Non-linear kinetic modelling of reversible bioconversions: Application to the transaminase catalyzed synthesis of chiral amino-alcohols

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Rios-Solis et al. – 2013 – Non-linear kinetic modelling of reversible bioconversions Application to the transaminase catalyzed synthesis of chiral amino-alcohols

Biochemical Engineering Journal 73 (2013) 38– 48

L. Rios-Solis a, N. Bayira, M. Halima, C. Dua, J.M. Wardb, F. Baganza, G.J. Lyea,∗
a Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK
b Institute of Structural and Molecular Biology, Darwin Building, University College London, Gower Street, London WC1E 6BT, UK


This work describes the establishment of a full kinetic model, including values of apparent kinetic
parameters, for the whole cell E. coli mediated synthesis of the chiral amino-alcohol (2S,3R)-2-amino-
1,3,4-butanetriol (ABT), using (S)-(−)--methylbenzylamine (MBA) as amino donor. The whole cell
biocatalyst expressed the CV2025 -transaminase from Chromobacterium violaceum. Establishment of
the most suitable reaction mechanism and determination of the complete forward and reverse kinetic
parameter values for the reversible bioconversion where obtained using a hybrid methodology. This
combined traditional initial rate experiments to identify a solution in the vicinity of the global minimum,
with nonlinear regression methods to determine the exact location of the solution. The systematic
procedure included selection and statistical evaluation of different kinetic models that best described
the measured reaction rates and which ultimately provided new insights into the reaction mechanism;
in particular the possible formation of a dead end complex between the amino donor and the cofactor
enzyme complex. The hybrid methodology was combined with a microscale experimental platform, to
significantly reduce both the number of experiments required as well as the time and material required
for full kinetic parameter estimation. The equilibrium constant was determined to be 849, and the forward
and reverse rate constants were found to be 97 and 13 min−1, respectively, which greatly favoured
the asymmetric synthesis of chiral ABT. Using the established kinetic model, the asymmetric synthesis
of ABT was simulated, and excellent agreement was found between the experimental and predicted data
over a range of reaction conditions. A sensitivity analysis combined with various simulations suggested
the crucial bottleneck of the reaction was the second half reaction of the ping pong bi–bi mechanism,
in part due to the low Michaelis constant of substrate l-erythrulose (ERY). The toxicity of MBA towards
the transaminase was identified as another major bottleneck. The kinetic model was useful to give early
insights into the most appropriate bioconversion conditions, which can improve the rate and yield of ABT
formation, as well as minimizing the toxicity and inhibition effects of the substrates and products. The
systematic methodology developed here is considered

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