Modulation of protein-saccharide interactions by deep-sea osmolytes under high pressure stress.

Deep-sea organisms must cope with high hydrostatic pressures (HHP) up to the kbar regime to control their biomolecular processes. To alleviate the adverse effects of HHP on protein stability most organisms use high amounts of osmolytes. Little is known about the effects of these high concentrations on ligand binding. We studied the effect of the deep-sea osmolytes trimethylamine-N-oxide, glycine, and glycine betaine on the binding between lysozyme and the tri-saccharide NAG3, employing experimental and theoretical tools to reveal the combined effect of osmolytes and HHP on the conformational dynamics, hydration changes, and thermodynamics of the binding process. Due to their different chemical makeup, these cosolutes modulate the protein-sugar interaction in different ways, leading to significant changes in the binding constant and its pressure dependence. These findings suggest that deep-sea organisms may down- and up-regulate reactions in response to HHP stress by altering the concentration and type of the intracellular osmolyte.

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.

Enhanced Polyphenols Recovery from Grape Pomace: A Comparison of Pressurized and Atmospheric Extractions with Deep Eutectic Solvent Aqueous Mixtures.

Deep eutectic solvents (DES) are emerging as potent polyphenol extractors under normal atmospheric conditions. Yet, their effectiveness in hot pressurized liquid extraction (HPLE) must be studied more. We explored the ability of various water/DES and water/hydrogen bond donors (HBDs) mixtures in both atmospheric solid liquid extraction (ASLE) and HPLE (50%, 90 °C) for isolating specific polyphenol families from Carménère grape pomace. We assessed extraction yields based on total polyphenols, antioxidant capacity, and recovery of targeted polyphenols. The HBDs ethylene glycol and glycerol outperformed DES in atmospheric and pressurized extractions. Ethylene glycol exhibited a higher affinity for phenolic acids and flavonols, while flavanols preferred glycerol. Quantum chemical computations indicated that a high-water content in DES mixtures led to the formation of new hydrogen bonds, thereby reducing polyphenol-solvent interactions. HPLE was found to be superior to ASLE across all tested solvents. The elevated pressure in HPLE has caused significant improvement in the recovery of flavanols (17-89%), phenolic acids (17-1000%), and flavonols (81-258%). Scanning electron microscopy analysis of post-extraction residues suggested that high pressures collapse the plant matrix, thus easing polyphenol release.

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.

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.

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.