The research on alternative solvents and co-solvents is a relevant aspect when envisioning the improvement of biocatalytic reactions. Among these solvents and co-solvents, deep eutectic solvents (DES) may be considered as customizable new reaction media for biocatalysis. Accordingly, in this work, sixteen DES aqueous solutions, as well as of the individual DES components at the same conditions have been investigated in laccase-catalyzed reactions. Cholinium- and betaine-based DES formed with polyols at different molar ratio and concentrations were evaluated. The results reported show that in presence of most DES the laccase activity is preserved and, with a particular DES, enhanced up to 200%. Molecular docking studies demonstrated that while most DES components establish hydrogen-bonds with the enzyme amino acids, those that establish stronger interactions with the enzyme (expressed by absolute values of docking affinity energies) lead to an enhanced laccase activity. Finally, the laccase stability was evaluated in additional tests under extreme storage temperatures (60 ºC and -80 ºC). Although no significant protection to high temperatures was afforded by DES, an enhanced laccase activity when stored at low temperatures was found, at least up to 20 days. Combining experimental results and molecular docking this work shows that DES can be designed as co-solvents to improve biocatalytic reactions.

Many applications involving ionic liquids (ILs) require the knowledge of their interfacial behaviour, such as wettability and adhesion. In this context, herein, two approaches were combined aiming at understanding the impact of the IL chemical structures on their wettability on both polar and non-polar surfaces, namely: (i) the experimental determination of the contact angles of a broad range of ILs (covering a wide number of anions of variable polarity, cations, and cation alkyl side chain lengths) on polar and non-polar solid substrates (glass, Al-plate, and poly-(tetrafluoroethylene) (PTFE)); and (ii) the correlation of the experimental contact angles with the cation–anion pair interaction energies generated by the Conductor-like Screening Model for Real Solvents (COSMO-RS). The combined results reveal that the hydrogen-bond basicity of ILs, and thus the IL anion, plays a major role through their wettability on both polar and non-polar surfaces. The increase of the IL hydrogen-bond accepting ability leads to an improved wettability of more polar surfaces (lower contact angles) while the opposite trend is observed on non-polar surfaces. The cation nature and alkyl side chain lengths have however a smaller impact on the wetting ability of ILs. Linear correlations were found between the experimental contact angles and the cation–anion hydrogen-bonding and cation ring energies, estimated using COSMO-RS, suggesting that these features primarily control the wetting ability of ILs. Furthermore, two-descriptor correlations are proposed here to predict the contact angles of a wide variety of ILs on glass, Al-plate, and PTFE surfaces. A new extended list is provided for the contact angles of ILs on three surfaces, which can be used as a priori information to choose appropriate ILs before a given application.

There is considerable interest in the use of structurally stable and catalytically active enzymes, such as cytochrome c (Cyt c), in the pharmaceutical and fine chemicals industries. However, harsh process conditions, such as temperature, pH, and the presence of organic solvents, are the major barrier to the effective use of enzymes in biocatalysis. We demonstrate the suitability of cholinium-based ionic liquids (ILs) formed by the cholinium cation and dicarboxylate-based anions as potential media for enzymes, in which remarkable enhanced activity and improved stability of Cyt c against multiple stresses were obtained. Among the several ILs studied, an exceptionally high catalytic activity (>50-fold) of Cyt c was observed in the aqueous solutions of cholinium glutarate ([Ch][Glu]; at 1 : 1 weight ratio of IL : H₂O) compared to the commonly used phosphate buffer solutions (pH 7.2), and >25-fold compared to the aqueous solutions of cholinium dihydrogen phosphate ([Ch][Dhp]; 1 : 2 weight ratio of IL : water)—the best known IL for long term stability of Cyt c. The catalytic activity of the enzyme in the presence of ILs was retained against several external stimuli, such as chemical denaturants (H₂O₂ and GuHCl) and temperatures of up to 120 °C. The observed enzyme activity is in agreement with its structural stability, as confirmed by UV–vis, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy. Taking advantage of the multi-ionization states of di/tri-carboxylic acids, the pH was switched from acidic to basic by the addition of the corresponding carboxylic acid and choline hydroxide, respectively. The activity was found to be maximum at a 1 : 1 molar ratio of [Ch][carboxylate], with a pH in the range from 3 to 5.5. Moreover, it was found that the cholinium-based ILs studied herein protect the enzyme against protease digestion and allow long-term storage (at least for 21 weeks) at room temperature. An attempt by molecular docking was also made to better understand the efficacy of the investigated cholinium-based ILs towards the enhanced activity and long term stability of Cyt c. The results showed that dicarboxylate anions interact with the active site's amino acids of the enzyme through H-bonding and electrostatic interactions, which are responsible for the observed enhancement of the catalytic activity. Finally, it is demonstrated that Cyt c can be successfully recovered from the aqueous solution of ILs and reused without compromising its yield, structural integrity and catalytic activity, thereby overcoming the major limitations in the use of IL–protein systems in biocatalysis.