JPH01180451A - Adsorptive body for affinity separation - Google Patents

Abstract

PURPOSE:To obtain an adsorptive elution characteristic capable of sufficiently dealing with purification of a large amt. by using at least one among salicylic acid, phthalic acid and the deriv. thereof in which at least one carboxylic acid is left without being modified as a ligand. CONSTITUTION:The ligand is directly immobilized on a carrier or a material which can form said ligand is synthesized on the carrier. At least one more than functional group which can be easily brought into bonding reaction with the carrier such as amino group or hydroxyl group is required to be incorporated into the molecules of the ligand in the case of immobilizing the ligand directly to the carrier. The functional group of the ligand which can bond the carrier to be used and the ligand is usable; for example, an amino group, carboxyl group, formyl group, etc., are exemplified but are not limited thereto. There are various kinds of such ligands and are not particularly limited. 5-salicylic acid, phthalic monoamide, etc., are synthesized in the case of synthesizing the ligand on the carrier and general synthesizing methods such as dehydration condensation and diazo coupling may be adapted as the synthesizing method.

Description

DETAILED DESCRIPTION OF THE INVENTION (1) Industrial Application Field The present invention relates to an adsorbent for affinity separation useful for separation, purification, etc. of proteolytic enzymes (f-lothiase).

(2) Conventional techniques and problems In recent years, methods using affinity separation have been widely used as one of the protein purification methods, and various adsorbents for affinity chromatography have been developed for this purpose. It has been put into practical use.

However, most of these affinity separation methods are used to purify products that have been purified to some extent, that is, in the final step of the overall purification process;
The entire purification process consists of many steps, making it complex and resulting in a low recovery rate of the target product. Until now, there has been no affinity separation method that is a relatively simple process and is suitable for industrial large-scale purification.

In addition, in the field of enzyme purification, particularly in the field of alkaline glothiase purification, various ligands such as amino acids, amino acid derivatives, proteinaceous inhibitors, and pegylated inhibitors have been developed, but it is difficult to obtain practical elution characteristics. The elution conditions for this are often harsh for enzymes; for example, most elution conditions use organic solvents such as alcohol and acetone, high or low solvent systems, and protein denaturant systems such as urea and guanidine hydrochloride. In these methods, the protease becomes unstable in the eluent, which limits the elution time and temperature, making it unsuitable for large-scale industrial purification. In addition, since it is necessary to transfer the protease to stable conditions, an organic solvent recovery system, P
This method requires a H neutralization tank, a denaturing agent exclusion system, etc., and has the disadvantage of complicating the process.

On the other hand, phenylarsonic acid-based and phenylboronic acid-based ligands have been known to be able to elute alkaline protease under mild conditions, but phenylarsonic acid-based ligands contain arsenic and are toxic. Therefore, it is dangerous to handle large quantities when preparing an adsorbent, and it is also difficult to completely prevent desorption of the ligand during protease purification. Therefore, this method of handling the ligand requires a detoxification system and has drawbacks such as complicating the process. In addition, phenylboric acid-based ligands have low selectivity for adsorption of proteases, so it is necessary to sufficiently exclude substances that compete with protease adsorption, and during elution, proteases adsorbed with this ligand are difficult to elute. Therefore, it has drawbacks such as requiring a large amount of eluent and requiring a long elution time.

An object of the present invention is to overcome the drawbacks of the conventional adsorbent for affinity separation, and to make it possible to elute proteases, such as alkaline glothiase, with a neutral to weakly basic aqueous solution.
It is an object of the present invention to provide a safe adsorbent for affinity separation having adsorption/elution characteristics sufficient for large-scale purification, and a method for purifying protease using the adsorbent.

(3) Means for Solving the Problems According to the present invention, an adsorbent for affinity separation in which the ligand is a derivative of salicylic acid, phthalic acid, or cholera in which at least one carboxyl group remains unmodified. is provided.

The adsorbent for affinity separation of the present invention is one in which the above-mentioned ligand is directly immobilized on a carrier, or one in which a substance that can serve as the ligand is synthesized on the carrier. When immobilizing a ligand directly on a carrier, amine groups,
It is necessary to contain at least one functional group (hereinafter abbreviated as "functional group" unless otherwise specified) that is likely to cause a bonding reaction with a carrier, such as a hydroxyl group.

The functional group of the ligand used in the present invention may be any functional group as long as it can bind the carrier used and the ligand, such as an amino group, a carboxyl group, and a formyl group.

Examples include, but are not limited to, hydroxyl groups. There are various types of these ligands, and they are not particularly limited. For example, when the functional group is an amino group, 5-(4-aminophenylazo)salicylic acid,
4-Amine salicylic acid.

Examples include phthalic acid, 4-aminophthalic acid, 2,3-dihydrobenzoic acid, 2,4-dihydrobenzoic acid, orceric acid, and quinolinic acid.

When the ligand of the present invention is synthesized on a carrier, 5-(4-
Aminophenylazo)salicylic acid, phthalic acid monoamide, etc. can be synthesized, and general synthesis methods such as dehydration condensation and diazo coupling can be applied.

