JPH0213372A - Method for purifying superoxide-dismutase - Google Patents

Abstract

PURPOSE:To readily separate and purify superoxide.dismutase(SOD) by bringing an SOD-containing solution into contact with a polyacrylic acid ester-based nonionic synthetic adsorbent, adsorbing the SOD on the afore-mentioned adsorbent and eluting the SOD. CONSTITUTION:Water-soluble salts, such as preferably ammonium sulfate, are added to an SOD-containing solution and the resultant solution is brought into contact with a polyacrylic acid ester-based synthetic adsorbent, preferably by a column method to adsorb the SOD on the afore-mentioned adsorbent. The SOD is then eluted with water, etc., to separate and purify the SOD from the SOD-containing solution.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to superoxide dismutase (hereinafter referred to as S
This invention relates to a novel separation and purification method for OD (abbreviated as OD).

[Prior art and issues] SOD exists widely in the biological world, and is a superoxide anion radical (
In recent years, it has been used as an anti-inflammatory agent to treat various inflammations, osteoarthritis, rheumatoid arthritis, ischemic diseases,
It has been recognized to have pharmacological effects in the treatment of side effects caused by radiation irradiation and certain urinary disorders. Until now SO
D is heat treatment, salting out with ammonium sulfate, resolution with organic solvent, DEAE-cellulose [Journal of Biological Chemistry (J, Biol.

Chew, ), 244.6049 (1969)], ]
DEAE-Sephadex Journal of Biochemistry (J, Biochem), 85.139
7 (1979)],] CMC Luulose CM-Sephadex [Journal of Biological Chemistry (J, Biol, Chem.

), 248.35g2 (1973)] and hydroxyapatite [Journal of Biological Chemistry (J, Biol, Chem.
, ), 252°6421 (1977)], but these methods require desalting operations during the purification process and require a long time for adsorption and elution. There are problems such as: In addition, SOD using cross-linked agarose gel bonded with phenyl groups and octyl groups
SO using a purification method (Japanese Patent Publication No. 60-12025) and a polyvinyl-based synthetic polymer gel bonded with butyl groups.
A purification method for D (Japanese Patent Application Laid-Open No. 63-49078) is also known, but these methods have difficulty operating at high flow rates due to the weak mechanical strength of the gel, and are difficult to achieve high resolution because the gel particles tend to be non-uniform. There are problems such as being difficult to obtain and expensive. Furthermore, a method for purifying SOD using a porous insoluble carrier bonded with copper or zinc chelate groups (Japanese Patent Publication No. 60-1202
No. 6) has also been proposed, but this requires a metal chelate group to be bonded to the carrier each time the carrier is used, which causes problems such as complicated operations and difficulty in achieving chromatographic reproducibility. There is. Therefore, these conventional SOD purification methods are considered unsuitable for industrially purifying SOD in large quantities. The present inventors have completed various studies to solve these problems, and as a result, have completed the present invention.

[Means for Solving the Problems] The present invention is characterized in that a solution containing SOD is brought into contact with a polyacrylic acid ester nonionic synthetic adsorbent to adsorb SOD, and then the adsorbed SOD is fractionally eluted. The present invention provides a method for purifying SOD.

The polyacrylic acid ester nonionic synthetic adsorbent used in the present invention is a porous nonionic synthetic adsorbent having a giant network structure made of a polymer of an alkyl ester of alkyl-substituted acrylic acid such as acrylic acid or methacrylic acid. In addition, adsorbents may include a sulfoxide etc. in a part of the alkyl ester moiety, and furthermore, those having a part of acrylic acid amide bond etc. in addition to the ester bond, and the polyacrylic acid As long as the ester composition is about 50% or more, a copolymer with, for example, acrylonitrile or the like may be used.

Furthermore, among these resins, those with polarity can be used more preferably than those with non-polar ones.

