Chinese Journal of Chemical Engineering, 19(5) 876—880 (2011)
Isolation of Lysozyme from Chicken Egg White Using
Polyacrylamide-based Cation-exchange Cryogel*
YAN Luding (晏禄丁), SHEN Shaochuan (沈绍传), YUN Junxian (贠军贤) and YAO Kejian
(姚克俭)**
State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, College of Chemical Engineering
and Materials Science, Zhejiang University of Technology, Hangzhou 310032, China
Abstract An effective cation-exchange chromatographic method for lysozyme isolation from chicken egg white
is presented, using supermacroporous cryogel grafted with sulfo functional groups. The chromatographic processes
were carried out by one-step and sequential elution, respectively. Sodium phosphate buffer (pH 7.8) containing different concentrations of NaCl is used as elution agent. The corresponding breakthrough characteristics and elution
behaviors in the cryogel bed were investigated and analyzed. Purity of lysozyme in the elution effluent was assayed
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The maximum purity of the obtained
lysozyme was about 96%, and the cryogel is demonstrated as a potential separation medium for purification of
high-purity lysozyme from chicken egg white.
Keywords cation-exchange cryogel, lysozyme, isolation, sequential elution, chicken egg white
1
INTRODUCTION
Lysozyme is an important valuable enzyme that
destroys certain bacteria by cleaving the β-linkages
between the N-acetyl-muramic acid and N-acetylglucosamine of the peptidoglycan in the bacterial cell
walls [1]. Nowadays, lysozyme is widely used as food
preservative and antimicrobial agent [1-3]. The therapeutic potential and preventive role of egg white lysozyme as an anticancer drug has also been reported [4, 5].
The effective separation and purification of lysozyme
from chicken egg white (CEW) is an interesting work
due to its powerful applications, and different methods
or techniques have been developed in recent years.
Derazshamshir et al. [6] prepared Zn2+-chelated poly
[2-hydroxyethyl
methacrylate-N-methacryloyl-(L)histidine methyl ester (HEMA-MAH)] cryogel for the
purification of lysozyme from egg white. The purity of
the desorbed lysozyme was about 88.6% with recovery
of about 80.5%. Akgöl et al. [7] reported that the purity
of lysozyme eluted from magnetic-poly [HEMA-Nmethacryloyl-l-phenylalanine (HEMA-MAPA)] nanobeads was about 96.03% with the recovery of 78.57%.
Ghosh et al. [8] utilized cascade ultrafiltration systems
for separation of lysozyme from certain pretreated egg
white solution. The purities of lysozyme products
were 96% and 97%, while the recoveries were 75%
and 71% for the three-stage and four-stage cascade
systems, respectively. Dembczyński et al. [9] used
aqueous two-phase system composed of ethylene oxide (EO), propylene oxide (PO) and potassium phosphate in the isolation of lysozyme from CEW. The
high yield of lysozyme was 85% with a purification
factor of 16.9 in two extraction steps.
Compared with conventional packed-bed columns, polyacrylamide-based cation-exchange supermacroporous cryogels have lots of large-sized pores
(about 10-100 μm), which permit chromatography to
be performed with low mass transfer resistance for
both adsorption and elution stages [10]. The size of
pores is close to the un-grafted cryogels [11, 12], and
several advantages of cryogels are easy preparation,
versatile surface modification and high theoretical
plates. The properties of pure lysozyme adsorption/
desorption onto/from the cation-exchange cryogel
with sulfo groups have been reported in our previous
work [10, 13], but the practical application of cationexchange cryogel on lysozyme purification from CEW
has not been studied so far.
In this work, we will present the application of
chromatographic separation of high-purity lysozyme
from CEW solution using the cation-exchange cryogel
above mentioned. The breakthrough characteristics
and elution performances of lysozyme from CEW solution, as well as the purities and recoveries of lysozyme from the elution effluent fractions under different elution conditions in the cryogel column will
also be investigated.
