JP2631099B2 - Purification of isocitrate dehydrogenase - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to a method for purifying isocitrate dehydrogenase, and more particularly, to a method for purifying isocitrate dehydrogenase, the method comprising the steps of: It is intended to be purified by ion exchange chromatography using a resin.

Isocitrate dehydrogenase (hereinafter sometimes referred to as “ICDH”) is an enzyme that comes into reversible contact with the oxidative decarboxylation reaction of D-isocitrate. Depending on the required coenzyme, ICDH (NAD ) (EC1.1.1.41) and ICDH (NADP) (EC
1.1.1.42). The present invention is applicable to the purification of any type of ICDH, especially NADP.
Is suitable for the latter type of ICDH using as a coenzyme.

Isocitrate dehydrogenase plays an important role in the citrate cycle, which is the most ubiquitous pathway in the metabolism of intracellular substances in living organisms, and is used in pharmaceutical applications such as improving metabolism and in the technical field of foods such as nutritional foods. It plays an important role. In addition, this isocitrate dehydrogenase can be used for the determination of isocitrate and oxaloacetate, and for the determination of NAD, NADH, NADP, and NADPH. It has been used for the elimination of ammonia and is very important as a clinical test reagent in the field of analysis and measurement such as diagnosis of diseases and particularly measurement of biological trace substances.

(Prior art) As a method for purifying isocitrate dehydrogenase, a method combining a conventional method for enzyme purification such as ammonium sulfate fractionation, heat treatment, and alcohol precipitation (A. Kornberg: Methods in Enzymo)
theory, vol 1, 705-709, 1955); Purification method using CM-cellulose (W. Clelard et al: Methods in Enzymology, vol.
8, 30-33, 1966); a purification method by affinity chromatography using blue dextran (Wagaoka et al; J. Bioc.
hem, 81, 71-78, 1977).

On the other hand, the present invention is to purify ICDH with high efficiency and extremely high recovery rate in a very short time, particularly using a weakly acidic cation exchange resin. No, it is new.

(Problems to be Solved by the Invention) Among these known purification methods, the first method is complicated in operation and has a low recovery rate. Is not at all suitable. Next, CM
-When trying to purify isocitrate dehydrogenase by ion exchange chromatography on an industrial scale using cellulose, the flow rate when passing the enzyme stream or elution stream through the column must be kept to SV 0.1 or less. However, there is a drawback that a pressure loss occurs and the flow stops flowing. On the other hand, at SV 0 or less, there is a problem that it takes a very long time to chromatographically analyze a large amount of enzyme stream. A carboxymethyl group was introduced from dextran and agarose. CM−
Even for Sephadex, CM-Sepharose (Pharmacia), when it was scaled up to an industrial scale, the problem of pressure loss could not be avoided.

Thus, all of these known purification methods were completely unsuitable for industrial large-scale processing.

(Means for Solving the Problems) The present invention has been made to solve these disadvantages, and is a method for purifying a large amount of an enzyme in a very short time, and at the same time, the activity of the enzyme is reduced at all. This method was developed for the purpose of newly developing a method of purifying at an excellent recovery rate without causing any purification.

The present inventors have conducted intensive studies to solve this problem, and by using a cation exchange resin as a chromatographic carrier, it is possible to process a large amount of enzyme stream in a short time, and the separation ability in chromatography is superior to CM-Sepharose. And found that it was not inferior.

Further, a search was conducted among cation exchange resins, and it was found that a weakly acidic cation exchange resin was effective. In particular, it has been found that, in the case of a weakly acidic exchange resin in which the functional group is a carboxylic acid group, the time required for chromatography can be shortened, the separation ability can be improved, and the recovery rate can be increased.

The present invention has been completed as a result of further research based on these new findings, and the use of a weakly acidic cation exchange resin is an important point.

As the weakly acidic cation resin, various types can be appropriately used, and a wide variety of (meth) acrylic acid-based resins having a carboxylic acid group or other weakly acidic resins can be used. For example, commercially available weak acidic cation exchange resins having a carboxylic acid group can be freely used,
Amberlite IRC-50, Amberlite CG-50 (manufactured by Organo) and Diaion WK10, WK11, WK20 (manufactured by Mitsubishi Kasei) can be advantageously used. The present invention is not limited to the above-mentioned ion exchange resins, but any weakly acidic cation exchange resin having a carboxylic acid group can be used.

In order to carry out the present invention, a conventional method can be applied.For example, after contacting the ion-exchange resin with an enzyme-containing solution to adsorb the enzyme, the target enzyme is isolated by eluting with an appropriate eluent. I just need. Specifically, for example, the enzyme solution is charged into a column filled with an ion exchange resin and then eluted, or after the resin is adsorbed by placing the resin in the enzyme solution, the resin is separated and eluted. Then, ICDH may be isolated and purified.

