Fig. 2: Fig. 2: Enzyme deactivation profile (  ) with model fitting (line) in the absence of benzaldehyde (a) and in the presence of benzaldehyde of various concentrations: (b) 9.49 mM, (c) 19.0 mM, (d) 31.6 mM, (e) 41.1 mM, (f) 51.4 mM, (g) 60.0 mM, (h) 101 mM, (i) 153 mM and (j) 202 mM. The experiment was performed in glass vials containing 2.5 M MOPS buffer (pH 7.0), 1 mM MgSO 4 and 1 mM TPP with initial enzyme activity in the range of 2.8 – 3.2 U ml -1 at 6˚C. The enzyme activity of each data point was an average from three replicates. Error bars show the lowest and highest values. Model development of pyruvate decarboxylase deactivation kinetics by benzaldehyde N. Leksawasdi 1,2, M. Breuer 3, B. Hauer 3, P. L. Rogers 1, B. Rosche 1 1 School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia 2 Department of Food Engineering, Faculty of Agro-Industry, Chiang Mai University, Chiang Mai, 50000, Thailand 3 BASF-AG, Fine Chemicals and Biocatalysis Research, Ludwigshafen, Germany RESULTS OBJECTIVE To develop a mathematical model for PDC (from Candida utilis) deactivation kinetics by benzaldehyde in the concentration range of 0 – 200 mM. RESULTS CONCLUSION A kinetic model has been developed which provides good fitting to the experimental deactivation of PDC in 2.5 M MOPS, 1 mM MgSO 4, 1 mM TPP, pH 7.0 at 6  C with various initial concentrations of benzaldehyde up to 200 mM. Using standard statistical analysis, it was established that the model provided a good overall fit for these deactivation profiles. The rate equation for PDC deactivation derived from this study will be used with other differential equations in modelling the enzymatic production of R-PAC from pyruvate and benzaldehyde. REFERENCES Bailey, J.E., Ollis, D.F. (1986) Biochemical Engineering Fundamentals, 2nd edn. Singapore: McGraw-Hill, pp Goetz, G., Iwan, P., Hauer, B., Breuer, M., Pohl, M. (2001) Biotechnol. Bioeng. 74, 317–325. Han, K., Levenspiel, O. (1988) Biotechnol. Bioeng. 32, 430 – 437. Iwan, P., Goetz, G., Schmitz, S., Hauer, B., Breuer, M., Pohl, M. (2001) J. Mol. Cat. B: Enzymatic. 11, Leksawasdi, N., Joachimsthal, E.L., Rogers, P.L. (2001) Biotechnol. Lett. 23, Long, A., Ward, O.P. (1989) Biotechnol. Bioeng. 34, 993 – 941. Rosche, B., Breuer, M., Hauer, B., Rogers, P.L. (2003) Biotechnol. Lett. 25, Rosche, B., Leksawasdi, N., Sandford, V., Breuer, M., Hauer, B., Rogers, P.L. (2002a) Appl. Microbiol. Biotechnol. 60, Rosche, B., Sandford, V., Breuer, M., Hauer, B., Rogers, P.L. (2001) Appl. Microbiol. Biotechnol. 57, Rosche, B., Sandford, V., Breuer, M., Hauer, B., Rogers, P.L. (2002b) J. Mol. Cat. B, Enzymatic 19, 109 – 115. Shin, H.S., Rogers, P.L. (1996) Biotechnol. Bioeng. 49, 52 – 62. INTRODUCTION Fig. 1 ( R)-phenylacetylcarbinol (R-PAC) is a key intermediate for the production of the anti-asthmatic and nasal decongestant compounds, ephedrine and pseudoephedrine. An enzymatic process for production of R-PAC via the biotransformation of benzaldehyde and pyruvate by pyruvate decarboxylase (PDC) is currently under development (Shin & Rogers 1996, Rosche et al. 2001, 2002a,b, 2003) and has shown significant improvements when compared to the traditional yeast-based fermentation process. The enzymatic process for the biotransformation is illustrated in Fig. 1. Benzaldehyde which supplies the aromatic ring to R-PAC is toxic (Goetz et al. 2001, Iwan et al. 2001). Continuous exposure of the enzyme to benzaldehyde results in irreversible deactivation of PDC (Long & Ward 1989). Understanding the deactivation kinetics of PDC by benzaldehyde is necessary in the optimisation of the biotransformation process as sufficient enzyme activity must be maintained for an extended period so that the production of R-PAC can be maximised. Fig. 1 Fig. 1: Formation of R-PAC from benzaldehyde & pyruvate by PDC Fig. 2(a)–2(j). Fig. 2(f) The experimental results of PDC deactivation for various benzaldehyde concentrations are shown in Fig. 2(a)–2(j). A lag period of more than 10 h occurred before enzyme deactivation when benzaldehyde concentrations were less than 40 mM. At higher concentrations of benzaldehyde, the lag time was shortened to about 5 h. The deactivation profile of PDC by 50 mM benzaldehyde (Fig. 2(f)) shows results from two different batches of enzyme. The similarity of both profiles suggests good experimental data reproducibility between enzyme batches. Experimental deactivation kinetics Equation (1) The general deactivation model proposed by Han & Levenspiel (1988) was modified by addition of an initial period of constant enzyme activity (t lag ). This initial phase of constant (or elevated) activity has been discussed previously (Rosche et al. 2002a) as possibly being due to enzyme refolding following its solubilization from the PDC acetone precipitate. In the present study, the overall deactivation kinetics given in Equation (1) incorporates the effect of t lag, the first order time dependency (k d1 ) and the first order benzaldehyde deactivation (k d2.b) to give a typical pattern of exponential enzyme decay as described by Bailey & Ollis (1986). Model for PDC deactivation Model fitting to experimental data Equation (1) Fig. 2(a) – 2(j) The optimal values of the kinetic constants derived from the modelling program (Leksawasdi et al. 2001) after fitting Equation (1) to experimental data points were as follows: k d1 = 2.64 x h -1, k d2 = 1.98 x mM -1 h -1, and t lag = 5.23 h. The average correlation coefficient (R 2 ), Residual Sum of Squares (RSS), and Mean Sum of Square for Residuals (MS) for the fittings were , 4936, and 91.4 respectively. The resultant simulation curves from the model are shown in Fig. 2(a) – 2(j) for comparison with experimental data points. Benzaldehyde concentration in mM