JPS61215330A - Purification of serrapeptase - Google Patents

Abstract

PURPOSE:To purify a serrapeptase useful as an anti-inflammatory agent, etc., in high purity, by contacting a serrapeptase-containing solution with an acrylate- type nonionic adsorption resin, and eluting the adsorbed serrapeptase with an elutant. CONSTITUTION:A solution containing serrapeptase is made to contact with an acrylate-type nonionic adsorption resin (e.g. Diaion HP-1MG), and the adsorbed serrapeptase is eluted with an elutant, preferably an elutant containing a water-soluble salt as a solute. The adsorbent resin has preferably a surface area of about 100-500m<2>/g and a pore volume of about 0.3-1.2ml/g. The resin has strong adsorptivity to serrapeptase and the adsorbability of serrapeptase to said resin can be improved remarkably in the presence of a water-soluble salt. Accordingly, a serrapeptase having high specific activity can be produced by this process.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides serrapeptase (
5errapeptase, International Proprietary Name)

Conventional technology Serrapeptase useful as an anti-inflammatory agent includes, for example, 10-tease produced by Serratia spp.
1-10981, U.S. Patent No. 8,691,014
(Refer to the publication) are listed.

Conventionally, purification methods for serrapeptase include salting-out method by addition of salts, fractional precipitation method by addition of water-soluble organic solvents, and chromatography method using various ion-exchange adsorbents. There is.

However, these methods require a large amount of raw materials such as salts and organic solvents, require a great deal of labor and time to collect fine precipitates, and do not use Sephadex, which has ion exchange groups. Although the purification method has advantages in terms of chromatographic separation, etc., it has drawbacks such as insufficient flow rate when performing column operation and problems as an industrial method.

In addition, in recent years, a purification method using a non-polar adsorbent based on a highly porous polymer, which is a copolymer of styrene and divinylbenzene, has been proposed for certain enzymes. However, this adsorbent has the disadvantage that the yield during adsorption and elution is poor and that serrapeptase is hardly adsorbed to these adsorbents.

As described above, conventional methods for purifying serrapeptase have various drawbacks, making it difficult to obtain serrapeptase with high specific activity.

In view of the above circumstances, the present inventors conducted various studies in order to establish a more efficient purification method for serrapeptase, and found that acrylic ester-based nonionic adsorption resin has strong adsorption power for serrapeptase. having, furthermore,
More surprisingly, when water-soluble salts are present during adsorption, the adsorption power to the adsorption resin does not increase significantly.
When serrapeptase is eluted from the adsorption resin on which serrapeptase is adsorbed, there is no possibility that the elution yield will be dramatically improved by coexisting water-soluble salts in the eluent, which are estimated to weaken the elution power. The present invention was completed as a result of further research based on the new findings of Kozu et al.

The present invention is directed to a serapephtase method, which is characterized in that a solution containing serapeftase is brought into contact with an acrylic ester-based nonionic adsorption resin to adsorb serrapeptase, and then the adsorbed serrapeptase is eluted with an eluent. “This is a method for purifying tase.

Examples of solutions containing serapeftase include Japanese Patent Publication No. 41-1.0981 and U.S. Patent No. 3.691.
, No. 014, etc., by culturing in a medium a microorganism belonging to the genus Serratia and having the ability to produce serrapeptase, or the culture solution is subjected to bacterial cell separation, salting out, solvent fractionation, or membrane separation. Methods such as (-) include an aqueous solution containing a partially purified crude enzyme.

Examples of microorganisms that belong to the genus Serratia and have the ability to produce serrapeptase include Serratia sp.
Five strains are listed, and the Serratia sp.
re Co11eition) (United States) in 196 A.D.
May 7, 1982, deposited with accession number ATCC 21074 and included in the collection's Catalog of Strains T, 15th edition, 1982 (The America
n Type Culture Co11ection
Catal-ogue of 5 trains (
Pifteenth Edition 1982)-2 has been published.

The concentration of serrapeptase in a solution containing serrapeptase (2) can be applied without any problems if it is in a normal aqueous solution state, preferably 10% or less, 0.0% or less.
If it is 1% or more, it is easy to apply. The pH of the solution is preferably about 4 to 9, which is the stable pH range of serrapeptase.

The acrylic ester nonionic adsorption resin used in the present invention is a porous nonionic adsorption resin having a giant network structure, which is a polymer of an alkyl ester of alkyl-substituted acrylic acid such as acrylic acid or methacrylic acid. In addition, a part of the alkyl ester moiety may contain sulfoxide, etc., and in addition to the carbon ester bond, a portion may have an acrylamide bond, etc. As long as the composition of the acrylic acid ester is about 50% or more, it may be a copolymer with, for example, acrylonitrile.

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

A specific example of an adsorption resin made only of acrylic ester is Diaion HP-4MG.

14P-2MG, HP-8MG (manufactured by Mitsubishi Chemical Industries, Ltd.: product name), Amberlite XAD-7, XAD-8
(manufactured by Rohm & Heath, USA: trade name), and the like.