Since this method is extremely efficient and highly resolving,
Crude enzyme solution of all origins other than ICDH crude enzyme solution extracted from animal and plant cells, microbial cells, fermentation broth by fermentation method can be treated freely, regardless of enzyme concentration and its origin. It is a method.

In addition, it is particularly noteworthy that yeast is an excellent source of ICDH. It was very difficult to separate ICDH from these enzymes. However, just astonishingly, when the yeast extract was subjected to ion exchange chromatography using Amberlite IRC-50, it was surprisingly found that glucose dehydrogenase, hexokinase, α-glucosidase in the yeast extract. The enzyme of aldose reductase was not adsorbed and passed through the column, but only isocitrate dehydrogenase (NADP) was specifically adsorbed to the column. Therefore, the present invention is a method which is particularly excellent as a method for purifying ICDH originating from a yeast extract. As the yeast, any yeast capable of producing ICDH can be used. For example, Saccharomyces, Candida, Endomyces, Hansenula, Kloe
Widely used are ckera, Pichia, Rhodotorula yeast and other ICDH yeasts.

Although the present invention has such a very high specificity and can selectively purify only the enzyme of interest (this excellent effect is also apparent from FIGS. 1 and 2), This is an extremely industrial method that can be carried out by a very simple operation of contacting, adsorbing, and eluting the enzyme solution with the resin. If necessary, this method may be combined with another purification method, or the method may be repeated as many times as necessary. According to the present invention, an enzyme having a high degree of purification can be obtained, so that it can be used freely as a medicine, a diagnostic agent, or a measurement and analysis reagent.

 Hereinafter, examples of the present invention will be described.

Example 1 60 ml of yeast extract was passed at a flow rate of SV10 through a 40 ml column of Amberlite IRC-50 (Organo) which had been equilibrated with 10 mM phosphate buffer (pH 6.5) in advance. Next, after washing with 60 ml of 10 mM phosphate buffer (pH 7.5), gradient elution was performed to increase the phosphate buffer concentration from 10 mM to 400 mM.

As a result, as shown in FIG. 1, only isocitrate dehydrogenase (NADP) specifically adsorbed to the resin, and andose reductase, α-glucosidase, glucose-
All contaminating enzymes such as 6-phosphate dehydrogenase and hexokinase passed through the column.

The ICDH (NAD) adsorbed on Amberlite IRC-50
When P) was eluted with a phosphate buffer, it was eluted at a concentration of about 100 mM. It was confirmed that the activity of the obtained enzyme was not reduced at all, and that the purity was excellent.

Example 2 Amberlite CG-5 instead of Amberlite IRC-50
The procedure was the same as in Example 1 except that 0 (Organo) was used, and the chromatogram in FIG. 2 was obtained.

As is clear from Fig. 2, Amberlite CG-50
Was also completely absorbed by isocitrate dehydrogenase (NADP), while most of aldose reductase, α-glucosidase, glucose-6-phosphate dehydrogenase and hexokinase passed through the column.

Then, in the same manner as in Example 1, ICDH (NADP) was eluted at a concentration of about 100 mM.

Example 3 CM-Sepharose (Pharmacia) was used instead of the exchange resin Amberlite IRC-50, and SV was changed to SV10.
Ion exchange chromatography was carried out in the same manner as in Example 1 except that the value was changed to 0.1, ICDH was isolated and purified, and the purity and recovery were compared with those in Example 1 to obtain the results shown in the following table.

As is clear from the results in the above table, according to the present method, not only the purity and the recovery rate are excellent, but also the flow rate is 100 times higher, in other words, the flow rate is extremely high, in a very short time and in a large amount. It can be seen that there is provided an excellent purification method that can be processed in a short time.

(Effects of the Invention) According to the present invention, for example,
In a short time of 1/100, a very large amount of the crude enzyme solution can be treated to isolate and purify ICDH, and at that time, the purity and the recovery rate are not reduced as compared with the conventional method. And the enzyme activity is not affected at all. Moreover, the processing operation does not require any complicated and delicate operations requiring skill.

Therefore, the present invention can be said to be an optimal method as an industrially superior method for purifying ICDH suitable for large-scale processing.

[Brief description of the drawings]

Figure 1 shows Amberlite as a weakly acidic cation exchange resin.
FIG. 2 is a chromatogram using IRC-50, and FIG. 2 is an ion exchange chromatogram using Amberlite CG-50.

Claims (2)

(57) [Claims] 1. A method for purifying isocitrate dehydrogenase of the type requiring NADP + as a coenzyme, wherein the isocitrate dehydrogenase is specifically adsorbed using a weakly acidic cation exchange resin and then eluted. A method for purifying said isocitrate dehydrogenase, comprising the steps of: 2. The method according to claim 1, wherein the weakly acidic cation exchange resin has a carboxylic acid group.
The method described in the section.