Study of Microbial Transglutaminase Partitioning in Thermo-pH–Responsive Aqueous Two-Phase Systems

The recyclable aqueous two-phase systems (ATPS) responding to environmental stimuli have been widely studied in the purification of biologics. In this study, a thermo-responsive polymer PNE was copolymerized after optimization of monomer ratio. In addition, its lower critical solution temperature (LCST, 31 °C) and first recovery (99.43%) were determined. Then, PNE was used to form two recyclable ATPS with another thermo-responsive polymer PNDBN and a pH-responsive polymer PADB4.91, which were polymers already prepared in the lab. Meanwhile, the partition behavior of microbial transglutaminase (MTG) was explored using these two ATPS. The result showed that the PNE/PADB4.91 ATPS was superior to PNDBN/PNE ATPS owing to its faster phase formation and better partition performance. In order to optimize the partition behavior, several parameters were investigated based on PNE/PADB4.91 ATPS. In the ATPS constructed with 2.5% (w/v) PNE and 3.5% (w/v) PADB4.91, the maximal partition coefficient (1/KE) and enzymatic recovery (ERB) of MTG were 12.9 and 95.21% in the presence of 10 mM KCl when the temperature, pH, and the addition amount of MTG were 25 °C, 7.0, and 2 mg/mL, respectively.