Journal
BIOTECHNOLOGY AND BIOENGINEERING
Volume 101, Issue 1, Pages 27-38Publisher
WILEY
DOI: 10.1002/bit.21867
Keywords
thiamine diphosphate; benzaldehyde lyase; kinetic modeling; parameter estimation; optimal experimental design; in silico experiments
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Funding
- Deutsche Forschungsgemeinschaft (Bonn, Germany)
- Collaborative Research Center [540]
- [GRK 1166]
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For reactions using thiamine diphosphate (ThDP)-dependent enzymes many empirically-derived kinetic models exist. However, there is a lack of mechanistic kinetic models. This is especially true for the synthesis of symmetric 2-hydroxy ketones from two identical aldehydes, with one substrate acting as the donor and the other as the acceptor. In this contribution, a systematic approach for deriving such a kinetic model for thiamine diphosphate (ThDP)-dependent enzymes is presented. The derived mechanistic kinetic model takes this donor-acceptor principle into account by containing two K-m-values even for identical substrate molecules. As example the stereoselective carbon-carbon coupling of two 3,5-dimethoxy-benzaldehyde molecules to (R)-3,3',5,5-tetramethoxy-benzoin using benzaldehyde lyase (EC 4.1.2.38) from Pseudomonas fluorescens is studied. The model is derived using a model-based experimental analysis method which includes parameter estimation, model analysis, optimal experimental design, in silico experiments, sensitivity analysis and model revision. It is shown that this approach leads to a robust kinetic model with accurate parameter estimates and an excellent prediction capability. Biotechnol. Bioeng. 2008;101: 27-38. (C) 2008 Wiley Periodicals, Inc.
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