ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY, 2017

VOL. 45, NO. 2, 380–388

http://dx.doi.org/10.3109/21691401.2016.1160404

Aqueous two-phase system purification for superoxide dismutase induced by

menadione from Phanerochaete chrysosporium
lu, Burcu Tongul and Leman Tarhan
Berna Kavakcıog
Department of Chemistry, Faculty of Science, University of Dokuz Eylul, Buca, Izmir, Turkey

ABSTRACT ARTICLE HISTORY

In the present work, the partitioning behavior of menadione-induced superoxide dismutase (SOD; EC Received 30 November 2015
1.15.1.1), an antioxidant enzyme that has various applications in the medical and cosmetic industries, Revised 26 January 2016
from the white rot fungus Phanerochaete chrysosporium has been characterized on different types of Accepted 26 February 2016
aqueous two-phase systems (ATPSs) (poly(ethylene glycol)/polypropylene glycol (PEG/PPG)-dextran, PEG- Published online 25 March
2016
salt and PPG-salt). PEG-salt combinations were found most optimal systems for the purification of SOD.
The best partition conditions were found using the PEG-3350 24% and K2HPO4 5% (w/w) with pH 7.0 at KEYWORDS
25  C. The partition coefficient of total SOD activity and total protein concentration observed in this sys- Aqueous two-phase systems;
tem were 0.17 and 6.65, respectively, with the recovery percentage as 78.90% in the bottom phase and partitioning; polymer/salt
13.17% in the top phase. The highest purification fold for SOD from P. chrysosporium was found as 6.04 system; purification;
in the bottom phase of PEG 3350%24 – K2HPO4%5 (w/w) system with pH 7.0. SOD purified from superoxide dismutase
P. chrysosporium was determined to be a homodimer in its native state with a molecular weight of
60 6 4 kDa. Consequently, simple and only one step PEG-salt ATPS system was developed for SOD puri-
fication from P. chrysosporium.

Introduction
Superoxide dismutase (SOD) is the first line of antioxidant
The phenomenon of protein partitioning in aqueous two- defence against reactive oxygen species (ROS) generated by res-
phase systems (ATPSs) was first introduced in 1956 by the piration and, among eukaryotic organisms; it is the only enzyme
pioneering work of Albertsson (1956). It has been established that can detoxify superoxide (O2  ) and prevents the formation
that ATPS forms when combinations of hydrophilic solutes of other more potent oxidants, such as peroxynitrite and
(polymers or polymer and certain salts) display incompatibility hydroxyl radical (Bowler et al. 2001). It has been stated that the
in aqueous solution above their critical concentrations enzyme reduces fibrosis (Warner 1994) and plays an important
(Palomares 2004, Walter 2012). Generally speaking, the roles in degenerative diseases, aging, and longevity (Nelson
researchs in ATPS can be categorized into two main areas, et al. 2005). Hence, from an industrial point of view, SOD has
including the elucidation of the mechanistic molecular under- found wide usage in cosmetic and supplementary products.
standing of solute partitioning in ATPS (Baskir et al. 1989, Besides, this enzyme is useful in prolonging the survival time of
Graber et al. 2000, Kavakçıog lu and Tarhan 2013, Lue and organs isolated for transplantation (Prieels et al. 1991), preserva-
Blankschtein 1996, Zaslavsky 1994) and the practical imple- tion of perishable materials, such as foodstuff, laundry composi-
mentation of the technique for the recovery of biological tions, for the removal of Amadori and Maillard products (Bafana
products from different sources (Antov et al. 2006, de Ara ujo et al. 2011), and also biosensors for (Tian et al. 2002). In fact,
et al. 2011, Gavasane and Gaikar 2003, Minami and Kilikian there are several techniques for SOD purification from various
1998, Nalinanon et al. 2009, Pan et al. 2001, Wu et al. 2001, sources, including human lung, plants, different bacterial strains,
Xu et al. 2002). It should be stated that due to the numerous, rat testis and chicken liver as ultrafiltration, extraction with
complex, and interrelated interactions among the system organic solvents and chromatography. But these techniques are
components and biomolecules, to get a general rule with time consumer and cause low recovery yield because the high
respect to solute partition is not possible and desirable parti- number of step is required (Duke and Salin 1985, Hayakawa
tion can be achieved with the manipulation of system by et al. 1985, Marklund 1982, Mruk et al. 1998, Ozt€ € urk-Urek
€ and
changing the composition, pH, and ionic strength. Although Tarhan 2001, Spiegelhalder et al. 1993). The design and develop-
additional knowledge is needed to fully understand this phe- ment of rapid, simple and more efficient bioprocesses for pro-
nomenon, it has been proved that practical application of tein purification are needed because of the increasing demand
ATPS for the recovery of biological products from different to biomolecule production. ATPS is effective for the primary
sources generates economic, robust, easy to scale and bio- recovery of proteins, nucleic acids, low-molecular-weight com-
compatible extraction process with short process time and pounds and cells, organelles and virus-like particles with the
low-energy consumption (Hatti-Kaul 2001). advantages, such as biocompatibility, low costs, ease to scale-up,