As shown in Table 1, the adsorbent resin used in the present invention has a surface area of about 100-500%27fl*pore volume of about 0.3-1.2ml/9. I prefer what I have.

Table - Physical properties of adsorption resin Surface area Pore volume/area (Schiff) Cm17g) Diaion HP-IMG 800 0.
9 Diamond Ion HP-2MG 500 1
．． 2 Diamond Ion HP-aMG 500
0.6Amberlite XAD-74500゜3Amberlite (manufactured by Rohm and Haas, USA).

Adsorption in the method of the present invention is carried out by conventional operations such as column method and patch method.

In the adsorption process, the serrapeptase-containing solution may be brought into contact with the adsorbent as it is, or may be brought into contact with the adsorbent in the presence of water-soluble salts. ability improves.

Examples of the water-soluble salts 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,
Among them, cations include ammonium ions, alkali metal ions such as sodium, potassium, and lithium, and magnesium.

Water-soluble salts made of alkaline earth metal ions such as calcium and metal ions such as zinc and cobalt are more preferred.

Also, the anion is sulfate ion.

Water-soluble salts consisting of inorganic acid ions such as phosphate ions, chloride ions, and nitrate ions, and organic acid ions such as acetate ions, tartrate ions, and citrate ions are more preferably used.

Specific examples of the water-soluble salt include sulfate (eg, sodium salt, potassium salt, magnesium salt, ammonium salt, etc.), chloride (eg, sodium chloride, potassium chloride, ammonium chloride, etc.).

Calcium chloride, cobalt chloride, etc.), acetates (e.g., sodium salts, ammonium salts, zinc salts, etc.), phosphates (e.g., sodium salts, potassium salts, etc.), nitrates (e.g., sodium salts, etc.), tartrates ( Examples include sodium salts, etc.), citrates (eg, sodium salts, etc.).

The amount of water-soluble salts that may be used in the solution containing serrapeptase is approximately 5 to 20% (W) based on the solution.
/V).

In the method of the present invention (= in the adsorption operation, the temperature is about 0
-30°C, preferably at about 0-10°C.

The adsorption operation involves applying the solution containing serrapeptase to a column filled with adsorption resin, for example, at SV=about 0.0.
A column method in which the liquid is passed through the solution at a flow rate of 5 to 20 times per hour (a flow rate of about 0.05 to 20 times the amount of the adsorption resin packed) to adsorb the serrapeptase. Any method such as a patch method of adsorption may be used.

Elution in the method of the present invention is carried out by treating the resin on which serrapeptase has been adsorbed, for example, with a water-containing organic solvent. Examples of the organic solvent include lower alcohols (e.g., methanol, ethanol, 10-panol,
Impropatol, phthanol, imbutanol, etc.)
Examples include ketones (eg, acetone, methyl ethyl ketone, etc.), esters (eg, methyl acetate, ethyl acetate, etc.), ethers (eg, dioxane, etc.).

During the elution (second), the water-containing organic solvent that is the eluent may further contain water-soluble salts. When the water-soluble salts are contained, the elution efficiency is improved. The water-soluble salts include the above-mentioned The same water-soluble salts that may be allowed to coexist during adsorption are mentioned.

The amount of the water-soluble salts used is about 0.5 to 10% (W/V) based on the amount of the water-containing organic solvent (2).The elution operation in the method of the present invention is carried out at a temperature of about 0 to 30°C, more preferably Approximately O
Performed at ~10°C. The elution operation is the adsorption operation C.
Any method such as a column method or a patch method may be used.

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 serrapeptase purification method of the present invention include:
Very good adsorption capacity 1: large, good elution yield,
It can be directly applied to crude enzyme solutions with many impurities, has high fractionation ability, and can greatly increase enzyme specific activity.
When performing column chromatography on an industrial scale, a high flow rate can be ensured, and the simple operations of adsorption and elution can greatly reduce labor. Therefore, the method of the present invention can be used on a page-by-page basis as a purification method for industrially producing serrapeptase.

EXAMPLES The following Examples and Examples (2) will further explain the present invention in more detail.The following operations were all carried out under conditions of about 10°C.

Experimental Example 1 500 mJ of a serrapeptase-containing aqueous solution (serrapeptase activity 5080 PU/m1. However, regarding PU, see Japanese Patent Publication No. 10198/1983) was applied to a column packed with 100 mJ of adsorption resin at a flow rate of 1 G 0ffll? /H to adsorb serrapeptase in the solution, the adsorption capacity of each resin was as shown in the fJr12 table.

From the above, the adsorbent resin whose matrix is a copolymer of styrene and divinylbenzene hardly adsorbs serrapeptase, but when an acrylic ester-based adsorption resin is used, serrapeptase can be adsorbed efficiently. Recognize.

Experimental Example 2 The same serrapeptase-containing aqueous solution C as in Experimental Example 1, to which ammonium disulfate was added, was adsorbed onto resin Amberly)
The relationship between the concentration of ammonium sulfate added and the adsorption power to Amberly XAD-8 was measured when the solution was passed through AD-8 and the 5% breakthrough point was determined. The results are shown in Table 3 (two copies).

Table 3 shows that adding 5 to 20% ammonium sulfate (as shown in Table 2) significantly improves the adsorption capacity.

