JPH021500A - Production of conalbumin and metal complex thereof - Google Patents

Abstract

PURPOSE:To obtain the subject compound useful as a reagent for animal cell culture, in high purity on an industrial scale at a low cost, by adsorbing conalbumin to a cation exchange material, eluting the adsorbed compound, adding a polyvalent metal, collecting the formed precipitate, again dissolving the precipitate and removing foreign proteins with a cation exchange material. CONSTITUTION:Conalbumin is adsorbed to a cation exchange resin and eluted by contacting with an aqueous solution of a neutral salt. A substance capable of supplying bivalent or trivalent metal ion is added to the eluted liquid to precipitate the metal complex of conalbumin. The precipitate is collected and dissolved in a basic aqueous solution. A cation exchange resin is added to the solution and the foreign proteins are removed to obtain a conalbumin-metal complex. The solution containing the complex is added with an acid to separate the metal ion from the conalbumin-metal complex and the metal salt in the solution is removed to obtain the objective conalbumin having high purity.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing highly pure conalbumin and its metal composite.

[Conventional technology]

Conalbumin (also known as ovotransferrin), which is present in egg white and accounts for approximately 13% of egg white protein, has the ability to bind metal ions such as iron, copper, manganese, and zinc. As a medium reagent to inhibit the growth of auxotrophic microorganisms.
It has also been used as a culture medium reagent for animal cells (Japanese Unexamined Patent Publication No. 111682/1982) because it can act as a serum replacement component in conjunction with Evidermal Growth Factor.

With the remarkable development of biotechnology in recent years, the current situation is that conalbumin with increasingly high purity is required not only in the above-mentioned conventional fields of use but also in future expanded use.

[Problem to be solved by the invention]

Conventional methods for producing conalbumin include organic solvent fractionation (Japanese Patent Publication No. 46-38544), ammonium sulfate fractionation (Ne
w Food Industry Vol, 24. No
, 1. p88-89 (1982)) etc. are well known.

In the organic solvent fractionation method, a solution of a substance capable of supplying trivalent iron ions is added to egg white together with an inorganic salt to form a conalbumin-iron salt complex, and then proteins other than conalbumin are first separated, typically with acetone. After precipitating and removing the organic substances, the conalbumin/iron salt combination is precipitated again with acetone, and conalbumin is fractionated in the form of such an i-polymer.

This method not only requires great care in handling as it uses an organic solvent such as acetone, but also the egg whites after conalbumin recovery with acetone can be used not only in the food field but also in other fields. The problem is that it is difficult to measure.

In addition, in the ammonium sulfate fractionation method, an equal amount of ammonium sulfate is added to egg white to separate albumin (furnace fluid) and globulin (precipitate).
After fractionating the albumin fraction, add acid to the albumin fraction to precipitate and remove ovalbumin, and then add ammonium sulfate to precipitate and separate the conalbumin fraction. After precipitation, ferric citrate is added to form a conalbumin/iron salt combination,
In this method, conalbumin is separated in the form of a complex by performing dialysis and ethanol precipitation again. It has been pointed out that although this salting-out method can achieve a rough fractionation, it is not possible to selectively precipitate only conalbumin from the many proteins present in egg white, and that there is a limit to high purity. has been done.

In these conventional methods, when the goal is to obtain a final product of higher purity (conalbumin/iron salt combination), for example, in the organic solvent fractionation method, the above-mentioned operations are usually repeated, but the purity obtained is at best 75%, and in the ammonium sulfate fractionation method, the above-mentioned operation is generally repeated or other purification methods such as gel filtration or ion exchange chromatography are used in combination; The purity achieved is approximately 80% at most. Moreover, all of them had the problem that the final yield decreased considerably as the purity increased.

Under these circumstances, the present invention provides a method for easily producing conalbumin on an industrial scale and with a higher purity than the conventional method, without the problems associated with the conventional method as described above. The purpose is to provide a method.

