Optimized extraction of polygalacturonase from Aspergillus aculeatus URM4953 by aqueous two-phase systems PEG/Citrate

Abstract

This study sets out to compare three techniques for extracting polygalacturonase (PG) from Aspergillus aculeatus URM4953, namely, precipitation by organic solvent (ethanol and acetone), precipitation by ammonium sulfate salt and extraction by aqueous two-phase systems (ATPS) PEG/Citrate using a factorial design 24. Afterwards, a central composite design (CCD) was used to optimize PG extraction by ATPS. The variables studied were concentrations of PEG (CPEG) and citrate (CCIT), PEG molar mass (MPEG) and pH. The responses analyzed were the partition coefficient (K), the activity yield (Y) and the purification factor (PF). The thermodynamic parameters of the optimized ATPS partition were estimated as a function of temperature. Among the techniques used, only the ATPS was able to pre-purify the PG (PF = 1.8) with an 85% yield. Optimization increased the PF value to 2.3. The best condition was 23% CCIT, 19% CPEG with the MPEG and pH set to 8000 g/mol and 8, respectively. The mathematical models of the CCD showed a high adjustment to predict the extraction of polygalacturonase from Aspergillus aculeatus URM4953 by ATPS. The thermodynamic parameters ΔG°m (−3.15 kJ/mol), ΔH°m (9.14 kJ/mol) and ΔS°m (41.2 J/Kmol) of partition showed that the preferential migration of almost all proteins from the crude extract to the salt rich-phase by increasing the temperature was spontaneous, while the PG preferred the PEG-rich phase. The ATPS showed the best performance as a first step for pre-purifying the PG from Aspergillus aculeatus URM4953.