CONTACT Leman Tarhan leman.tarhan@deu.edu.tr Department of Chemistry, Faculty of Science, University of Dokuz Eylul, Buca, Izmir, Turkey
ß 2016 Informa UK Limited, trading as Taylor & Francis Group
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 381

rapid phase separation, easy manipulation, possibility for con- performed at 28  C with agitation at 150 rpm for 6 h. The
tinuous operation, and suspended solids removal (Andrews and menadione-treated cells were harvested and washed several
Asenjo 2010, Benavides et al. 2008). Therefore, aqueous two- times with 20 mM potassium phosphate buffer (pH 7.4) at
phase systems (ATPS) is one of the alternative for the recovery 4  C, and then, crude extracts were prepared.
of SOD. The only known research on ATPS partition of SOD was
conducted by Simental-Martınez et al. (2014). In this study, the
Preparation of crude extract from P. Chrysosporium
highest purification fold was 4.0 6 0.17 with 85.2 6 3.5 U/mg for
The harvested cells were resuspended in 20 mM potassium
SOD from Kluyveromyces marxianus without considering ultrafil-
phosphate buffer (pH 7.4) in a volume equal to three times of
tration step.
the wet weight of the cells. The optimized homogenization
In the present work, it was aimed to purify the SOD in the
procedure was performed for 3 min at 9000 rpm with 30-s
crude extract from Phanerochaete chrysosporium, known as a
interval. The cell debris in the homogenate was removed by
type of basidiomycete, has the ability of the abundant aro-
centrifugation at 15,000 rpm and for 15 min at 4  C. The crude
matic polymer lignindegradation, composting and removal of
extract was not frozen before use.
pentachlorophenol in soil (Chen et al. 2015a, Chen et al.
2015b, Fernandez-Fueyo et al. 2012, Martinez et al. 2014,
Chen et al. 2016), after the induction of the enzyme activity Superoxide dismutase activity assay
with menadione by using different types of ATPS (ATPSs) The SOD enzyme activity was determined by spectrophotom-
(poly(ethylene glycol)/polypropylene glycol (PEG/PPG)-dextran, etry at 490 nm. The SOD activity assay was based on the
PEG-salt and PPG-salt) and manipulating the systems in terms measurement of autoxidation of 6-hydroxydopamine (6-
of molecular weights of polymers, compositions of the com- OHDA), which is inhibited by SOD (Crosti et al. 1987). One
ponents, pH and concentrations of neutral salts. unit is the amount of SOD required to inhibit the initial rate
of 6-OHDA autoxidation by 50%.
Materials and methods
Materials Total protein assay
Samples withdrawn from each phase were diluted 10-fold
PEG with average molecular masses of 1000, 2000, 3350,
with a known amount of distilled water and total protein con-
6000, and 8000; PPG with average molecular masses of 425, tents were measured with Bradford protein assay (Bradford
1000, 2700, and dextran with average molecular mass 1976). BSA was used as a standard. An identically diluted solu-
100,000 g/mol were purchased from Sigma Chemical Co. (St. tion of the corresponding phase from a system containing no
Louis, MO) and used without further purification. All other catalase extract was used as a blank.
used chemical reagents were of analytical grade. Distilled and
deionized water was used.
Preparation of aqueous two-phase systems
ATPSs were prepared by mixing required components at the
Methods
desired pH in 15-mL graduated tubes. The final weight of
Media and growth conditions of P. Chrysosporium the systems was adjusted to 5 g by the addition of double-
Cultures of P. chrysosporium (German Collection of distilled deionized water. Enzyme extract was added to the
Microorganisms and Cell Cultures, DSMZ-1547) were maintained system as the final component to avoid possible precipita-
in potato dextrose agar (PDA) medium (pH 5.6) as described by tion and denaturation risks. The tubes were mixed by vortex-
Beever and Bollard (1970). Sterilization of the medium was car- ing for 60 s to allow redistribution of the components.
ried out by autoclaving at 121  C for 20 min. Inoculation was Complete phase separation was achieved by low-speed
performed at 28  C for seven days in Petri dishes. batch centrifugation at 2500 rpm for 5 min. The systems
P. chrysosporium was cultured in modified Tien and Kirk were left standing at 25  C for 1 h after phase separation to
liquid medium (Tien and Kirk 1984) (g/L); KH2PO4 (2.0), CaCl2 reach equilibrium. The equilibrium state was characterized
(0.114), MgSO4 (0.7), NH4Cl (0.12), glucose (2.0), thiamin-HCl by the absence of turbidity in both the top and bottom
(1.103), Tween 80 (0.05), FeSO4.7H2O (70 lg), ZnSO4.7H2O phases. After the phases were carefully separated, both
(46 lg), MnSO42H2O (35 lg); CoCl2.6H2O (7 lg). The liquid phases were appropriately diluted for the determination of
medium was sterilized by autoclaving at 121  C for 20 min. SOD activities and total protein amounts. To correct for poly-
Incubation was carried out at 28  C for 10 days and with agi- mer and salt interference in the total protein assays, a blank
tation at 150 rpm in the 250-mL erlenmayer flasks containing system was prepared for each set of conditions, and the
90 mL liquid medium and 10 mL spore suspension (OD650; samples were read against the blanks. All partition experi-
0.500). ments were conducted in triplicate, and the averages were
used in the calculations.