[Means to solve the problem]

The inventors of the present invention conducted extensive research in line with the above objectives, and found that after adsorbing and extracting conalbumin from egg white using a cation exchange resin, conalbumin in the eluate was converted into a single metal complex. If the solution of this metal complex is purified again using a cation exchange resin, it is possible to obtain a conalbumin/metal complex, and eventually conalbumin, with a simpler operation than conventional methods and with extremely high purity. I discovered that.

The present invention was completed based on such knowledge, and the present invention first involves contacting a cation exchange resin that adsorbs conalbumin with a neutral salt aqueous solution to elute conalbumin. A substance capable of supplying divalent or trivalent metal ions is added to the collected conalbumin eluate to precipitate the conalbumin metal complex, and then the separated precipitate is dissolved in a basic aqueous solution. We present a method for producing a conalbumin-metal complex, which is characterized by adding a cation exchange resin to this solution to adsorb and remove contaminant proteins.

Furthermore, the second aspect of the present invention is to add an acid to the solution obtained after adsorbing and removing contaminant proteins by the above method, to separate metal ions from the conalbumin-metal complex, and then to remove metal salts in the solution. The present invention provides a method for producing conalbumin characterized by the following.

The present invention will be explained in detail below.

In the method of the present invention, the cation exchange resin that adsorbs conalbumin can be prepared by adsorbing and extracting conalbumin from egg white using a cation exchange resin, and the actual operation is optional. Generally, the pH is adjusted to 4.5 to 5.5, preferably 5.1 to 5.2, by adding an acid such as citric acid or hydrochloric acid; if the pH is lower than 4.5, egg white protein will precipitate. (I don't like it because it becomes easier)
Add about 1/1/2 of the volume to the sufficiently homogenized egg white liquid.
Add 3 times the volume of cation exchange resin and slowly stir for several hours (approximately 1 to 2 hours) to adsorb the conalbumin in the egg white to the cation exchange resin, then usually wash thoroughly with water to remove the conalbumin. Create adsorption resin. Here, the raw egg white is not limited to fresh egg white, but frozen egg white, dried egg white, or egg white after extracting lysozyme, avidin, etc. by a known method can also be used. It goes without saying that frozen egg whites should be thawed, and dried egg whites should be reconstituted into egg white liquid. Further, as the cation exchange resin, commercially available ones such as those shown below can be preferably used.

Amberlight IRC- manufactured by Roam and Burst
50, IRC-84, CG-50, Denio Lai 1-C5-101 manufactured by Diamond Jamrock Chemical Co.
, CC-3, ES-80, C-464V, DOWEX CCR-2 manufactured by Dow Chemical Company, and Revachit GNP manufactured by Bayer.

CNP-80, Diamondion WK-10, WK-11, WK-20, etc. manufactured by Mitsubishi Chemical Industries, Ltd.

In addition, in actual use, these cation exchange resins are treated with caustic soda or other alkaline agent before being added to the egg white liquid to give a concentration of about 0.8 to 1.1 milliequivalents/m! (resin) cation (Na5K excluding H).

When used as a substituted form (cations such as NH4), it is convenient for management because when added to pH-adjusted egg white liquid, the pH of the egg white liquid hardly changes.

According to the method of the present invention, the conalbumin adsorption resin obtained as described above is brought into contact with a neutral salt aqueous solution to elute conalbumin. The neutral salt aqueous solution used for elution is preferably an aqueous solution of sodium chloride, potassium chloride, ammonium chloride, sodium sulfate, etc., with a pH around neutrality, and usually at a salt concentration of about 4.0 to 8.0 parts. use ; If the concentration is lower than 4.096, the elution efficiency will decrease, but if it is within the above range, the conalbumin can be almost completely eluted.
There is no need for a concentration higher than IJ%. The elution operation is arbitrary as long as conalbumin can be eluted from the resin. Generally, a neutral salt aqueous solution may be added at a volume ratio of about 1 to 2 parts to 1 part of the resin in front of the eye and stirred for about 30 to 60 minutes.

The resin is then expanded in a conventional manner and, for example, filtered, to obtain a conalbumin eluate.

