Enzymatic Hydrolysis of Soybean Meals in Deep Eutectic Solvents and Ionic Liquids: Experimental and Molecular Insights

The enzymatic hydrolysis of soybean proteins was investigated using four enzymes (alcalase, flavourzyme, neutrase, and papain) in combination with different concentrations of natural deep eutectic solvents (NADESs) and ionic liquids (ILs). The goal was to assess the influence of these alternative green solvents on enzymatic efficiency. First, the hydrolysis kinetics were studied over a period of 240 min, in which the results showed an optimum time of 150 min, allowing high rates of soluble peptides without system saturation. The protein hydrolysis rate (PHR) was used as an indicator of enzymatic efficiency to evaluate the effect of NADESs based on cholinium chloride (ChCl) with hydrogen bond donors (urea, glycerol, lactic acid [Lac], and acetic acid [Ac]) and ILs formed with cholinium-cation and lactate or acetate anions. The results showed that the alcalase presented the highest hydrolysis values in the presence of ChCl:urea, ChCl:glycerol, and [Ch][Lac], in which a strong hydrolytic activity was observed at concentrations of 50% of the solvent. In contrast, solvents with acidic HBDs (ChCl:lactic acid and ChCl:acetic acid) and [Ch][Ac] showed strong inhibitory effects on enzymatic activity. Molecular docking revealed that while acetate directly interacted with Ser221 of alcalase, the nucleophilic residue in the catalytic triad, lactate formed more distributed and less disruptive interactions. Urea showed strong affinity with peripheral residues, preserving enzymatic structure and functionality. These results demonstrate that combining experimental data and molecular docking analysis constitutes a strategic approach for the rational design of green solvents, optimizing their application as cosolvents in biocatalytic reactions.