SOD activity induction with menadione as

an exogenous factor Calculations of the partition parameters
After 10 days culture, menadione (0.75 mM) was added to The partition coefficient is described as the ratio of the total
the growth medium that containing stationary phases enzyme activity or protein concentration in the top phase to
P. chrysosporium in 250-ml Erlenmayer flask. Incubation was that in the bottom phase.
382 B. KAVAKCıO
gLU ET AL.

Ke ¼ At/Ab, and Kp ¼ Ct/Cb, where At and Ab are the total SOD from P. chrysosporium in terms of its Ke, Kp, PF, %R values
enzyme activities, and Ct and Cb are the total protein concen- for different types of ATPSs (PEG/PPG – dextran 100,000 g/
trations of the top and bottom phases, respectively. mol, PEG-salt and PPG-salt). To induce the initial SOD activity
The specific activity of the enzyme (SA, expressed in U/mg in P. chrysosporium, cells were treated with menadione, which
protein), the purification fold (PF), and the recovery (R) in the is known as a superoxide anion radical producer and conse-
top and bottom phases were also calculated according to the quently inducer of SOD, in the range of 0.1–0.75 mM for 1–8 h
given equations to evaluation efficiency of the systems: (Tongul and Tarhan 2014). The observed highest activity was
found as 372 U/mg, with the treatment of 0.75 mM mena-
SAt ¼ At =Ct and SAb ¼ Ab =Cb dione at 6th h. Through this induction step, the initial activity
of the SOD extract that would applied to ATPSs reached to
5.4-fold of control.
PFt ¼ ðSAÞt =ðSAÞi and PFb ¼ ðSAÞb =ðSAÞi
In the case of biomolecule partition/purification with
ATPSs, the selection of phase system type and the investiga-
Rt ¼ At =Ai and Rb ¼ Ab =Ai tion of certain parameters as optimal pH and ionic strength
are significant steps. In order to select phase system type, dif-
Ai and SAi are the activity and the specific activity of the initial ferent polymer – polymer and polymer – salt systems were
enzyme, respectively. examined in terms of their compatibility for selective partition
The partition experiments were carried out in triplicate, of SOD (Table 1). As shown in Table 1, total enzyme activities
and the average results are the values reported in the present in both top and bottom phases of polymer – salt systems
work. were significantly higher in comparison with polymer – poly-
mer systems. It was observed that SOD activity gradually
decreased with the increases in molecular weights of PEG and
Native molecular weight determination of superoxide PPG in both phases of the polymer – polymer systems.
dismutase from P. Chrysosporium Consequently, PEG – dextran and PPG – dextran systems
In order to determine the native molecular weight of SOD were not found suitable for the purification of SOD due to
from P. chrysosporium, standard protein mixture instead of low recovery percentages compared to polymer – salt
sample was applied to the sephadex G-100 gel filtration col- systems.
umn (bed volume, 25 mL). The column was eluted (0.6 mL/ As is known to all, choosing the phase forming compo-
min) with 0.15 MNaCl in 5 mM potassium phosphate buffer nents types to the distributions of the target and residual bio-
(pH 7.4). In the column, albumin (66 kDa), carbonic anhydrase molecules is the second most important step following the
(29 kDa), cytochrome c (12.4 kDa), and aprotinin (6.5 kDa) were selection of the phase system type as polymer/salt (Hatti-Kaul
used as molecular weight markers with blue dextran (200 kDa) 2001). Polymer/salt systems especially using sulfates or phos-
for determining the void volume. The molecular weight for phates have proved particularly useful in sequential protein
SOD was estimated from the calibration curve. separation schemes because of their low cost and ease of
handling. In this type of systems, two-phase formation is
based on the reduction effect of the low-molecular-weight
Electrophoretic procedures solute on polymer solubility in water. As stated earlier, phos-
SDS–PAGE of crude extract, top and bottom phases was per- phates and sulfates are the most appropriate and commonly
formed according to the method of Laemmli and Favre used inorganic salts in enzyme purification with ATPS
(1973). Protein solutions were mixed at 1:4 (v/v) ratio with the (Gautam and Simon 2006, Klomklao et al. 2005, Porto et al.
SDS–PAGE sample buffer (0.125 M Tris–HCl (pH 6.8), 4% SDS, 2008, Su and Chiang 2006, Y€ €
ucekan and Onal 2011). On the
20% glycerol, 10% b-mercaptoethanol) and boiled for 5 min. other hand, it was stated in some papers that citrates could
The samples were loaded onto the gel made of 4% stacking use as a substitute for inorganic salts because the fact that
and 12% separating gels and subjected to electrophoresis at a inorganic salts lead to high phosphate or sulfate concentra-
constant current of 150 V per gel using a Protean II Cell appar- tions in the effluent streams and therefore to environmental
atus (Bio-Rad Laboratories, Richmond, CA). After electrophor- concern (Nitsawang et al. 2006). Unfortunately, we could not
esis, the gel was stained overnight with Coomassie Brilliant study with the citrate and sulfate salts due to problem
Blue R-250. Protein patterns were visualized after washing the encountered in the measurement of the enzyme activity
gel with 40% (v/v) methanol and 10% (v/v) acetic acid solu- because these salts hindered the autoxidation of 6-OHDA, the
tion for several times to remove Coomassie particles until substrate of the SOD (data not shown). On the other hand, it
clear background was observed. Spectra multicolor broad was observed that viscosities of the PPG – salt systems were
range protein ladders (10 to 260 kDa) were used as SDS–PAGE too high and this situation led to loss of SOD activity in a sig-
molecular weight standards. nificant manner when compared with PEG – salt systems
(Table 1). Additionally, PEG was selected as a polymer compo-
nent of the polymer – salt systems because of having lower
Results and discussion
cost and approaching equilibrium easier in comparison with
ATPSs provide a powerful method for the separation and puri- PPG.
fication of biomolecules mainly enzyme and proteins. In this In this research, the effects of the (1) molecular weight of
study, it was aimed to characterize the partition behaviour of PEG and composition of the system; (2) pH and finally (3)
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 383

