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928 Biotechnol. Prog.

2003, 19, 928935

Reverse Micellar Extraction and Precipitation of Lysozyme Using


Sodium Di(2-ethylhexyl) Sulfosuccinate
Youn-Ok Shin, Martin E. Weber, and Juan H. Vera*
Department of Chemical Engineering, McGill University, 3610 University Street,
Montreal, Quebec, Canada H3A 2B2

Sodium di(2-ethylhexyl) sulfosuccinate, referred to as Aerosol-OT or AOT, was used


to remove lysozyme from an aqueous phase via reverse micellar extraction and
precipitation method. For both methods, when the surfactant was in excess, a complete
removal of lysozyme from the aqueous phase was obtained at the values of pH below
the pI of lysozyme. However, for the reverse micellar method, a solubilization limit of
lysozyme in the organic phase was observed, and a white precipitate was formed at
the aqueous-organic interface. This observation suggested using AOT directly as a
precipitating ligand. The lysozyme precipitated with AOT was fully recovered, with
its original enzymatic activity, using acetone as a recovery solvent. A mechanism is
suggested to explain the solubilization of lysozyme in an AOT reverse micellar system.
It is shown that a direct precipitation method can be used with advantage instead of
using the reverse micellar extraction method to recover lysozyme from an aqueous
phase.

Introduction The ion distribution around the surfactant heads (16, 21,
22) and the reverse micellar size (23, 24) have been
Proteins are produced using bacterial cells, mam- considered to be factors that influence protein extraction.
malian cells, or plant extracts. Separation methods such The flexibility (25) and the surface charge (26) of protein
as crystallization, column chromatography, or electro- molecules have also been related to the solubilization
phoresis have been applied to purify these proteins (1). behavior.
As an alternative, the use of polymers coupled with an Once extracted, the proteins were recovered into a
affinity ligand was suggested (2-5). Upon a change in fresh aqueous phase with pH and salt concentration
temperature or pH, the protein-polymer-affinity ligand adjustment (12). This process is referred to as back
complex precipitates from the aqueous phase (6). The extraction. The rate of the back extraction is much slower
disadvantages of this method are that the percent than the rate of the forward extraction (11, 27, 28).
precipitation is limited by the solubility of the polymer Adjustment of pH and ionic strength alone may not be
in the aqueous phase and that a membrane separation sufficient to recover the protein from the reverse micelles
is required to separate protein from the polymer at the (29, 30). Ethanol (31), 2-propanol (32), ethyl acetate (33),
end of the process (2, 6). Furthermore, only proteins that and counter surfactants (34) have been added in attempts
can sustain elevated temperatures can be purified using to improve the recovery. The use of an acetone phase,
this method (5). instead of using a fresh aqueous phase, was suggested
Liquid-liquid extraction of proteins using reverse as an alternative to recover the proteins from the reverse
micellar phase was proposed as an alternative purifica- micellar phase (35, 36).
tion method (7). Ionic surfactants are usually considered One mechanism of protein loss, often found in studies
as a protein denaturant (8, 9). Thus, for the protein of reverse micellar extraction processes, is through the
purification, the use of commercially available ionic formation of a water-insoluble protein-surfactant com-
surfactants has been limited to reverse micellar extrac- plex that precipitates at the interface between the
tion. Upon contact of an organic phase containing the aqueous and organic phases (37, 38). The formation of
surfactant with an aqueous phase containing proteins this precipitate depended largely on the mass transfer
and electrolytes, the surfactant forms reverse micelles kinetics (39) and on the saturation limit of the protein
in the organic phase. As a result of their electrostatic in the reverse micellar phase (40). The protein-surfac-
interaction with the surfactant headgroup, proteins and tant complex which precipitated at the interface was
other charged species are solubilized inside the water viewed as an undesirable product, since the protein was
pools of the reverse micelles (10). This process is referred thought to be denatured (41). Attempts were made to
to as forward extraction. Many reverse micellar extrac- avoid the formation of a protein-surfactant complex by
tion studies used sodium di(2-ethylhexyl) sulfosuccinate using new surfactants to form the reverse micellar phases
(AOT) as an anionic surfactant (11-16) and trioctylm- (38, 42).
ethylammonium chloride (TOMAC) or cetyltrimethylam- Even though the technology was first suggested more
monium bromide (CTAB) as cationic surfactants (17-20). than 2 decades ago, reverse micellar extraction still
suffers from poor recovery efficiency and protein loss at
* To whom correspondence should be addressed. Fax: (514) 398- the aqueous-organic interface. In this work, it is dem-
6678. Email: juan.vera@mcgill.ca. onstrated that the lysozyme precipitated at the aqueous-
10.1021/bp025789r CCC: $25.00 2003 American Chemical Society and American Institute of Chemical Engineers
Published on Web 04/12/2003
Biotechnol. Prog., 2003, Vol. 19, No. 3 929

