Recombinant d-galactose dehydrogenase partitioning in aqueous two-phase systems: effect of pH and concentration of PEG and ammonium sulfate

Abstract

Background

d-Galactose dehydrogenase (GalDH; EC 1.1.1.48) belongs to the family of oxidoreductases that catalyzes the reaction of β-d-galactopyranose in the presence of NAD⁺ to d-galacto-1,5-lactone and NADH. The enzyme has been used in diagnostic kits to neonatal screen for galactosemia diseases. This article reports the partitioning optimization of recombinant Pseudomonas fluorescens GalDH in aqueous two-phase systems (ATPS).

Methods

Preliminary two-phase experiments exhibited that the polyethylene glycol (PEG) concentration, pH value, and concentration of salt had a significant influence on the partitioning efficiency of recombinant enzyme. According to these data, response surface methodology (RSM) with a central composite rotatable design (CCRD) was performed to condition optimization.

Results

The optimal partition conditions were found using the 14.33% PEG-4000 and 11.79% ammonium sulfate with pH 7.48 at 25°C. Yield, purity, recovery, and specific activity were achieved 92.8%, 58.9, 268.75%, and 373.9 U/mg, respectively. PEG and ammonium sulfate concentration as well as pH indicated to have a significant effect on GalDH partitioning. Enzyme activity assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated the suitability of predicted optimal ATPS as well. The K_m and molecular weight values for the purified GalDH were 0.32 mM and 34 kDa, respectively.

Conclusions

Ultimately, our data showed the feasibility of using ATPS for partitioning and recovery of recombinant GalDH enzyme.

Keywords

Aqueous two-phase systems (ATPS) / d-Galactose dehydrogenase (GalDH) / Response surface methodology (RSM) / Partition / Pseudomonas fluorescens

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