JPS635039B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for purifying S-adenosyl-L-methionine (hereinafter abbreviated as SAM), and more specifically, to a method for efficiently isolating and purifying highly purified SAM from a crude SAM-containing solution containing impurities. SAM is an important substance involved in the metabolism of fats, proteins, sugars, etc. in vivo. Recently, it has been discovered that SAM has a therapeutic effect on hepatemia, hyperlipidemia, arteriosclerosis, depression and neurological manifestations of psychosis, degenerative joint disease, neurological pain sensation, insomnia, etc. mass production is expected. Conventionally, many methods have been known for purifying SAM, including a method that combines a process of treatment with a strongly acidic cation exchange resin and a process of treatment with activated carbon (Japanese Patent Publication No. 46-13680).
), a method of treatment with a weakly acidic cation exchange resin (Enzymologia Vol. 29, p. 283), a method of combining a process of treatment with an H ⁺ type weakly acidic cation exchange resin and a process of treatment with activated carbon (Unexamined Japanese Patent Publication No. (Sho 56-145299),
Method of treatment with chelate resin (Special Publication 1973-
20998, etc.), a method via a salt of SAM and picric acid or picroronic acid (Special Publication No. 1973-
No. 21079, No. 52-35727) are known. Among these methods, the process has the advantage of being very simple and economical compared to the method described above, but the separation of SAM and impurities is incomplete and it cannot be used as a medicine. The major drawback is that high purity SAM cannot be obtained.
In addition, in the case of the method developed as an improved method, although the purity is improved, the recovery rate of SAM decreases due to the strong adsorption of SAM by activated carbon, and in order to increase the recovery rate, the amount of organic solvent in the eluate is increased. This has the disadvantage that separation from impurities is insufficient. Therefore, the present inventors conducted intensive studies to improve these shortcomings in the conventional technology, and as a result, the rough
When isolating and purifying SAM from a SAM-containing solution
The inventors have discovered that it is extremely effective to combine treatment with an H ⁺ -type weakly acidic cation exchange resin and treatment with a porous synthetic resin adsorbent, and have completed the present invention. That is, an object of the present invention is to provide a method for efficiently isolating and purifying highly pure SAM,
The object of the present invention is to process a crude SAM-containing liquid through a purification process in which a treatment process using (A) an H ⁺ -type weakly acidic cation exchange resin and (B) a porous synthetic resin adsorbent are combined at least once in any order. This is achieved by supplying and refining. The method for producing the crude SAM-containing liquid used in the present invention is not particularly limited, and for example,
Candida, a genus of Saccharomyses with SAM-producing ability
Microorganisms belonging to the genus Mucor, etc. are cultured in a methionine-containing medium, and the intracellular and/or
Alternatively, a method in which SAM is generated and accumulated outside the bacterial body and extracted using an extractant such as perchloric acid, hydrochloric acid, sulfuric acid, sacrificial acid, or phosphoric acid, or adenosine triphosphate and methionine are extracted using methionine-adenosyltransferase. An example is a method of carrying out an enzymatic reaction in the presence of. In the present invention, when treating such a crude SAM-containing liquid, (A) treatment with an H ⁺ type weakly acidic cation exchange resin and (B) treatment with a porous synthetic resin adsorbent are carried out at least once each in an arbitrary combination. It is an essential requirement to do so. Here, the process (A) is performed as follows. In other words, the pH of the crude SAM-containing liquid is usually 3.5 ~
Adjust to 6.5, preferably 4 to 6.5. At this time, roughly
If the pH of the SAM-containing solution is too low, it will be difficult for SAM to be retained and adsorbed by the ion exchange resin, and if the pH is high, SAM
becomes easier to decompose. The method for adjusting the pH is not particularly limited, but it is preferable to use a combination of acid and alkali that forms precipitates that are poorly soluble or insoluble in water, or to use an anion exchange resin (OH ^- type). Next, the crude SAM-containing solution is brought into contact with an H ⁺ -type weakly acidic cation exchange resin to selectively adsorb positively charged SAM and remove neutral and negatively charged impurities. The weakly acidic cation exchange resin used may be one having a carboxylic acid group as an ion exchange group, and a specific example is Amberlite IRC.
-50, IRC-84 (manufactured by Rohm and Haas),
Examples include Diaion WK20 (manufactured by Mitsubishi Kasei Corporation). The contact method may be either a batch method or a column method, but the column method is more preferred in terms of operability and ease of removing impurities. Next, the SAM adsorbed on the ion exchange resin is separated by fractional elution with an aqueous solution of an inorganic or organic acid having a pH of usually 3.0 or less, preferably 0.2 to 2.0. The acid used is not particularly limited, and examples include hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, and P-toluenesulfonic acid. Further, if necessary, trace amounts of impurities can be removed by washing with water or a dilute acid aqueous solution (for example, pH 3.5 or higher) prior to the fractional elution of SAM. On the other hand, the process (B) above is performed as follows. That is, after adjusting the pH of the crude SAM-containing liquid to 6.5 or lower, it is brought into contact with a porous synthetic resin adsorbent.