2
2.1
MATERIALS AND METHODS
Materials
Fresh chicken eggs were purchased from a local
market. N,N′-methylene-bis-acrylamide (MBAAm,
99%), 2-acrylamido-2-methyl-1-propane-sulfonic acid
(AMPSA, 99%) and lysozyme (from CEW) were bought
from Sigma-Aldrich (Steinheim, Germany). Acrylamide
Received 2010-01-21, accepted 2011-04-20.
* Supported by the National Natural Science Foundation of China (21036005, 20876145), the Science and Technology Cooperation Project between China-Europe Country’s Governments from the Ministry of Science and Technology of China (1017) and
the Natural Science Foundation of Zhejiang Provincial (Y4080326).
** To whom correspondence should be addressed. E-mail: [email protected]
Chin. J. Chem. Eng., Vol. 19, No. 5, October 2011
(AAm, 99.9%), Coomassie brilliant blue R-250 and
protein marker (a mixture of six proteins: 14.4, 20, 31,
45, 66.2 and 98 kDa) were purchased from Biobasic
(Toronto, Canada). N,N,N′,N′-tetramethyl-ethylenediamine
(TEMED, 99%) was purchased from Amresco (Ohio,
USA). Ammonium persulfate (APS, 98%) and other
chemicals were of analytical grade from local sources.
The basic polyacrylamide-based cryogel was
prepared in 10 mm inner-diameter glass column under
freezing-temperature variation condition (route B) [12],
then grafted in-situ with 2 mol·L−1 AMPSA as reported
in our previous work [10, 13, 14].
2.2
Preparation of egg white solution
The egg white was manually collected from fresh
eggs. CEW solution was prepared using the method of
Guérin-Dubiard et al. [15] with a few modifications.
10 ml of egg white was diluted with 10 ml of ultrapure
water (Milli-Q system, Millipore, USA), and the mixture was adjusted to pH 6 with 1 mol·L−1 HCl. The solution was stirred gently at 2 °C overnight, then centrifuged at 4 °C and 8000 r·min−1 for 8 min to remove the
precipitate. Prior to chromatography, the solution was
adjusted to pH 7.8 with 1 mol·L−1 NaOH, and diluted
to 160 ml with 20 mmol·L−1 phosphate buffer (pH 7.8),
then centrifuged at 12,000 r·min−1 for 15 min. The
supernatant was used as a lysozyme source for the
following chromatographic processes.
2.3
Chromatography of lysozyme by cationexchange cryogel
The chromatographic purification of lysozyme
from the CEW solution using cation-exchange cryogel
were carried out at a constant flow velocity of 2 cm·min−1,
according to our previous work [10], using 20 mmol·L−1
sodium phosphate buffer (pH 7.8) as a running buffer.
The column was equilibrated with running buffer.
Thereafter, a feedstock of 27.2 ml was loaded and the
column was washed with the same buffer.
One-step and sequential elution were performed
with 0.5 mol·L−1 NaCl and various concentrations of
NaCl, respectively, in running buffer. The chromatography processes were monitored using an on-line flowthrough UV spectrometer at 280 nm as reported in our
previous work [12]. The column effluent was recorded
over time and collected by 1.6 or 0.8 ml of each aliquot for further analysis. Finally, column cleanings
were performed with 0.1 mol·L−1 HCl, 1.5 mol·L−1
NaCl and deionized water.
2.4
Analytical method
Sodium dodecyl sulfate (SDS)-polyacrylamide
gel electrophoresis (PAGE), according to the method
of Laemmli [16], was performed to analyze the fractions of the column effluent using 14% separating gel
877
and 4% stacking gel (Mini-PROTEAN Tetra Cell system, Bio-Rad, USA). The protein samples were dissolved in sample buffer, and heated at 95 °C for 3 min.
