprocessing Conference. The Garvan Institute for Medical Research, Sydney, Australia, 14 – 15 April, poster presentation p. 52, ISBN 0 7334 2023 0. C. Gunawan, M. Breuer, B. Hauer, P.L. Rogers, B. Rosche. (2003). Process Development for R-phenylacetylcarbinol (PAC) Production in Aqueous/Organic Two- Phase Biotransformation. 6th International Symposium on Biocatalysis and Biotransformations. BIOTRANS 2003, Palacky University, Olomouc, Czech Republic, 28 June – 3 July, poster presentation number 245, p. 507, ISSN 0009-2770. Publications v C. Gunawan, M. Breuer, B. Hauer, P.L. Rogers, B. Rosche. (2003). Investigation on R- phenylacetylcarbinol (PAC) Production in Aqueous/Organic Two-Phase Biotransformation, School of Biotechnology and Biomolecular Sciences Third Annual Symposium, Sydney, Australia, 7 November 2003, poster presentation P-12, ISBN 0 7334 1581 4. C. Gunawan, M. Breuer, B. Hauer, P.L. Rogers, B. Rosche. (2004). Effect of Organic to Aqueous Phase Volume Ratio in Two-Phase System for R-phenylacetylcarbinol Biosynthesis, Fermentation and Bioprocessing Conference. UQ Centre University of Queensland, Brisbane, Australia, 5 – 6 July 2004, poster presentation number 21, p.55, ISBN 0 646 43707 0. C. Gunawan, M. Breuer, B. Hauer, P.L. Rogers, B. Rosche. (2004). Bioprocess Development for R-phenylacetylcarbinol (PAC) Production in Aqueous/Organic Two- Phase System, School of Biotechnology and Biomolecular Sciences Third Annual Symposium, Sydney, Australia, 5 November 2004, oral presentation 2-1, ISBN 0 7334 2162 8. C. Gunawan, M. Breuer, B. Hauer, P.L. Rogers, B. Rosche. (2005). Optimization of Aqueous/Organic Two-Phase System for (R)-phenylacetylcarbinol (PAC) Biosynthesis, 7th International Symposium on Biocatalysis and Biotransformations. BIOTRANS 2005, TU Delft, Delft, The Netherlands, 3 – 8 July, poster presentation number 147, p. 147, ISBN 90 809691 17. G. Satianegara, C. Gunawan, A.K. Chen, M. Breuer, B. Hauer, P.L. Rogers, B. Rosche. (2003). R-phenylacetylcarbinol (R-PAC) Production and Stability Study with Pyruvate Decarboxylase from Four Yeast Strains, Fermentation and Bioprocessing Conference. The Garvan Institute for Medical Research, Sydney, Australia, 14 – 15 April, poster presentation p. 63, ISBN 0 7334 2023 0. Publications vi G. Satianegara, C. Gunawan, A.K. Chen, M. Breuer, B. Hauer, P.L. Rogers, B. Rosche. Comparison of Four Yeast Pyruvate Decarboxylase for R-phenylacetylcarbinol Production, 6th International Symposium on Biocatalysis and Biotransformations. BIOTRANS 2003, Palacky University, Olomouc, Czech Republic, 28 June – 3 July, poster presentation number 119, p. 430, ISSN 0009-2770. B. Rosche, V. Sandford, N. Leksawasdi, A. Chen, G. Satianegara, C. Gunawan, M. Breuer, B. Hauer, P.L. Rogers. (2003). Bioprocess development for ephedrine production, 6th International Symposium on Biocatalysis and Biotransformations. BIOTRANS 2003, Palacky University, Olomouc, Czech Republic, 28 June – 3 July, poster presentation P224, ISSN 0009-2770. Table of Contents vii TABLE OF CONTENTS ACKNOWLEDGEMENTS............................................................................................i ABSTRACT...................................................................................................................ii PUBLICATIONS..........................................................................................................iv TABLE OF CONTENTS ...........................................................................................viii LIST OF TABLES....................................................................................................xivv
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