Model-based experimental analysis of enzyme kinetics in aqueous-organic biphasic systems

  • Modellgestützte experimentelle Analyse von Enzymkinetiken in wässrig-organischen Zweiphasensystemen

Zavrel, Michael; Büchs, Jochen (Thesis advisor)

Aachen : Publikationsserver der RWTH Aachen University (2009)
Dissertation / PhD Thesis

Aachen, Techn. Hochsch., Diss., 2009


Immobilization of biocatalysts in hydrogel beads which are suspended in organic solvents is a promising approach for the production of hydrophobic fine chemicals. Due to the superposition of mass transfer, diffusion, and enzyme reaction the rational design of such immobilizates is rather complex. For this reason, a mechanistic kinetic model considering all three phenomena was derived. As an example, the stereoselective carboligation of two 3,5-dimethoxy-benzaldehyde (DMBA) molecules to (R)-3,3',5,5'-tetramethoxy-benzoin (TMB) catalyzed by the enzyme benzaldehyde lyase (BAL) was investigated. BAL was immobilized in kappa-carrageenan hydrogel beads which were surrounded by an organic solvent. In the first step the phenomena enzyme reaction, mass transfer, and diffusion were studied in separate systems. Each system was individually investigated using the model-based experimental analysis (MEXA) approach including a priori simulations, sensitivity analysis, and optimal experimental design. This supports not only the development of mechanistic kinetic models, but also the determination of optimal measurement methods and the development of new experimental setups. Subsequently, the derived individual kinetic models were successively coupled and finally combined to a kinetic model for the gel-stabilized aqueous-organic biphasic reaction system. Using the MEXA approach, not only the kinetic parameters could be estimated with high precision, but also new mechanistic models could be developed which revealed possible limitations and bottlenecks. Accordingly, the release of the product was identified as rate-limiting step in the enzymatic mechanism of BAL, whereas the binding of the substrate turned out as the catalytic bottleneck in the mechanism of benzoylformate decarboxylase (BFD). Furthermore, a new approach for solvent selection was developed in order to optimize the extractable yield of the carboligation reaction in a biphasic system. Consequently, methyl-iso-butyl-ketone was chosen as organic solvent. The diffusion of the reactants in the hydrogel bead was investigated in the bead center, which was identified as the optimal measurement position using a priori simulations. It was demonstrated on the example of propionic acid that Nernst-Planck law is superior to Fick's law for modeling the diffusion of dissociating species in hydrogel beads. The coupling of the sub-systems revealed that sophisticated experimental design is crucial to avoid limitations due to the superposition of the phenomena. For instance, in a stirred biphasic system the enzyme kinetic parameters can only be identified if the reaction is rate-limiting. Furthermore, measurements in both phases are required. Emulsification should be avoided since the formation of aggregates was observed which indicates enzyme precipitation due to phase toxicity. Finally, the parameter estimates for the gel-stabilized aqueous-organic biphasic reaction system were compared to those obtained for the individual and coupled systems. No significant influence of the reaction system on the enzyme kinetic parameters, on the partition coefficients, and on the effective diffusion coefficient of the substrate could be observed. However, the estimated values of the mass transfer coefficients and the effective diffusion coefficient of the product deviated from system to system. Therefore, for the rational design of enzyme immobilizates the model parameters have to be determined using the complete system. Adopting the parameter estimates obtained from individual systems may lead to an incorrect model prediction and an inefficient process design. The derived mechanistic kinetic model for the gel-stabilized aqueous-organic system allows to detect limitations caused by diffusion or mass transfer, and paves the way for the rational design of enzyme immobilizates and for the optimization of such processes. It can easily be adapted to other reaction systems, biocatalysts, solvents, and geometries of immobilizates. Moreover, the identification of catalytic bottlenecks in the enzyme mechanisms of BAL and BFD will certainly support the development of new enzyme variants with enhanced activities.