Electrophoresis was run at 75 V in stacking gel and
120 V in separating gel using an electrophoretic buffer
of Tris-Glycine containing 0.1% SDS. The gels were
stained with 0.12% Coomassie Brilliant Blue R-250
for detection of protein, destained with 50% water,
40% ethanol and 10% acetic acid, and finally scanned
using a Tanon-3500 digital gel imaging system (Tanon
Science & Technology, China). Purities and molecular
weights of the proteins were estimated by the Quantity
One software (Version 4.2, Bio-Rad, USA).
The protein concentrations in the CEW solution
and each aliquot of the collected column effluent were
analyzed by Bradford method [17] at 595 nm (Ultrospec 3300 pro, GE Healthcare, UK).
3
3.1
RESULTS AND DISCUSSION
Properties of cation-exchange cryogel
The properties of the cation-exchange cryogel
were determined according to Yao et al [10, 12, 13, 18, 19].
Fig. 1 shows the values of height equivalent to theoretical plate (H) at the flow velocities of 1-10
cm·min−1. It can be seen that the values of H of the
un-grafted cryogel column range from 0.08 to 0.10 cm,
and after grafting polymerization the values of H are
in the range of 0.13 to 0.16 cm, which are slightly
lower than 0.16 to 0.19 cm of the cryogel reported
previously [10]. The porosity of grafted cryogel is
82.8%, and the water permeability based on Darcy’s
law is 7.6×10−12 m2. The axial dispersion coefficient
(Dax) increases with the increase of superficial liquid
velocity (UL) in an exponential form. From the obtained data, Dax = 0.012U L1.31 .
Figure 1 Values of H at various liquid velocities in the
cation-exchange cryogel bed (10 mm diameter, 78 mm length)
−1
● before the grafting polymerization by 2 mol·L AMPSA;
−1
○ after the grafting polymerization by 2 mol·L AMPSA
3.2 One-step elution of the adsorbed lysozyme
from cation-exchange cryogel
The binding capacity of the cation-exchange
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Chin. J. Chem. Eng., Vol. 19, No. 5, October 2011
cryogel was 2.4 mg per ml wet cryogel bed, using lysozyme as a model protein, being close to that of the
cryogel reported previously (2.5 mg·ml−1) [10, 13]. The
chromatographic profile of CEW solution with
one-step elution is shown in Fig. 2, where c and c0 are
the protein concentrations in the collected column
effluent and the CEW solution, and V is the loaded
volume, respectively. The column effluents were collected with 1.6 ml per aliquot from 0 to 115.6 ml and
0.8 ml per aliquot from 115.6 to 148.4 ml. It can be
seen that there was a single chromatographic peak in
the whole stage of elution, indicating that the adsorbed
proteins were eluted effectively by 0.5 mol·L−1 NaCl
in running buffer.
Figure 3 SDS-PAGE analysis of chromatographic isolation process of lysozyme from CEW by cation-exchange
cryogel with one-step elution
M, molecular mass markers; Lane 1, loading (8.4-10 ml); Lane
2, loading (24.4-29.2 ml); Lane 3, washing; Lane 4, peak fraction from elution 1; Lane 5, fraction from elution 2; Lane 6,
blank; Lane 7, CEW solution (feed)
3.3 Purification of lysozyme by cation-exchange
cryogel with sequential elution
Figure 2 Chromatographic behaviour of lysozyme isolation from CEW by cation-exchange cryogel with one-step
elution
The cryogel column was equilibrated and washed with 20
mmol·L−1 phosphate buffer, pH 7.8 (running buffer), eluted
with 0.5 mol·L−1 NaCl (elution 1) followed by 1.5 mol·L−1
NaCl in running buffer (elution 2). The superficial liquid velocity was 2 cm·min−1.
