Assessing the Influence of Betaine-Based Natural Deep Eutectic Systems on Horseradish Peroxidase.

To validate the use of horseradish peroxidase (HRP) in natural deep eutectic systems (NADES), five different betaine-based NADES were characterized in terms of water content, water activity, density, and viscosity experimentally and by thermodynamic modeling. The results show that the NADES under study have a water activity of about 0.4 at 37 °C for water contents between 14 and 22 wt %. The densities of the studied NADES had values between 1.2 and 1.3 g.cm-3 at 20 °C. The density was modeled with a state-of-the-art equation of state; an excellent agreement with the experimental density data was achieved, allowing reasonable predictions for water activities. The system betaine:glycerol (1:2) was found to be the most viscous with a dynamic viscosity of ∼600 mPa.s at 40 °C, while all the other systems had viscosities <350 mPa.s at 40 °C. The impact of the NADES on the enzymatic activity, as well as on, conformational and thermal stability was assessed. The system betaine/sorbitol:water (1:1:3) showed the highest benefit for enzymatic activity, increasing it by two-folds. Moreover, upon NADES addition, thermal stability was increased followed by an increment in a-helix secondary structure content.

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.

Boosting the kinetic efficiency of formate dehydrogenase by combining the effects of temperature, high pressure and co-solvent mixtures.

The application of co-solvents and high pressure has been shown to be an efficient means to modify the kinetics of enzyme-catalyzed reactions without compromising enzyme stability, which is often limited by temperature modulation. In this work, the high-pressure stopped-flow methodology was applied in conjunction with fast UV/Vis detection to investigate kinetic parameters of formate dehydrogenase reaction (FDH), which is used in biotechnology for cofactor recycling systems. Complementary FTIR spectroscopic and differential scanning fluorimetric studies were performed to reveal pressure and temperature effects on the structure and stability of the FDH. In neat buffer solution, the kinetic efficiency increases by one order of magnitude by increasing the temperature from 25° to 45 °C and the pressure from ambient up to the kbar range. The addition of particular co-solvents further doubled the kinetic efficiency of the reaction, in particular the compatible osmolyte trimethylamine-N-oxide and its mixtures with the macromolecular crowding agent dextran. The thermodynamic model PC-SAFT was successfully applied within a simplified activity-based Michaelis-Menten framework to predict the effects of co-solvents on the kinetic efficiency by accounting for interactions involving substrate, co-solvent, water, and FDH. Especially mixtures of the co-solvents at high concentrations were beneficial for the kinetic efficiency and for the unfolding temperature.