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Kinetics and Modelling of Enzymatic Process for R-Phenylacetylcarbinol (PAC) Production

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Kinetics and Modelling of Enzymatic Process for R-Phenylacetylcarbinol (PAC) Production Kinetics and Modelling of Enzymatic Process for R-phenylacetylcarbinol (PAC) Production Noppol Leksawasdi A thesis submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy School of Biotechnology and Biomolecular Sciences University of New South Wales Sydney, Australia August, 2004 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. ___________________________ Noppol Leksawasdi For The Royal Thai Government i Acknowledgements Acknowledgements I wish to express my greatest gratitude and thanks to my supervisor, Professor Peter L. Rogers and my co-supervisor, Dr. Bettina Rosche for their excellent guidance, invaluable advice, support and encouragement throughout the course of this study and preparation of this thesis. I am very grateful to the Royal Thai Government (RTG) for the award of this scholarship and for providing me with the opportunity to undertake this study. I will always be grateful for the reassurance of continual financial support throughout my study from the RTG when our country faced an economic crisis during 1997-1998. I would like to thank the sponsors of the project, BASF Ludwigschafen, for their financial support for equipment and materials. My sincere thanks are also due to Dr. Russell Cail, Malcolm Noble, Martin Zarka, Wolfgang Nittel and Dr. Christopher Marquis for their technical assistance and thoughtful suggestions. A very special thank you to Alan, Vanessa, Allen, Gernalia, Cindy, Richard, Charles, Nick, Eny, Jae, Apple, Jeab, Mallika, Ronachai, Olarn, Astutiati, Caleb and Fiona for their help and suggestions. I wish to extend my thanks also to the students and staffs of the School of Biotechnology and Biomolecular Sciences (BABS) for their valued friendship. My heartfelt appreciation goes to my parents and my brothers for their understanding, encouragement and endurance during the years. I am also grateful to the kindness and generosity of my landlord and landlady, Arthur and Marge Keeble as well as my former landlady, Wendy Ryan. Last but not least, I wish to thank Onn for her kindness, friendship, support, wonderful meals and innumerable help. Noppol Leksawasdi PhD Thesis 2004 ii Abstract Abstract R-phenylacetylcarbinol (PAC) is used as a precursor for production of ephedrine and pseudoephedrine, which are anti-asthmatics and nasal decongestants. PAC is produced from benzaldehyde and pyruvate mediated by pyruvate decarboxylase (PDC). A strain of Rhizopus javanicus was evaluated for its production of PDC. The morphology of R. javanicus was influenced by the degree of aeration/agitation. A relatively high specific PDC activity (328 U decarboxylase g-1 mycelium) was achieved when aeration/agitation were reduced significantly in the latter stages of cultivation. The stability of partially purified PDC and crude extract from R. javanicus were evaluated by examining the enzyme deactivation kinetic in various conditions. R. javanicus PDC was less stable than Candida utilis PDC currently used in our group. A kinetic model for the deactivation of partially purified PDC extracted from C. utilis by benzaldehyde (0–200 mM) in 2.5 M MOPS buffer has been developed. An initial lag period prior to deactivation was found to occur, with first order dependencies of PDC deactivation on exposure time and on benzaldehyde concentration. A mathematical model for the enzymatic biotransformation of PAC and its associated by-products has been developed using a schematic method devised by King and Altman (1956) for deriving the rate equations. The rate equations for substrates, product and by- products have been derived from the patterns for yeast PDC and combined with a deactivation model for PDC from C. utilis. Initial rate and biotransformation studies were applied to refine and validate a mathematical model for PAC production. The rate of PAC formation was directly proportional to the enzyme activity level up to 5.0 U carboligase ml-1. Michaelis- Menten kinetics were determined for the effect of pyruvate concentration on the Noppol Leksawasdi PhD Thesis 2004 iii Abstract reaction rate. The effect of benzaldehyde on the rate of PAC production followed the sigmoidal shape of the Monod-Wyman-Changeux (MWC) model. The biotransformation model, which also included a term for PDC inactivation by benzaldehyde, was used to determine the overall