Purification and characterization of a novel Aspergillus heteromorphus URM 0269 protease extracted by aqueous two-phase systems PEG/citrate

Abstract

A novel Aspergillus heteromorphus URM 0269 protease (EC 3.4.21) was extracted by aqueous two-phase systems PEG/citrate (ATPS). Extraction was performed by factorial design 24 for analyzing the independent variable effects named PEG molar mass (MPEG), PEG concentration (CPEG), sodium citrate concentration (CCIT) and pH. Afterwards, extracted protease was characterized in terms of kinetic and thermodynamic parameters. Protease partitioned preferentially for the PEG rich phase displaying the highest purification factor of 7.83 with a 157.53% yielded. Despite the enzyme acted optimally at 50 °C and pH 8.0, it was more stable in lower temperatures (10–40 °C) and pH conditions (5.0–10.0). The kinetic parameters of activation for casein hydrolysis revealed higher affinity by casein (KM = 2.8 mg/mL) with maximum catalysis rate of 45.0 U/mL. The reaction activation energy and the standard enthalpy variation of enzyme unfolding were 24.2 and 54.2 kJ/mol, respectively. Protease catalyzed casein hydrolysis at 25 °C, showed activation Gibbs free energy, enthalpy and entropy of 65.8 kJ/mol, 21.8 kJ/mol and −146.7 J/mol·K, respectively. The thermodynamic parameters of protease thermoinactivation suggested a predominating mechanism of reversible unfolding since the inactivation Gibbs free energy increased from 91.0 to 102.3 kJ/mol. These results displayed the great potential of the protease to be exploited in industrial applications.