According to the method of the present invention, a substance capable of supplying 21ilf or 3g6 of metal ions is added to the conalbumin eluate thus obtained to precipitate the metal complex of conalbumin. Here, the substances capable of supplying divalent or trivalent metal ions include inorganic or Means an organic metal salt. Examples of such substances include metal salts that can supply Fe3+, which has a particularly strong binding property to conalbumin, specifically ferric nitrate, ferric chloride, ferric sulfate, Ferric citrate, ferric succinate, etc. are preferably used.In actual use, these metal salts are generally prepared in the form of an aqueous solution with a salt concentration of about 1 to 10 parts per part of eluate by volume. Te1150~1
It is preferable to use the ratio of about /70 parts. Only conalbumin in egg white protein contains the above metal ions, especially Fe.
``Since H has the property of bonding to form a complex,
This operation makes it possible to separate and remove egg white proteins, such as lysozyme and avidin, which are thought to be present in the eluate and whose isoelectric points are on the more alkaline side than that of fecal albumin. Furthermore, even if egg white from which lysozyme, avidin, etc. have already been extracted is used as a raw material for egg white, it is thought that a small amount of egg white proteins such as lysozyme and avidin still remain in the eluate. Therefore, the above operation is necessary.

According to the method of the present invention, the resulting conalbumin-metal complex precipitate is then separated and dissolved in a basic aqueous solution, and a cation exchange resin is added to this solution to further remove other contaminant proteins. Remove by adsorption. Here, the precipitate may be separated according to a conventional method, for example, by filtration. The present inventors did not immediately convert the precipitate thus collected into the desired conalbumin or metal complex, but instead dissolved it in a basic aqueous solution and then treated it with a Baidu cation exchange resin. It has been found that other contaminant proteins (contaminant proteins whose isoelectric points are on the alkaline side) can be adsorbed and removed. Note that the term "dissolved" herein includes partially dissolved, dispersed, or partially dispersed states.

This waste basic aqueous solution may be an aqueous solution of a common alkaline agent such as sodium hydroxide or potassium hydroxide, which exhibits basicity when dissolved in water. In the actual operation, the method is ff-as long as it is possible to finally dissolve the precipitate in a basic aqueous solution. For example, after dissolving the precipitate in water, sodium hydroxide, potassium hydroxide etc. to make it from around neutral to basic, preferably pH 6
, 5 to 6.8.

Moreover, this waste cation exchange resin is no different from the waste cation exchange resin used to adsorb and extract conalbumin from egg white. In addition, when using the cation exchange resin, it should be kept in a neutral to basic aqueous solution, preferably at pH 6.5 to 6.8.
, it is convenient for pH control to treat it with R. caustic soda or other alkaline agent to form a cation-substituted form of about 1.0 to 1.4 milliequivalents/ml (resin).

Although the cation exchange resin used here is somewhat colored after use, it can be reused after being decolored by ordinary acid or alkali treatment or heat treatment. It goes without saying that the disused resin for adsorbing and extracting conalbumin from egg whites can also be appropriately regenerated into a cation-substituted form and then reused at Uni.

The actual adsorption removal operation using such a cation exchange resin is not particularly limited; This may be carried out by stirring for ~1 hour.

The contaminant protein adsorbed and removed by the above procedure was examined by electrophoresis and had a molecular weight of 0. 00(1~6
('), 000 egg white proteins, but it has not yet been determined what they are. perhaps,
It is thought that it is a modified substance of ovalbumin or conalbumin.

According to the method of the present invention, the cation exchange resin on which the contaminant proteins have been adsorbed is then separated by a conventional method, e.g., by furnace separation, and the resulting liquid can be used as is, if desired.
Alternatively, it can be subjected to further drying treatment, such as freeze-drying, to produce a product as a conalbumin/metal complex, but usually an acid is added to this furnace liquid to produce a conalbumin/metal composite.
After separating the metal ion from the metal complex, the resulting metal salt is removed from the solution to produce conalbumin.

