Recombinant d-galactose dehydrogenase partitioning in aqueous two-phase systems: effect of pH and concentration of PEG and ammonium sulfate
© Kianmehr et al; Licensee Springer 2014
Received: 15 March 2014
Accepted: 2 June 2014
Published: 23 July 2014
d-Galactose dehydrogenase (GalDH; EC 18.104.22.168) 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.
Liquid-liquid extraction using aqueous two-phase systems (ATPS) has been applied for recovery and purification of many industrial enzymes [,]. When two aqueous solutions of certain incompatible substances, such polyethylene glycol (PEG) and dextran or PEG and salt, are mixed above a critical concentration, two-phase separation occurs. Separation techniques based on two-phase partitioning have proved to be suitable tools for recovery of biomolecules. Compared with the traditional techniques, ATPS have the advantages such as ensuring high values of the purification parameters, preserving the targeted biomolecules, yielding separation performance, and ease to scale-up. Successful applications of ATPS for downstream processing of proteins on industrial scales have been demonstrated [,].
d-Galactose dehydrogenase (GalDH; d-galactose: NAD+ oxidoreductase; EC 22.214.171.124) belongs to the family of oxidoreductases that catalyzes the dehydrogenation reaction of β-d-galactopyranose in the presence of NAD+ to d-galacto-1,5-lactone and NADH. The kinetic mechanism of Bi-Bi has been determined for this enzyme, with the NAD+ binding first to the enzyme. The substrates of GalDH are d-galactose and NAD+, whereas its products are D-galactono-1,4-lactone, NADH, and H+ []. GalDH has been identified in plants (e.g., green peas and Arabidopsis thaliana), algae (e.g. Iridophycus flaccidum), bacteria, and mammals. However, GalDH from Pseudomonas fluorescens bacterium is the best investigated enzyme, as its recombinant form has been produced in Escherichia coli [,]. GalDH is a significant tool for the measurement of β-d-galactose, α-d-galactose, and lactose as well. The enzyme has been used in diagnostic kits to screen blood serum of neonates for galactosemia diseases []. Galactosemia is an inborn metabolic disorder that without strict dietary control results in mental retardation, microcephaly, and seizures. Newborn screening using GalDH is a simple method which has proved sensitive, reliable, rapid, and cheap compared to other methodologies []. This enzyme has been purified by conventional methods including ammonium sulfate precipitation followed by chromatography which are usually time-consuming and expensive [,]. Owing to the commercial importance of GalDH, developing the efficient and scalable alternative methods for downstream processing is of great interest. In this work, we aimed to use ATPS technology for partitioning of P. fluorescens GalDH. The best partition conditions are generally achieved by systematic variation of different parameters such as temperature, pH, size and concentration, and type of polymer and salt. However, despite the apparent simplicity, partition of compounds is very complex due to the several factors involved. In fact, 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 []. For these reasons, mathematical modeling has been utilized to identify parameters mainly those that affect the partition of proteins in ATPS [,]. An effective statistical technique is the response surface methodology (RSM) which is a useful statistical tool for studying of systems where several independent variables influence the responses []. In recent years, the use of RSM in performing biological process has gained importance. The main advantage of RSM is the reduced number of tests needed to calculate multiple factors and their interactions []. In this communication, the RSM was applied to identify the suitable operating conditions for partitioning of recombinant P. fluorescens GalDH in ATPS.
Polyethylene glycols with different molecular weights were purchased from Merck (Darmstadt, Germany). d-Galactose and NAD+ were obtained from Sigma-Aldrich (St. Louis, MO, USA) and utilized in enzyme activity assay. The salts and all other chemicals were of analytical grade. P. fluorescens strain which produces GalDH enzyme has been isolated form a soil sample by Anvarsadat Kianmehr.
Construction of expression plasmid for P. fluorescens GalDH
Primers for polymerase chain reaction (PCR) amplification were designed based on the available nucleotide sequence of GalDH of the P. fluorescens genome using DNASIS MAX software (DNASIS version 3.0, Hitachi Software Engineering Co., Ltd., Tokyo, Japan). The gdh gene was amplified from the genomic DNA with specific primers GDHFw (5′-TGGATCC ATGCAACCGATTCGTCTCG-3′) and GDHRev (5′-GCGAAGCTT TTAATCGTAGAACGGC-3′), which contained the restriction sites for Bam HI and Hin dIII, respectively. PCR amplification was performed under condition: preincubation at 95°C for 1 min and then 30 cycles of 95°C for 1 min, 61°C for 1 min and 72°C for 2 min. The PCR reaction product was cut with Bam HI and Hin dIII and then ligated into the pET-28a (+) expression vector. The construct bearing the gdh gene was named pET28aGDH and transformed into E. coli BL-21 (DE3).
