Abstract
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). 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. 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 Km and molecular weight values for the purified GalDH were 0.32 mM and 34 kDa, respectively. Ultimately, our data showed the feasibility of using ATPS for partitioning and recovery of recombinant GalDH enzyme.
Highlights
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
Optimization of GalDH partition process In order to elucidate the main factors that will be included in recombinant P. fluorescens GalDH partition in aqueous two-phase systems (ATPS), a series of preliminary studies were performed
The increase of polyethylene glycol (PEG) MW from 4,000 to 8,000 daltons resulted in less available space of GalDH in the upper phase, which led to the decrease of partition coefficient
Summary
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. This article reports the partitioning optimization of recombinant Pseudomonas fluorescens GalDH in aqueous two-phase systems (ATPS). Newborn screening using GalDH is a simple method which has proved sensitive, reliable, rapid, and cheap compared to other methodologies [9] This enzyme has been purified by conventional methods including ammonium sulfate precipitation followed by chromatography which are usually time-consuming and expensive [6,8]. The classical optimization approach varying the level of one parameter at a time, while holding the rest of the variables constant, is generally time-consuming [10] For these reasons, mathematical modeling has been utilized to identify parameters mainly those that affect the partition of proteins in ATPS [11,12]. The main advantage of RSM is the reduced number of tests needed to calculate multiple factors and their interactions [14] In this communication, the RSM was applied to identify the suitable operating conditions for partitioning of recombinant P. fluorescens GalDH in ATPS
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
