Process for eluting egg white lysozyme

In the process of eluting egg white lysozyme by contacting a weakly acidic cation exchange resin having egg white lysozyme adsorbed thereon with an eluting agent comprising a salt solution, an alkali agent is added to the eluate containing the weakly acidic cation exchange resin having egg white lysozyme adsorbed thereon whereby the egg white lysozyme can be eluted more effectively. From the eluate thus obtained, lysozyme can be collected in a high yield by an ordinary collecting method such as salting out.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a process for eluting egg white lysozyme 
from a weakly acidic cation exchange resin having egg white lysozyme 
adsorbed thereon. 
2. Description of the Prior Art 
As one process for extracting from egg white lysozyme which is known as a 
bacteriolytic enzyme present in the egg white in trace amounts, it has 
previously been proposed to cause a weakly acidic cation exchange resin 
such as Amberlite, Duolite or Diaion to adsorb the lysozyme and then to 
elute the lysozyme therefrom. Ordinarily, the lysozyme is eluted from the 
resin by a method which involves first packing the resin having the 
lysozyme adsorbed thereon in a column, and thereafter eluting the lysozyme 
with a salt solution having a pH substantially in the neutral range, e.g., 
of the order of 6.5 to 7.5, such as an aqueous solution of sodium chloride 
or ammonium secondary phosphate as is disclosed, for example, in Japanese 
Patent Pub. No. 7828/1966. The salt solution includes, in addition to a 
solution obtained by simply dissolving a salt in water, a so-called buffer 
solution obtained by admixing a salt with an acid or an alkali having 
either one of the ions constituting the salt (anion or cation). 
However, the column method, while ensuring process efficiency due to a 
continuous operation system, cannot effectively promote intimate contact 
between the resin having lysozyme adsorbed thereon and the salt solution 
and thus is unsuitable especially in the case where the resin to be 
treated is in only a small quantity. For this reason, a batch process is 
preferred in order to elute lysozyme effectively from a resin having 
lysozyme adsorbed thereon. 
However, when a salt solution of a pH value near the neutral range is 
actually used in a similar manner for eluting lysozyme from a resin by a 
batch process in accordance with the elution process described above, it 
is difficult, even when the elution operation is repeated several times 
and the lysozyme is collected from each of the eluates thus obtained by a 
step such as salting out, to increase the recovery, i.e., the yield, in 
spite of the troublesome elution operation repeated several times. 
Presumably this is because the concentration of the lysozyme in the eluate 
has become lower. A solution for this problem has long been sought, but 
has not been found to date. 
As a result of our extensive research, it has been found that, in the above 
described batch process for eluting egg white lysozyme adsorbed on a resin 
by contacting the resin with a salt solution, the lysozyme can be eluted 
more effectively by adding an alkali agent to the eluate containing the 
resin having lysozyme adsorbed thereon. 
SUMMARY OF THE INVENTION 
An object of the present invention is to provide a process for eluting egg 
white lysozyme adsorbed on a weakly acidic cation exchange resin 
effectively in a high yield on the basis of the findings mentioned above. 
The present invention which has achieved this object provides a process for 
eluting egg white lysozyme by contacting a weakly acidic cation exchange 
resin having egg white lysozyme adsorbed thereon with an eluting agent 
comprising a salt solution, which process comprises adding an alkali agent 
to the eluate containing the weakly acidic cation exchange resin having 
egg white lysozyme adsorbed thereon.

DETAILED DESCRIPTION OF THE INVENTION 
In the process of the present invention, the step of causing the weakly 
acidic cation exchange resin to adsorb egg white lysozyme may be 
accomplished in the same manner as the conventional adsorption step of 
this type. For instance, to a thoroughly homogenized egg white solution 
having a pH adjusted to fall substantially in the neutral range (ca. 6.5 
to 7.5) with addition of acid, is added a weakly acidic cation exchange 
resin in a quantity of about 20 to 30% by volume of the egg white 
solution, and the mixture is stirred slowly for several hours (ca. 1 to 8 
hours), causing the cation exchange resin to adsorb the lysozyme in the 
egg white. Ordinarily, the resulting resin is then thoroughly washed with 
water to obtain a resin having lysozyme adsorbed thereon. The weakly 
acidic cation exchange resins which can be used herein are commercially 
available resins such as, for example: 
Amberlite IRC-50, Amberlite IRC-84, and Amberlite CG-50, supplied by Rohm & 
Haas Co.; 
Duolite CS-101, Duolite CC-3, and Duolite ES-80, supplied by Diamond 
Shamrock Chemial Co.; 
Dowex CCR-2, supplied by the Dow Chemical Co.; 
Lebatit CNP and Lebatit CNP-80, supplied by Bayer Co.; and 
Diaion WK-10, Diaion WK-20, and Diaion WK-11, supplied by Mitsubishi Kasei 
Kogyo K.K., Japan. 
The weakly acidic cation exchange resin, when actually used, is 
advantageously converted by treatment with caustic soda or other alkalis 
into a resin substituted with about 1.2 to 1.4 meq./ml resin of a cation 
except for H.sup.+, such as Na.sup.+, K.sup.+ and NH.sub.4.sup.+, prior to 
the addition to the egg white solution since the pH of the egg white 
solution which has been adjusted to neutrality does not appreciably vary 
when the cation substituted resin is added thereto. 