Experimental Example a Using the same method as Experimental Example 2, using Diaion HP-IMG as the adsorption resin, coexistence of water-soluble salts (10% salts)
The adsorption ability of serrapeptase was investigated by adding w/v (double addition). The results are shown in Table 4 (d) (The adsorption capacity when no salts are added is expressed as 1.) As shown in Table 4, by adding water-soluble salts, the adsorption capacity when no salts are added is It can be seen that the adsorption capacity of serrapeptase is significantly improved compared to the above.

Experimental Example 4 500ml of serrapeptase-containing aqueous solution similar to Experimental Example 1
Ammonium sulfate 51 was added to the solution, dissolved and touched, and the solution was passed through a column packed with XAD-81001M to adsorb serrapeptase. Next, 150mA of 10% ammonium sulfate water! After washing with water, the elution yield of serrapeptase was investigated when it was eluted with an eluent prepared by adding ammonium sulfate to a concentration of 0 to 10% in 40% methanol water. The results are shown in Table 5.

From Table 5 above, ammonium sulfate concentration 0.5-1O%
It can be seen that the elution yield is significantly improved when eluting with 40% methanol water.

Example 1 Serrapeptase producing bacterium Serratia sp. Ets (5e
rratia sp E, ATCC21074) in Agricultural and Biological Chemistry (Agricultural and B
iological Che-mistry)
34, 110-818 (1970), the culture solution obtained by culturing in the same manner as described in 2 was centrifuged to obtain supernatant 51.
(serrapeptase activity 4800 PU/ml).

Ammonium sulfate 7501 was added to this supernatant to dissolve it. Add this liquid to Diaion HP-IMG 500.
Serrapeptase was adsorbed by passing the solution through a column filled with Il+/ at a flow rate of 10,001 rrl/H. After adsorption 1
Wash with 500ml of 5% ammonium sulfate water, then
% ammonium sulfate/50% methanol water 500M
Column C: Serrapeptase was eluted at a flow rate of 1/H.

Collect active fractions and purify serrapeptase solution 750
m1 (81500 PU/m) was obtained. The enzyme specific activity was higher than that in the centrifuged supernatant.

Example 2 Sodium sulfate '15011 was added to and dissolved in the centrifugal supernatant 151 obtained in the same manner as in Example 1. This liquid was poured into a column (241/
After passing through the solution at a flow rate of H, and then passing through 5% sodium sulfate water to remove impurities, 0.5% sodium sulfate.4
5% methanol water was passed through the solution at a flow rate of 217H to elute the adsorbed serrapeptase and collect the active fraction. Purified serrapeptase solution 5.91 (9760 PU/ml) f
Obtained.

This liquid was circulated through a polysulfone-based ultrafiltration membrane PM-10 (manufactured by Romicon, Inc., USA) and concentrated to 50 ON (10°C, 1.5 kg/cm, circulation flow rate: 90 J/H).
, membrane area 0.1fi"). 500 g of water was added to the concentrated solution and ultra-superconcentration was performed again. The same operation was repeated 5 times to obtain 500 ml of purified concentrated solution. 1 sg of lactose was added and dissolved in this solution. After that, it was freeze-dried to obtain purified serrapeptase powder 19.1.9 (2400 PU/η).The enzyme specific activity of this purified powder was 15 times higher than that of the centrifuged supernatant.

Example 3 Centrifugal supernatant 81+ obtained in the same manner as in Example 1 = 600 g of ammonium sulfate was dissolved to form Diaion HP-2MG.
80omy! Serrapeptase was adsorbed by passing the solution through the column packed with the solution at a flow rate of 300 mA/H.

Next, it was washed with 15% ammonium sulfate water and eluted with 10% sodium chloride/45% ethanol water to obtain 6701 rLg of purified serrapeptase solution. (16400PU/me)
.

Example 4 Serrapeptase adsorbed on Diaion HP-IMG in Example was eluted using 05% ammonium acetate/40% acetone water instead of 4% ammonium sulfate/50% methanol water as an eluent to obtain a purified enzyme solution. 1.6
1 (12750 PU/ml). The enzyme specific activity was improved by a factor of 100% compared to the centrifuged supernatant.

Effects of the Invention In the purification method of the present invention, an acrylic acid ester nonionic adsorption resin is used as an adsorbent (Secondly, the adsorption capacity of serrapeptase can be dramatically improved. Furthermore, C: Here [ Second, by coexisting water-soluble salts in the serrapeptase-containing solution brought into contact with the resin or in the eluate used during the elution operation, the adsorption or elution of serrapeptase can be dramatically improved.

Claims (3)

[Claims] (1) A method for purifying serrapeptase, which comprises bringing a solution containing serrapeptase into contact with an acrylate ester nonionic adsorption resin to adsorb serrapeptase, and then eluting the adsorbed serrapeptase with an eluent. (2) The purification method according to claim 1, wherein the solution containing serrapeptase is brought into contact with the resin in the presence of water-soluble salts. (3) The purification method according to claim 1, which uses an eluent in which water-soluble salts are dissolved.