At this time, the same methods as in (A) can be used for adjusting the pH and contacting with the adsorbent. By contact with such an adsorbent, impurities such as amines, methylthioadenosine (a decomposition product of SAM), and pigments are selectively adsorbed. In addition, SAM can be allowed to pass through or be adsorbed by selecting conditions, and when adsorbed, SAM can be fractionated and eluted with an aqueous solution of an inorganic or organic acid with a pH of 3.5 or less, preferably 0.2 to 3.0. can be selectively separated. The inorganic acids and organic acids used may be any of those in the same range as in the case of (A), and methanol, ethanol, n-propanol may be used as necessary as long as a uniform solution can be formed. ,
Organic solvents such as isopropanol, acetone, methyl ethyl ketone, methyl formate, ethyl acetate, dioxane, toluene, etc. can be used in combination. Furthermore, if desired, trace amounts of impurities can be removed by washing with water or a dilute acid aqueous solution prior to fractional elution, as in the case of (A). Furthermore, the porous synthetic resin adsorbent used in the present invention is water-insoluble and has a giant network structure.
Specific examples include non-polar adsorbents that have a styrene-divinylbenzene copolymer as a core, such as Amberlite XAD-2, XAD-4 (manufactured by Rohm and Haas), Diaion HP-10, HP-
20, HP-30, HP-40, HP-50 (manufactured by Mitsubishi Chemical Corporation)
Intermediate polar adsorbents whose core is a polymer of acrylic ester and/or methacrylic ester, or a copolymer of these monomers and a non-polar monomer such as styrene or divinylbenzene; For example, Amberlight XAD-7, XAD-8
(manufactured by Rohm and Haas), Diaion
Examples include HP-ZMG (manufactured by Mitsubishi Kasei Corporation).
These can be used in combination as desired. These adsorbents have a common feature in that they selectively adsorb impurities such as amines and dyes in crude SAM-containing liquids, but their ability to adsorb SAM is selective depending on the type; In the case of type adsorbent
SAM is not adsorbed, and in the case of intermediate polarity adsorbents, it depends on the PH range, with adsorption occurring in relatively weakly acidic regions and no adsorption in relatively strongly acidic regions. In the present invention, each of the processes (A) and (B) is performed at least once. The order of processing can be combined as appropriate; a specific example is (A) - (B), (B)
- (A), (A) - (B) - (A), (B) - (A) - (B), etc., and if necessary, the above treatment steps (A) and (B) can be carried out. can be added. However, as the number of processing steps increases, the process becomes more complex and economical efficiency decreases.
Usually, a combination of the above four types is selected. Furthermore, when performing treatment (B) prior to treatment (A), it is operationally advantageous to set conditions that allow the synthetic resin adsorbent to selectively adsorb only impurities without adsorbing SAM. Specific methods include, for example, a method using a non-polar adsorbent, a method using an intermediate polar adsorbent and contacting in an acidic region of PH 3.5 or less, preferably 0.2 to 3.0. . Furthermore, when carrying out the treatment (B) at the final stage of the purification process, SAM may be adsorbed on a synthetic resin adsorbent and then separated, or it may be allowed to pass through without being adsorbed. In the present invention, the SAM elution fraction eluted from such a purification step is concentrated under reduced pressure as necessary, and then subjected to methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, methoxyethanol, acetone, methyl ethyl ketone, By contacting with an organic solvent such as methyl acid, ethyl acetate, methyl acetate, ethyl acetate, butyl acetate, dioxane, etc.
A precipitate of a salt consisting of SAM and an inorganic or organic acid is obtained. In addition, the SAM eluate can be used with anion exchange resin (OH ^- type) or SAM without using an organic solvent.
After removing the excess acid using an alkali that forms a water-insoluble salt with the acid used for fractional elution, the solvent is evaporated to dryness under reduced pressure. powder can be obtained. Furthermore, since the H ⁺ type weakly acidic ion exchange resin used in the present invention is regenerated into the H ⁺ type at the stage of eluting SAM, special regeneration treatment is not necessarily necessary, and it can be used repeatedly by simply washing with water. Furthermore, the synthetic adsorbent can be easily regenerated by simply washing it with, for example, a 50% aqueous methanol solution and then washing with water. Thus, according to the present invention, SAM of extremely high purity can be produced with simple operations using easily regenerated processing means.
can be obtained efficiently. The present invention will be explained in more detail with reference to Examples below. Example 1 Medium of Schlenk.F. et al. [Journal of Biological Chemistry (J.
