Leveraging liquid-liquid phase separation and volume modulation to regulate the enzymatic activity of formate dehydrogenase.

Engineering of reaction media is an exciting alternative for modulating kinetic properties of biocatalytic reactions. We addressed the combined effect of an aqueous two-phase system (ATPS) and high hydrostatic pressure on the kinetics of the Candida boidinii formate dehydrogenase-catalyzed oxidation of formate to CO2. Pressurization was found to lead to an increase of the binding affinity (decrease of KM, respectively) and a decrease of the turnover number, kcat. The experimental approach was supported using thermodynamic modeling with the electrolyte Perturbed-Chain Statistical Associating Fluid Theory (ePC-SAFT) equation of state to predict the liquid-liquid phase separation and the molecular crowding effect of the ATPS on the kinetic properties. The ePC-SAFT was able to quantitatively predict the KM-values of the substrate in both phases at 1 bar as well as up to a pressure of 1000 bar. The framework presented enables significant advances in bioprocess engineering, including the design of processes with significantly fewer experiments and trial-and-error approaches.

Simultaneous Predictions of Chemical and Phase Equilibria in Systems with an Esterification Reaction Using PC-SAFT.

The study of chemical reactions in multiple liquid phase systems is becoming more and more relevant in industry and academia. The ability to predict combined chemical and phase equilibria is interesting from a scientific point of view but is also crucial to design innovative separation processes. In this work, an algorithm to perform the combined chemical and liquid-liquid phase equilibrium calculation was implemented in the PC-SAFT framework in order to predict the thermodynamic equilibrium behavior of two multicomponent esterification systems. Esterification reactions involve hydrophobic reacting agents and water, which might cause liquid-liquid phase separation along the reaction coordinate, especially if long-chain alcoholic reactants are used. As test systems, the two quaternary esterification systems starting from the reactants acetic acid + 1-pentanol and from the reactants acetic acid + 1-hexanol were chosen. It is known that both quaternary systems exhibit composition regions of overlapped chemical and liquid-liquid equilibrium. To the best of our knowledge, this is the first time that PC-SAFT was used to calculate simultaneous chemical and liquid-liquid equilibria. All the binary subsystems were studied prior to evaluating the predictive capability of PC-SAFT toward the simultaneous chemical equilibria and phase equilibria. Overall, PC-SAFT proved its excellent capabilities toward predicting chemical equilibrium composition in the homogeneous composition range of the investigated systems as well as liquid-liquid phase behavior. This study highlights the potential of a physical sound model to perform thermodynamic-based modeling of chemical reacting systems undergoing liquid-liquid phase separation.

Osmolyte effect on enzymatic stability and reaction equilibrium of formate dehydrogenase.

Osmolytes are well-known biocatalyst stabilisers as they promote the folded state of proteins, and a stabilised biocatalyst might also improve reaction kinetics. In this work, the influence of four osmolytes (betaine, glycerol, trehalose, and trimethylamine N-oxide) on the activity and stability of Candida bondinii formate dehydrogenase cbFDH was studied experimentally and theoretically. Scanning differential fluorimetric studies were performed to assess the thermal stability of cbFDH, while UV detection was used to reveal changes in cbFDH activity and reaction equilibrium at osmolyte concentrations between 0.25 and 1 mol kg-1. The thermodynamic model ePC-SAFT advanced allowed predicting the effects of osmolyte on the reaction equilibrium by accounting for interactions involving osmolyte, products, substrates, and water. The results show that osmolytes at low concentrations were beneficial for both, thermal stability and cbFDH activity, while keeping the equilibrium yield at high level. Molecular dynamics simulations were used to describe the solvation around the cbFDH surface and the volume exclusion effect, proofing the beneficial effect of the osmolytes on cbFDH activity, especially at low concentrations of trimethylamine N-oxide and betaine. Different mechanisms of stabilisation (dependent on the osmolyte) show the importance of studying solvent-protein dynamics towards the design of optimised biocatalytic processes.

Prediction of pH in multiphase multicomponent systems with ePC-SAFT advanced.

Proton activity, which is usually expressed as a pH value, is among the most important properties in the design of chemical and biochemical processes as it determines the dissociation of species in aqueous mixtures. This article addresses the prediction of pH values in multiphase systems based on the IUPAC definition via proton activity. The required proton activity coefficients were predicted using the thermodynamic equation of state ePC-SAFT advanced. The developed framework considers reaction equilibria and phase equilibria (vapor-liquid and liquid-liquid) to predict pH in the equilibrated liquid phases.