Research Article

Received: 30 November 2011 Revised: 19 March 2012 Accepted: 11 May 2012 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jctb.3868

The optimization of aqueous two-phase

extraction of lysozyme from crude hen egg
white using response surface methodology
Yanmin Lu, Wenjuan Lu, Wei Wang, Qingwei Guo and Yanzhao Yang∗

Abstract
BACKGROUND: Aqueous two-phase extraction is a versatile method for separating biological particles and macromolecules.
In the present wok, the feasibility of using PEG 4000/potassium citrate aqueous two-phase system (ATPS) for recovering and
purifying lysozyme was investigated. Response surface methodology was used to determine an optimized ATPS for purification
of lysozyme from crude hen egg white.

RESULTS: Mathematical models concerning the purification of lysozyme from chicken egg white in polyethylene glycol 4000 (PEG
4000)/potassium citrate ATPS are established using response surface methodology. Screening experiments using fractional
factorial designs show that the pH of the system significantly affects the recovery and purification of lysozyme. An optimized
ATPS was proved to be at pH 5.5 and 30 ◦ C and contained 18% (w/w) PEG, 16% (w/w) potassium citrate, 3.75% (w/w) potassium
chloride (KCl). Under those conditions, the specific activity, purification factor and activity yield for lysozyme were 31100 U
mg−1 , 21.11 and 103%, respectively.

CONCLUSION: The PEG 4000/potassium citrate ATPS has the potential to be applied to establish bioprocesses for the primary
recovery and partial purification of lysozyme.

c 2012 Society of Chemical Industry

Keywords: aqueous two-phase system; bioseparations; extraction; purification; statistical design

INTRODUCTION yield of 70% of lysozyme were obtained by Su and Chiang.15

Egg white is a natural protein source with exceptional nutritional
and biological potential, as well as biotechnological interest.1
Among egg white proteins, lysozyme (14.4 KDa) is a hydrophilic
protein that has been well characterized and widely used as a
model for numerous scientific studies, such as enzymatic reactivity,
protein stability and crystallization.2,3,4 Moreover, lysozyme is
an enzyme which has bactericidal and bacteriostatic properties.
It has been commonly used as a natural preservative in
the food industry.5 Different methods, such as ion exchange
chromatography,6 gel-filtration chromatography,7 dye-binding
chromatography,8 membrane separation,9 reverse micelles,10
magnetic cation exchange11 and ethanol precipitation12,13 can
be applied to separate lysozyme from hen egg white in order
to prepare commercial product. However, only a few have been
implemented at industrial scale. It is desirable to develop new
techniques that could improve or even replace some stages of the
current purification procedures.
Partitioning in aqueous two phase systems (ATPSs) has been
proved to be a valuable technique for separating and purifying
mixtures of biomolecules. Compared with other separation
techniques, aqueous two phase extraction offers many advantages
such as a short processing time, low energy consumption,
biocompatible environment and the relative ease of its scale-up.14
Aqueous two-phase systems are hence a promising technique
and have been successfully employed to isolate lysozyme from
hen egg white. A specific activity of about 39 500 U mg−1 and
The lysozyme is able to preferentially interact with hydrophobic
polymers such as PEG.16 It is also verified that the hydrophobic
interaction is the main driving force for lysozyme partitioning in
polymer/salts ATPS.17 Dembczynski separated the lysozyme from
egg white using EO50PO50/K2 HPO4 ATPS with a high yield of
85% and specific activity of 32 300 U mg−1 .18,19 The partitioning
behavior of both lysozyme and conalbumin proteins was discussed
in PEG/citrate and PEG/sulfate ATPSs by Sousa.20 It was found that
lysozyme migrates toward the polymeric phase and conalbumin
to the saline phase in the two systems.
Lysozyme has the highest total surface hydrophobicity of the
proteins in chicken egg white.15,16 Protein hydrophobicity played
a greater role in protein partitioning in PEG 4000/citrate systems
than in the two other PEG/salt systems (PEG 4000/phosphate
system and PEG 4000/sulfate system).21 The partition coefficients
of lysozyme are higher in the PEG/citrate systems than that in
PEG/sulfate systems.20 Since citrates are biodegradable and non-
toxic, PEG + citrate salts could form environmentally safe ATPS,
∗
Correspondence to: Yanzhao Yang, School of Chemistry and Chemical
Engineering, Shandong University, 27 Shanda Nanlu, Jinan, PR China.
E-mail: yzhyang@sdu.edu.cn
Key Laboratory for Special Functional Aggregated Materials of Education
Ministry, School of Chemistry and Chemical Engineering, Shandong University,
Jinan 250100, P.R. China