As the acid added to the conalbumin/metal complex solution after removing contaminant proteins, any inorganic or organic acid can be used, but citric acid, nitric acid, hydrochloric acid, etc. are preferably used in the membrane. .

These acids are usually used as an aqueous solution of about 5 to 20%, and the volume ratio is generally 1/20 to 1/35 to 1 part of the above solution.
It is preferable to use it at a ratio of about 1.5 parts. This operation is usually carried out with continuous stirring for about 0.5 to 1 hour after addition of the acid solution.

The metal ions separated from the complex by the above operation are immediately converted into metal salts by the added acid, and the metal salts thus formed are then removed from the solution. This removal operation is not particularly limited, and any method such as a suction removal method using an anion exchange resin or an ultrafiltration method may be used. A suction removal method using an anion exchange resin is preferred because it is easy to operate and has good efficiency. Commercially available anion exchange resins such as Amberlite IRA-
45, IRA-410; Duolite A-561, A-
368; DOWEX MWA-1, WGR, DIAION WAIO1 WA20, WA30, etc. are preferably used. In the adsorption removal operation using such a resin, the adsorption resin is placed in the membrane at a volume ratio of 1/3 to 1 part of the target solution.
It may be added at a ratio of about 15 parts and stirred for about 20 to 40 minutes. At this time, in order to increase the adsorption efficiency, for example, I
The pH of the solution may be maintained at 4.0-4.5 using NMCI.

The anion exchange resin on which the metal salts have been adsorbed is then separated, e.g. filtered, by a conventional method, and the resulting solution can be used as is or subjected to further desalination operations such as electrodialysis, mosaic charged membrane desalination, membrane desalination, etc. plan? After performing a combined operation with filtration and the like, it can be subjected to a drying treatment, for example freeze-drying, to produce a conalbumin product as a powder.

The conalbumin obtained by the method of the present invention as described above is usually a product with a purity of 94-95% or more, and the yield from the raw material egg white is usually 0.796-0.796%.
It is about 896. As is clear from the results of the test examples described later, the purity and yield of the conventional white organic solvent fractionation method are at most 75% and 0.6%, respectively, and the purity and yield of the ammonium sulfate fractionation method are 75% and 0.6%, respectively. at most 80% and 0.5 respectively
%, it can be seen that it is extremely high.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples. In the present invention, % means weight %.

Example I A. Preparation of conalbumin adsorption resin: 5000 ml of egg white liquid (egg white liquid after lysozyme and avidin extraction) which had been adjusted to pH 5.2 by adding 1NHCl and was sufficiently homogenized was treated with alkali in advance. 1. Cation exchange resin (Diaion WK-10) in Na-form with O ml/ml (resin) 1
500 ml was added and held for 2 hours with gentle stirring. Next, the egg white liquid was removed by decanting and filtration, and then thoroughly washed with clean water to obtain a conalbumin adsorption resin.

B. Elution of conalbumin: Conalbumin adsorption resin 15 obtained as above
3000 ml of 55 saline solution was added to QOml, and conalbumin was eluted into the solution while stirring gently for 30 minutes. Next, 500 ml of saline solution of the same concentration was added once again to the resin from which the eluate had been separated, extrusion was performed, and this liquid and the previous eluate were combined.

C. Production of conalbumin/metal complex: 60 ml of a 5% ferric nitrate aqueous solution was added to the thus obtained conalbumin eluate 350 ('1m+) to precipitate the conalbumin/iron salt complex. Then, it was filtered. After separating the precipitate from the lever, it was dissolved in 10,100O fresh water, and the pH was adjusted to 6.5 with an IN aqueous solution of sodium hydroxide.This basic solution was preliminarily treated with an alkali to 10,100O to give 1.2 milliequivalents/ml ( 1000 ml of a cation exchange resin (Diaion WK-10) in the Na-form of the resin (resin) was added, and the mixture was maintained for 30 minutes with gentle stirring.