Table 1. Effects of molecular weight of PEG and system composition in PEG–K2HPO4 on menadione induced SOD partitioning at 25  C
Sample no. System At (U) Ab (U) Ke Kp PFt PFb Rt (%) Rb (%)
1 PEG 1000 10% – K2HPO4 20% 87.12 31.39 2.77 0.25 1.67 0.15 34.47 12.06
2 PEG 1000 15% – K2HPO4 15% 86.99 71.79 1.21 0.57 0.92 0.43 33.42 27.58
3 PEG 2000 10% – K2HPO4 20% 46.11 38.75 1.19 0.27 0.83 0.19 17.72 14.89
4 PEG 2000 15% – K2HPO4 15% 45.38 59.28 0.76 0.55 0.49 0.35 17.43 22.77
5 PEG 3350 10% – K2HPO4 20% 37.45 41.91 0.89 0.25 0.72 0.20 14.39 16.11
6 PEG 3350 15% – K2HPO4 15% 41.31 53.48 0.77 0.48 0.49 0.30 15.87 20.55
7 PEG 6000 10% – K2HPO4 20% 32.82 48.71 0.67 0.14 1.02 0.21 12.61 18.71
8 PEG 6000 15% – K2HPO4 15% 27.54 53.61 0.51 0.18 0.69 0.24 10.58 20.59
9 PEG 8000 10% – K2HPO4 20% 19.83 54.43 0.36 0.09 0.92 0.23 7.62 20.92
10 PEG 8000 15% – K2HPO4 15% 13.12 60.58 0.22 0.12 0.47 0.26 5.04 23.28
About 0.5 mL crude extract with SOD activity of 260.4 U and total protein amount of 700 mg was present in each tube.

