JPH0283394A - Purification of s-adenosyl-l-homocystein - Google Patents

Abstract

PURPOSE:To obtain the title cysteine free from coloring by dye in high purity and recovery by treating a liquid containing crude S-adenosyl-L-homocystein with a weakly acidic cation exchange resin and porous synthetic absorbent. CONSTITUTION:A liquid containing crude S-adenosyl-L-homocystein is parified by a process obtained by combining a process treating the above-mentioned L-homocystein with a weakly acidic cation exchange resin with a process treating the above-mentioned L-homocystein with a porous synthetic resin absorbent each one or more times in arbitrary order.

Description

Detailed Description of the Invention The present invention provides S-adenosyl-L-homocysteine (hereinafter referred to as
For more details regarding the purification method of SAH (abbreviated as SAH),
The present invention relates to a method for efficiently isolating and purifying highly pure SAH from a crude SAH-containing liquid containing impurities.

SAH is an important physiologically active substance that is generated in vivo through a methyl group-donating reaction involving S-adenosyl-L-methionine (hereinafter abbreviated as SAM). Recently, SAH has been found to be effective as a sedative, sleep-inducing agent, etc., and its mass production is expected.

As a method for purifying SAH, there is a method in which SAH is precipitated by adding phosphotungstic acid to a crude SAH-containing solution (Meth
ods in ENZYMOLOGY 6,573.1
963), (1) A method in which SAH is adsorbed on a strongly acidic cation exchange resin, eluted with sulfuric acid, and phosphotungstic acid is added to the eluate to precipitate SAH (J, Nut, r.

Sci, Vitaminol, 23.73.197
7),■ After adsorbing SAH on activated carbon, ethanol/
A method of elution with water/concentrated ammonia water (50:50:1), concentrating the eluate under reduced pressure, adjusting the pH of the concentrate to 7 with #acid, and then releasing SAH at 0°C (Tokuko Showa). 45-3
No. 7536), etc. are known.

However, in the method (2), the purity of the precipitated SAH is low and a more complicated recrystallization process is required.On the other hand, in the methods (2) and (2), the recovery rate of SAH is low, and the SAH and impurities such as coloring components are mixed. However, the separation of SAH is incomplete and SAH of high purity that can be used as a medicine cannot be obtained.

Therefore, the present inventors conducted intensive studies to improve these shortcomings in the conventional technology, and as a result, SAH was extracted from the crude SAH-containing liquid.
In isolating and purifying AH, the inventors have discovered that it is extremely effective to combine treatment with a 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 high-purity SAH, and an object of the present invention is to process a crude SAH-containing liquid with (a) a weakly acidic cation exchange resin. (b) This is accomplished by supplying the treatment steps using a porous synthetic resin adsorbent in any order to a combined purification step at least once, and purification.

The method for producing the crude SAH-containing liquid used in the present invention is not particularly limited. A method for producing SAH by contacting and reacting with SAM (Japanese Unexamined Patent Publication No. 146595/1982), a method for producing SAH by enzymatic demethylation from SAM (J, Biol, Chem.
240.2512.1965) or SA
A method of manufacturing by chemically demethylating M
5-37536) and the like.

In the present invention, when treating such a crude SAH-containing liquid, (a) treatment with a weakly acidic cation exchange resin and (b) treatment with a weakly acidic cation exchange resin are performed.
) It is essential that treatment with a porous synthetic resin adsorbent be performed at least once in any combination.

Here, the process (a) is performed as follows.

That is, the pH of the crude SAH-containing liquid is usually adjusted to 3.5-8, preferably 5-7. At this time, if the pH of the crude SAH-containing liquid is outside the above range, SAH will be difficult to be retained and adsorbed by the ion exchange resin. The method for adjusting the pH is not particularly limited, but it is preferable to use a combination of acid and alkali that forms a precipitate that is soluble or insoluble in water, or to use an anion exchange resin (OR-type).

Next, the crude SAH-containing liquid is brought into contact with a weakly acidic cation exchange resin to selectively adsorb positively charged SAH 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 specific examples include Amberlite IRC-50, IRC-84 (
(manufactured by Rohm and Haas), Diaion WK20
(manufactured by Mitsubishi Kasei Corporation). At this time, the resin is
It may be either the Na'' form or the NH4'' form, but preferably the H+ form is used.

The contact method may be either a batch method or a column method, but
A column method is more preferred in terms of operability and ease of removing impurities.

The SAH adsorbed onto the ion exchange resin is then typically at a pH of 3.
, 0 or less, preferably 0.2 to 2.0, by fractional elution with an aqueous solution of an inorganic or organic acid.

