Expression and response surface optimization of the recovery and purification of recombinant D-galactose dehydrogenase from Pseudomonas fluorescens

摘要

The enzyme D-galactose dehydrogenase (GalDH) has been used in diagnostic kits to screen blood serum of neonates forgalactosemia. 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 fromPseudomonas fluorescens in aqueous two-phase systems (ATPS). The identified GalDH gene was amplified by PCR andconfirmed 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 concentrationsand pH value were selected as variables to analyze purification of GalDH. To build mathematical models, RSM with a centralcomposite design was applied based on the conditions for the highest separation. The recombinant GalDH enzyme wasexpressed after induction with IPTG. It showed NAD+-dependent dehydrogenase activity towards D-Galactose. According tothe RSM modeling, an optimal ATPS was composed of PEG-2000 14.0% (w/w) and ammonium sulfate 12.0% (w/w) at pH7.5. Under these conditions, GalDH preferentially concentrated in the top PEG-rich phase. The enzyme activity, purificationfactor (PF) and recovery (R) were 1400 U/ml, 60.0% and 270.0%, respectively. The PEG and salt concentrations were found tohave significant effect on the recovery of enzyme. Briefly, our data showed that RSM could be an appropriate tool to define thebest ATPS for recombinant P. fluorescens GalDH recovery