concentrations of neutral salts as an additional component on were about 9.5. The prepared systems were listed in Tables 1
the distribution of menadione-induced SOD from P. chrysospo- and 2 together with detailed results.
rium in PEG – K2HPO4 systems between top and bottom It is known that the miscibility of PEG below 1000 g/mol
phases were investigated. increases too much, which leads to higher concentrations for
inducing a two-phase system. It was also emphasized by
another group that when the smaller PEG size used, the more
Effects of PEG molecular weight and system composition polymer is needed in order to enter into the two-phase
on the partitioning behaviors of menadione-induced region in the phase diagram (Forciniti et al. 1991). The
SOD from P. Chrysosporium another problem with small PEG sizes is low selectivity arising
Although the mechanism governing the partition of biomole- from contaminants that mainly accumulate in the top phase
cules in ATPS is not fully understood, it is believed that the together with target biomolecule. Sharjahan et al stated that
surface properties are probably the most important factors lower molecular weight PEG is not a good option for purifica-
affecting the separation performance (Gautam and Simon tion as the low molecular weight of the polymer may draw all
2006, Hatti-Kaul 2001, Klomklao et al. 2005, Nitsawang et al. proteins to the top phase that will cause poor separation and
2006, Porto et al. 2008, Rahimpour and Baharvand 2009, Su low purification factors of the target protein (Johansson et al.
€ 2008). On the other hand, we also observed that molecular
and Chiang 2006, Y€ ucekan and Onal 2011). The partitions of
weights above 8000 g/mol caused to strongly increased vis-
the target protein and the other molecules in the extract pri-
cosity which is not suitable for ATPS. From this point of view,
marily depend on the PEG molecular weight and the system
molecular weights of PEG were chosen in the range of
composition because these parameters influence the protein
1000–8000 g/mol.
partition by changing the number of van der Waals, hydro-
In the beginning of the research, PEG 10%–K2HPO4 20%
phobic, hydrogen bond and ionic interactions. It was also
and PEG 15%–K2HPO4 15% systems were prepared with five
emphasized in the study conducted by Qiang Lin et al. that
different molecular weights of PEG as stated earlier. As can
among a broad array of the factors, the influence of phase-
be seen from Table 1, while SOD activity in the polymer-rich
forming polymers should be concerned firstly, including poly-
top phase generally decreased with increasing concentration
mer concentration and molecular weight (Han and Lee 1997).
and also molecular weight of PEG, the separate comparison
The effect of polymer concentration on biomaterial partition-
of each PEG molecular weight showed the activity increases
ing is obvious, and it is straightforward to design partitioning in the salt rich bottom phase with decreasing K2HPO4 con-
experiments to test trends with polymer concentration. centration. Theoretically, high Kp value along with high Ke
However, the polymer molecular weights influence partition- indicates that most of proteins as well as target one were
ing both by altering the phase diagram and by changing the more partitioning to the top phase, while high Ke with low
polymer–protein interaction; thus, the testing of the trend Kp implies that especially the target enzyme was more parti-
with polymer molecular weight is less straightforward. tioned into the top phase. According to the results, while Ke
Moreover, researchers cannot isolate the effect of polymer values were close to 1 except the systems prepared by
molecular weight from the effect of polymer concentration using 10% of PEG 1000 and both concentrations of PEG
(Lin et al. 2003). Thereby, it can be concluded that these two 6000 and 8000, Kp values were under 1 in all of the systems.
factors should be considered simultaneously for modelling of Therefore, the results can be interpreted as that while target
protein partitioning in ATPS. Accordingly, in order to select a protein SOD almost equally distributed between top and
suitable molecular weight of PEG and also phase composition bottom phases especially in the PEG 2000 and 3350 systems,
for the initial purification of menadione-induced SOD from total proteins predominantly accumulated in the bottom
P. chrysosporium, ATPSs were performed with five different phase of all the systems. In addition, it was clear that both
molecular weights of PEG (MW; 1000, 2000, 3350, 6000, and the target and total proteins predominantly distributed in
8000) and with different PEG and salt concentrations the bottom phases of PEG 6000 and 8000 systems in par-
(3.5–22%) in the beginning of the research. Other parameters ticular. This situation can be the result of volume excluding
including phase-forming salt type, enzyme extract amount effect of polymers with high molecular weights. On the
added to the systems and total system weights were kept other hand, that even the maximum Rb value of these PEG
constant. The pH values of the systems were the original and 6000 and 8000 systems was found as 23.28% clearly showed
384 B. KAVAKCıO
gLU ET AL.