organic interface during the reverse micellar extraction A small amount of NaCl solution (normally less than 10
process can be recovered by dissolving it in a polar L of 0.1 M NaCl) was then added to the acetone phase
organic solvent such as acetone. On the basis of this in order to neutralize the charges and dissociate the
finding, a new method that uses AOT as a precipitating lysozyme from the surfactant. The surfactant-free
ligand is proposed. The precipitation of lysozyme is lysozyme precipitated as a solid 1-2 min after the
studied, and the yields are compared with the results addition of NaCl, while the surfactant remained in the
obtained with its reverse micellar extraction. Comparison acetone phase. The recovered solid lysozyme was washed
of solubilization of the lysozyme-surfactant complex in with acetone to remove residual surfactant. The qualita-
reverse micelles with the direct extraction of lysozyme tive and quantitative analyses of the final product were
into a reverse micellar phase suggests a mechanism to carried out by dissolving the recovered-solid lysozyme
explain the latter process. into a fresh aqueous phase.
Solubility Product Measurement of Lysozyme-
Materials and Methods AOT Complex. For the measurement of the solubility
product of the lysozyme-AOT complex, the lysozyme-
Materials. Sodium di(2-ethylhexyl) sulfosuccinate AOT complex formed by direct addition of AOT was
(AOT), lysozyme (chicken egg white, pI ) 11.0, MW ) collected by centrifugation, washed three times with
14.3 kDa), and Micrococcus lysodeikticus were purchased distilled water, and then dispersed in 5 mL of fresh
from Sigma (Oakville, ON). The purchased lysozyme distilled water. The mixture was stirred for 24 h at room
contained 5-10% buffer: sodium acetate/sodium chloride/ temperature. An excess solid phase was visible in the
hydrochloric acid. Phosphate buffer (pH 6.2) purchased bottom of the test tube. The concentrations of lysozyme
from Anachemia (Oakville, ON) was used for enzymatic and AOT in the aqueous phase were measured after
activity measurements. Reagent grade isooctane and filtering the solution though a 0.45 m filter.
decanol, HPLC grade acetonitrile and triflouric acid,
sodium chloride, and 1 N NaOH were obtained from Analytical Techniques. The concentrations of ly-
Fisher Scientific (Montreal, QC). sozyme and AOT in the aqueous phase were measured
using an Agilent high performance liquid chromatograph
Extraction of Lysozyme into Reverse Micellar (HPLC)-1100 with a Zorbax 300SB-C8 (4.6 mm i.d. 150
Phase. A volume of 10 mL of an aqueous solution mm length) column. The mobile phase consisted of two
containing lysozyme and 0.1 M NaCl was contacted with solvents, 5% acetonitrile and 0.1% triflouric acid in water
an equal volume of an isooctane phase containing a (solvent A), and 5% water and 0.085% triflouric acid in
specified concentration of AOT. Without NaCl in the acetonitrile (solvent B). The sample volume was set at 5
aqueous phase, no reverse micelles were formed. The L, and the wavelength of the detector was set at 210
mixture was vortexed for 5 s and left for phase separation nm.
for 12 h at room temperature (21 ( 1 C). The pH of the
The lysozyme concentration in the organic phase was
initial aqueous lysozyme solution, adjusted to 6.0 ( 0.5
measured on a Cary Varian 1/3 spectrophotometer at 280
using 1 N NaOH, was measured using an accupHast
nm (A280nm) and at 310 nm (A310nm). The reading measured
