Integrated preservation of water activity as key to intensified chemoenzymatic synthesis of bio-based styrene derivatives.

The valorization of lignin-derived feedstocks by catalytic means enables their defunctionalization and upgrading to valuable products. However, the development of productive, safe, and low-waste processes remains challenging. This paper explores the industrial potential of a chemoenzymatic reaction performing the decarboxylation of bio-based phenolic acids in wet cyclopentyl methyl ether (CPME) by immobilized phenolic acid decarboxylase from Bacillus subtilis, followed by a base-catalyzed acylation. Key-to-success is the continuous control of water activity, which fluctuates along the reaction progress, particularly at high substrate loadings (triggered by different hydrophilicities of substrate and product). A combination of experimentation, thermodynamic equilibrium calculations, and MD simulations revealed the change in water activity which guided the integration of water reservoirs and allowed process intensification of the previously limiting enzymatic step. With this, the highly concentrated sequential two-step cascade (400 g·L-1) achieves full conversions and affords products in less than 3 h. The chemical step is versatile, accepting different acyl donors, leading to a range of industrially sound products. Importantly, the finding that water activity changes in intensified processes is an academic insight that might explain other deactivations of enzymes when used in non-conventional media.

Investigating Biomolecules in Deep Eutectic Solvents with Molecular Dynamics Simulations: Current State, Challenges and Future Perspectives.

Deep eutectic solvents (DESs) have recently gained increased attention for their potential in biotechnological applications. DESs are binary mixtures often consisting of a hydrogen bond acceptor and a hydrogen bond donor, which allows for tailoring their properties for particular applications. If produced from sustainable resources, they can provide a greener alternative to many traditional organic solvents for usage in various applications (e.g., as reaction environment, crystallization agent, or storage medium). To navigate this large design space, it is crucial to comprehend the behavior of biomolecules (e.g., enzymes, proteins, cofactors, and DNA) in DESs and the impact of their individual components. Molecular dynamics (MD) simulations offer a powerful tool for understanding thermodynamic and transport processes at the atomic level and offer insights into their fundamental phenomena, which may not be accessible through experiments. While the experimental investigation of DESs for various biotechnological applications is well progressed, a thorough investigation of biomolecules in DESs via MD simulations has only gained popularity in recent years. Within this work, we aim to provide an overview of the current state of modeling biomolecules with MD simulations in DESs and discuss future directions with a focus for optimizing the molecular simulations and increasing our fundamental knowledge.

Comparison and Validation of Force Fields for Deep Eutectic Solvents in Combination with Water and Alcohol Dehydrogenase.

Deep eutectic solvents (DESs) have become popular as environmental-friendly solvents for biocatalysis. Molecular dynamics (MD) simulations offer an in-depth analysis of enzymes in DESs, but their performance depends on the force field chosen. Here, we present a comprehensive validation of three biomolecular force fields (CHARMM, Amber, and OPLS) for simulations of alcohol dehydrogenase (ADH) in DESs composed of choline chloride and glycerol/ethylene glycol with varying water contents. Different properties (e.g., protein structure and flexibility, solvation layer, and H-bonds) were used for validation. For two properties (viscosity and water activity) also experiments were performed. The viscosity was calculated with the periodic perturbation method, whereby its parameter dependency is disclosed. A modification of Amber was identified as the best-performing model for low water contents, whereas CHARMM outperforms the other models at larger water concentrations. An analysis of ADH's structure and interactions with the DESs revealed similar predictions for Amber and CHARMM.

Modeling Alcohol Dehydrogenase Catalysis in Deep Eutectic Solvent/Water Mixtures.

The use of oxidoreductases (EC1) in non-conventional reaction media has been increasingly explored. In particular, deep eutectic solvents (DESs) have emerged as a novel class of solvents. Herein, an in-depth study of bioreduction with an alcohol dehydrogenase (ADH) in the DES glyceline is presented. The activity and stability of ADH in mixtures of glyceline/water with varying water contents were measured. Furthermore, the thermodynamic water activity and viscosity of mixtures of glyceline/water have been determined. For a better understanding of the observations, molecular dynamics simulations were performed to quantify the molecular flexibility, hydration layer, and intraprotein hydrogen bonds of ADH. The behavior of the enzyme in DESs follows the classic dependence of water activity (aW ) in non-conventional media. At low aW values (<0.2), ADH does not show any activity; at higher aW values, the activity was still lower than that in pure water due to the high viscosities of the DES. These findings could be further explained by increased enzyme flexibility with increasing water content.