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<span style="font-size:11.0pt;mso-bidi-font-size: 10.0pt;font-family:"Times New Roman";mso-fareast-font-family:"Times New Roman"; mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-GB;mso-fareast-language: EN-US;mso-bidi-language:AR-SA" lang="EN-GB">Expression and response surface optimization of the recovery and purification of recombinant D-galactose dehydrogenase from<i> Pseudomonas fluorescens</i></span>

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Title Expression and response surface optimization of the recovery and purification of recombinant D-galactose dehydrogenase from Pseudomonas fluorescens
 
Creator Azar, Shadi Rokhsartalab
Naiebi, Raika
Homami, Ameneh
Akbari, Zahra
Kianmehr, Anvarsadat
Mahdizadehdehosta, Rahman
Najafzadeh, Faezeh
 
Subject Aqueous two-phase systems
D-Galactose dehydrogenase
Expression
Response surface methodology
Purification
 
Description 68-74
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.


 
Date 2015-03-30T05:12:23Z
2015-03-30T05:12:23Z
2015-02
 
Type Article
 
Identifier 0975-0959 (Online); 0301-1208 (Print)
http://hdl.handle.net/123456789/31264
 
Language en_US
 
Rights CC Attribution-Noncommercial-No Derivative Works 2.5 India
 
Publisher NISCAIR-CSIR, India
 
Source IJBB Vol.52(1) [February 2015]