Zaira Maugeri

Novel choline-chloride-based deep-eutectic-solvents with renewable hydrogen bond donors: levulinic acid and sugar-based polyols

Choline chloride (ChCl) has been combined with renewable hydrogen bond donors (HBD) to form novel deep eutectic solvents (DES) with melting points lower than 100 °C. In some cases (e.g. sorbitol or isosorbide as HBDs) chiral DES can be easily produced. Moreover, the addition of glycerol decreases the viscosity of DES significantly. In the ChCl : levulinic acid DES case, singular properties were observed. Thus, equimolecular mixtures of ChCl : levulinic acid (1 : 1) formed a crystal at room temperature, rapidly melting in contact with air humidity. Addition of excess of acetone to ChCl : levulinic acid (1 : 2) leads to the almost quantitative precipitation of choline chloride, which can be used again to form a DES.

Reengineering CelA2 cellulase for hydrolysis in aqueous solutions of deep eutectic solvents and concentrated seawater

Cellulases are promising catalysts for the depolymerization of cellulose under mild conditions. Reengineered cellulases are required to match application demands in biorefineries and to avoid cost-intensive downstream processing. This manuscript provides a novel fluorescence-based high throughput screening method for directed evolution of cellulases, based on 4-methylumbelliferyl-β-D-cellobioside (4-MUC). The 4-MUC high throughput screening system was successfully employed to identify CelA2 variants with enhanced stability and activity in mixtures of water with deep eutectic solvents like choline chloride : glycerol (ChCl : Gly), and seawater. The cellulase variant 4D1 (L21P; L184Q; H288R; K299I; D330G; N442D) was isolated and showed, compared to wild type, an increase in specific activity in 30% (v/v) ChCl : Gly (7.5-fold; 0.4 to 3.0 U mg−1) and in concentrated seawater (1.6-fold; 5.5 to 9.3 U mg−1). In addition, the residual activity of 4D1 in the presence of 3-fold concentrated seawater is unaffected whereas CelA2 wild type loses >50% of its activity. Furthermore, the position H288 was identified as a key position for activity and resistance in 4D1.

Whole-Cell Biocatalysis in Deep-Eutectic-Solvents/Aqueous Mixtures

Whole‐cell biocatalysis with the use of baker’s yeast is demonstrated in different mixtures of water with deep eutectic solvents (DESs; choline chloride/glycerol, 1:2 mol/mol). Enantioselective ketone reduction is observed for long reaction times (>200 h), which suggests that the whole cells remain stable in these neoteric solvents. By changing the proportion of the DES added, a complete inversion of enantioselectivity is observed, from approximately 95 % enantiomeric excess (ee) (S) in pure water to approximately 95 % ee (R) in the pure DES. Presumably, some (S)‐oxidoreductases present in baker’s yeast are inhibited by DESs.