JPS59109190A - Extractive separation of s-adenosyl-l-methionine - Google Patents

Abstract

PURPOSE:To extract and to separate high-purity S-adenosyl-L-methionine efficiently, by freezing a mold containing S-adenosyl-L-methionine by a safe and simple process. CONSTITUTION:A yeast mold accumulating S-adenosyl-L-methionine in its body obtained by cultivating a yeast belonging to the genus Saccharomyces, etc. is collected, it is suspended in 1-100pts.wt., preferably 3-10pts.wt. water based on 1pt.wt. wet mold amount to give a suspension, which is brought into contact with an aqueous solution of a bivalent ion, frozen, the frozen substance is melted by a conventional procedure, and the mold residue is removed from the prepared melted solution by a conventional procedure.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to S-adenosyl-L-methionine (hereinafter referred to as SA
For more details regarding the extraction and separation method of
This invention relates to a method for efficiently extracting and separating SAM with a small amount of impurities (high purity) from a microbial cell capable of producing SAM.

SAM is an important substance involved in the metabolism of fats, proteins, sugars, etc. in vivo. Recent SAMs include lipidemia, hyperlipidemia, arteriosclerosis, depression, degenerative joint disease,
It has been found to have a therapeutic effect on neurological pain sensation, insomnia, etc., and its mass production is expected.

Conventionally, many methods have been known for extracting and separating SAM from microbial cells capable of producing SAM. For example, SAM-containing microbial cells are extracted with perchloric acid.

Cell walls are disrupted by treatment with drugs such as inorganic acids such as hydrochloric acid and sulfuric acid, organic acids such as @acid and trichloroacetic acid, and esters such as methyl acetate and ethyl acetate, and the existing SAM is extracted into a solution. Examples include purification methods using exchange resins, activated carbon, chelate resins, etc.
No. 35726, JP-A-52-125194, JP-A-5
No. 6-92899, etc.).

However, these methods tend to cause decomposition of SAM because they use drugs to treat bacterial cells, and there are health and safety issues.In addition, the separation of SAM and impurities is incomplete, making it difficult to use it as a medicine. There is a problem in that it is difficult to obtain SAM with high purity, and attempts to improve the purity result in a decrease in the recovery rate of SAM.

Therefore, the present inventors conducted intensive studies to improve this shortcoming of the conventional technology, and as a result, they discovered that it is not necessary to freeze and thaw the bacterial cells when extracting and separating highly pure SAM from the bacterial cells capable of producing SAM. It has been found that this is extremely effective, and that the effect is further improved if the bacterial cells are treated with an aqueous divalent steel ion solution prior to freezing and thawing, leading to the completion of the present invention.

In other words, an object of the present invention is to provide SAM with safe and simple operation.
It is an object of the present invention to provide a method for efficiently separating and extracting highly purified SAM from cells containing SA.
This is achieved by bringing yeast cells that have accumulated M in the body into contact with an aqueous divalent copper ion solution, if desired, and then freezing them in water and thawing them to extract and separate KSAM outside the cells.

The method for producing the 5Ali-containing microbial cells used in the present invention is not particularly limited (for example, Saccharomyces having SAM-producing ability).
Examples include a method in which yeast belonging to the genus Candida or the like is cultured in a methionine-containing medium to produce and accumulate SAM within the bacterial cells.

Bacteria are collected from the SAM-containing microbial culture solution obtained in this manner by a conventional method, washed if necessary, and then resuspended in water. The amount of water to be used should be selected appropriately.)
(Usually 1 to 100 parts by weight per 1 part by weight of wet bacteria,
Desirably, 3 to 10 layers can be used. If the amount of water is too small, the extraction rate of SAM outside the image body will not be sufficient, and if it is too large, the degree of SAMJ in the aqueous solution will decrease, unnecessarily burdening the subsequent purification and separation process.

The water used is not particularly limited as long as it can freeze the bacterial cells, but from the viewpoint of extraction efficiency, it is preferable to use distilled water. However, as long as the effects of the present invention are not essentially impaired, a hydrophilic organic solvent may be used in combination, or an inorganic salt such as common salt may be used.

Freezing of the suspension then takes place. There are no particular limitations on the temperature, time, etc. of the freezing method, and any method may be used as long as the suspension is uniformly frozen.

The frozen product thus obtained is then thawed according to a conventional method. The melting temperature may be selected appropriately, but is usually 0 to 25
℃ range is desirable; if the temperature is too high, SAM
decomposes and the yield decreases.

By removing bacterial cell residue from the resulting molten liquid using a conventional method (e.g., centrifugation), SAM can be removed in the subsequent purification step.
A SAM aqueous solution of good purity with few impurities that interfere with the behavior can be obtained.