J Chem Technol Biotechnol (2012) www.soci.org

c 2012 Society of Chemical Industry
 www.soci.org
which are more suitable for the extraction of biological materials.22
However, there is no research on recovery and purification of
lysozyme from crude hen egg white using PEG/citrate ATPS.
In the present work, an ATPS composed of PEG and potassium
citrate was employed. The major goals of this work were to
investigate the feasibility of using PEG4000/potassium citrate
ATPS for recovering and purifying lysozyme from crude hen egg
white and to optimize the operating conditions of this process.
The optimum conditions were obtained at low cost and in short
time using response surface methodology (RSM). The lysozyme
had transferred into the PEG-rich upper phase, while the majority
of other concomitants remained in the bottom phase. This ATPS
was used to purify lysozyme successfully from egg white. The
lysozyme was recovered in the PEG-rich top phase with an activity
yield of 103% and a purification factor of 21.11.
MATERIALS AND METHODS
Materials
Hen eggs were purchased from a market. Potassium citrate, citric
acid and Coomassie Blue G250 (Sinopharm Chemical Reagent
Co., Ltd), PEG4000 (Kermel), lysozyme (Amresco), Micrococcus
lysodeikticus (Sigma-Aldrich), and BCA (Aladdin) were used
without further purification. All the reagents were of analytical
grade. Pure water was used throughout this research. The pure
water was prepared by aquaprince ultrapure water machine
(AJF-0501, china). The conductivity of pure water is 2.1 µS cm−1
(conductivity meter: DDS-307, Shanghai Precision & Scientific
Instrument Co., Ltd).
Preparation of the aqueous two-phase systems (ATPS)
The potassium citrate buffers were prepared by adding a small
amount of 40% (w/w) citric acid solution to 40% (w/w) potassium
citrate solution to reach the desired pH. ATPSs were prepared
by weighing out appropriate amounts of 50% (w/w) PEG stock
solution, 40% (w/w) potassium citrate buffer solution, lysozyme
solution and water in order to achieve the desired final system
composition. All systems were mixed using a vortex mixer and
centrifuged for 10 min at 1500 rpm after being retained at a
specified temperature for 30 min. The volume of each phase
was measured when the system turned clear. Top and bottom
phases were separated with pipettes and assayed for protein
concentration and protease activity.
The activity yield (Y1 ) was determined as the ratio of the total
activity in the top phase to that of the initial extract. The specific
activity (Y2 ) was expressed in units per milligram of protein (U
mg−1 ) in the top phase. The purification factor (PF) was calculated
as the ratio of the specific activity in the top phase to the specific
activity in the egg white.
Response surface experimental design and data analysis
Response surface design, modeling and analysis were carried out
using SAS software (Version 9.2 in Windows; SAS Institute Inc. Cary,
NC, USA).
Screening experiment by fractional factorial designs were
performed initially. Five independent variables, including PEG
concentration (X1 ), potassium citrate concentration (X2 ), potassium
chloride (X3 ), pH (X4 ) and temperature (X5 ) were taken into account.
The purification factor (PF) and activity yield (Y1 ) of lysozyme were
set as the response variables. On the basis of the first-order
wileyonlinelibrary.com/jctb

c 2012 Society of Chemical Industry
Y Lu et al.
model (1) obtained by the factorial design used, a new series of
experiments was done in the direction of steepest ascent.


k
Y = β0 + βi Xi + ε (1)
i=1
where Y is the response (in per cent), β0 and βi are regression
coefficients of the model, and Xi is the variable that represents the
experimental factor.
Response surface methodology (RSM) was used to optimize the
separation system and better evaluate the interaction among the
factors. The second-order model for predicting the optimum was
expressed according to the following equation:


k

k


Y = β0 + βi Xi + βii Xi2 + βij Xi Xj + ε (2)
i=1 i=1 i<j
where Y is the response (in per cent), β0 is the value for the fixed
response at the central point of the experiment, βi , βii and βij are
the linear, quadratic and cross-product coefficients, respectively,
and Xi and Xj are the coded independent variables.
Analytical methods
The total protein concentration was analyzed using the BCA assay
(562 nm), using BSA as the standard.23 The activity of the lysozyme
was determined by turbidimetric assay, based on the lysis of M.
lysodeikticus by the enzyme.24 One unit of lysozyme activity is
equal to a decrease in turbidity of 0.001 min−1 at 450 nm at pH
6.24 and 30 ◦ C. The activity of the lysozyme was expressed in units
per milligram of protein (U mg−1 ). To avoid interference from PEG
and citrate, all samples were analyzed against blanks containing
the same phase composition but without protein.
Size exclusion chromatography (SEC)
SEC experiments were carried out on LC-10ATvp (Shimadzu). An
ultrahydrogel linear column (7.8 mm I.D. × 30 cm) was used.
Assays were performed at room temperature, at a flow rate
of 0.5 mL min−1 . A 20 µL sample was eluted with 0.05 mol L−1
sodium phosphate buffer and 0.2 mol L−1 NaCl at pH 7. The
chromatographic mobile phase prior to use, and all samples before
injection into the column, were filtered through a membrane filter
(0.45 mm, Millipore). Protein was monitored by measuring the
absorbance at 280 nm.
Electrophoresis
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) was carried out according to the method of Laemmli,25
using a 12% polyacrylamide separating gel and a 5% stacking gel.
Commercial pure lysozyme (activity of 30 000 U mg−1 , Sigma) was
used to identify the lysozyme bands. Electrophoresis was run at a
constant voltage of 100 V. The protein samples migrated towards
the cathode during electrophoresis. Proteins bands were stained
with Coomassie Blue R250 according to the method of Meyer.26
RESULTS AND DISCUSSION
Analysis of screening experiments
Screening experiments were carried out to determine the
important factors in protein partitioning. Based on the phase
J Chem Technol Biotechnol (2012)
Extraction of lysozyme from chicken egg white www.soci.org

Table 1. Conditions and results of the fractional factorial design Table 2. Experimental design of the ascent and corresponding results
selected for lysozyme extraction by PEG4000/potassium citrate ATPS.
The significance of the variables is indicated in footnotes∗ X1 /% X2 /% X3 /% Y1 /% PF Y2 (U mg−1 )

X1 /% X2 /% X3 /% X4 X5 / ◦ C Y1 /% PF 0 18 17.5 5 74.78 14.49 21377

1 17.7 17.3 4.6 75.06 14.49 21375
1 15 15 2 6 45 71.20 19.18 2 17.4 17.1 4.2 85.82 14.92 22000
2 21 15 2 6 25 84.24 19.00 3 17.1 16.9 3.8 84.32 18.89 27862
3 15 20 2 6 25 82.13 19.33 4 16.8 16.7 3.4 88.09 19.02 28048
4 21 20 2 6 45 75.03 18.82 5 16.5 16.5 3 89.63 19.43 28667
5 15 15 8 6 25 94.36 16.21 6 16.2 16.3 2.6 87.23 20.53 30288
6 21 15 8 6 45 84.78 12.93 7 15.9 16.1 2.2 95.39 22.09 32582
7 15 20 8 6 45 82.16 16.26 8 15.6 15.9 1.8 84.11 20.09 29637
8 21 20 8 6 25 9.50 2.20 9 15.3 15.7 1.4 68.28 20.01 29512
9 15 15 2 9 25 89.61 12.97 10 15 15.5 1 68.42 17.53 25855
10 21 15 2 9 45 59.63 11.68 11 14.7 15.3 0.6 57.13 17.33 25561
11 15 20 2 9 45 54.22 13.09 12 14.4 15.1 0.2 53.98 13.94 20562
12 21 20 2 9 25 8.75 6.37
13 15 15 8 9 45 95.85 13.82 X1 : PEG4000 concentration (w/w); X2 : potassium citrate concentration
14 21 15 8 9 25 19.49 6.34 (w/w); X3 : potassium chloride concentration (w/w); Y1 : activity yield; PF:
purification factor; Y2 : specific activity.
15 15 20 8 9 25 7.04 2.68
16 21 20 8 9 45 9.97 3.91
17 18 17.5 5 7.5 35 63.15 11.31
18 18 17.5 5 7.5 35 67.71 11.59 the system was fixed at 5.5 for subsequent experiments. According
19 18 17.5 5 7.5 35 66.73 10.44 to the regression equations, both activity yield and purification
∗
factor slightly increase as the temperature (X5 ) increases. In order
X1 : PEG4000 concentration (w/w); X2 : potassium citrate concentration to facilitate the subsequent operation, the temperature of aqueous
(w/w); X3 : potassium chloride concentration (w/w); X4 : system pH; X5 :
system temperature; Y1 : activity yield; PF: purification factor. two phase extraction was fixed at 30 ◦ C.