229, p. 1037 (1957)] and Saccharomyces Cerevisiae .
210g of bacterial cells that have accumulated SAM by culturing 1FO2044
Suspended in 1000ml of 1.5N perchloric acid and incubated at room temperature for 1 hour.
Shaking extraction was performed. Next, potassium bicarbonate was added to the extract from which bacterial cell residue had been removed by centrifugation, and the pH was adjusted to 5.0.The resulting potassium perchlorate precipitate was removed by suction, and 1.15 g of SAM was obtained.
1080 ml of extract containing . This extract was added to a weakly acidic cation exchange resin Amberlite IRC-50 (H ⁺
mold) (product name, manufactured by Rohm and Haas) 200
The SAM was retained and adsorbed by passing through a column packed with ml.
After washing the column with 400ml of 0.0001N acetic acid,
Separately elute SAM with sulfuric acid and collect 630ml of SAM elution fraction.
I got it. This SAM elution fraction was adjusted to pH 4.5 using a weakly basic anion exchange resin Amberlite IRA-45 (OH ^- type) (trade name, manufactured by Rohm & Haas), and then treated with an acrylic ester synthetic resin. The SAM was retained and adsorbed by passing through a column packed with adsorbent Amberlite XAD-7 (trade name, manufactured by Rohm and Haas) 1.5. After washing the column with 0.0001N acetic acid 2, SAM was fractionally eluted with 0.1N sulfuric acid to obtain 1440 ml of SAM elution fraction. After concentrating this SAM elution fraction under reduced pressure to a total volume of 200 ml,
800 ml of acetone was added to obtain a precipitate of SAM sulfate. A precipitate was obtained by centrifugation, dissolved in a small amount of water, and then lyophilized to obtain 174 g of SAM sulfate as a white powder that was homogeneous in paper chromatography and silica gel thin layer chromatography. SAM
The recovery rate and purity are shown in Table 1. Example 2 1000 ml of an extract containing 1.06 g of SAM obtained in the same manner as in Example 1 was obtained. This extract was passed through a column packed with 200 ml of weakly acidic cation exchange resin Amberlite IRC-84 (H ⁺ type) (trade name, manufactured by Rohm and Haas) to retain and adsorb SAM. After washing the column with 400 ml of 0.0001N hydrochloric acid, SAM was fractionally eluted with 0.2N hydrochloric acid to obtain 640 ml of SAM elution fraction. This SAM elution fraction was treated with a weakly basic anion exchange resin Amberlite IRA-45 (OH ^- type) at pH 4.8.
Synthetic adsorbent Amberlite XAD after adjusting to
-7 (Product name, manufactured by Rohm and Haas) 1.5
The SAM was retained and adsorbed through a column packed with After washing the column with 0.0001N acetic acid 2,
SAM was fractionally eluted through a mixed solvent of 0.1N hydrochloric acid and acetone (1:0.1 volume ratio), and the SAM elution fraction was
Obtained 1120ml. After concentrating this SAM elution fraction under reduced pressure until the total solution amounted to 200 ml, weakly basic anion exchange resin Amberlite IRA-45 (OH ^- type) was added to adjust the pH to 2.0, and the resin was removed by suction. was concentrated under reduced pressure.
This concentrated solution was freeze-dried to obtain 1.19 g of SAM hydrochloride as a white powder that was uniform in paper chromatography and silica gel thin layer chromatography.
The SAM recovery rate and purity are shown in Table 1. Example 3 In the same manner as in Example 1, 950 ml of an extract containing 0.98 g of SAM was obtained. This extract is applied to the styrene-divinylbenzene based synthetic resin adsorbent Amberlite.
It was passed through a column packed with 200 ml of XAD-2 (trade name, manufactured by Rohm and Haas) without adsorbing SAM. Fractions containing SAM were collected and potassium bicarbonate was added to adjust the pH to 5.0, and the resulting potassium perchlorate precipitate was removed by suction filtration to obtain a SAM-containing solution. Next, this SAM-containing solution was passed through a column packed with 200 ml of weakly acidic cation exchange resin Amberlite IRC-50 (H ⁺ type) to retain and adsorb SAM. After washing the column with 400 ml of 0.0001N acetic acid, SAM was fractionally eluted with 0.1N sulfuric acid to obtain 630 ml of SAM elution fraction. After concentrating this SAM elution fraction under reduced pressure to a total volume of 200 ml, 800 ml of acetone was added to the SAM.
