JPH0117410B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to an adsorbent and a method for purifying an enzyme using the same, and more specifically, an adsorbent having a hydrophobic group and a method for purifying an enzyme by hydrophobic chromatography using the same. It is in. (Prior art) Hydrophobic chromatography as referred to in the present invention utilizes hydrophobic bonds formed between hydrophobic groups contained in a chromatography adsorbent and hydrophobic groups on the surface of protein molecules as adsorption force. It is based on the principles of chromatography and has recently been studied at the research level, but there are still relatively few practical examples. Chromatography adsorbents with hydrophobic groups and enzyme purification methods using them are fundamentally different from conventional purification methods such as ion-exchange chromatography, so they may be difficult to purify when conventional purification methods are not effective. It is particularly effective for Furthermore, since oxygen is adsorbed even in the presence of a high salt concentration, it has the advantage that desalination treatment as a preliminary treatment is not necessary. (Problems to be Solved by the Invention) Conventionally reported methods, for example, methods for purifying enzymes by hydrophobic chromatography using a chromatographic carrier containing a linear aliphatic hydrocarbon, have the following problems. be. This hydrophobic chromatography has too strong adsorption and requires a highly concentrated organic solvent solution to elute the adsorbed enzyme, which may cause deactivation of the enzyme or still result in poor elution. There are drawbacks such as insufficient adsorption and residual adsorption within the column.
There are a few other cases in which affinity chromatography has been attempted using chromatography carriers immobilized with steroid hormones, but the hormones that are the raw materials for chromatography carriers are extremely expensive; The problem is that it is unsuitable for large-scale use. In view of the above-mentioned conventional drawbacks, the present inventors have conducted extensive research and have invented a novel chromatographic adsorbent and a method for purifying enzymes using the same. That is, the present invention

[Formula] An adsorbent for protein purification consisting of dehydrocholic acid cepharose with a group immobilized on a support.

The present invention provides a method for purifying an enzyme by hydrophobic chromatography using an adsorbent consisting of dehydrocholic acid cepharose having a group fixed to a support. In the present invention

[Formula] Examples of the support for immobilizing the group include the following. Examples include agarose gel, dexrin gel, cellulose, porous glass beads, porous silica gel, porous synthetic resin, etc. In the present invention, water-insoluble polysaccharides such as agarose gel are particularly preferred. In the present invention

[Formula] As a method for fixing the group to the above-mentioned support, a commonly used carboxyimide method can be used. against the support