Table 2. Effects of molecular weight of PEG and system composition in PEG–K2HPO4 on menadione-induced SOD partitioning at 25  C
Sample no. System At (U) Ab (U) Ke Kp PFt PFb Rt (%) Rb (%)
1 PEG 1000 20% - K2HPO4 8% 88.12 133.19 0.66 1.33 0.59 1.19 33.86 51.18
2 PEG 1000 22% - K2HPO4 7% 87.12 134.14 0.65 1.06 0.65 1.06 33.47 51.55
3 PEG 2000 20% - K2HPO4 8% 47.37 139.25 0.34 1.69 0.28 1.44 18.20 53.51
4 PEG 2000 22% - K2HPO4 7% 45.77 145.18 0.32 1.44 0.29 1.36 17.59 55.79
5 PEG 3350%20 - K2HPO4%5 43.39 234.17 0.19 0.68 0.41 1.51 16.67 89.98
6 PEG 3350%22 - K2HPO4%4 44.51 176.75 0.25 0.16 1.22 0.79 17.10 67.92
7 PEG 3350%22 - K2HPO4%3 — No phase formation —
8 PEG 6000%20 - K2HPO4%5 34.03 87.64 0.39 0.13 1.14 0.38 13.08 33.68
9 PEG 6000%22 - K2HPO4%4 27.58 46.50 0.59 0.17 0.72 0.21 10.60 17.87
10 PEG 6000%22 - K2HPO4%3.5 31.09 39.10 0.80 0.29 0.53 0.19 11.95 15.02
11 PEG 6000%20 - K2HPO4%3 — No phase formation —
12 PEG 6000%22 - K2HPO4%3
13 PEG 8000%20 - K2HPO4%5 18.92 46.42 0.41 0.11 0.73 0.19 7.27 17.84
14 PEG 8000%22 - K2HPO4%4 19.49 45.85 0.42 0.14 0.61 0.20 7.48 17.62
15 PEG 8000%22 - K2HPO4%3.5 16.19 44.82 0.36 0.15 0.48 0.19 6.22 17.22
16 PEG 8000%20 - K2HPO4%3 — No phase formation —
17 PEG 8000%22 - K2HPO4%3
0.5 mL crude extract with SOD activity of 260.4 U and total protein amount of 700 mg was present in each tube.

Table 3. Effects of PEG 3350 concentration and system pH on menadione induced SOD partitioning at 25  C
Sample no. System pH At (U) Ab (U) Ke Kp PFt PFb Rt (%) Rb (%)
1 PEG 3350%20 - K2HPO4%5 9.46 43.39 234.17 0.19 0.68 0.41 1.51 16.67 89.98
2 PEG 3350%20 - K2HPO4%5 7.68 102.23 147.13 0.69 2.38 0.55 1.91 39.26 56.54
3 PEG 3350%22 - K2HPO4%5 9.5 50.44 229.62 0.22 0.80 0.44 1.59 19.38 88.24
4 PEG 3350%22 - K2HPO4%5 7.98 105.52 143.80 0.73 3.04 0.54 2.23 40.55 55.26
5 PEG 3350%24 - K2HPO4%5 9.37 51.70 223.60 0.23 1.12 0.38 1.82 19.87 85.92
6 PEG 3350%24 - K2HPO4%5 7.67 105.74 143.58 0.74 5.15 0.49 3.39 40.63 55.17
7 PEG 3350%26 - K2HPO4%5 9.73 44.34 226.63 0.19 1.88 0.26 2.51 17.04 87.09
8 PEG 3350%26 - K2HPO4%5 7.43 100.93 148.39 0.68 3.48 0.49 2.55 38.79 57.02
0.5 mL crude extract with SOD activity of 260.4 U and total protein amount of 700 mg was present in each tube.

the denaturation effects, most likely due to the high salt Effects of PEG concentration and pH on the partitioning
concentrations. behaviors of menadione-induced SOD from
As shown in Table 2, to prevent enzyme denaturation, P. Chrysosporium in PEG 3350 – K2HPO4%5 systems
the systems were prepared by lowering the salt concentra-
tions from 15–20% to 3.5–8% and keeping the PEG amounts Because the best result was obtained in the PEG 3350%20 –
at minimum levels required to formation of two phases in K2HPO4%5 system, this system was analysed further more in
the second step of the study. Unlike the systems presented terms of polymer composition and pH under the conditions
in Table 1, Ke values were generally under 1 in all of the which salt amount was kept constant (Table 3). In these sys-
systems, which shows the bottom phase distribution of the tems, pH values were adjusted to pH 7.5 6 0.4 by using 1 M
target enzyme. According to the results, it was determined HCl or 1 M NaOH. As can be seen from Table 3, it can be said
that while Ke values slightly decreased with increasing con- for all investigated systems that adjustment of pH values
centrations of polymer and decreasing concentrations of salt approximately to 7.5 caused to distribution of target enzyme
in the PEG 1000 and 2000 systems, the significant increases almost equally between phases and consequently SOD activity
were observed in higher PEG molecular weights including increases in the top phase when compared with the systems
3350 and 6000 g/mol (Table 2). Although there are differen- in their original pH values. In addition, high Kp values along
ces among the species, generally, the molecular weight of with low Ke were obtained via pH adjustment and also opti-
SOD is in the range of 30–70 kDa (Ali et al. 2014, Benavides mization of polymer concentration. It was observed that
et al. 2008, Lee et al. 1981, Moran et al. 2003). It can be although SOD activities were slightly decreased in the bottom
thought that unlike the proteins with high molecular phase with the increasing percentages of polymer up to 24%,
weights, relatively small protein SOD more preferred the top PEG 3350%24 – K2HPO4%5 – pH 7.67 system was found as
phase with increasing molecular weight and also concentra- the best system in terms of its PFb value of 3.39 due to high
tion of PEG due to the hydrophobic interactions. However, selectiveness with the Ke and Kp values of 0.74 and 5.15,
the observed denaturation effect especially in PEG 6000 and respectively.
PEG 8000 systems may be due to the high viscosity of these In summary, it is obvious that (1) lowering pH values
systems. On the other hand, when considered in terms of Kp approximately to 7.5 resulted in increased SOD activities and
values, it can be said that the selectivities of the initial sys- accumulation of total proteins in the top phase (2) increasing
tems improved by manipulating system composition. The PEG 3350 percentage up to 24% contributed the top-phase
maximum PFb was found as 1.51 in PEG 3350%20 – distribution of total proteins together with slight increases in
K2HPO4%5 system with the Ke of 0.19, Kp of 0.68, and Rb of
Ke values. From all these points of view, PEG 3350%24 –
89.98%.
K2HPO4%5 system was chosen for further optimization of the
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 385