A part of the collected effluents was analyzed by
SDS-PAGE, as shown in Fig. 3. Lysozyme in CEW
solution (Lane 7) disappeared in Lanes 1-3, indicating
that lysozyme was adsorbed onto the cation-exchange
cryogel in the breakthrough process. At least two proteins were desorbed by 0.5 mol·L−1 NaCl in running
buffer since two protein bands were observed in Lane
4. According to the band analysis of molecular mass,
the lower band is lysozyme and the upper band is ovoglobulin (molecular mass of ovoglobulin G2 and ovoglobulin G3, were 47 and 50 kDa, respectively [20]).
In the chromatographic condition (pH 7.8), lysozyme and avidin are the only two positively charged
proteins (isoelectric point of 10.7 and 10.0, respectively [20]) in the CEW solution, and able to be adsorbed onto the cation-exchange cryogel by static
electrical interactions. However, the avidin band was
not observed in Fig. 3, indicating that the avidin had
been removed in the process of preparation of the
CEW solution. The SDS-PAGE results also showed
that the adsorbed proteins were eluted completely
during the elution 1 (Fig. 2) because no band was
detected in Lane 5 (Fig. 3). The purity of the eluted
lysozyme, as determined by SDS-PAGE, was about
70%.
In order to enhance the purity of lysozyme, three
chromatographic profiles were carried out using different 3-stage sequential elution with a common
breakthrough condition, as shown in Fig. 4. In the first
elution stage (elution 1), the cryogel was eluted with
0.08 mol·L−1 (profile 1), 0.1 mol·L−1 (profile 2), and
0.12 mol·L−1 (profile 3) of NaCl in running buffer,
respectively. Then, running buffers containing 0.5
mol·L−1 NaCl (elution 2) and 1.5 mol·L−1 NaCl (elution 3) were sequentially used as elution agent in the
next two stages of elution. As can be seen, one chromatographic peak was observed in each stage of elution 1 and 2, referring to corresponding proteins desorbed from the cryogel. In elution 3, no distinct
chromatographic peak appeared, indicating that the
first two stages of consecutive elution ensured the
complete desorption of lysozyme.
SDS-PAGE analysis of the collected effluents of
the three chromatographies were presented in Figs.
5-7. In the breakthrough process (Lanes 1-3), the majority of the impurities passed through the cryogel bed
freely and lysozyme together with the minority of
impurities was adsorbed by the cryogel. The protein
impurities were eluted effectively in elution 1 (Lane 4)
and the target lysozyme was desorbed in elution 2
(Lane 5). The concentration of NaCl in running buffer
for eluting lysozyme was higher than that for the protein impurities, indicating the strong ionic interactions
between the positively charged surfaces of lysozyme
and the negatively charged surfaces of the cryogel pore
walls under present chromatographic conditions. The
purities of the eluted lysozyme (elution 2) for the three
chromatographic profiles, as analyzed by SDS-PAGE,
reached 86%, 94%, and 96%, respectively.
The effluent samples eluted with 0.5 mol·L−1
NaCl were diluted in different ratios and the concentrations of lysozyme in the effluent samples were also
analyzed by the Bradford method. The recovery of
lysozyme (defined as the ratio of the eluted pure lysozyme to that theoretically contained in CEW solution,
Chin. J. Chem. Eng., Vol. 19, No. 5, October 2011
879
Figure 4 Profiles of protein concentrations during the chromatographic purification of lysozyme from CEW by
cation-exchange cryogel with sequential elution
The cryogel column was equilibrated and washed with 20 mmol·L−1 phosphate buffer, pH 7.8 (running buffer). The liquid velocity
was 2 cm·min−1. (■, profile 1) eluted with 0.08 mol·L−1 NaCl (elution 1), followed by 0.5 mol·L−1 NaCl (elution 2) and 1.5 mol·L−1
NaCl in running buffer (elution 3); (○, profile 2) eluted with 0.1 mol·L−1 NaCl (elution 1), followed by 0.5 mol·L−1 NaCl (elution 2)