reference/pH probe, purchased from Fisher Scientific
at 310 nm was subtracted from the reading measured at
(Montreal, QC). Since the pH of the initial lysozyme
280 nm to eliminate the noise caused by surfactant. A
solution varied between 4 and 6, the concentration of
linear calibration curve of lysozyme in the AOT reverse
NaOH in the initial lysozyme solution was less than 0.04
micellar phase was obtained with standards containing
mM.
from 0 to 0.7 g/L lysozyme. The samples with higher
Formation of Lysozyme-AOT Complex by Direct lysozyme concentration were diluted using an identical
Addition of AOT. An aqueous solution containing AOT AOT reverse micellar phase containing no lysozyme.
was added to 10 mL of an aqueous solution, which
The enzymatic activity of lysozyme was measured
contained from 0 to 5 g/L lysozyme. No pH adjustment
according to the method described by Davis et al. (45).
was made to the initial AOT or lysozyme-containing
The concentrations of sodium and chloride ions in the
aqueous solutions. The solubility of AOT in water was
aqueous phase were measured using atomic absorption
reported to be 15 g/L or 33.8 mM in the temperature
spectroscopy and ion chromatography, respectively. All
range from 20 to 70 C (43). The critical micelle concen-
experiments were performed using two to six replicates,
tration (CMC) of AOT in water is in the range from 2.5
and the standard deviation of the measurements is
to 4.1 mM at 25 C (44). The initial concentration of AOT
indicated in the figures by error bars.
in the lysozyme solution was fixed between 0.02 and 1.5
mM, to avoid the formation of micelles. The volume of
the AOT solution added to the lysozyme solution was less
Results and Discussion
than 0.2 mL. As soon as the AOT solution was added, Reverse Micellar Extraction of Lysozyme as a
the insoluble lysozyme-AOT complex formed. The mix- Function of AOT Concentration. The effect of AOT
ture was vortexed for 5 s and centrifuged, and then the concentration on the solubilization of lysozyme into the
supernatant was removed. The lysozyme-ligand complex reverse micellar phase was examined using an aqueous
was washed with water by adding 10 mL of distilled phase containing from 0 to 15 g/L (0 to 11 mM) lysozyme
water to the test tube containing precipitated lysozyme- and an organic phase containing from 0 to 100 mM AOT.
ligand complex, followed by a second centrifugation. The salt concentration in the lysoyzme solution was 0.1
Lysozyme Recovery from Lysozyme-AOT Com- M NaCl. Figure 1 shows the concentration of lysozyme
plex. Lysozyme was recovered from the white precipitate in the reverse micellar phase as a function of the initial
formed at the aqueous-organic interface during the lysozyme concentration in the aqueous phase. As the
reverse micellar extraction process and from the surfactant concentration in the organic phase increased,
lysozyme-AOT complex formed by direct addition of AOT the amount of lysozyme solubilized into the reverse
using acetone as a recovery solvent. Ten milliliters of micellar phase increased. For the case of 0 mM AOT
acetone was added to the test tubes containing the reverse micellar phase, no lysozyme was solubilized in
precipitated lysozyme-AOT complex. The solid dissolved the organic phase, confirming that lysozyme was in-
immediately in acetone and resulted in one clear solution. soluble in isooctane. At 20 mM AOT, the maximum
930 Biotechnol. Prog., 2003, Vol. 19, No. 3