Lysozyme is eluted from the resin thus obtained. 
The salt solution used for the elution of lysozyme may be any of 
conventional salt solutions, such as, for example, an aqueous solution of 
sodium, potassium and ammonium salts, more specifically, an aqueous 
solution of sodium chloride, potassium chloride, sodium sulfate and 
ammonium secondary phosphate having a pH in the vicinity of neutrality, or 
a buffer solution obtained by admixing any of these salts with an aqueous 
solution of an acid or an alkali having either one of the ions (anion or 
cation) constituting the salt. 
The salt solution is used ordinarily at a concentration of about 0.3M or 
higher so that the lysozyme adsorbed on the resin can be easily eluted 
from the resin. It is preferable from the viewpoint of ease in handling 
that the elution operation be carried out at a salt solution: resin ratio 
of 100:10 to 100 parts by volume. 
The unique feature of the present invention, which relates to a process for 
eluting lysozyme by contacting a weakly acidic cation exchange resin 
having egg white lysozyme adsorbed thereon with an eluting agent 
comprising a salt solution, is the addition of an alkali agent to the 
eluate containing the resin. 
The alkali agent to be added must be one having a strong alkalinity such 
that the pH of its aqueous solution of 1 mole concentration is 13 or 
higher, such as caustic soda or caustic potash, and is preferably used in 
the form of an aqueous solution of a concentration of the order of 0.1N to 
2N. If a solution of lower concentration of the alkali agent is added to 
the eluate, the overall quantity of the resultant solution mixture will be 
increased, while a solution of higher concentration tends to result in 
denaturation of lysozyme. 
The quantity of the alkali agent to be added may be of an order such that 
the pH of the eluate can be maintained in the neutral range, i.e., about 
6.5 to 8.0. In the case where this quantity is so small that the pH of the 
eluate becomes lower than 6.5, lysozyme cannot easily be eluted from the 
resin. Conversely, the addition of the alkali agent in an excessive 
quantity such as to make the pH of the eluate higher than 8.0 tends to 
result in partial denaturation of lysozyme. 
The rate at which the alkali agent is added depends on the state in which 
lysozyme is adsorbed on the resin and thus cannot be determined 
definitively. Generally speaking, the alkali agent may be added in a 1N 
solution at a rate of about 0.5 to 7 ml/min., preferably about 3 to 4 
ml/min., per liter of the resin; in a 0.5N solution at a rate of about 1 
to 14 ml/min., preferably about 6 to 8 ml/min.; and in a 2N solution at a 
rate of about 0.3 to 4 ml, preferably about 2 to 3 ml/min., with stirring 
for about 5 to 30 minutes. 
In order to prevent a rapid local change of the pH of the eluate and to 
ensure uniform pH throughout the eluate, it is desirable that the alkali 
agent be added while the eluate is stirred at a speed, for example, of 30 
r.p.m. The addition of the alkali agent and stirring of the eluate may be 
conducted intermittently provided that the pH of the eluate is maintained 
under the conditions specified hereinbefore, but continuous addition and 
stirring is preferred in order to maintain constant pH conditions. 
By adding the alkali agent to the eluate in the process of eluting lysozyme 
from the resin, lysozyme adsorbed on the resin can be eluted effectively 
in a high yield as will be apparent from the Example of Experiment 
described hereinlater. Presumably, this is because the addition of the 
alkali agent prevents the lowering of the pH of the eluate caused by the 
substitution of lysozyme for a salt (ion exchange), thereby promoting the 
elution of the lysozyme. 
The effectiveness of the solution process of the present invention in 
eluting lysozyme and hence in improving the yield of extraction of 
lysozyme from egg white will now be shown by the following Example of 
Experiment in which lysozyme was actually eluted and then collected. 
EXAMPLE OF EXPERIMENT 
A. Preparation of Resin Having Lysozyme Adsorbed Thereon 
To 1,000 ml of a thoroughly homogenized egg white solution having a pH 
adjusted to 6.5 with addition of hydrochloric acid was added 200 ml of a 
cation exchange resin (Dowex CCR-2) which had been converted by alkali 
treatment into a resin substituted with 1.3 meq./ml resin of Na.sup.+, and 
the resulting mixture was stirred slowly for 300 minutes. The egg white 
solution was then removed by decantation and filtration and the residue 
was washed three times with 800 ml of pure water to obtain a resin having 
lysozyme adsorbed thereon. 
B. Elution of Lysozyme 
To about 200 ml of the resin obtained in the manner described above was 
added 800 ml of a 3% aqueous sodium chloride solution. To this mixture was 
further added a 1N NaOH solution at a rate of about 0.6 ml/min. over a 
period of 10 minutes with stirring at a speed of 30 r.p.m. to maintain the 
pH of the eluate at about 7.0 and to elute the lysozyme into the salt 
solution. 