The acid used is not particularly limited, and examples thereof include hydrochloric acid, sulfuric acid, phosphoric acid, #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 SAH.

On the other hand, the process (b) above is performed as follows. That is, after adjusting the pH of the crude SAH-containing liquid as necessary, preferably to around neutrality, it is brought into contact with the porous synthetic resin adsorbent. At this time, the same methods as in (a) above can be used for the pH adjustment method and the contact method with the adsorbent. The dye is selectively adsorbed by contact with such an adsorbent.

Furthermore, the porous synthetic resin adsorbent used in the present invention is water-insoluble and has a giant network structure, and a specific example thereof is a non-polar type adsorbent having a core of styrene-divinylbenzene copolymer, such as Amberlite. XAD-2,XA
D-4 (manufactured by Rohm and Haas), Diaion HP-10, HP-20, HP-30, HP-40, H
P-50 (manufactured by Mitsubishi Kasei Corporation), Sepabeads SP-20
6, S P-207 (manufactured by Mitsubishi Kasei Corporation), polymers of acrylic esters and/or methacrylic esters, or copolymerization of these monomers with nonpolar monomers such as styrene, divinylbenzene, etc. Intermediate polar adsorbent with coalescence as its core, such as Amberlite XAD-7, X
Examples include AD-8 (manufactured by Rohm and Haas) and Diaion HP-2MG (manufactured by Mitsubishi Kasei). These can be used in combination as desired.

In the present invention, each of the processes (a> and (b)) is performed at least once.The order of III processes may be combined as appropriate, and specific examples thereof include ■(a)→(b),
■(b) → (a), ■(a) → (b) → (a), ■(b
) → (a) → (b), etc., and the treatment steps (a) and (b) can be added as necessary. However, as the number of processing steps increases, the process becomes more complicated and economical efficiency decreases, so a combination of the above four types is usually selected.

In the present invention, SAH eluted from such purification step
After the eluted fraction is concentrated by vacuum concentration or a method using a reverse osmosis membrane, it is left to stand at a low temperature to extract SAH. Thereafter, SAH is collected by filtration or centrifugation, and water is removed by freeze drying, spray drying, air drying, or the like to obtain a crystalline SAH powder. Further, if necessary, the purity of SAH can be further increased by redissolving the SAH crystal in hot water and then recrystallizing it at a low temperature.

Further, since the weakly acidic ion exchange resin used in the present invention is regenerated into H° type at the stage of eluting SAH, 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, for example, by simply washing it with a 50% aqueous methanol solution and then washing it with water.

Thus, according to the present invention, SAH of extremely high purity can be efficiently obtained with a simple operation using a processing means that is easy to regenerate.

The present invention will be explained in more detail with reference to Examples below.

In addition, the determination of SAH in the examples was performed using high performance liquid chromatography (manufactured by JASCO Corporation, TRIROTAR-V type, column: Cosmoci15C18, Detecjer
:UV260nm).

Example 1 Glucose Ig/dR, peptone 1.5g/clR1 yeast extract 0.3g82, K2HPOA 0.3g/dQ,
NaC10,2g/dR1MgSO4・7t(200,
02g/JR1 agar 2g/d9. Pseudomonas putida IFO12996 or Alcaligenes
1 platinum loop of Faecalis IF012699, glucose Ig/clR, peptone 1.5g/dQ, yeast extract 0
．． 3g/clQ, 2HPO40, 3g/cB! , Na
C10,2g/dR1MgSO+ ・711200.0
The cells were inoculated into 500 mR of a heat-sterilized liquid medium containing 2 g/dΩ, adjusted to pH 7.0, and cultured with shaking at 28° C. for 40 hours. Bacteria were collected by centrifugation, washed with 0.1 M potassium phosphate buffer (PH8, 0), and centrifuged again to obtain warm bacterial cells. Next, 3 g of wet bacterial cells of Pseudomonas putida IFO12996 and 1 wet bacterial cell of Alcaligenes faecalis IFO12699 were added.
Adenosine 10mM, DL-homocysteine 2
0mM, potassium phosphate buffer (pH 8,0) 1
Suspend in 100ml of substrate solution consisting of 00mM,
The mixture was shaken and reacted at 7°C for 7 hours. Then 100℃10
After heating for a minute, distilled water was added to bring the volume to about 300 mQ, and bacterial cell residue was removed by centrifugation to obtain 300 mQ of SAH-containing liquid (pH 7.5) containing 1.20 g of SAM.