Cell cultivation and production of recombinant GalDH
A recombinant strain of E. coli BL21 (DE3) was grown overnight in Luria-Bertani (LB) medium containing 40 µg/mL of kanamycine at 37°C and 150 rpm. When cell density reached an OD600 of 0.8, GalDH enzyme was expressed by the addition of 0.7 mM sterile isopropyl-β-d-thiogalactopyranoside (IPTG). After 5 h of induction at 30°C, cells were harvested and stored at −20°C for further use. Pelleted E. coli cells were suspended in lysis buffer (50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, pH 8.0), mechanically broken by sonication using a pulse sequence of 15 s on and 10 s off and clarified by centrifugation at 4,000 rpm at 4°C for 1 h. The supernatant was employed as a crude enzyme in partition experiments [].
ATPS were prepared in 15-mL graduated tubes by mixing the appropriate amounts of PEG-4000, (NH4)2SO4, and enzyme solution. A final weight of 10-g system was obtained by adding a sufficient amount of 0.1 M potassium phosphate buffer (pH 8.0). Systems were agitated for 1 h at room temperature and then centrifuged at 3,000 rpm at 25°C for 40 min to speed up the phase separation. The volumes of the phases were determined, and the samples from the two phases were carefully tested for enzyme assay and total protein concentration. To avoid interference of the phase components, samples were analyzed against blanks containing the same compositions, but without enzyme []. In this work, all partition experiments were done at 25°C.
Enzyme activity was determined by monitoring the reduction of NAD+ at 340 nm. Mixture assay contained 10 mM d-galactose, 100 mM Tris-HCl buffer (pH 8.6), 2.5 mM NAD+, and the enzyme solution in a total volume of 1 mL. The change of absorbance at 340 nm was measured and corrected for blank values not including d-galactose. One unit of GalDH activity (U) is defined as the amount of enzyme catalyzing the formation of 1 μmol NADH per minute under the assay conditions []. The total protein concentration was determined by a Bio-Rad protein assay kit with bovine serum albumin (BSA) as a standard []. The purity of recombinant enzyme in ATPS was analyzed by a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Samples were diluted in a sample loading buffer and heated at 100°C for 5 min prior to being loaded into electrophoretic gel. After separation, the gel was stained with Coomassie Brilliant Blue R-250 and then destained by diffusion in a solution containing 40% (v/v) methanol and 10% (v/v) acetic acid []. The kinetic parameters of the final purified enzyme were calculated from the secondary plots of intercepts versus reciprocal concentrations of the other substrate.
Determination of partition parameters
To evaluate the partition performance of GalDH, different parameters were defined []. These include the partition coefficient (KE or KP), which is calculated as the ratio of the enzyme activity or protein concentration in the top phase divided by the correspondent value in the bottom phase. Specific activity (SA), which is defined as the enzyme activity (U/ml) in the phase sample divided by the total protein concentration (mg/ml) and is expressed in U/mg of protein.
Design of experiments and statistical analysis
Factors and value levels used in the central composite design
PEG (%, w/w)
Salt (%, w/w)
Experimental results from the central composite design
Results and discussion
Optimization of GalDH partition process
Results of ANOVA for the influence of variables on SA in GalDH partitioning using PEG-4000/(NH 4 ) 2 SO 4 ATPS
Lack of fit
SDS-PAGE and kinetic analysis of recombinant GalDH
The work presented here showed the potential application of ATPS for partitioning and recovery of recombinant GalDH in a single step. The RSM combined to a proper factorial experimental design proved to be a powerful tool in designing and modeling the best two-phase condition for enzyme partitioning. It was concluded that the ATPS consisting 14.33% (w/w) PEG-4000 and 11.79% (w/w) (NH4)2SO4, pH 7.48 at 25°C were the most optimum system to perform GalDH partition. Under these experimental conditions, the response values for PF, R, Y, and SA were 58.9, 268.75%, 92.8%, and 373.9 U/mg, respectively, and these results were also confirmed by the evaluation of activity assay and purity of final product.
We would like to express our thanks to Dr. Hamid Shahbaz Mohammadi for revising the text.
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