C. Collection of Eluted Lysozyme 
The solution containing the resin was then filtered through a filter cloth 
of 80 mesh, and the pH of the filtrate thus obtained was adjusted to 9.5 
with addition of 0.5N NaOH solution. To this filtrate was added sodium 
chloride until the sodium chloride concentration thereof reached 5%, and 
lysozyme was collected by the salting out method. The quantity of the 
lysozyme thus collected was 9.2 g in cake form which was found, on 
conventional potency analysis, to correspond to 3.1 g of pure crystalline 
lysozyme. 
As a control, the following elution process was conducted. To about 200 ml 
of the resin with lysozyme adsorbed thereon prepared in accordance with 
the procedure A described above, was added 800 ml of a 0.5M sodium 
phosphate buffer solution (pH 7.0) containing 3% of sodium chloride, and 
the mixture was stirred for 10 minutes at a speed of 30 r.p.m. to elute 
the lysozyme into the salt solution. This resin containing solution was 
filtered through a filter cloth of 80 mesh, and lysozyme was collected 
from the filtrate thus obtained under the same conditions as in the 
procedure C. In this case, the filtered resin was further subjected to 
elution and collection three times, respectively, whereupon lysozyme was 
collected in the following quantities. 
______________________________________ 
Pure crystalline 
Lysozyme in 
lysozyme (g) on potency 
cake form (g) 
analysis 
______________________________________ 
1st time 4.5 1.5 
2nd time 1.1 0.3 
3rd time 0.5 0.1 
4th time not precipitat- 
0.0 
ed 
Total 6.1 1.9 
______________________________________ 
It will be noted from the results obtained in the above Example of 
Experiment that, in the process of eluting with a salt solution lysozyme 
from a resin having lysozyme adsorbed thereon, the lysozyme can be eluted 
in higher yields by the process of the present invention in which a salt 
solution is employed and also an alkali agent is added than by a 
conventional process in which a salt solution having a pH substantially in 
the neutral range is utilized. 
In order to indicate more fully the nature and utility of this invention, 
the following specific examples of practice constituting preferred 
embodiments of the invention are set forth, it being understood that these 
examples are presented as illustrative only and are not intended to limit 
the scope of the invention. 
EXAMPLES 
Example 1 
A resin having lysozyme adsorbed thereon was prepared as in the Example of 
Experiment described above except that Na-form Dowex CCR-2 was replaced by 
the same quantity of Amberlite IRC-50 which had been converted by alkali 
treatment into a resin substituted with 1.2 meg./ml resin of K.sup.+. The 
resin obtained was then subjected to elution and collection in accordance 
with the procedures B and C of the Example of Experiment, whereupon 8.9 g 
of lysozyme was collected in cake form which was found, on potency 
analysis, to correspond to 2.9 g of pure crystalline lysozyme. The pH of 
the eluate was maintained at about 7.5 throughout the elution process. 
Example 2 
Lysozyme was eluted by the procedure B of the Example of Experiment except 
that the 3% aqueous sodium chloride solution used as a salt solution and 
the 1N NaOH solution were replaced respectively by a 3% aqueous potassium 
chloride solution and a 1N KOH solution. The eluate thus obtained was 
subsequently treated by the procedure C of the Example of Experiment, 
whereupon 8.7 g of lysozyme was collected in cake form which was found, on 
potency analysis, to correspond to 2.8 g of pure crystalline lysozyme. The 
pH of the eluate was maintained at about 6.7 throughout the elution 
process. 
Example 3 
Lysozyme was collected under the same conditions as in the Example of 
Experiment except that lysozyme was eluted into a salt solution containing 
a resin to which was added a 1N NaOH solution at a rate of about 0.8 
ml/min. over a period of 10 minutes with stirring at a speed of 40 r.p.m. 
The quantity of the lysozyme thus collected was 9.0 g in cake form which 
was found, on potency analysis, to correspond to 3.0 g of pure crystalline 
lysozyme. The pH of the eluate was maintained at about 7.3 throughout the 
elution process. 
Example 4 
A resin having lysozyme adsorbed thereon was prepared as in the Example of 
Experiment except that Na-form Dowex CCR-2 was replaced by the same 
quantity of Diaion WK-10 which had been converted by alkali treatment into 
a resin substituted with 1.2 meq./ml resin of K.sup.+. The resin obtained 
was then subjected to elution and collection in accordance with the 
procedures B and C of the Example of Experiment, whereupon 9.2 g of 
lysozyme was collected in cake form which was found, on potency analysis, 
to correspond to 3.0 g of pure crystalline lysozyme. The pH of the eluate 
was maintained at about 7.4 throughout the elution process. 
Example 5 
Lysozyme was eluted into a salt solution by the procedure B of the Example 
of Experiment except that the 3% aqueous sodium chloride solution used as 
a salt solution was replaced by a 0.5M sodium phosphate buffer solution 
containing 3% of sodium chloride and having a pH of 7.0. The eluate 
obtained was then treated by the procedure C of the Example of Experiment, 
whereupon 9.1 g of lysozyme was collected in cake form which was found, on 
potency analysis, to correspond to 2.9 g of pure crystalline lysozyme. The 
pH of the eluate was maintained at about 7.0 throughout the elution 
process.