As a synthetic adsorbent consisting only of polyacrylic acid ester, for example, the product name Diaion I (P-IMG, HP
-2MGSHP-3MG (manufactured by Mitsubishi Chemical Corporation),
There are commercially available adsorbents such as Amberlite XAD-7 and XAD-8 (manufactured by Rohm and Haas, Inc., USA). Also,
An example of a synthetic adsorbent containing a sulfoxide in a part of the alkyl ester moiety of a polyacrylic ester is an adsorbent under the trade name Amberlite XAD-9 (manufactured by Rohm and Haas, Inc., USA).

As the SOD targeted by the present invention, for example, SOD derived from mammals and SOD derived from microorganisms, which are distributed over a wide range in the biological world, can be used as appropriate, and the origin is not particularly limited. Preferably S derived from microorganisms
It is better to use OD.

In the present invention, the SOD solution to be adsorbed onto the polyacrylic acid ester nonionic synthetic adsorbent is prepared in advance by 2 to 3
Add M salts before use.

The salts used here include relatively highly water-soluble salts consisting of cations such as ammonium ions or metal ions and anions such as various inorganic acids or organic acids, and the cations include ammonium ions, sodium, Water-soluble salts consisting of alkali metal ions such as potassium and lithium, alkaline earth metal ions such as magnesium and calcium, and metal ions such as zinc and chloride are more preferable. Also, as anions, chloride ions,
Water-soluble salts consisting of inorganic acid ions such as sulfuric acid, phosphoric acid, and nitrate ions and organic acid ions such as acetic acid, tartaric acid, and citrate ions are more preferably used.

Specific examples of water-soluble salts include ammonium sulfate, sodium sulfate, magnesium sulfate, potassium sulfate, sodium phosphate, potassium phosphate, ammonium phosphate, sodium chloride, potassium chloride, and ammonium chloride. If the pH falls outside the range of 5 to 9 due to the addition of salts, it may be adjusted with sodium hydroxide, sodium carbonate, hydrochloric acid, sulfuric acid, or the like.

SOD can be adsorbed onto the synthetic adsorbent by a column method in which the liquid is passed through a column filled with a synthetic adsorbent, or by a column method in which SOD is adsorbed by passing the liquid through a column packed with a synthetic adsorbent, or by
This is carried out by a method such as a batch method in which a synthetic adsorbent is added to solution D and adsorbed while stirring, but it is more preferable to use a column method.

The specific velocity of the flowing liquid (flow rate per hour/adsorbent amount) for adsorption by the column method is preferably 1 or less.

The adsorbed SOD is eluted by stepwise decreasing the salt concentration of water or an aqueous solution containing water-soluble salts. Further, elution can be performed using a water-soluble organic solvent (for example, methanol, ethanol, butanol, propatool, acetone, methyl acetate, dioxane, etc.) or a water-soluble organic solvent in which a water-soluble salt is coexisting. The water-soluble salts used here include the same water-soluble salts that may be allowed to coexist during adsorption.

The adsorption and elution operations are carried out at temperatures of about 0-30°C, more preferably about 0-10°C. After use, the synthetic adsorbent can be used repeatedly by washing with sodium hydroxide and water. Furthermore, it is of course possible to perform the purification method of the present invention in combination with conventional salting-out methods, solvent fractionation methods, chromatography methods, and even membrane separation methods.

Characteristic advantages of the purification method of the present invention include (1) the synthetic adsorbent has a high SOD adsorption capacity;

(2) Since the adsorption is carried out at a high salt concentration, pre-operations such as sample desalting and dialysis are not necessary, and the purification process can be considerably shortened.

(3) A high flow rate can be obtained and large-scale purification can be performed.

(4) It has high separation ability and greatly increases SOD specific activity, making it easy to purify SOD.

(5) Can be directly applied to crude SOD solutions containing many impurities.

etc., and the present invention is extremely useful as a method for industrially mass-purifying SOD.

[Examples] Examples are shown below, but the present invention is not limited to the scope of these examples.

The SOD activity measurement method uses superoxide (0
? Ishiguro et al.'s method utilizes SOD's inhibition of the reduction of neotetrazolium by ) [Chemical and Pharmaceutical Bulletin (Chew, Ph.
arn, Bull, ), 22.2935 (1974
)]. Protein was expressed as absorbance at 280 nm.