A sulfate precipitate was obtained. Obtain the precipitate by centrifugation, dissolve it in a small amount of water, and then lyophilize it to obtain SAM sulfate as a white powder that is uniform in paper chromatography and silica gel thin layer chromatography.
1.50g was obtained. The recovery rate and purity of SAM are shown in Table 1. Example 4 200 g of bacterial cells that had accumulated SAM in the same manner as in Example 3 were suspended in 1000 ml of 0.1N formic acid, heated at 60°C for 10 minutes, and then immediately cooled. 1020 ml of the extract containing 0.91 g of SAM obtained by removing bacterial cells by centrifugation was mixed with 200 ml of the acrylic acid ester synthetic resin adsorbent Amberlite XAD-8 (trade name, manufactured by Rohm and Haas). Pass it through a packed column,
The SAM was passed through the column without being adsorbed to the resin. The fractions containing SAM were collected and treated with a weakly basic anion exchange resin Amberlite IRA-45 (OH ^- type).
After adjusting the pH to 5.0, it was passed through a column packed with 200 ml of weakly acidic cation exchange resin Amberlite IRC-84 (H ⁺ type) to retain and adsorb SAM. column
After washing with 400ml of 0.0001N hydrochloric acid, wash with 0.2N hydrochloric acid.
SAM was fractionally eluted to obtain 710 ml of SAM elution fraction. A weakly basic anion exchange resin Amberlite IRA-45 (OH ^- type) was added to this SAM extraction fraction.
The pH was adjusted to 2.0, the resin was removed by suction, and the liquid was concentrated under reduced pressure. This concentrated solution was freeze-dried to obtain 1.01 g of SAM hydrochloride as a white powder, which was uniform in paper chromatography and silica gel thin layer chromatography. The recovery rate and purity of SAM are the first
Shown in the table. Example 5 SAM elution fraction obtained by treatment with a styrene-divinylbenzene-based synthetic resin adsorbent and an H ⁺ type weakly acidic cation exchange resin in the same manner as in Example 3.
630 ml was further passed through a column packed with 200 ml of acrylic acid ester-based synthetic resin adsorbent Amberlite XAD-7, and the SAM was allowed to pass through the column without being adsorbed to the resin. Collect the fractions containing SAM,
After concentrating under reduced pressure until the total volume is 200ml, add acetone.
800 ml was added to obtain a precipitate of SAM sulfate. A precipitate was obtained by centrifugation, dissolved in a small amount of water, and then lyophilized to obtain 1.46 g of SAM sulfate as a white powder that was homogeneous by paper chromatography.
The recovery rate and purity of SAM are shown in Table 1. Comparative Example 1 Potassium hydrogen carbonate was added to 950 ml of an extract containing 0.98 g of SAM obtained in the same manner as in Example 1.
The pH was adjusted to 5.0, and the resulting potassium perchlorate precipitate was removed by suction to obtain a SAM-containing solution. This SAM-containing solution was passed through a column packed with 200 ml of weakly acidic cation exchange resin Amberlite IRC-50 (H ⁺ type) to retain and adsorb SAM. After washing the column with 400ml of 0.0001N acetic acid, separate and elute SAM with 0.1N sulfuric acid. 610ml of SAM elution fraction.
I got it. This SAM elution fraction was passed through a column packed with 200 ml of activated carbon for chromatography to adsorb SAM. After washing the column with 600ml of 0.2N sulfuric acid,
SAM was fractionally eluted through a mixed solvent of 1.0N sulfuric acid and methanol (1:1 volume ratio) to obtain a SAM elution fraction. This SAM elution fraction was concentrated under reduced pressure to a total volume of 200 ml, and then 800 ml of acetone was added to precipitate SAM sulfate. Obtain the precipitate by centrifugation, dissolve it in a small amount of water, and freeze-dry to obtain 1.28 g of SAM sulfate as a white powder that is homogeneous by paper chromatography and silica gel thin layer chromatography.
I got it. The recovery rate and purity of SAM are shown in Table 1. Comparative example 2 Weakly acidic cation exchange resin Amberlite IRC-
An experiment was conducted in the same manner as in Example 1, except that the treatment was limited to 50 (H ⁺ type). Results first
Shown in the table.

【table】

[Table] From the results, it is clear that the method according to the present invention allows highly purified SAM to be obtained at a higher recovery rate than the known method.

Claims (1)

[Claims] 1. In a method for purifying S-adenosyl-L-methionine by supplying a crude S-adenosyl-L-methionine-containing liquid to a purification step, the purification step comprises (A)
Process of treatment with H ⁺ type weakly acidic cation exchange resin
(B) A method for purifying S-adenosyl-L-methionine, which comprises combining the steps of treatment with a porous synthetic resin adsorbent at least once in any order.