Figure 2. Initial pH of the aqueous solution as a function of


Figure 1. Solubilization of lysozyme into AOT reverse micellar lysozyme concentration at room temperature.
phases; initial aqueous phase, 0.1 M NaCl, pH ) 6.0 ( 0.5
adjusted with NaOH; initial volumes of aqueous and organic (i) Upon contact, lysozyme forms a complex with the
phases ) 10 mL.
ligand as a result of electrostatic interactions.
(ii) The lysozyme-AOT complex has a low solubility
concentration of lysozyme in the reverse micellar phase in water and in isooctane, since no solubilization of
was 0.09 mM. The samples prepared with an aqueous lysozyme was observed in the pure isooctane phase, as
phase containing from 0.1 to 0.15 mM lysozyme showed shown in Figure 1.
a lysozyme concentration of 0.09 mM in the organic (iii) The complex solubilizes in a reverse micellar phase
phase, indicating that this is the saturation limit of if present; otherwise, the complex remains as solid.
lysozyme in the 20 mM AOT reverse micellar phase. With (iv) The solubilization of the lysozyme-AOT complex
an initial lysozyme concentration of 0.18 mM, the reverse takes place until the saturation limit of lysozyme in the
micellar phase contained about 0.07 mM lysozyme. On reverse micellar phase is reached. The excess lysozyme-
the other hand, the equilibrium lysozyme concentration AOT complex remains at the aqueous-organic interface.
in the aqueous phase after contact with the reverse To verify this hypothesis, two-step experiments were
micellar phase was below the detection limit of HPLC performed. First the lysozyme was directly precipitated
for all samples, indicating essentially complete removal from the aqueous phase using AOT as a precipitation
of lysoyzme from the aqueous phase. The lysozyme ligand. This precipitate was then solubilized into reverse
removed from the aqueous phase but not solubilized into micellar phases containing different concentrations of
the reverse micellar phase formed a white precipitate at AOT.
the aqueous-organic interface. Formation of Lysozyme-AOT Complex by Direct
An increase in AOT concentration in the organic phase Addition of AOT. The precipitation of lysozyme was
further increased the solubility limit of lysoyzme in the performed using an initial lysozyme aqueous solution
AOT reverse micellar phase. At 30 mM AOT, the extrac- containing 0.15-5 g/L (0-0.36 mM) lysozyme. No pH and
tion of lysozyme into the reverse micellar phase was salt concentration adjustment was made to the lysozyme
complete up to an initial lysozyme concentration of 0.38 solution. Figure 2 shows the pH of the initial lysozyme
mM. The samples prepared with an aqueous phase aqueous solution. The pH of the lysozyme solution,
containing 0.5 mM lysozyme resulted in a sudden de- containing the buffer coming with the commercial
crease in the lysozyme concentration in the 30 mM AOT lysozyme, decreased from 5.8 to 4.2 as the lysozyme
reverse micellar phase. Similarly, a solubilization limit concentration increased from 0.01 to 0.36 mM. Two sets
of lysozyme in the 70 and 100 mM AOT reverse micellar of experiments were performed, and the difference in the
phase was 0.75 and 0.83 mM, respectively. When the pH between the replicates was about (0.1.
initial lysozyme concentration in the aqueous phase was An aqueous phase containing AOT was then pipetted
greater than the saturation limit value, a noticeable into the initial lysozyme aqueous solution to form a
decrease in the lysozyme concentration in the reverse lysozyme-AOT precipitate. Figure 3 shows the percent
micellar phase was obtained, and a white precipitate at removal of lysozyme from the aqueous solution as a
the aqueous-organic interface was observed. A similar function of the molar ratio, R, between AOT and lysozyme
phenomenon was observed in a previous study using an initially added to the solution. Increasing the molar ratio
AOT (37) or a dioctyldimethylammonium chloride reverse increased the percent removal until a molar ratio of about
micellar phase (40). 10, at which 100% percent removal of lysozyme was
Solubilization Mechanism of Lysozyme into a obtained. When R was greater than 10, the excess AOT
Reverse Micellar Phase. On the basis of the results remained in the aqueous phase. When R was less than
in Figure 1, a mechanism of lysozyme extraction into a 10, the concentration of AOT remaining in the solution
reverse micellar phase is proposed: was below the detection limit of HPLC, indicating that
Biotechnol. Prog., 2003, Vol. 19, No. 3 931

Figure 4. Equilibrium pH of the aqueous phase upon removal


of lysozyme as an insoluble lysozyme-AOT complex: no salt
Figure 3. Percent of lysozyme precipitated with AOT: no salt added, no pH adjustment.
added, no pH adjustment.