When the thawing method is used, the cytoplasmic membrane and vacuolar membrane are ruptured without completely destroying the cell wall, and as a result, the elution of various proteins and nucleic acids contained in the cytoplasm is reduced by a wide margin. It can be assumed that a highly pure SAM aqueous solution can be obtained. When the SAM aqueous solution thus obtained is treated with an ion exchange resin, activated carbon, etc. in a conventional manner, SAM with good purity can be isolated.

In addition, in the present invention, prior to freezing the Kokutai, -5= bacterial cells are brought into contact with an aqueous divalent copper ion solution to selectively excrete low-molecular compounds in the cytoplasm φ, and as a result, SAM purity is significantly increased. can be improved. The treatment with an aqueous copper ion solution is usually carried out by suspending the bacterial cells in water or a buffer solution, adding the aqueous copper ion solution, and then allowing the suspension to stand or stirring. The pH at this time is usually 5 to 8, preferably 5.5 to 7, and the bacterial cell content in the suspension is usually 1 to 50% by weight based on the wet bacterial weight.
, preferably 5 to 30 F-, and the contact temperature and contact time are usually 0 to 50°C for 10 minutes to 3 hours, preferably 2
The temperature is 0 to 40°C for 30 minutes to 2 hours.

The source of copper ions used may be any water-soluble copper ion compound, and specific examples include cupric chloride, cupric bromide, cupric sulfate, cupric acetate, etc. It will be done. The amount of copper ions added is usually 5 μM or more in terms of copper ion concentration in the suspension, preferably 10 to 50 μM.
0 l1M.

Thus, according to the present invention, it is possible to obtain a highly purified SAM aqueous solution very efficiently with simple operations.

The present invention will be further specifically described below with reference to Examples.

Example 1 Medium of Schlenk (F.) et al. [Journal of Biological Gummy Science (J.

Biol, chem,) Volume 229, Page 1037 (1
957)) and 'Saccbar'
omyces.

SAM-containing bacterial cells 109 obtained by culturing S. cerevisiae) I FO2044 (wet cells were washed twice with distilled water. After that, the cells were sufficiently suspended in 50°C of distilled water, and then incubated at -20°C for 5 hours. Leave to freeze. Next, completely thaw the frozen material by indirectly heating it with water at 20°C. The thawed liquid is centrifuged to remove residual bacterial cells, and the SAM content and ODzao When the relative purity of SAM in the material was measured, it was found that 1.72 g of SAM-containing phosphorus;
A value of 42.8% SAM relative purity was obtained.

The purity of SAM is determined by taking a portion of the test solution, developing it with two-dimensional paper chromatography, detecting the shM spot, and detecting the SAM concentration in the test solution with an ultraviolet detector.
It was calculated from the measurement of OI)tao of the test solution using the following formula.

Comparative Example 1 10 g of SAM-containing bacterial cells identical to those used in Example 1 (
The wet product t) was suspended in 50 tit of 1.5N perfiic acid and extracted by shaking at room temperature for 1 hour. Next, potassium hydrogen carbonate was added to the extract from which residual bacterial cells had been removed by centrifugation, and the pH was adjusted to 5.0, and the resulting potassium perchlorate precipitate was removed by suction, and the same procedure as in Example 1 was carried out. When the content and relative purity of SAM were measured using a method, it was found that SAM
The content was 1.73.9, and the SAM relative purity was 15.6%.

Example 2 After washing 10 g of SAM-containing bacterial cells (wet fi:), which were the same as those used in Example 1, twice with distilled water, tris(hydroxymethyl)aminoethane-2- was adjusted to pH 6.4.
[N-morpholino]ethanesulfonic acid buffer 100 parts (
It was suspended in a buffer solution (concentration: 10 mmolar).

Next, cupric chloride was added to the solution at a concentration of 0.1 mmIJ molar, and the mixture was gently stirred at 30"C for 1 hour to expel low-molecular contaminants from the cells. The bacteria were collected, and the mixture was poured with distilled water. After that, freeze-thaw in the same manner as in Example].
Insoluble substances were removed from the resulting suspension by centrifugation, and the content and purity of SAM contained in the supernatant were measured. SAM content 't1.70.9, SAM relative purity 90.
．． It was 9chi.

Percentage Applicant: Nippon Zeon Co., Ltd. 9-

Claims (1)

[Scope of Claims] 1. A method for extracting and separating S-adenosyl-methionine, which comprises freezing yeast cells that have accumulated S-azonone-luciemethionine in water and then thawing them. Yeast cells that have accumulated 2S-adeno/room-methionine in the body are brought into contact with an aqueous divalent copper ion solution in advance, and then,
A method for extracting and separating S-adenosyl-methionine, which is characterized by freezing in water and then thawing.