Examples of carriers used in the present invention include agarose, cellulose, dextran, and polyacrylamide.

All carriers commonly used in affinity separation, such as polyvinyl alcohol, chitosan, and porous glass, can be used without exception. Specific commercial products of agarose-based carriers include, for example, Sephadex (Pharmaci
m company registered trademark, hereinafter abbreviated as ■).

Bioglu A (BIO-RAD ■), etc. Cellulose-based products include Cellulofine (Chisso Corporation ■), and dextran-based products include Sephadex (Pharmacia ■) and Polymer. Examples of acrylamide-based products include Endefix P (Wako Tsumugi Pharmaceutical @)■), and examples of polyvinyl alcohol-based products include Toyo Pearl (Toyo Soda Co., Ltd.).
■), SeA? Beads (Mitsubishi Kasei Co., Ltd.), chitosan-based products such as
Co., Ltd. ■) and the like are commercially available, but the carrier for chromatography is not limited to these. In addition, there are various types of these carriers, and they are not particularly limited. For example, when the functional group is an amine group, the carrier has CNB.
r-activated Sepharose 4B, CH-Sepharose 4B.

Activated CH-Sepharose 4B, epoxy activated Sepharose 4B, etc. can also be used.

When immobilizing a ligand on a carrier, it depends on the type of carrier, that is, the active group and functional group on the carrier side, but in any case, general methods for preparing immobilized carriers can be applied.

On the other hand, enzymes that can be purified using the adsorbent for affinity separation of the present invention include Alkaline Groteapi,
subtilisin BPN', subtilisin Carlsberg, Kazusase (Showa Denko@)■).

Alcalase (NoVO), Savinase (NoVO)
Examples include, but are not limited to, Esperase (NoVO Company ■), Chymotrigsin, and the like.

(4) Function Since the adsorbent for affinity separation obtained by the present invention can use a general carrier, it can be put to practical use in a moving bed system such as a packed column system or a fluidized bed system.

Moreover, the ligand of the present invention has the advantage and feature that it can be adsorbed and eluted with an aqueous solution.

Therefore, the process is much simpler than conventional separation and purification processes using organic solvents for elution, or processes using arsonic acid-based ligands that require detoxification equipment. This is an industrially advantageous method because it is easier to elute and obtain a higher recovery rate than those using acid-based ligands.

(5) Examples Examples are given below. Enzyme quantification in the examples was performed using TN
This was done using the BS method.

Example 1 Immobilization of 5-(4-aminophenylazo)salicylic acid activated CH-cephalo-y, 4B (Pharmac
Add ice-cooled 10 mM HCL aqueous solution 15- to 3S, stir for about 15 minutes to swell the resin, transfer the resin onto a glass filter, and add ice-cooled 10 mM HCL aqueous solution 15- to 3S.
Washed with 600 ml of HCt aqueous solution. Then ice-cooled 1
Wash quickly with 20ml of 00mM sodium bicarbonate aqueous solution, and immediately transfer the resin to 100mM sodium hydrogencarbonate aqueous solution 1(p)18.0) containing 10mM 5-(4-aminophenylazo)salicylic acid (p), and stir overnight at room temperature. did. After the reaction, the resin was transferred onto a glass filter again and washed with an aqueous solution of 100 mM hydrogen carbonate until the P solution lost its color, to obtain the desired adsorbent. This adsorbent contains IQ-'mQtS (resin)
5-(4-aminophenylazo)salicylic acid was immobilized.

Example 2 Synthesis of 5-(4-aminophenylazo)salicylic acid on a carrier and beads CM-13 (100 g) were mixed with 7,500 m of dioxa
1 and the resin was transferred into 300 ml of dioxane. To this were added dicyclohexylcaldodiimide 5I and 10 g of p-phenylenediamine, and the mixture was stirred at room temperature all day and night. The resin was then treated with dioxane soomz and water 5
The resin was washed three times with QQmJ, and the resin was washed with ice-cooled 1M hydrochloric acid 5.
Sodium nitrite 29 was added little by little while keeping the liquid temperature at 4°C, and the mixture was reacted for about 3 hours. After the reaction, the resin was washed with 5 QQm of ice-cold water, and the resin was transferred to 300 ml of water, and while maintaining the liquid temperature at 10 DEG C. or less and the pH at 8 to 9, 5 g of salicylic acid was added, and the mixture was allowed to react for about 3 hours.

After reaction, add 10mM sodium hydroxide solution and 10mM hydrochloric acid.

The target adsorbent was obtained by sequentially washing with water for 500 d each. This adsorbent has 10-4 to 10-5 mot/m
1 (resin) of 5-(4-aminophenylazo)salicylic acid was immobilized.

Example 3 Synthesis of phthalic acid monoamide on a carrier Sepa beads 襥-13100N were washed three times with 500 ml of dioxane, and a 10% dioxane solution of phthalic anhydride 300
The resin was transferred into rnl and stirred at room temperature overnight. next,
Resin f, 3 times, 500 ml of dioxane, then 500 ml of water
00 ml to obtain the target adsorbent. Phthalic acid monoamide of 10-' to 10-' mol/ml (resin) was immobilized on this adsorbent.

