Optimization of aqueous two-phase partitioning for purification of recombinant Eupenicillium terrenum fructosyl peptide oxidase

Abstract

Abstract Fructosyl peptide oxidase (FPOX), a flavoenzyme belonging to the class of oxidoreductases, has been used as a diagnostic enzyme for HbA1c measurement test. Design of experiments methodology was applied to evaluate the partition behavior of recombinant Eupenicillium terrenum FPOX in aqueous two-phase systems (ATPS). Different ATPS parameters including polyethylene glycol (PEG) molecular weight, PEG concentration, salt type, salt concentration, pH, system buffer type, system buffer concentration, temperature, neutral salt type, neutral salt concentration, and crude enzyme loading were investigated using a Plackett–Burman design (PBD). Taking into consideration an increase in partition coefficient (KE), PEG concentration, pH, system buffer concentration, and NaCl concentration, which played significant roles in enzyme partition, were chosen for further optimization by Box–Behnken design (BBD) in response surface methodology (RSM). The optimized condition led to maximum recovery (106.8%), yield (95.1%) and purification factor (42.4) values which were predicted to be achieved in ATPS formed by 14.0% (w/w) PEG-4000, 15.0% (w/w) Na2CO3, 5.0% (w/w) NaCl and 55.0 mM potassium phosphate buffer with pH 8.0. The partitioned recombinant FPOX was obtained as a single band into the upper PEG-rich phase and its specific activity was calculated to be 201.23 ± 2.1 U/mg. In summary, our data showed that the ATPS optimization using design of experiments can be an appropriate method for recovery of recombinant FPOX.