Table 4. Effects of system pH on menadione induced SOD partitioning at 25  C

Sample no. System pH At (U) Ab (U) Ke Kp PFt PFb Rt (%) Rb (%)
1 PEG 3350%24 - K2HPO4%5 9.00 36.19 132.24 0.27 1.22 0.35 1.58 19.41 71.11
2 PEG 3350%24 - K2HPO4%5 8.50 35.76 113.91 0.31 3.56 0.25 2.79 19.23 61.24
3 PEG 3350%24 - K2HPO4%5 8.00 44.50 97.22 0.46 4.56 0.29 2.91 23.93 52.27
4 PEG 3350%24 - K2HPO4%5 7.75 61.96 85.53 0.72 4.32 0.41 2.45 33.30 45.99
5 PEG 3350%24 - K2HPO4%5 7.50 51.17 108.06 0.47 5.87 0.32 3.99 27.51 58.10
6 PEG 3350%24 - K2HPO4%5 7.25 41.69 132.08 0.32 6.32 0.26 5.19 22.41 71.01
7 PEG 3350%24 - K2HPO4%5 7.00 24.50 146.75 0.17 6.65 0.15 6.04 13.17 78.90
8 PEG 3350%24 - K2HPO4%5 6.75 24.31 112.42 0.22 5.12 0.16 3.69 13.07 60.44
9 PEG 3350%24 - K2HPO4%5 6.50 22.35 89.54 0.25 4.48 0.15 2.64 12.01 48.14
0.5 mL crude extract with SOD activity of 186 U and total protein amount of 500 mg was present in each tube.