Path of steepest ascent

diagram, PEG4000 and potassium citrate levels were selected.27
Fractional factorial designs and the results are illustrated in Table 1.
On the basis of the data obtained by this design, the models
obtained for each of the response variables (Y1 and PF) can be
expressed by the following equations (in uncoded values):
Y1 = 319.15 − 4.69X1 − 6.76X2 − 2.53X3 − 9.95X4 + 0.86X5
(3)
PF = 53.02 − 0.67X1 − 0.74X2 − 0.96X3 − 2.21X4 + 0.15X5
(4)
In general, the magnitude of the coefficients of the linear
regression models could be used to evaluate the contribution
of the corresponding independent variables to the dependent
variables.15 Increasing the PEG concentration causes a rise in
intermolecular interactions between the protein and the polymer.
Therefore the lysozyme tends to partition to the upper phase.20
However, according to the regression equations, the effect of PEG
concentration (X1 ) is quite the opposite of the result reported in
the literature. It can be explained that the denaturation of protein
encountered in the system due to the high concentrations of PEG
and salts, such as the experiment runs 8, 12, 15 and 16 in Table 1.
According to the significance test, the concentration of potassium
citrate (X2 , P = 0.0161) and potassium chloride (X3 , P = 0.0008)
are significant factors (P < 0.05) for PF.
The regression equations also suggested that pH (X4 ) is a
significant factor for the two response variables. Y1 and PF decrease
as the pH of the system is increased. It can be explained that the
lysozyme was stable under acidic conditions.28 After denaturation
of lysozyme under acidic conditions, natively folded protein could
be detected in the soluble fraction of the sample. Conversely, under
basic conditions, no native lysozyme was detected.29 The pH of
Based on the first-order model equation, the path of steepest
ascent was to improve the activity yield (Y1 ) and purification factor
(PF) of lysozyme. The specific activity (Y2 ) of lysozyme was also
calculated. Details of the experiments and results are shown in
Table 2. It is obvious that ATPS 7 is in the general vicinity of the
optimum. An ATPS containing 2.5% (w/w) KCl, 16% (w/w) PEG and
16% (w/w) potassium citrate was chosen for further experiments.
Response surface method experiments
A central composite face-centred design (CCF) was used to
optimize the aqueous two phase extraction process for lysozyme
purification. The response variables were activity yield (Y1 ),
purification factor (PF) and specific activity (Y2 ) of lysozyme. Details
of the design and experimental results are given in Table 3. The
experimental results of the central composite face-centred design
are fitted to a second-order-prediction function. The models were
expressed as follows:
Y1 = 64.97 + 0.70X3 − 0.65X12 + 1.46X1 X2 − 0.66X1 X3
−0.79X22 + 0.97X2 X3 (5)
PF = 8.94 + 0.51X1 − 0.00075X22 + 0.0068X2 X3 (6)
Y2 = 13 185.98 + 748.41X1 − 1.10X22 + 10.03X2 X3 (7)
The high values of R-squared (R2 = 0.967 for Y1 ; R2 = 0.893
for PF and Y2 ) and the adjusted R-squares (R2adj = 0.947 for Y1 ;
R2adj = 0.868 for PF and Y2 ) were calculated separately, which
indicates that there is a good degree of correlation between actual
and predicted data.30,31 Statistical analysis of lysozyme purification
using a regression model was checked by Fisher’s F-test for analysis
of variance (ANOVA) and the results are shown in Table 4. The
ANOVA, which was performed on the prediction models, shows

J Chem Technol Biotechnol (2012)

c 2012 Society of Chemical Industry wileyonlinelibrary.com/jctb
 www.soci.org Y Lu et al.

Table 3. Uncoded values of the variables used in the different

experimental assays of the central composite design and the
corresponding experimental results

X1 /% X2 /% X3 /% Y1 /% PF Y2

1 14 15 0 71.00 15.18 22388

2 18 15 0 69.43 16.89 24915
3 14 17 0 52.41 13.69 20197
4 18 17 0 73.96 15.67 23116
5 14 15 5 94.74 15.23 22463
6 18 15 5 88.84 17.54 25877
7 14 17 5 95.76 15.13 22319
8 18 17 5 95.18 17.61 25977
9 14 16 2.5 95.62 19.35 28539
10 18 16 2.5 98.42 21.01 30990
11 16 15 2.5 73.73 15.55 22936
12 16 17 2.5 74.11 16.79 24762
13 16 15 0 63.44 17.07 25183
14 16 15 5 91.21 15.35 22647
15 16 16 2.5 88.99 18.41 27150
16 16 16 2.5 91.45 20.01 29512
17 16 16 2.5 91.26 20.87 30785
X1 : PEG4000 concentration (w/w); X2 : potassium citrate concentration
(w/w); X3 : potassium chloride concentration (w/w); Y1 : activity yield; PF:
purification factor; Y2 : specific activity.