Example 4 Purification by Affinity Chromatography 1) Adsorption Separation Affinity chromatography was performed on each of the adsorbents K of Examples 1 to 3.

A column of 1.2 cWIφ×40 liters was filled with the resin, the column was equilibrated in advance with 20-phosphate buffer (pH 9), and affinity chromatography was started. 1 Chikazusase prepared with pH9Fc (Showa Denko K.K.)
) Enzyme log aqueous solution 1000111/'i was added to the column as a crude sample to adsorb the enzyme onto the resin.

Next, wash 500ml Tekara A with 20mM phosphate buffer (pH 9) and add 20mM phosphate buffer (pH 9) containing 20mM sodium sulfate.
M phosphate buffer (pH 6) 50 omJ (Example 3 only 10
The enzyme was eluted at 00 m/). The results are shown in Table IK.0 2) Separation conditions The adsorbent of Example 2 was subjected to affinity chromatography in the same manner as in Example 4-1) by changing the volume, salt concentration, and type of salt of the crude sample and eluate. I did it. Note that the column equilibration and washing instructions were the same as those for the crude sample. The results are shown in Tables 2 and 3. 3) The specific activity of the enzyme purified by Kempo is 300 n.
k/A was 28o or more, and an enzyme of extremely high purity was obtained. The present enzyme solution is colorless and transparent, and the freeze-dried powder is white, and when used in enzyme granules, a product that is clean enough to withstand use without the use of cosmetics can be produced.

Example 6 The enzyme was separated using the column of Example 1 using Alcalase (NoVO). As a result, it was adsorbed at pH 9, eluted with an eluent of pH 6 and sodium sulfate 200 mM, and the enzyme adsorption amount was 40 In9/ff1A' ( Resin) was collected at a recovery rate of 99% and dried to obtain a white enzyme bulk powder.

Example 7 Enzyme was separated using the column of Example 1 using Sptilisin BPN' (SIGMA). As a result, it was adsorbed at -9, eluted with an eluent of pH 6 and sodium sulfate 200mM, and the enzyme adsorption amount was 42In9/ The resin was collected at a recovery rate of 98 cm (resin) and dried to obtain a white enzyme bulk powder.

Example 8 As a result of separating the enzyme using α-chymotrypsin (SIGMA) in the same manner using the column of Example 1, p)1
8, pH 6, sodium sulfate Zo.

- Elute with an eluent of -, enzyme adsorption amount 241n97ml (
Resin) 9 was recovered with a recovery rate of 88%.

Table 1 Enzyme separation results using each affinity separation agent Example 1 28 101 7
8 Example 3 33 98
72 Example 4 51 90
69 Note 1) 0 recovery rate = eluted enzyme i/adsorbed fermentation amount Note 2
), Purification rate = specific activity of eluted enzyme / specific activity of crude sample (specific activity of crude sample = 4.4 nkat/A280) Table 2 Effect of adsorption conditions - Enzyme adsorption amount of crude sample (m9 protein/AT resin) Table 3
Effect of elution conditions Added salt to eluate Added salt concentration (mM) Elution rate (%) 9 Sodium chloride 100
986 #tt
739 Sodium sulfate
50 666 1/
100 959 #
500 91 Note 1), added salt is 20
Refers to salts added in addition to mM phosphate buffer.

Note 2), Elution rate = Amount of eluted enzyme / Amount of adsorbed enzyme (7) Effects of the invention The adsorbent of the present invention has a concentration of 2 enzyme proteins per 1 ml of the adsorbent.
It can adsorb 0 to 30 cm or more, and has an adsorption capacity of 2 to several tens of times that of conventional affinity adsorbents. Conventionally, the affinity separation method was not a sufficiently efficient method for large-scale purification of enzymes and had not been put to practical use. However, if the adsorbent of the present invention is used, large-scale purification of enzymes for detergents, etc. is no longer necessary. Affinity separation methods can be fully utilized even for substances that

The adsorbent of the present invention adsorbs enzymes at a pH of at least 6 to 10, preferably at a pH of 6 to 9, and at least at a pH of 6 to 9.
Elution is possible by adding 1100mM or more of salt to the eluate of ~9.

As described above, the affinity separation method using the adsorbent of the present invention has sufficient adsorption capacity for large-scale purification, and it is possible to separate alkaline glothiase from crude enzyme solution etc. using only an aqueous salt solution without using an organic solvent or the like. A major feature is that it can selectively separate and purify.

The enzyme obtained by water droplet has a considerably high specific activity and contains few impurities (for example, color components), so it is extremely useful as an enzyme for various uses, especially as an enzyme for liquid detergents.

Patent applicant: Showa Denko Co., Ltd.

Claims (1)

[Claims] An adsorbent for affinity separation of protease, which uses salicylic acid, phthalic acid, or a derivative thereof in which at least one carboxyl group remains unmodified as a ligand.