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

Paper ID Volume ID Publish Year Pages File Format Full-Text
3351 165 2013 11 PDF Available
Title
Non-linear kinetic modelling of reversible bioconversions: Application to the transaminase catalyzed synthesis of chiral amino-alcohols
Abstract

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)-(−)-α-methylbenzylamine (MBA) as amino donor. The whole cell biocatalyst expressed the CV2025 ω-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 to be generic and useful in regard to speeding up bioconversion process design and optimization.

► Created a rapid methodology to determine kinetic parameters for complex enzymatic reactions. ► Used statistical analysis to allow selection of the most appropriate kinetic model. ► Quantified kinetic parameters for the transaminase synthesis of chiral amino alcohols. ► Used microscale methods for rapid and efficient collection of enzyme kinetic data. ► Identified potential dead-end complex formation and bottlenecks for bioprocess design.

Keywords
Nonlinear kinetic modelling; Transaminase; Reversible bioconversions; Amino-alcohols; Microscale high-throughput; Reaction mechanism selection
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Non-linear kinetic modelling of reversible bioconversions: Application to the transaminase catalyzed synthesis of chiral amino-alcohols
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biochemical Engineering Journal - Volume 73, 15 April 2013, Pages 38–48
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us