all of the ligand added to the solution formed lysozyme-


ligand complex and precipitated. The slope of the line in
Figure 3 is 10, indicating that 1 mol of precipitated
lysozyme was complexed with 10 mol of AOT. A similar
precipitating behavior of cytochrome c was observed
where the cytochrome c complexed with AOT at a fixed
molar ratio of 11 and precipitated from the aqueous phase
(41).
Figure 4 shows the equilibrium pH after the lysozyme-
ligand complex was formed. The pH of the solution
increased as more lysozyme was removed from the
aqueous phase. When lysozyme was removed completely
(R g 10), the excess ligand remained in the solution, and
the pH was approximately 6.
Effect of Salt on the Formation of Lysozyme-AOT
Complex. The effects of salt on the precipitation of
lysozyme were studied using an aqueous phase contain-
ing 0.1-1 M NaCl and lysozyme. Figure 5 shows the
percent precipitation of lysozyme as a function of the
molar ratio when NaCl was present in the initial lysozyme
solution. At 0.1 M NaCl, the results were the same as
those depicted in Figure 3. At higher salt concentrations,
the amount of lysozyme complexed with AOT decreased.
At 0.3 M NaCl, only 60% of the lysozyme was precipitated
at R ) 10, and a value of R ) 20 was required for a 95% Figure 5. Effect of salt on the percent lysozyme precipitated
precipitation. A salt concentration of 1 M decreased the with AOT.
percent precipitation to 30-35% with little effect of the
molar ratio. Increasing the salt concentration increased phase containing 0.1 M NaCl and contacted with an equal
the concentration of chloride counterions competing with volume of an isooctane phase containing 5-100 mM AOT.
AOT to bind with the positively charged lysozyme. In The mixture was stirred vigorously for 72 h to ensure
addition, as the salt concentration increased, the dis- the complete solubilization of lysozyme-AOT complex.
sociation of the AOT anion from its counterion (sodium) After a phase separation of 12 h, the organic and aqueous
decreased, and complex formation between lysozyme and phase samples were collected for lysozyme analysis.
AOT decreased. The decreasing behavior of the percent Figure 6 shows the concentration of lysozyme achieved
precipitation of cytochrome c was also observed as the in an AOT reverse micellar phase using lysozyme-ligand
result of an increase in the salt concentration in the complex, compared with the reverse micellar extraction
aqueous phase (41). result from Figure 1. The error bars indicate the 95%
Solubilization of Lysozyme-AOT Complex into An confidence interval. As shown in Figure 6, for both
AOT Reverse Micellar Phase. The dried lysozyme- experiments, the concentration of lysozyme increased in
ligand complex, formed without addition of salt to the the reverse micellar phase as the concentration of AOT
initial lysozyme aqueous phase, was added to an aqueous increased. For the reverse micellar extraction, duplicate
932 Biotechnol. Prog., 2003, Vol. 19, No. 3