This SAW-containing solution was passed through a column packed with 100 mQ of weakly acidic cation exchange resin Amberlite IRC-50 (H+ type) (manufactured by Rohm and Haas) to retain and adsorb SAH. Column 0.002N#M 400ml
After washing with 0. SAH was fractionally eluted with IN sulfuric acid.
110 fractions of A and H elution fractions were obtained. Barium hydroxide was added to this SAH elution fraction to adjust the pH to 7.0, and the resulting barium sulfate precipitate was removed by suction filtration.
206 (manufactured by Mitsubishi Kasei Corporation) was added, and the mixture was brought into contact with the mixture for 30 minutes with stirring. Next, the synthetic resin adsorbent was removed by suction filtration, and the filtrate was concentrated under reduced pressure to a total volume of 40%, and then left at 5°C for 24 hours to crystallize SAH, which was recovered by suction filtration and freeze-dried. 1.09 g of white SAH powder was obtained.

The recovery rate and purity of SAH are shown in Table 1.

Example 2 In the same manner as in Example 1, 315 SAH-containing liquids containing 1.15 g of SAH were obtained. This SAH-containing solution was passed through a column packed with a weakly acidic cation exchange resin Amberlite IRC-84 (H+ type, manufactured by Rohm and Haas) 100- to retain and adsorb SAH. Column 0.002N
After washing with acetic acid 400-0. SAH was fractionally eluted with IN hydrochloric acid to obtain SAH elution fraction 130-. This SA
After adjusting the H elution fraction to pH 6.8 using a weakly basic anion exchange resin Amberlite IRA-45 (OH-type), the styrene-divinylbenzene-based synthetic resin adsorbent Diaion HP-20 (manufactured by Mitsubishi Kasei Corporation) was added. The sample was passed through a column packed with 50-glue without adsorbing SAH. Both components containing SAH were collected, concentrated under reduced pressure to a liquid volume of 40-40°C, and kept at 5°C for 2
The SAH was allowed to stand for 4 hours, recovered by suction filtration, and lyophilized to obtain 1.04 g of white SAH powder. The recovery rate and purity of SAH are shown in Table 1.

Example 3 In the same manner as in Example 1, 300 d of SAH-containing liquid containing 1.03 g of SAH was obtained. This SAH-containing liquid is mixed with styrene-divinylbenzene based synthetic resin adsorbent Amberlite XAD.
-2 (manufactured by Rohm and Haas) through a column packed with 100 m9 of SAH without adsorbing it. Next, the fractions containing SAH were collected and passed through a column packed with weakly acidic cation exchange resin Amber 945 RC-50 (H'' type) (manufactured by Rohm & Haas) 100+T19 to retain and adsorb SAH. 0.
After washing with 400 d of 0.002N acetic acid, S with IN sulfuric acid
AH was fractionally eluted to obtain S AH elution fraction 120-. Barium hydroxide was added to this SAH elution fraction and pi(
The filtrate was adjusted to 7.0 and the barium sulfate precipitate produced by suction filtration was removed, and the filtrate was concentrated under reduced pressure to a total volume of 50 liters. The concentrated solution was left at 5°C for 24 hours to extract SAH, which was recovered by suction filtration and freeze-dried to obtain 0.94 g of white SAH powder. The recovery rate and purity of SAH are shown in Table 1.

Comparative Example In the same manner as in Example 1, 300 ml of SAH-containing liquid containing 1.10 g of SAH was obtained. This SAH-containing liquid was passed through a column packed with 50 m9 of activated carbon for chromatography to remove SAH.
was adsorbed. After washing the column with 200 mQ of distilled water, ethanol/water/concentrated ammonia water (65:35:2)
SAH was eluted through a mixed solvent of 1 to obtain an SAH elution fraction.

This SAH elution fraction was concentrated under reduced pressure to 20 d, adjusted to p) 17.0 with acetic acid, and left at 0°C for 48 hours to allow SA
H was extracted, collected by suction filtration, and freeze-dried to obtain SAH powder 1. Obtained OOg. The recovery rate and purity of SAH are shown in Table 1.

Table 1 Note 1; SAH purity: SAH detected at 260 nm in high performance liquid chromatography analysis Note 2; SAH
Recovery rate: The ratio of the amount of SAH in the crystals after purification to the amount of SAH in the crude SAH-containing liquid was calculated from the quantitative value determined by high performance liquid chromatography.

From these results, it is clear that by using the method according to the present invention, SAH with higher purity and without coloration due to dyes can be obtained at a higher recovery rate than with known methods.

Claims (1)

[Claims] 1. In a method for purifying S-adenosyl-L-homocysteine by supplying a crude S-adenosyl-L-homocysteine-containing liquid to a purification step, the purification step comprises (a) treatment with a weakly acidic cation exchange resin. A method for purifying S-adenosyl-L-homocysteine, characterized in that the steps of step (b) and (b) treatment with a porous synthetic resin adsorbent are combined at least once each in any order.