Next, the resin adsorbed with contaminant proteins was separated to obtain a solution containing conalbumin/iron salt combination.

D. Production of conalbumin: 30 ml of a 20% aqueous citric acid solution was added to the conalbumin/iron salt combined solution after removing contaminant proteins, and stirred for 1 hour. Next, 300 ml of anion exchange resin (Diaion WA-10) was added, and gentle stirring was continued for 30 minutes. At this time, the pH of the solution was maintained at 4.0 to 4.5 using 1NHCl.

The anion exchange resin adsorbed with metal salts was separated in a furnace, and the resulting liquid was further subjected to electrodialysis and membrane filtration (('1.45
μm) and then lyophilized according to a conventional method to obtain a powder (purity 95%) at 38K (yield 0 from raw egg white liquid).
, 76%). Note that the purity was measured by electrophoresis.

Example 2 A. Preparation of conalbumin adsorption resin: Egg white liquid whose pH was adjusted to 5.2 with INHCl and sufficiently homogenized (egg white liquid obtained by separating the egg yolk after breaking the egg)
To 5,400 ml, add 2,000 ml of a cation exchange resin (Diaion WK-10) that has been previously treated with alkali to form Na-form at 0.9 milliequivalents/ml (resin).
The mixture was maintained for 2 hours with gentle stirring. Next, the egg white liquid was removed by decanting and filtration, and then thoroughly washed with clean water to obtain a conalbumin adsorption resin.

B. Elution of conalbumin 2 Conalbumin adsorption resin 20 obtained as above
00 ml L 596 Add 2000 ml of saline solution,
Conalbumin was eluted into the liquid while stirring gently for 30 minutes.

C. Production of conalbumin/metal composite: 2000 ml of conalbumin eluate obtained by furnace separation of the resin: 1%
300 ml of ferric nitrate aqueous solution was added to precipitate the conalbumin/iron salt combination.

Next, after filtering and separating this precipitate, Shimizu 1010
0O and pH adjusted with IN aqueous sodium hydroxide solution.
was set at 6.4. This basic solution was preliminarily treated with an alkali to about 10,100O to give a concentration of 1.2 meq/ml (resin)
A-form cation exchange resin (Diaion WK-
10) Add 1000 ml and hold for 30 minutes while stirring gently.

Next, the resin adsorbed with contaminant proteins was separated in a furnace to obtain a solution containing conalbumin/iron salt combination.

D. Production of conalbumin: 40 ml of 20% citric acid aqueous solution was added to the conalbumin/iron salt combined solution after removing contaminant proteins, and stirred for 30 minutes. Next, 300 ml of anion exchange resin (Diaion WA-10) was added, and gentle stirring was continued for 30 minutes. At this time, the pH of the solution was maintained at 4.5 using 1NHCl.

The anion-exchange resin adsorbed with metal salts is separated from each other, and the resulting solution is further subjected to mosaic QT and membrane desalting treatment, and then freeze-dried according to a conventional method to obtain a powder (95% purity).
) 37 g (yield 0.7% from raw egg white liquid) was obtained.

The purity was measured by electrophoresis in the same manner as in Example 1 above.

〔Effect of the invention〕

A comparative study with the conventional organic solvent fractionation method or ammonium sulfate fractionation method demonstrated that the method of the present invention yields a conalbumin-metal complex and/or conalbumin with higher purity than conventional methods. This will be demonstrated using the results obtained.

Comparative Test ■6 Method of the Present Invention The conalbumin/iron salt combined solution obtained by carrying out steps A to C of Example 2 above was freeze-dried according to a conventional method,
Conalbumin/iron salt combined powder was obtained. The purity of this product was determined to be 9426 by electrophoresis.

- Also, the yield from the raw egg white liquid was 0.896.

Next, a 20% aqueous solution of this conalbumin/iron salt combined powder was prepared, and conalbumin was produced as a powder from this solution under the same conditions as in Step D of Example 2, and the purity of this product was as follows. At 95%, the yield from raw egg white liquid is 0. It was 7%.