[Formula] The proportion of the group is preferably 1 to 500 μg equivalent per ml. (Function) Enzymes are purified using the hydrophobic chromatography adsorbent thus obtained, but the enzymes that can be purified by the present invention are not particularly limited, and the present invention is applicable to all enzymes. Among these, it is particularly useful for purifying enzymes such as steroid metabolic enzymes such as 3α-hydroxysteroid dehydrogenase and β-hydroxysteroid dehydrogenase. Examples of methods for using the hydrophobic chromatography adsorbent according to the present invention for enzyme purification include the following methods. (a) An aqueous enzyme solution is passed through an adsorbent column, and the adsorbed enzyme is eluted with an organic solvent solution such as dimethylformamide, ethyl alcohol, or glycerin. The organic solvent concentration at this time varies depending on the type of organic solvent and oxygen, but
A range of 6% (V/V) is desirable. (b) The enzyme adsorbed on the column is eluted with an aqueous solution of a surfactant such as Cholate Triton X-100 (trade name). The concentration of the surfactant at this time varies depending on the type of surfactant and enzyme, but is preferably in the range of 0.01 to 5% (W/V). (c) Enzyme is adsorbed in the presence of a coenzyme such as NAD ⁺ or NADP ⁺ and eluted by reducing the concentration of the coenzyme. The concentration of coenzyme at this time is
Although it varies depending on the type of coenzyme and enzyme, 1 to
A range of 500 μM is desirable. (d) adsorbing the enzyme in the presence of a coenzyme and an organic solvent and eluting the enzyme by reducing the concentration of the coenzyme in the presence of the organic solvent; The preferred concentration ranges of the coenzyme and organic solvent at this time are the same as above. (Example) Examples of the present invention will be shown and explained in detail below. Example 1 15 g of AH-Sepharose 4B (Pharmacia: trade name) was washed with a 0.5M NaCl solution, and water was removed by filtration. After adding 60 ml of water and 120 ml of dioxane containing 1 g of dehydrocholic acid, the pH was adjusted.
Adjust to 4.5-6.0. Add 1.5 g of carbodiimide (EDC) (Peptide Institute Co., Ltd.) to this with gentle stirring, and continue stirring for 24 hours while maintaining the pH between 4.5 and 6.0. After reaction, 50% (V/
V) was washed with dioxane and further washed with water.
Approximately 40 ml of adsorbent (cepharose dehydrocholic acid) was obtained through the above operations. Its basic structural formula is shown in Figure 1.
- Shown in A. Example 2 A column (10 ml) of the adsorbent of Example 1 above was installed in a 20 m
After equilibration with M phosphate buffer (PH7.5) (hereinafter simply referred to as buffer), 40 ml of the above buffer containing 1 mM NADH was poured. A solution of hydroxysteroid-dehydrogenase crude enzyme (Sigma, grade) of Cydomonastematosterone (3α-hydroxysteroid dehydrogenase)
20.9U/ml, β-hydroxysteroid dehydrogenase 26.7U/ml, protein 21.1mg/ml and 1
7 ml of the above buffer containing mM NADH is applied to the column, followed by washing with 100 ml of the above buffer containing 1 mM NADH. Next, 3α-hydroxysteroid dehydrogenase (3α-enzyme) was eluted by flowing 200 ml of the above buffer solution (1 in FIG. 2). Then 5% dimethylformamide and 1mM
Wash the column with 100 ml of the above buffer containing NAD ⁺ ,
Subsequently, β-hydroxysteroid dehydrogenase (β-enzyme) was eluted with 200 ml of the above buffer containing 5% dimethylformamide (2 in Figure 2). As a result, the recovery rate of the 3α-enzyme was approximately 67%, and the peak specific activity was 310 U/mg protein (310 times purified). Furthermore, the recovery rate of β-enzyme was approximately 61%, and the peak specific activity was 61 U/mg (47-fold purification). Incidentally, when similar chromatography was attempted using a sepharose acetate column as shown in FIG. 1-B, no oxygen was adsorbed onto the column at all. According to affinity chromatography performed by Battais et al. using esterone aminocaproate agarose as an adsorbent, the purification results of the same enzyme sample as above showed that the 3α-enzyme had a specific activity of 13.3 U/mg.
The protein was purified 6.5 times to 86.5 U/mg protein, and the recovery rate was 68%. In addition, the β-enzyme
The purification was 12.8 times from 0.26 U/mg protein to 3.33 U/mg protein, and the recovery rate was 69%. (Effects) As shown in the results of the examples, the method of the present invention is not much different in recovery rate than the conventional method, but is extremely superior in purification effect and final purity.

[Brief explanation of drawings]

In the figure, FIG. 1 shows the structural formula, where A is the adsorbent of the present invention (cephalose dehydrocholic acid) and B is the conventional adsorbent (cephalose acetate). FIG. 2 shows the hydrophobic chromatography fractions of the present invention, where 1 is the 3α-enzyme and 2 is the β-enzyme.

Claims (1)

[Scope of Claims] 1. An adsorbent for protein purification comprising cephalose dehydrocholic acid having a group of the formula fixed to a support. 2 A method for purifying an enzyme by hydrophobic chromatography using an adsorbent consisting of dehydrocholic acid cephalose with the [Formula] group immobilized on a support.