carried out using acetone as a recovery solvent. First we


tested the purity and the activity of lysozyme recovered
from the white precipitate formed at the aqueous-
organic interface during the reverse micellar extraction
process. Upon the addition of acetone, the white lysozyme-
AOT precipitate was dissolved within 1-2 min. Ligand-
free lysozyme precipitated as a solid from this solution,
while the AOT remained in the acetone phase. We then
tested the recovery of lysozyme from the lysozyme-AOT
precipitate formed by adding AOT directly as a precipi-
tating ligand. In both cases, the results were similar. The
percent recovery of lysozyme was (70 ( 18)%. Some loss
of lysozyme occurred during washing. The final product
was analyzed for its enzymatic activity by dissolving the
washed ligand-free lysozyme precipitate in distilled
water. The activity of lysozyme solubilized in the final
aqueous phase was between 45,000 and 51,000 units/mg
protein. The activity of the original lysozyme solution,
before the contact with AOT, was 48,000 ( 3,000 units/
mg protein. The use of acetone did not affect the lysozyme
activity, and the recovered lysozyme showed the same
activity as the initial lysozyme. Clearly, the formation
of the lysozyme-ligand complex and the recovery of the
lysozyme using acetone did not denature the lysozyme.
Figure 6. Concentration of lysozyme in AOT reverse micellar For comparison, the recovery of lysozyme from AOT
phase initially introduced as insoluble lysozyme-AOT complex reverse micellar phase using acetone showed 75% recov-
and extracted from an aqueous phase using a reverse micellar ery (30). This again suggests that the mechanism of
phase. reverse micellar extraction and direct precipitation of
lysozyme are similar.
results showed less than 1% standard deviation, and the On the basis of these observations and on the results
error bars are not visible in the figure, indicating the shown in Figure 6, we conclude that the reverse micellar
excellent reproducibility of data. For the solubilization extraction of lysozyme using AOT surfactant can be
of the lysozyme-AOT complex into the reverse micellar simplified by using a direct precipitation method. This
phase, the 95% confidence interval was more noticeable. direct method eliminates the use of an isooctane organic
This is believed to be due to the vigorous stirring and phase to form reverse micellar phase. The amount of AOT
the time taken to solubilize the dry lysozyme-ligand required per mole of purified lysozyme was 1 order of
complex into a reverse micellar phase, which could cause magnitude less using a direct precipitation method. For
physical changes to the lysozyme. Nevertheless, the the reverse micellar extraction, about 100 mol of AOT
concentration of lysozyme in the reverse micellar phase were required to extract 1 mol of lysozyme into the
obtained by solubilizing the lysozyme-AOT complex reverse micellar phase. In comparison, for the direct
showed the same trend as the reverse micellar extraction precipitation method, only 10 mol of AOT were sufficient
experiment. On the other hand, the lysozyme concentra- to extract 1 mol of lysozyme from the aqueous phase.
tion in the aqueous phase was below the detection limit As a practical application, the use of AOT as a
of the HPLC, confirming the insolubility of lysozyme- precipitating ligand was studied to test the selectivity of
AOT complex in the aqueous phase. On the basis of this the method. Lysozyme was successfully purified from hen
result, we believe that the mechanism for the reverse egg white, with 100% purity without any denaturing (46).
micellar extraction of lysozyme consists of two steps: first The potential use of this direct precipitation method for
the formation of the water insoluble lysozyme-AOT other proteins using AOT is presently under study.
complex that precipitates out of the aqueous phase and
Modeling of Lysozyme-AOT Complex Formation.
then the solubilization of this lysozyme-AOT complex
For modeling, the term ligand is used here for AOT
in the reverse micellar phase. Once the reverse micellar
because it was used as a ligand to form insoluble
phase reaches the saturation limit, as shown in Figure
compound rather than as a surface-active agent. The ter
1, the lysozyme-AOT complex remains as a precipitate
crystallized lysozyme indicates the bulk lysozyme pur-
at the interface.
chased from the supplier (Sigma, ON). Lysozyme-ligand
A small-angle X-ray light scattering study of cyto-
complex indicates the white insoluble complex formed
chrome c in an AOT reverse micellar phase showed that
upon the addition of AOT to the initial lysozyme aqueous
the protein is located at the interface of the AOT reverse
solution, and ligand-free lysozyme indicates the AOT-free
micelles (41). It is believed that the presence of both
solid lysozyme precipitated with acetone.
hydrophobic and hydrophilic domains is necessary for
solubilization of the protein-AOT complex. In water or The model proposed here is based on the following
a pure organic phase, no solubilization of lysozyme-AOT assumptions:
complex was observed. In the presence of reverse mi- (1) The commercially available crystallized lysozyme
celles, which contain both hydrophobic and hydrophilic contains hydrochloric acid (chlorohydrate compounds).
domains, the lysozyme-AOT complex may be solubilized Thus, the positively charged lysozyme has chloride ions
in the isooctane phase. However, the shape of the reverse as counterions in an aqueous phase.
micelles and the location of proteins in a reverse micellar (2) Lysozyme forms an insoluble complex when associ-
phase are not fully understood. ated with a well-defined number, Z, of anionic ligand
Recovery of Lysozyme from Lysozyme-AOT Com- residues. Lysozyme complexed with less than Z number
plex. The recovery of lysozyme from precipitate was of the ligands is soluble in the aqueous phase.
Biotechnol. Prog., 2003, Vol. 19, No. 3 933

