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Equilibrium of enzyme-catalyzed reactions and enzyme stability under influence of ionic liquids

The presence of ionic liquids (ILs) in the reaction medium of enzyme-catalyzed reactions allows increasing water solubility of reactants and conversion or selectivity significantly. However, ILs affect both, the thermodynamic reaction equilibrium and enzyme stability. These two phenomena are considered separately in this work.




Enzyme-catalyzed reactions become increasingly important for the development of new alternative synthetic pathways in industrial production processes. However, chemical and biochemical reactions often have multiple bottlenecks, e.g. low yield, selectivity, or solubility of the reactants in the reaction system. Thus, usually the reaction conditions (e.g. addition of solvents, changing pH or temperature) are varied in order to increase yield, selectivity, or reactant solubility.
One category of substances that can be added to the reaction medium is the class of ionic liquids (ILs) [1,2,3]. These may be used in single-phase reaction systems, either as pure solvents or as co-solvents. ILs are organic salts that are usually liquid at moderate temperatures (<100 °C) and have extremely low vapor pressures. Physical properties such as density, solubility or viscosity of ILs depend on the IL-cation and IL-anion that are the constituents of an IL. Since these properties can be adapted to specific applications by targeted modification of the IL-cation and IL-anion, ILs are also called "tailor-made solvents". Due to their non-flammable and thermally stable properties, ILs are also referred to as "green solvents" and have shown great potential to replace traditional, flammable and toxic organic solvents [2,3].
The focus of previous publications is on the effect of ILs on reaction kinetics [2]. However, ILs also affect both, the thermodynamic reaction equilibrium and enzyme stability [4,6]. Thermodynamic models are promising tools to describe the influence of ILs on enzyme-catalyzed reactions. However, the complexity and the ionic character of the biomolecules and the ILs represent an enormous demand for thermodynamic modelling [6,7]. Further, the catalytic activity of the enzyme will be influenced by the presence of ILs. Thus, considering reactions in IL-containing medium must involve enzyme-activity investigations [1].
The aim of this work is to investigate the effect of type and concentration of different ILs on the thermodynamic equilibrium of enzyme-catalyzed reactions and the effect on the enzyme stability [5].
For that purpose the alanine aminotransferase-reaction (ALAT: L-alanine + 2-oxoglutarate ↔ L-glutamate + pyruvate) as well as the hydrogenation of acetophenone catalyzed by an alcohol dehydrogenase (ADH: acetophenone ↔ phenylethanol) will be investigated. The equilibrium concentrations of the reactants and products will be measured at different IL and substrate concentrations. The equilibrium concentrations allow determining the Kx values as a function of concentrations of ILs and substrates. From that dependence the Kgamma,exp. values which are defined by the activity coefficients of the products and reactants can be determined. In all experiments the reaction-medium conditions such as pH and ionic strength are controlled accurately since they play an important role especially in biological reactions. Kgamma,calc. values of the reaction will be predicted using the thermodynamic model ePC-SAFT and compared with experimental Kgamma,exp. values.
Besides the influence of ILs on the reaction equilibrium, the knowledge of the influence on the stability of the investigated enzyme is extremely important. In order to characterize the effect of type and concentration of the IL on the stability of enzymes, the unfolding temperature (TN->U) of the native enzyme in media containing IL is determined experimentally. The combined investigation of reaction equilibria and enzyme stability will contribute to make use of ILs as solvents/cosolvents in enzyme-catalyzed reactions accessible.



[1] S. Dreyer and U. Kragl:
"Ionic Liquids for Aqueous Two-Phase Extraction and Stabilization of Enzymes."
Biotechnology and Bioengineering, vol. 99, pp. 1416-1424, 2008
[2] U. Kragl, M. Eckstein and N. Kaftzik:
"Enzyme catalysis in ionic liquids."
Current Opinion in Biotechnology, vol. 13, pp. 565-571, 2002
[3] M. Moniruzzaman, K. Nakashima, N. Kamiya and M. Goto:
"Recent advances of enzymatic reactions in ionic liquids."
Biochemical Engineering Journal, vol. 48, pp. 295-314, 2010
[4] P. Attri and P. Venkatesu:
"Exploring the thermal stability of α-chymotrypsin in protic ionic liquids."
Process Biochemistry, vol. 48, pp. 462-470, 2013
[5] H. Passos, I. Khan, F. Mutelet, M. Oliveira, L. Santos, C. Held, G. Sadowski, M. Freire and J. Coutinho:
"Vapor−Liquid Equilibria of Water + Alkylimidazolium-Based Ionic Liquids: Measurements and Perturbed Chain Statistical Associating Fluid Theory Modeling."
Industrial & Engineering Chemistry Research, vol. 53, pp. 3737−3748, 2014



Foto von PD Dr.-Ing. Christoph Held

PD Dr.-Ing. Christoph Held


TU Dortmund
Fakultät Bio- und Chemieingenieurwesen
Emil-Figge-Str. 70
44227 Dortmund

Raum G2-513