rate constants for the formation of PAC, acetaldehyde and acetoin. Implementation of digital pH control for PAC production in a well-stirred organic- aqueous two-phase biotransformation system with 20 mM MOPS and 2.5 M dipropylene glycol (DPG) in aqueous phase resulted in similar level of PAC production [1.01 M (151 g l-1) in an organic phase and 115 mM (17.2 g l-1) in an aqueous phase after 47 h] to the system with a more expensive 2.5 M MOPS buffer. Noppol Leksawasdi PhD Thesis 2004 iv Publications Publications Published papers (1) PAC production Rosche, B., Leksawasdi, N., Sandford, V., Breuer, M., Hauer, B. and Rogers, P.L. (2002). Enzymatic (R)-phenylacetylcarbinol production in benzaldehyde emulsions, Applied Microbiology and Biotechnology 60: 94-100. Leksawasdi, N., Breuer, M., Hauer, B., Rosche, B. and Rogers, P.L. (2003). Kinetics of pyruvate decarboxylase deactivation by benzaldehyde, Biocatalysis and Biotransformation 21: 315-320. Leksawasdi, N., Chow, Y.Y.S., Breuer, M., Hauer, M., Rosche, B. and Rogers, P.L. (2004). Kinetic studies and model validation of enzymatic (R)-phenylacetylcarbinol batch biotransformation process, Journal of Biotechnology, 111: 179-189. (2) Others Leksawasdi, N., Joachimsthal, E.L. and Rogers, P.L. (2001). Mathematical modelling of ethanol production from glucose/xylose mixtures by recombinant Zymomonas mobilis, Biotechnology Letters 23: 1087–1093. Boonmee, M., Leksawasdi, N., Bridge, W. and Rogers, P.L. (2003). Batch and continuous culture of Lactococcus lactis NZ133: experimental data and model development, Biochemical Engineering Journal 14: 127-135. Noppol Leksawasdi PhD Thesis 2004 v Publications Pulsawat, W., Leksawasdi, N., Rogers, P.L. and Foster, L.J.R. (2003). Anion effects on biosorption of Mn(II) by extracellular polymeric substance (EPS) from Rhizobium etli, Biotechnology Letters 25: 1267-1270. Papers in preparation (1) PAC production Leksawasdi, N., Breuer, M., Hauer, B., Rosche, B. and Rogers, P.L. (2004). Production of pyruvate decarboxylase from Rhizopus javanicus and its deactivation kinetics, Manuscript Submitted (Enzyme and Microbial Technology). Leksawasdi, N., Breuer, M., Hauer, B., Rosche, B. and Rogers, P.L. (2004). Mathematical model for kinetics of enzymatic conversion of benzaldehyde and pyruvate to (R)-phenylacetylcarbinol, Manuscript Submitted (Biochemical Engineering Journal). Leksawasdi, N., Breuer, M., Hauer, B., Rosche, B. and Rogers, P.L. (2004). Cost effective development of two-phase biotransformation for (R)-phenylacetylcarbinol production, Manuscript in preparation. Sandford, V., Leksawasdi, N., Breuer, M., Hauer, B., Rosche B. and Rogers, P.L. (2004). Factors affecting product formation and enzyme stability in the production of (R)-phenylacetylcarbinol by Candida utilis pyruvate decarboxylase, Manuscript in preparation. (2) Others Boonmee, M., Leksawasdi, N., Bridge, W. and Rogers, P.L. (2004). Evaluation of electrodialysis for lactate removal and pH control in the production of the dairy starter culture Lactococcus lactis NZ133, Manuscript Submitted (Journal of Membrane Science). Noppol Leksawasdi PhD Thesis 2004 vi Publications Boonmee, M., Leksawasdi, N., Bridge, W. and Rogers, P.L. (2004). Use of anion exchange resin for lactate removal and pH control in the production of the dairy starter culture Lactococcus lactis NZ133, Manuscript in preparation. Conference Proceedings Borompichaichartkul, C., Moran, G., Srzednicki, G., Driscoll, R., Leksawasdi, N. and Ball, G. (2001). Studies of physical state of water in frozen china maize (Huangmo 417) using NMR technique-deuterium relaxation and DSC, Quality management and market access: Proceedings of the 20th ASEAN/2nd APEC, seminar on postharvest technology. Lotus Hotel Pang Suan Kaew, Chiang Mai, Thailand, 11–14 September, pp. 215-220, ISBN 974 436 249 9. Poster and oral presentations (1) PAC production Leksawasdi, N., Rosche, B. and Rogers, P.L. (2002). Mathematical model development of R-PAC biotransformation in benzaldehyde emulsions, School of Biotechnology and Biomolecular Sciences First Annual Symposium, Sydney, Australia, 8 November 2002, oral presentation III-2, ISBN 0 7334 1581 4. Leksawasdi, N., Breuer, M, Hauer, B., Rosche, B. and Rogers, P.L. (2003). Model development of pyruvate decarboxylase deactivation kinetics by benzaldehyde, School of Biotechnology and Biomolecular Sciences Second Annual Symposium, Sydney, Australia, 7 November 2003, poster presentation P-22, ISBN 0 7334 1581 4. Leksawasdi, N., Breuer, M., Hauer, B., Rogers,
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