EFFECT OF AGITATION ON OBTAINING PEPTIDES BY ENZYMATIC PROTEOLYSIS IN DEEP EUTECTIC SOLVENTS

Deep eutectic solvents (DES) are a new generation of sustainable solvents. The intrinsic characteristics of DES make these solvents ideal media for the performance of enzymatic reactions, including hydrolyses. DES are often prepared with sugars, organic acids, polyalcohols, choline derivatives or amino acids and, mostly, present high viscosity. In a previous assay, it was not possible to detect proteolytic activity of commercial proteases in DES media on neither soy protein isolate nor bovine serum albumin, under the assay conditions. Among the hypotheses for the lack of hydrolytic activity were the high viscosity of the solvent and the consequent difficulty in homogenizing this reaction medium. The goal of the present work was to evaluate different agitation speeds to verify their effect on proteolytic activity in DES medium. Therefore, the reaction medium was composed of equal molar concentrations (1:1) of choline chloride and glycerol (pH = 6.35), and the commercial proteases applied were Protamex, Alcalase, Neutrase and Flavourzyme. The enzymatic reactions occurred at 60°C (Protamex, Alcalase and Neutrase) and 50°C (Flavourzyme) for 60 min, and stirring speed was tested at 500, 1000 and 1500 rpm. For each assay, 1% protein was dissolved in 0.5 mL of DES containing 30% water. The reaction started with the addition of the enzyme solution (0.1 mL of 1% solution, resulting in a total addition of 50% water to the DES) and was stopped by the addition of 0.5 mL of TCA (0.44 M), which also caused precipitation of the non-hydrolyzed protein. The proteolytic activity was verified by measuring increase in the absorbance at 280 nm of the media's supernatant in comparison to a blank assay, without added enzyme. According to the literature, the most recommended way of decreasing DES viscosity is by increasing the temperature. However, the reaction temperature is among the factors that most affect enzyme stability. Thus, in the present work, another way of overcoming the high viscosity of DES was evaluated, by increasing the agitation of the reaction medium. It was observed, though, that increasing the stirring speed was not enough to achieve the desired homogenization of the medium, as no proteolytic activity could be detected in either assay. Thus, other ways of enabling the occurrence of enzymatic proteolysis in DES need to be evaluated, including increasing the temperature associated with other forms of homogenization, other than stirring.