Liquid–liquid extraction of proteases from fermented broth by PEG/citrate aqueous two-phase system

This work deals with the use of an aqueous two-phase system (ATPS) of PEG/citrate to remove proteases from a Clostridium perfringens fermentation broth. To plan the experimental tests and evaluate the corresponding results, three successive experimental designs were employed, for which the PEG molar mass (MPEG) and concentration (CPEG), the citrate concentration (CC) and the pH were selected as independent variables, while the purification factor (PF), the partition coefficient (K), the activity yield (Y) and the selectivity (S) were selected as responses. PF of proteases in the top phase was shown to increase with increasing MPEG and decreasing CC, whereas a completely opposite trend was observed for K. On the other hand, Y was favored by simultaneous decreases in both these variables, while S decreased with increasing CC. Therefore, selecting a simultaneous increase in PF and Y as the most desirable result, the best performance of the system was obtained using MPEG = 10,000 g/mol, CPEG = 22% (w/w) and CC = 8.0% (w/w) at pH 8.5. Under these conditions, the activity yield was very high (131%) but the purification factor (4.2) and the selectivity (4.3) were lower than those ensured by more selective purification methods. According to these results, the ATPS seems to be an interesting alternative primary concentration/decontamination step for vaccine preparation from C. perfringens fermented broth.