Example 1 Serratia marcescens (serratiamarc)
essens ATCC21074) in Agricultural and Biological Chemistry [Agr.
ic.

Biol, Chem, 34.310 (1970)
Bacterial cells were collected by centrifugation from the culture solution obtained by culturing according to the method described in . The bacterial cells were suspended in water and Dynomil (manufactured by Wiley-Ko. Bachofen, Switzerland) was used.
The bacterial cells were crushed using a device. Ammonium sulfate was added to the disrupted solution at 30% (w/v) i11 degrees to dissolve it, and a clear supernatant containing SOD was obtained by centrifugation. This supernatant liquid 121
2 in advance with 30% (W/V) ammonium sulfate water (
Amberlite XAD-8 column (6C1 diameter, 36mm length) equilibrated with pH 7.5 with sodium hydroxide
cz, packed m 112) at a flow rate of 500xC/hour to adsorb SOD. Then, the column was
% (W/V) After washing with ammonium sulfate water,
It was eluted at a flow rate of 0.00 m12/hr and the fractions with SOD activity were collected. This elution pattern is shown in FIG. With this operation, the specific activity of the eluate was 405 (U/A280)
The concentration was 18.4 times higher than that of the supernatant. Moreover, the activity yield was 96%.

Example 2 Amberlite XAD-8 in which supernatant liquid 12& containing the same SOD as used in Example 1 was equilibrated in advance with 30% (W/V) ammonium sulfate water (corrected to 87.5 l with sodium hydroxide). Column (diameter 6cm, length 36cm)
cm, filling volume 1 (2) at a flow rate of 5003112/hour to adsorb SOD. Then, the column was
After washing with 0% (W/V) ammonium sulfate water,
% (W/V) ammonium sulfate water at a flow rate of 500 yQ/hour, and a fraction with SOD activity was collected. This elution pattern is shown in FIG. The specific activity of this eluate was +270 (U/A280), which was 5% compared to that of the supernatant.
It had increased 7.7 times. The activity yield in this operation is 8
It was 7%.

Example 3 Amberlite
SOD was adsorbed by passing through an AD-8 column (diameter 10 cm, length 40 cm shoulder, packing amount 3 g) at a flow rate of 1.512/hour. Next, the column was washed with the above 30% (w/v) ammonium sulfate water, and then eluted with 20% (w/v) ammonium sulfate water at a flow rate of 1,5 Q/wait time and SO
Fraxifun with D activity was collected. The activity yield in this operation was 85%, and the specific activity of the eluate was 1400 (U/
A280), which was 63.6 times higher than the supernatant.

Furthermore, this eluate was filtered through an ultrafiltration device (manufactured by Amicon, USA;
By repeating the operation of concentration and adding water 5 times using the filtration membrane YM-5 and Manryo Molecule fi5000), its specific activity was 27.
50 (U/A280), which was 125 times higher than the supernatant.

Example 4 Diamond ion HP instead of Amberlight XAD-8
The same operation as in Example 1 was performed except that -2MG was used. With this operation, the specific activity of the eluate was 390 (U/A 2
80), a 17.7-fold increase from the supernatant, and the activity yield was 92%.

Example 5 The same procedure as in Example 1 was carried out except that the eluent was 40% (v/v) ethanol water containing 5% (w/v) ammonium sulfate instead of "C" water. The specific activity of the eluate was 365 (U/A280), 16.6 times higher than the supernatant, and the activity yield was 90%.

[Effects of the Invention] According to the present invention, a purification method suitable for industrial large-scale purification of SOD can be provided.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the elution pattern by water in Example 1 of the present invention, and FIG. 2 is a drawing showing the elution pattern by water in Example 2.
V) It is a drawing showing the elution pattern by ammonium sulfate water.

Claims (1)

[Claims] (1) A solution containing superoxide dismutase is brought into contact with a polyacrylate-based nonionic synthetic adsorbent to adsorb superoxide dismutase, and then the adsorbed superoxide dismutase is fractionally eluted. Characteristic method for purifying superoxide dismutase.