SOD purification in terms of pH. The chosen ATPS was pre- a molecular weight (MW) of 30 6 2.5 kDa as the monomer
pared in the pH values ranging from 9.00 to 6.50. According subunit. It can be certainly said that although the crude P.
to the results shown in Table 4, SOD activities gradually chrysosporium extract contained various proteins having differ-
increased in the top phase of the systems up to pH 7.75 and ent molecular weights (line 2), considerable amount of con-
redecreased in higher pH with the value of 36.19 U in pH taminating proteins were eliminated after ATPS purification.
9.00. On the other hand, maximum SOD activity in the bot- According to the results, the initial SOD activity of the
tom phase was found for pH 7.00 system as 146.75 U. In add- crude extract from P. chrysosporium was induced with
ition, the best PFb as 6.04 was obtained in this system due to 0.75 mM menadione from about 69 to 372 U/mg and reached
high selectivity to the target enzyme. It can be said that at to 2246.88 U/mg with 6.04-fold purification by one step PEG
neutral pH, SOD partition better behaviours compared to 3350%24 – K2HPO4%5 (pH 7.00) system. When available litera-
acidic or alkaline conditions. pH changed the partition behav- ture has taken into consideration, there is only a paper on
iour of SOD as evidenced by lower purification folds on sys- ATPS partition of SOD (Fernandez-Fueyo et al. 2012). In the
tems with pH 6.5 and 9.0 compared to pH 7.0. The study conducted by Simental-Martinez et al., SOD from clari-
assumption that as the pH increased above the isoelectric fied extract of K. marxianus was purified 4.0 6 0.17-fold after
point (pI) of a protein, it became negatively charged, its inter- PEG 3350 18.8% – phosphate 15% with 0.5 M NaCl ATPS frac-
action with PEG became stronger and the partition coefficient tionation and specific activity was reached to 85.2 6 3.5
increased has been stated in numerous studies. However, that (Simental-Martınez et al. 2014). When these results were com-
Ke values did not show increasing trend with the increase in pared with our results, purification fold and the last specific
pH from 6.5 to 9.0 may be the indication of dominant hydro- activity were enhanced 1.51- and 26.4-fold.
philicity of the target protein. This presumption was also sup- On the other hand, in the researches on SOD purification
ported in the study investigating the partition behaviour by using traditional chromatographic steps, it was determined
of glucose-6-phospate dehydrogenase (G6PDH) in ATPS that initial SOD activities were range from 1.8 to 150.2 U/mg
(Johansson et al. 2008). The researchers stated that although in the crude extracts from different resources such as human
the G6PDH was strongly negatively charged in studied condi- and mouse lung, rat liver, Neurospora crassa, Streptomyces
tions, Ke values were under 1 because hydrophilicity of the spp., Escherichia coli, Helicobacter pylori, Crithidia fasciculata,
protein led to an effective repulsion from the PEG phase. Kluyveromyces marxianus, and piper betle leaf (Fattman et al.
The selected PEG 3350%24 – K2HPO4%5 (pH 7.00) system 2000, Fernandez-Fueyo et al. 2012, Hayakawa et al. 1985,
was investigated for the evaluation of the effect of neutral Keele et al. 1970, Le Trant et al. 1983, Liu et al. 2015, Misra
salts (NaCl and KCl) on SOD partitioning. Adjustment of salt and Fridovich 1972, Mruk et al. 1998, Reiss and Gershon 1976,
content in the system was made by addition of salts (solid Youn et al. 1996). Menadione-induced SOD activity of the
form) into the system to obtain concentrations of 3, 5, 7, 9, crude extract from P. chrysosporium was about 2.48-fold
and 11% (w/w). Unfortunately, the result of PF suggests that higher than the value observed in Streptomyces spp. extract
addition of neutral salts could not improve the partitioning of which was the highest one among the investigated literature
SOD in the bottom phase of the selected system (data not (Liu et al. 2015). Additionally, it should also be stated that
shown). Based on all these results, it was decided to choose even after the complex purification processes including two
PEG 3350%24 – K2HPO4%5 (pH 7.00) system as the optimized or more time-consuming chromatographic steps aiming at
ATPS for the initial purification of P. chrysosporium superoxide isolated SOD from bacteria as Escherichia coli, Helicobacter
dismutase. pylori, Nocardia asteroides, and Desulfovibrio gigas, fungi as
Neurospora crassa and tissues as rat liver purified SOD activ-
ities were close or lower than ATPS purified SOD from
Native molecular weight and protein pattern of
P. chrysosporium (Beaman et al. 1983, Dos Santos et al. 2000,
Phanerochaete chrysosporium superoxide dismutase
Johansson et al. 2008, Reiss and Gershon 1976, Spiegelhalder
partitioned with ATPS
et al. 1993, Yost and Fridovich 1973). On the other hand,
The molecular weight of the native form of the SOD was there were also outstanding results in the range of
found to be 60 6 4 kDa by using Sephadex G-100 gel filtration 10,930–64,200 U/mg but particularly in the limited number of
chromatography (Figure 1). On the other hand, as seen from researches using at least five or more chromatographic and
Figure 2, bottom phase (lane 4) contained a major band with required dialysis steps (Baum et al. 1983, Marklund 1982).
386 B. KAVAKCıO
gLU ET AL.

Figure 1. (A) Elution profile of SOD from Sephadex G-100 column; (B) Molecular weight calibration curve; standard proteins (––) and ATPS purified SOD (–䊊–).

Figure 2. SDS–PAGE profiles of P. chrysosporium SOD purified by partitioning in ATPS. Lane 1: molecular weight markers (15–260 kDa) (10 lg), lane 2: top phase of the
optimized system (25 lg), lane 3: crude SOD extract (25 lg) and lane 4: bottom phase of the optimized system (25 lg).

In comparison, the ATPS developed in this study could Conclusions

remove the contaminant protein while concentrating SOD,
omitting the steps of precipitation and chromatography used The partitioning and purification of SOD from P. chrysosporium
by the others to remove the contaminants. in various ATPSs have been reported in this paper. The opti-
From these points of view, compared to many other purifi- mized system for the purification of SOD was PEG 3350 24%
cation studies, the results as 6.04 purification fold and – K2HPO4 5% without NaCl addition at pH 7.0 with Ke 0.17; Kp
2246.88 U/mg last specific activity obtained from developed 6.65; Rb 78.90 and PFb 6.04. From the studied systems, it can
one step PEG-salt ATPS purification after menadione induction be concluded for the partition of SOD in PEG–phosphate sys-
of SOD in P. chrysosporium were absolutely attractive. tems that high PF values in the bottom phases could be
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 387

achieved by keeping K2HPO4 concentration low and pH close de Ara ujo RFF, Porto TS, Martins DBG, Dutra RF, Porto ALF, de Lima Filho
to neutral pH values. Addition of non-phase forming salt JL. 2011. Partitioning of lactate dehydrogenase from bovine heart
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