(2) White solvent fractionation method This conventional method was carried out as follows in accordance with the method of Example 2 disclosed in Japanese Patent Publication No. 46-38544.

In the same way as the egg white liquid used in Example 2 above, 160 g of common salt was added to 5000 ml of the egg white liquid obtained by separating the egg yolk after breaking the eggs, and then 125 ml of ferric citrate aqueous solution was gradually added and stirred. After adjusting this solution to pH 8.5, 2.51 g of acetone was gradually added, the resulting precipitate was removed, and the resulting furnace solution was adjusted to pH 8.0, and then 2.5 g of acetone was added gradually.
5j? was added with stirring to precipitate the conalbumin/iron salt combination. This precipitate was poured into a furnace, purified by a centration and elution method using Celite, and then freeze-dried to obtain 85 g of a red dry powder.

The purity of the conalbumin/iron salt combined product thus obtained was determined to be 70% by electrophoresis. Also, the yield from raw egg white liquid is 0. It was 796.

Next, a 20% aqueous solution of this conalbumin/iron salt combination product was prepared, and conalbumin was produced as a powder from this solution under the same conditions as in Step D of Example 2, and the purity of this product was as follows. At 75%, the yield from raw egg white liquid is 0. It was 6%.

■The conventional method of ammonium sulfate fractionation is New Food IndusLry
Vol, 24. No.

+, p8g-89 (1982), as follows.

In the same way as the egg white used in Example 2 above, an equal volume of saturated ammonium sulfate solution was added to 5,400 ml of the egg white liquid obtained by separating the egg yolk after cracking, the resulting precipitate was removed, and then 0.5M sulfuric acid After adjusting the pH to 4.6 with a solution, 865 g of ammonium sulfate was further added, and the resulting precipitate was collected and dialyzed in a cold place.

After removing a small amount of precipitate that had formed, the pH was adjusted to 6.0, and salt was added to the solution to give a concentration of 0.02M. Ethanol was then added to a concentration of 20v/v% to remove the precipitate. brought about. This precipitate was separated, and after adding 60 ml of 5% ferric citrate aqueous solution to it, the pH was adjusted to 9.0 and dialyzed, then the pH was adjusted to 6.0, and the concentration was further adjusted to 0.0.
Salt was added to give a concentration of 0.02M. Then 15v/v
% of ethanol was added to obtain a red conalbumin/iron preparation.

Next, a 20% aqueous solution of the thus obtained conalbumin/iron salt combined product was prepared, and conalbumin was produced as a powder from this solution under the same conditions as in Step D of Example 2 above. The purity of the product was 80%, and the yield from the raw egg white liquid was 0.59o.

From the results of the above comparative test examples and Examples 1 and 2, it is clear that the method of the present invention can produce a conalbumin-metal complex, and by extension, conalbumin, with a higher purity than the conventional method. It is. Moreover, since the method of the present invention does not use any organic solvent, there is no need to worry about the complexity of its handling, and the raw material egg white is not contaminated by it, so the advantage is that the egg white after conalbumin recovery can be used for 9 purposes. There is. Moreover, according to the method of the present invention, a highly purified product can be obtained in a single operation, and the operation is simple, so that it can be carried out on an industrial scale.

Applicant's agent: Mr. Sato

Claims (1)

[Claims] 1. Contacting a cation exchange resin that adsorbs conalbumin with a neutral salt aqueous solution to elute conalbumin, and supplying divalent or trivalent metal ions to the resulting conalbumin eluate. The metal complex of conalbumin is precipitated by adding a substance that can be used to remove conalbumin, and then the separated precipitate is dissolved in a basic aqueous solution, and a cation exchange resin is added to this solution to adsorb and remove contaminant proteins. A method for producing a conaluminiumbumin/metal composite, characterized by: 2. A compound characterized by adding an acid to the solution obtained after adsorbing and removing contaminant proteins by the method of claim 1, separating metal ions from the conalbumin-metal complex, and then removing metal salts in the solution. Method for producing albumin.