(717h) Overcoming Thermodynamic Limitation on the Yield of Aminotransferase Reactions

The maximum yield of many enzyme-catalyzed reactions, as of aminotransferase reactions, is limited by thermodynamic equilibrium. Therefore, the position of the thermodynamic reaction equilibrium has to be known. Reaction equilibria can be described by the thermodynamic (activity-based) equilibrium constant K_a*, which is accessible by the concentrations of products and reactants (K values) and their activity coefficients (K_γ* values) at reaction equilibrium. For an optimal process design, the influence of reaction conditions (T, pH, reactant molalities) on the reaction equilibrium (K values) has to be known. Enzymatic reactions can also be influenced by additives, e.g. ionic liquids or osmolytes. In those cases, the effect of additives on the stability of enzymes and on the reaction equilibrium (and thus the maximum yield) has to be known for process optimization.

In this work the thermodynamic equilibrium of the reaction (1)

L-alanine + 2-oxoglutarate â?? L-glutamate + pyruvate

catalyzed by alanine aminotransferase (ALAT) in aqueous media has been investigated. The thermodynamic equilibrium constant K_a* of reaction (1) was determined. K values were measured in the absence of additives between 25 and 37 °C at 5<pH<9 for different initial molalities of the reactants. Further, the influence of additives (1-butyl-3-methylimidazolium trifluoro­methane­sulfonate, 1-butyl-3-methyl­imidazolium chloride, 1-ethyl-3-methylimidazolium chloride, choline chloride, trimethylamine N-oxide (TMAO), urea) on the K values was investigated. Prior to that, spectroscopic measurements (UV+FTIR) were carried out to identify the effect of these additives on the thermodynamic stability of ALAT. Finally, reaction-equilibrium measurements in the presence of additives were performed for conditions that assured ALAT stability.

It was found that the thermodynamic equilibrium constant of reaction (1) is in the order of unity (K_a*(37 °C) = 0.75) and thus, the maximum yield is strongly limited by thermodynamic equilibrium. The experimental results also showed that equilibrium position (K value) is influenced by pH, reaction temperature, and initial molalities of the reacting agents. The K values of reaction (1) increased (shifting the reaction equilibrium to the right-hand side) with increasing temperature, increasing pH, increasing initial molalities of L-alanine and 2-oxoglutarate (equimolal conditions), as well as increasing ratio of their initial molalities. The presence of the additives decreased the stability of the enzyme except TMAO. It was also found that all additives in reaction media shifted the equilibrium towards the left-hand side of reaction (1) leading to decreased K values.

Finally, activity coefficients of reacting agents were predicted with ePC-SAFT. This allowed predicting the effect of the reaction conditions (temperature, pH, concentration ratio, additives) on K in good agreement with the experimental data. This work showed the importance of considering the influence of reaction conditions and additives on reaction equilibria to overcome thermodynamic limitations and increase the maximum yield of aminotransferase reactions.