Abstract
The (R)-phenylacetylcarbinol (PAC) batch biotransformation kinetics for partially purified Candida tropicalis TISTR 5350 pyruvate decarboxylase (PDC) were determined to validate a comprehensive mathematical model in 250 mL scale with 250 mM phosphate buffer/pH 7.0. PDC could convert initial 100/120 mM benzaldehyde/pyruvate substrates to the statistical significantly highest (p ≤ 0.05) maximum PAC concentration (95.8 ± 0.1 mM) and production rate (0.639 ± 0.001 mM min−1). A parameter search strategy aimed at minimizing overall residual sum of square (RSST) based on a system of six ordinary differential equations was applied to PAC biotransformation profiles with initial benzaldehyde/pyruvate concentration of 100/120 and 30/36 mM. Ten important biotransformation kinetic parameters were then elucidated including the zeroth order activation rate constant due to phosphate buffer species (ka) of (9.38 ± < 0.01) × 10–6% relative PDC activity min−1 mM−1. The validation of this model to independent biotransformation kinetics with initial benzaldehyde/pyruvate concentration of 50/60 mM resulted in relatively good fitting with RSST, mean sum of square error (MSE), and coefficient of determination (R2) values of 662, 17.4, and 0.9863, respectively.
Highlights
List of symbols A Pyruvate concentration B Benzaldehyde concentration convergence search criterion (CSC) Convergence search criterion
The PAC concentration could generally be increased through fed-batch processes with either pyruvate or benzaldehyde dosing protocols using 2.5 M or 20 mM of 3–morpholinopropane–1–sulfonic acid (MOPS) b uffer[18,19,20,33]
C. tropicalis TISTR 5350 was chosen based on optimal PAC production and volumetric pyruvate decarboxylase (PDC) carboligase activity in Yeast—Malt (YM) m edium[16,21,22,23,34]
Summary
List of symbols A Pyruvate concentration (mM) B Benzaldehyde concentration (mM) CSC Convergence search criterion (no unit). PAC could be produced through in vivo direct microbial transformation process with some strategies of benzaldehyde feeding using growing cells of yeasts, fungi, and bacteria[28,29,30] This biotransformation process can be conducted in vitro by using non-viable whole cells[15,17,24] and partially purified pyruvate decarboxylase (PDC) e nzyme[18,20,28,29,31,32,33,34]. Advantages of using partially purified PDC include prevention of benzyl alcohol or PAC-diol formation These are by-products that are often formed when PAC biotransformation is carried out in parallel with microbial cultivation process[32,35]. The heat labile property and cost-prohibitive nature of MOPS buffer (USD 1.09/g in comparison with only USD 0.02/g for phosphate buffer) were considered major obstacles to the industrial scale application of this buffering c ompound[34]
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