Table 4. Analysis of Variance (ANOVA) for the quadratic models

predicted for each response variable
Response
variable Source DF SS MS F Pr>F

Y1 Model 6 2941.586 490.2644 48.84762 <0.0001

Error 10 100.3661 10.03661
Lack of fit 8 96.60846 12.07606 6.427515 0.1416
Pure error 2 3.757613 1.878807
Total 16 30.41952
PF Model 3 67.37261 22.45754 36.03206 <0.0001
Error 13 8.10245 0.623265
Lack of fit 11 4.974731 0.452248 0.289187 0.9316
Pure error 2 3.127719 1.56386
Total 16 75.47506
Y2 Model 3 146570000 48855546 36.03484 <0.0001
Error 13 17625224 1355786 Figure 1. Response surface plot for a PEG4000/potassium citrate ATPS for
Lack of fit 11 10820480 983680 0.289116 0.9317 the separation of lysozyme from egg white. The actual levels corresponding
Pure error 2 6804744 3402372 to the coded levels of each variable: Cpotassium citrate (w/w) ∼ (–1: 15%, 1:
17%); Cpotassium chloride (w/w) ∼ (−1: 0, 1: 5%). Other conditions used in the
Total 16 164190000 study: 18% (w/w) PEG4000, T = 30 ◦ C, pH = 5.5.
Y1 : activity yield; PF: purification factor; Y2 : specific activity.

The response surface plots generated by Wolfram Mathematica

that all models are statistically reliable, with P-values much lower
than 0.05. The Fisher F-test with a very low probability value (Pr >
F < 0.0001) demonstrates very high significance for the regression
model and confirmed the adequacy of the quadratic model.32,33
This result indicates that it is statistically significant at the 99.999%
confidence level.34 Moreover, the P-value of the lack of fit is higher
than 0.05. The F-values and ‘probability > F’ values of all the
regression equations show that these models are significant. All
of the above considerations indicate excellent adequacy of the
quadratic models to the experimental data.
7 are shown in Fig. 1 and Fig. 2. These plots provide a method to
visualize the relationship between responses and test variables.35
Through the response surface plots, the interactions between two
variables and their optimum ranges can be well understood.
A linear increase in activity yield is observed with increase
in KCl concentration (Fig. 1, X3 ) at high PEG concentration.
According to the Hofmeister series, the choride ions are referred
to as chaotropes (‘water structure breakers’) and preferentially
partitioned to the polymer-rich phase. The phenomenon was
reported by Berggren.36 The lysozyme (pI = 10.7) is positively
charged at pH 5.5 applied in the present work. The recovery of the

wileyonlinelibrary.com/jctb

c 2012 Society of Chemical Industry J Chem Technol Biotechnol (2012)
Extraction of lysozyme from chicken egg white
Figure 2. Response surface plot for a PEG4000/potassium citrate ATPS for
the separation of lysozyme from egg white. The actual levels corresponding
to the coded levels of each variable: CPEG4000 (w/w) ∼ (−1: 14%, 1: 18%);
Cpotassium citrate (w/w) ∼ (−1: 15%, 1: 17%). Other conditions used in the
study: 3.75% (w/w) potassium chloride, T = 30 ◦ C, pH = 5.5.
lysozyme increases as the concentration of KCl increases. However,
the specific activity and the purification factor of lysozyme have
no obvious change.
Figure 1 and Fig. 2 indicate that the maximum response
variables (Y1 , PF and Y2 ) were recorded at the ‘0’ level (X2 : 16%
(w/w) potassium citrate). The response variables increase as the
concentration of potassium citrate increases at the beginning.
This could be due to the ‘salt out effect’, which will lead
to increased partitioning of the proteins to the top phase.37
Moreover, the lysozyme and conalbumin tend to enrich in the
J Chem Technol Biotechnol (2012)

