title: Expression and response surface optimization of the recovery and purification of recombinant D-galactose dehydrogenase from Pseudomonas fluorescens.
abstract: The enzyme D-galactose dehydrogenase (GalDH) has been used in diagnostic kits to screen blood serum of neonates for galactosemia. It is also a significant tool for the measurement of β-D-galactose, α-D-galactose and lactose as well. In this study, response surface methodology (RSM) was used to identify the suitable conditions for recovery of recombinant GalDH from Pseudomonas fluorescens in aqueous two-phase systems (ATPS). The identified GalDH gene was amplified by PCR and confirmed by further cloning and sequencing. E. coli BL-21 (DE3) containing the GalDH gene on a plasmid (pET28aGDH) was used to express and purify the recombinant enzyme. The polyethylene glycol (PEG) and ammonium sulfate concentrations and pH value were selected as variables to analyze purification of GalDH. To build mathematical models, RSM with a central composite design was applied based on the conditions for the highest separation. The recombinant GalDH enzyme was expressed after induction with IPTG. It showed NAD'-dependent dehydrogenase activity towards D-Galactose. According to the RSM modeling, an optimal ATPS was composed of PEG-2000 14.0% (w/w) and ammonium sulfate 12.0% (w/w) at pH 7.5. Under these conditions, GalDH preferentially concentrated in the top PEG-rich phase. The enzyme activity, purification factor (PF) and recovery (R) were 1400 U/ml, 60.0% and 270.0%, respectively. The PEG and salt concentrations were found to have significant effect on the recovery of enzyme. Briefly, our data showed that RSM could be an appropriate tool to define the best ATPS for recombinant P. fluorescens GalDH recovery.
authors.forename: Shadi Rokhsartalab
authors.initials: SR
authors.lastname: Azar
authors.forename: Raika
authors.initials: R
authors.lastname: Naiebi
authors.forename: Ameneh
authors.initials: A
authors.lastname: Homami
authors.forename: Zahra
authors.initials: Z
authors.lastname: Akbari
authors.forename: Anvarsadat
authors.initials: A
authors.lastname: Kianmehr
authors.forename: Rahman
authors.initials: R
authors.lastname: Mahdizadehdehosta
authors.forename: Faezeh
authors.initials: F
authors.lastname: Najafzadeh
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D005691:Galactose Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D005691:Galactose Dehydrogenases; C047959:galactose dehydrogenase