(3) No inorganic salts are removed from the aqueous assumed to contain 5% NaCl and 5% C3H2ONa. The
phase with the insoluble lysozyme-ligand complex. equilibrium pH of an aqueous solution containing an
(4) With hydrogen ion rather than sodium as its initial concentration of lysozyme, C0Lys, can be calculated
couterion, AOT is insoluble in water. from a charge balance:
(5) Sodium acetate and sodium chloride are fully
ionized in lysozyme solution. C0LysZ + CH+ + CNa+ ) COH- + CCl- + CC2H3O2- (2)
The first assumption is based on a study of the
variation of the pH of the solution as lysozyme is
dissolved in water as shown in Figure 2. The nature of where CC2H3O2- indicates the acetate ion concentration.
the crystallized lysozyme depends on the type of acid The value of COH- is obtained using the ion product for
solution used in the crystallization process (47). It was water, Kw, taken as 10-14 (53), and the dissociation
assumed that hydrochloric acid was used to precipitate constant of acidic acid, Ka, taken as 1.8 10-5 (54):
the commercial lysozyme.
Assumption (2) is based on the results shown in Figure Kw ) CH+COH- (3)
3. When the lysozyme was in excess, all of the added AOT
formed an insoluble complex with the lysozyme, and the CH+CC2H3O2-
concentration of surfactant in the aqueous phase was Ka ) (4)
CHC2H3O2
negligible. When AOT was in excess, all lysozyme was
removed from the aqueous phase. Thus, the insoluble
complex forms only when a mole of lysozyme binds with Equations 1 and 2 were solved for CH+ at different
Z moles of ligand. concentrations of lysozyme, and these values were used
Assumption (3) is based on the results of the sodium to calculate the pH values. These results are shown in
and chloride ion balances before and after the formation Figure 2. If the presence of HCl in lysozyme is ignored,
of the insoluble complex. When measured with atomic the equations fail to reproduce the variation pH shown
absorption spectroscopy and ion chromatography, the in the figure.
change in the sodium and chloride ion concentrations, Formation of Lysozyme-Ligand Complex. At a
before and after the formation of lysozyme-ligand in- pH less than its pI, lysozyme has an overall positive
soluble complex, was undetectable within experimental surface charge (Z > 0). Assuming Cl- as counterion, the
error. dissociation constant of lysozyme can be written as
Assumption (4) is suggested by the pH change in the
aqueous phase upon the addition of AOT solution, as CLys+10(CCl-)10
shown in Figure 4. KLysCl10 ) (5)
CLysCl10
Surface Charge of Lysozyme. Lysozyme contains 14
acidic (aspartic, tyrosine, and glutamic) amino acid
residues and 19 basic (arginine, histidine, and lysine) By the common ion effect, the solubility of AOT is
amino acid residues (48). Barlow and Thornton (49) limited in an acidic solution or in a salt solution, which
reported the ionic groups of lysozyme as 11 acidic groups is written as
excluding Tyrosine, and 18 basic groups excluding His-
KLH ) CH+CL- (6)
tidine. The dissociation constants of most of these ion-
isable groups are reported in the literature (48, 50, 51).
One carboxylic group is in the interior of the protein
KLNa ) CNa+CL- (7)
molecule, and it does not ionize in solution (50). Kuramit-
su and Hamaguchi (50) suggested the following equation where L- is the negatively charged group of AOT, or
for the net surface charge, Z, of chicken egg white ligand. The dissociated lysozyme reacts with L- forming
lysozyme: an insoluble lysozyme-ligand complex. The solubility
product of this complex, KLysL10, is
32 KiR
Z ) 19 - +
(1) KLysL10 ) (CLys+10)(CL-)10 (8)
i)1 H + KiR
The solubility product of AOT, KLNa ) 1.1 10-3 (M2),
where Ki is the dissociation constant of hydrogen from was obtained from the data reported by Caryl (43). The
group i, and R is a parameter describing the electrostatic solubility product of the lysozyme-ligand complex, KLysL10,
interactions between a protein molecule containing a net was determined experimentally to be 4 10-55 (M11). The
surface charge of Z and a proton (50, 52). The equation values of KLysCl10 and KLH were adjusted to fit the data.
considers 32 ionic groups, the 33 reported by Sakakibara Values of KLysCl10 ) 2.5 10-23 (M10), and KLH ) 9.2
and Hamaguchi (48) less one interior carboxylic group 10-9 (M2) gave a good fit to the data depicted in the
(50). The net surface charge of a protein is an average figures.
over a large number of individual forms of molecule The values of KLysCl10 and KLysL10 are very small because
constantly giving up and taking on protons, and its actual the concentration of one of the ions is raised to the power
net charge at any moment can be greater or less than 10 in eqs 5 and 8. These values indicate that the lysozyme
the average value of Z (52). is associated with chloride in solution unless the ligand,
The net surface charge of lysozyme calculated from eq AOT, is present. The solubility product of lysozyme-AOT
1 was between +9 and +11 in the pH range between 5.8 is in the order of 10-55 indicating that the complex is
and 4.2, shown in Figure 1. For simplicity, the net surface virtually insoluble in water and precipitates upon forma-
charge of lysozyme was considered to be constant at +10 tion.
in the model. The moles of lysozyme-ligand complex formed in the
The crystallized lysozyme obtained from the manufac- aqueous phase, nLysL10, can then be calculated using the
turer contained about 5-10 wt % sodium acetate and mole balances on the lysozyme and the ligand written
sodium chloride (Sigma, ON). The initial lysozyme was as
934 Biotechnol. Prog., 2003, Vol. 19, No. 3

nLysL10 (2) Dainiak, M. B.; Izumrudov, V. A.; Muronetz, V. I.; Galaev,


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A new method for lysozyme purification is proposed.
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