c 2012 Society of Chemical Industry
www.soci.org
Table 5. Extraction of lysozyme from egg white used
PEG4000/potassium citrate ATPS at the optimum conditions
Y1 PF Y2
1 109.26 22.67 33439
2 101.54 20.68 30504
3 104.32 21.56 31805
4 103.92 21.33 31464
5 105.11 21.63 31897
6 98.58 20.01 29517
7 96.20 19.89 29345
Average 102.70 21.11 31139
Conditions used in the study: 18% (w/w) PEG4000, 16% (w/w) potassium
citrate, 3.75% (w/w) potassium chloride, T = 30 ◦ C, pH = 5.5. Y1 : activity
yield; PF: purification factor; Y2 : specific activity.
upper phase and bottom phase, respectively, of the PEG–citrate
system.20 Therefore, the lysozyme may transfer to the upper phase
preferentially due to the ‘salt out effect’. Thus, PF and Y2 of lysozyme
in the upper phase are enhanced. However, the opposite trends
are shown when the concentration of potassium citrate is higher
than 16% (w/w). This could be caused by the ‘exclusion volume
effect’ strengthening, which leads to movement of the protein
from the top phase to the bottom phase.38,39
Furthermore, the PF and specific activity are increased as the
concentration of PEG 4000 is increased (Fig. 2). This is due to the
fact that the extraction process of lysozme is controlled by the
enthalpy.20 The intermolecular interactions between the protein
and the polymer are improved and the self-energy of the upper
phase is enhanced with the PEG concentration increasing. Both
facts are in favor of lysozyme extraction. By analyzing the plots,
the estimated specific activity (31 200 U mg−1 ), purification factor
(21.15) and activity yield (104%) for lysozyme were obtained at
the global solution of 18% (w/w) PEG4000, 16% (w/w) potassium
citrate and 3.75% (w/w) KCl.
To verify the calculated optimum, seven parallel experiments
were carried out at the factor levels determined by the RSM. The
average of specific activity, purification factor and activity yield
for lysozyme were 31 100 U mg−1 , 21.11 and 103%, respectively.
The results are shown in Table 5. A strong similarity is observed
between the predicted and experimental results reflecting the
adequacy of RSM to optimize lysozyme purification using ATPS
and the accuracy of the models to predict the desired responses.40
Evaluation of lysozyme purity by SEC and SDS-PAGE
Under the optimum extraction conditions, the purity of the
lysozyme was analyzed by SEC and SDS–PAGE (Fig. 3 and Fig. 4).
All the lysozyme in the egg white is transferred into PEG phase
(Fig. 3 – trace c and Fig. 4 – line 4). As shown in Fig. 3, the retention
time of Lysozyme is 26.5 min (trace a), and the high molecular
weight proteins present in egg white are retained in the bottom
phase (trace c). The results are consistent with the analysis of
SDS–PAGE. However, some traces of contaminant proteins are
still present in PEG-rich phase (Fig. 4 – line 3) and a commercial
lysozyme (Fig. 4 - line 1).
CONCLUSIONS
The extraction of lysozyme from egg white has been proved
to be promising as it has wide application in various industrial
wileyonlinelibrary.com/jctb
 www.soci.org
Figure 3. Size-exclusion chromatograms at 280 nm of (a) lysozyme stan-
dard at the concentration of 5 mg mL−1 , (b) the PEG-rich phase product
after extraction, (c) the bottom phase product after extraction, (d) hen egg
white three times diluted.
Figure 4. SDS-PAGE profile of lysozyme during purification by
PEG4000/potassium citrate ATPS. Samples on the gel are arranged as
followed: (1) lysozyme standard, (2) hen egg white, (3) the PEG-rich phase
product after extraction, (4) the bottom phase product after extraction.
fields. For instance, the primary recovery and partial purification
of the lysozyme could serve as a food preservative to protect
against L. monocytogenes contamination. It can also be used as a
drug for the treatment of ulcers and infections. The potential
applications for this egg white derived enzyme are diverse.
Further research and development are ongoing. In this work,
PEG4000/potassium citrate ATPS was employed at laboratory
scale to purify lysozyme from egg white. It is considered to be an
efficient, inexpensive, biodegradable and non-toxic system. Using
a fractional factorial design followed by RSM design, an optimized
ATPS was proved to be at pH 5.5 and 30 ◦ C and contained 18%
(w/w) PEG, 16% (w/w) potassium citrate, 3.75% (w/w) KCl. Under
these conditions, the lysozyme is recovered in the PEG-rich top
phase with an activity yield of 103% and a purification factor
of 21.11 in one-step extraction. The molecular weight of the
isolated enzyme (lysozyme) was also determined as 14.4 kDa by
SDS-PAGE along with protein marker (standard lysozyme). The
PEG4000/potassium citrate ATPS has the potential to be applied
wileyonlinelibrary.com/jctb