and 1.5 mol·L−1 NaCl (elution 3); (◆, profile 3) eluted with 0.12 mol·L−1 NaCl (elution 1), followed by 0.5 mol·L−1 NaCl (elution 2)
and 1.5 mol·L−1 NaCl (elution 3)
Figure 5 SDS-PAGE analysis of fractions obtained by
cation-exchange cryogel chromatography of lysozyme purification from CEW with sequential elution. The cryogel
column was eluted with 0.08 mol·L−1 NaCl (elution 1) followed by 0.5 mol·L−1 NaCl (elution 2), and 1.5 mol·L−1
NaCl (elution 3) in running buffer
M, molecular mass markers; Lane 1, loading (8.4-10 ml);
Lane 2, loading (24.4-29.2 ml); Lane 3, washing; Lane 4,
peak fraction from elution 1; Lane 5, peak fraction from elution 2; Lane 6, CEW solution (feed)
Figure 6 SDS-PAGE analysis of fractions obtained by
cation-exchange cryogel chromatography of lysozyme purification from CEW with sequential elution. The cryogel
column was eluted with 0.1 mol·L−1 NaCl (elution 1) followed by 0.5 mol·L−1 NaCl (elution 2), and 1.5 mol·L−1
NaCl (elution 3) in running buffer
M, molecular mass markers; Lane 1, loading (8.4-10 ml);
Lane 2, loading (24.4-29.2 ml); Lane 3, washing; Lane 4,
peak fraction from elution 1; Lane 5, peak fraction from elution 2; Lane 6, CEW solution (feed)
3.4% of the egg white [21], loaded onto the cryogel
column) in elution 2 under the present three cases were
estimated approximately from the quantitative analysis
results and the obtained values were 81%, 75% and 45%,
respectively. From the above mentioned results, the
amount of the eluted impurities and lysozyme (elution 1)
increased with the concentration increasing of NaCl in
running buffer, which caused the decreasing recovery
of the lysozyme (elution 2). In this work, high-purity
of lysozyme can be achieved by the chromatographic
separation with a loss of recovery. The purities and
recoveries of the obtained lysozyme from the three
chromatographic runs were shown in Fig. 8.
4
CONCLUSIONS
Isolation of lysozyme from CEW solution using
cation-exchange supermacroporous cryogel was carried out using one-step elution. The purity of the lysozyme, as determined by SDS-PAGE, was about 70%.
In order to obtain lysozyme with high purity, chromatographies of CEW solution were performed by sequential elution with different salt concentrations in
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Chin. J. Chem. Eng., Vol. 19, No. 5, October 2011
UL
superficial liquid velocity, cm·min−1
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1
2
3
Figure 7 SDS-PAGE analysis of fractions obtained by
cation-exchange cryogel chromatography of lysozyme purification from CEW with sequential elution. The cryogel
column was eluted with 0.12 mol·L−1 NaCl (elution 1) followed by 0.5 mol·L−1 NaCl (elution 2), and 1.5 mol·L−1
NaCl (elution 3) in running buffer
M, molecular mass markers; Lane 1, loading (8.4-10 ml);
Lane 2, loading (24.4-29.2 ml); Lane 3, washing; Lane 4,
peak fraction from elution 1; Lane 5, peak fraction from elution 2; Lane 6, CEW solution (feed)
4
5
6
7
8
9
10
Figure 8 The purity and recovery of the obtained lysozyme
by cation-exchange cryogel with sequential elution
a—eluted with 0.08 mol·L−1 NaCl (elution 1), followed by 0.5
mol·L−1 NaCl (elution 2) and 1.5 mol·L−1 NaCl in running
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1), followed by 0.5 mol·L−1 NaCl (elution 2) and 1.5 mol·L−1
NaCl (elution 3)
11
12
13
sodium phosphate buffer. In the elution procedure,
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impacting on the desorption of lysozyme and the corresponding purity in the isolation process.
14
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19
NOMENCLATURE
20
Dax
H
axial dispersion coefficient, m2·s−1
height equivalent to theoretical plate, cm
21
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