c 2012 Society of Chemical Industry
Y Lu et al.
to establish bioprocesses for the primary recovery and partial
purification of lysozyme.
ACKNOWLEDGEMENT
The authors acknowledge the financial support for this work
from the National Natural Science Foundation of China (grant
no. 20876089 and 21076115) and Natural Science Foundation of
Shandong province (No. ZR2010BM019).
REFERENCES
1 Awade AC, Moreau S, Molle D, Brulé G, and Maubois JL, Two-step
chromatographic procedure for the purification of hen egg
white ovomucin, lysozyme, ovotransferrin and ovalbumin and
characterization of purified proteins. J Chromatogr A 677:279–288
(1994).
2 Mine S, Tate S, Ueda T, Kainosho M and Imoto T, Analysis of the
relationship between enzyme activity and its internal motion using
nuclear magnetic resonance: 15 N relaxation studies of wild-type
and mutant lysozyme. J Mol Biol 286:1547–1565 (1999).
3 Singh S and Singh J, Effect of polyols on the conformational stability
and biological activity of a model protein lysozyme. AAPS Pharm Sci
Tech 4:E42 (2003).
4 Durbin SD and Feher G, Crystal growth studies of lysozyme as a model
for protein crystallization. J Cryst Growth 76:583–592 (1986).
5 Mecitoglu C, Yemenicioglu A, Arslanoglu A, Elmaci ZS, Korel F and
Cetin AE, Incorporation of partially purified hen egg white lysozyme
into zein films for antimicrobial food packaging. Food Res Int
39:12–21 (2006).
6 Jiang CM, Wang MC, Chang WH and Chang HM, Isolation of lysozyme
from hen albumen by alcohol-insoluble cross linked pea pod solid
ion-exchange chromatography. J Food Sci 66:1089–1092 (2001).
7 Islam R, Kite J, Baker AS, Ching Jr A and Islam MR, Affinity purification
of hen egg lysozyme using sephadex G75. Afr J Biotechnol
5:1902–1908 (2006).
8 Tejeda-Mansir A, Montesinos RM, Magana-Plaza I and Guzman R,
Breakthrough performance of stacks of dye-cellulosic fabric
in affinity chromatography of lysozyme. Bioproc Biosyst Eng
25:235–242 (2003).
9 Chiu HC, Lin CW and Suen SY, Isolation of lysozyme from hen egg
albumen using glass fiber-based cation-exchange membranes.
J Membr Sci 290:259–266 (2007).
10 Noh KM and Imm JY, One-step separation of lysozyme
by reverse micelles formed by the cationic surfactant,
cetyldimethylammonium bromide. Food Chem 93:95–101 (2005).
11 Safarik I, Sabatkova Z, Tokar O and Safarikova M, Magnetic cation
exchange isolation of lysozyme from native hen egg white. Food
Technol Biotechnol 45:355–359 (2007).
12 Gemili S, Umdu ES, Yaprak N, Ustok FI, Yener FYG and Gucbilmez CM,
Partial purification of hen egg white lysozyme by ethanol
precipitation method and determination of the thermal stability
of its lyophilized form. Turk J Agric For 31:125–134 (2007).
13 Jin MF, Davidson PM, Zivanovic S and Zhong QX, Production of corn
zein microparticles with loaded lysozyme directly extracted from
hen egg white using spray drying: extraction studies. Food Chem
115:509–514 (2009).
14 Balasubramaniam D, Wilkinson C, Van Cott K and Zhang C, Tobacco
protein separation by aqueous two-phase extraction. J Chromatogr
A 989:119–129 (2003).
15 Su CK and Chiang BH, Partitioning and purification of lysozyme from
chicken egg white using aqueous two-phase system. Process
Biochem 41:257–263 (2006).
16 Franco TT, Andrews AT and Asenjo JA, Use of chemically modified
proteins to study the effect of a single protein property on
partitioning in aqueous two-phase systems: effect of surface
hydrophobicity. Biotechnol Bioeng 49:300–308 (1996).
17 Johansson HO, Magaldi FM, Feitosa E and Pessoa Jr A, Protein
partitioning in poly(ethylene glycol)/sodium polyacrylate aqueous
two-phase systems. J Chromatogr A 1178:145–153 (2008).
18 Dembczyński R, Białas W and Jankowski T, Recycling of phase
components during lysozyme extraction from hen egg white in
J Chem Technol Biotechnol (2012)
Extraction of lysozyme from chicken egg white
the EO50PO50/K2 HPO4 aqueous two-phase system. Biochem Eng J
51:24–31 (2010).
19 Dembczyński R, Białas W, Regulski K and Jankowski T, Lysozyme
extraction from egg white in an aqueous two-phase system
composed of ehthylene oxide-propylene oxide thermoseparating
copolymer and potassium phosphate. Process Biochem 45:369–374
(2010).
20 Sousa RCS, Coimbra JSR, Silva LHM, Silva MCH, Rojas EEG and
Vicente AAA, Thermodynamic studies of partitioning behavior
of lysozyme and conalbumin in aqueous two-phase systems.
J Chromatogr B 877:2579–2584 (2009).
21 Andrews BA, Schmidt AS and Asenjo JA, Correlation for the partition
behavior of proteins in aqueous two-phase systems: effect of surface
hydrophobicity and charge. Biotechnol Bioeng 90:380–390 (2005).
22 Perumalsamy M and Murugesan T, Prediction of liquid–liquid
equilibria for PEG 2000–sodium citrate based aqueous two-phase
systems. Fluid Phase Equilib 244:52–61 (2006).
23 Smith PK, Krohn RI, Hemansson GT, Mallia AK, Gartner FH,
Provenzano MD, et al, Measurement of protein using bichoninic
acid. Anal Biochem 150:76–85 (1985).
24 Shugar D, Determination of lysozyme. BiochimBiophysActa 8:302–309
(1952).
25 Laemmli UK, Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 227:680–685 (1970).
26 Meyer TS and Lamberts Bl, Use of coomassie brilliant blue R250 for the
electrophoresis of microgram quantities of parotid saliva proteins
on acrylamide-gel strips. Biochim Biophys Acta 107:144–145 (1965).
27 Lu YM, Yang YZ, Zhao XD and Xia CB, Bovine serum albumin
partitioning in polyethylene glycol (PEG)/potassium citrate aqueous
two-phase systems. Food Bioprod Process 88:40–46 (2010).
28 Ellison RT and Giehl TJ, Killing of gram-negative bacteria by lactoferrin
and lysozyme. J Clin Investig 88:1080–1091 (1991).
29 Mamontov E, O’Neill H and Zhang Q, Mean-squared atomic
displacements in hydrated lysozyme, native and denatured. J Biol
Phys 36:291–297 (2010).
30 Su SN, Nie HL, Zhu LM and Chen TX, Optimization of adsorption
conditions of papain on dye affinity membrane using response
surface methodology. Bioresource Technol 100:2336–2340 (2009).
J Chem Technol Biotechnol (2012)

c 2012 Society of Chemical Industry
www.soci.org
31 Ahmad AL, Derek CJC and Zulkali MMD, Optimization of affinity
partitioning conditions of papain in aqueous two-phase system
using response surface methodology. Sep Purif Technol 62:702–708
(2008).
32 Box GEP, Hunter WG and Hunter JS, Statistics for Experiments: an
Introduction to Design, Data Analysis, and Model Building. John
Wiley & Sons, New York, pp. 91 (1978).
33 Sen R, Response surface optimization of the critical media components
for the production of surfactin. J Chem Tech Biotechnol 68:263–270
(1997).
34 Kiliç M, Bayraktar E, Ateş S and Mehmetoglu U, Investigation
of extractive citric acid fermentation using response-surface
methodology. Process Biochem 37:759–767 (2002).
35 Qiao DL, Hu B, Gan D, Sun Y, Ye H and Zeng XX, Extraction optimized
by using response surface methodology, purification and
preliminary characterization of polysaccharides from Hyriopsis
cumingii. Carbohyd Polym 76:422–429 (2009).
36 Berggren K, Johansson HO and Tjerneld F, Effects of salts and
the surface hydrophobicity of proteins on partitioning in
aqueous two-phase systems containing thermoseparating ethylene
oxide–propylene oxide copolymers. J Chromatogr A 718:67–79
(1996).
37 Ravindra Babu B, Rastogi NK and Raghavarao KSMS, Liquid–liquid
extraction of bromelain and polyphenol oxidase using aqueous
two-phase system. Chem Eng Process 47:83–89 (2008).
38 Almedia MC, Venancio A, Teixeira JA and Aires-Barros MR, Cutinase
purification on poly (ethylene-glycol-hydroxy propyl) starch
aqueous two phase systems. J Chromatogr B 711:151–159 (1998).
39 Rabelo APB, Tombourgi EB and Pressoa A Jr, Bromelain partitioning
in two phase aqueous systems containing PEO–PPO–PEO block
copolymer. J Chromatogr B 807:61–68 (2004).
40 Kammoun R, Chouayekh H, Abid H, Naili B and Bejar S, Purification of
CBS 819.72 α-amylase by aqueous two-phase systems: modelling
using response surface methodology. Biochem Eng J 46:306–312
(2009).
wileyonlinelibrary.com/jctb