JPH0335917B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for extracting and separating S-adenosyl-L-methionine (hereinafter abbreviated as SAM). This article relates to a method for efficiently extracting and separating SAM.

SAM is an important substance involved in the metabolism of fats, proteins, sugars, etc. in vivo. Recently, it has been discovered that SAM has therapeutic effects on hepatemia, hyperlipidemia, arteriosclerosis, depression, degenerative joint disease, neurological pain sensation, insomnia, etc.
Mass production is expected.

Conventionally, many methods have been known for extracting and separating SAM from bacterial cells capable of producing SAM.
Cell walls are disrupted by treatment with drugs such as inorganic acids such as hydrochloric acid and sulfuric acid, organic acids such as formic acid and trichloroacetic acid, and esters such as methyl acetate and ethyl acetate, and the existing SAM is extracted into a solution, followed by ion exchange. Examples include purification methods using resins, activated carbon, chelate resins, etc. (Japanese Patent Publication No. 52-35726,
No. 52-125194, JP-A-56-92899, etc.).

However, since these methods use chemicals to treat bacterial cells, they tend to cause decomposition of SAM, which poses health and safety issues, and the separation of SAM and impurities is incomplete, making it difficult to use them as medicines. There was a problem in that it was difficult to obtain SAM of high purity, and attempts to improve the purity would 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, when extracting and separating high-purity SAM from bacterial cells capable of producing SAM, the bacterial cells were pretreated with an aqueous divalent copper ion solution. They found that freezing and thawing after treatment is extremely effective, leading to the completion of the present invention.

That is, an object of the present invention is to provide a method for efficiently separating and extracting highly purified SAM from SAM-containing bacterial cells using a safe and simple operation. This is achieved by bringing the bacterial cells into contact with an aqueous solution of divalent copper ions, then freezing them in water and thawing them to extract and separate SAM from the bacterial cells.

The method for producing SAM-containing microbial cells used in the present invention is not particularly limited, and for example, Saccharomyces that has SAM-producing ability
Yeast belonging to the genus Candida, etc. are cultured in a methionine-containing medium, and
Examples include methods for generating and accumulating SAM.

The cells are collected from the SAM-containing cell culture solution obtained in this manner by a conventional method, washed if necessary, and then resuspended in water. The amount of water used may be appropriately selected, but it is usually 1 to 100 parts by weight, preferably 3 to 10 parts by weight, per 1 part by weight of the wet bacteria. If the amount of water is too small, the extraction rate of SAM outside the bacterial cells will not be sufficient, and if it is too large, the SAM concentration in the aqueous solution will decrease, unnecessarily putting a burden on 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, a hydrophilic organic solvent may be used in combination or an inorganic salt such as common salt may be dissolved within a range that does not essentially impair the effects of the present invention.

The suspension is then frozen. 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 in a conventional manner. The melting temperature may be selected appropriately, but
Usually, a range of 0 to 25°C is desirable; if the temperature is too high, SAM will decompose and the yield will decrease.

By removing bacterial cell residue from the resulting molten liquid using a conventional method (e.g., centrifugation), a highly pure SAM aqueous solution with few impurities that behaves the same as SAM in the subsequent purification process can be obtained. .

In general, SAM-containing bacterial cells contain SAM in the vacuole within the bacterial body.
By using the above-mentioned freezing and thawing method, the cytoplasmic membrane and vacuolar membrane are ruptured without completely destroying the cell wall, and as a result, various proteins contained within the cytoplasm are concentrated. It can be assumed that as a result of significantly reducing the elution of nucleic acids and nucleic acids, a highly pure SAM aqueous solution can be obtained. The SAM aqueous solution thus obtained was treated with an ion exchange resin,
SAM with good purity can be isolated by treatment with activated carbon.

In addition, in the present invention, prior to freezing the bacterial cells,
By bringing the bacterial cells into contact with an aqueous divalent copper ion solution, low molecular weight compounds in the cytoplasm are selectively excreted. As a result, SAM purity is significantly improved.
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 leaving the solution 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, preferably 5 to 30% by weight, based on the wet bacterial weight.
% by weight, and the contact temperature and contact time are usually 0 to 0.
The temperature is 50°C for 10 minutes to 3 hours, preferably 20 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 thereof include cupric chloride, cupric bromide, cupric sulfate, cupric acetate, etc. It will be done. The amount of copper ions added is usually determined by the copper ion concentration in the suspension.
It is 5 μM or more, preferably 10 to 500 μM.

Thus, according to the present invention, a highly purified SAM aqueous solution can be obtained very efficiently with simple operations.

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

Comparative Example 1 Medium of Schlenk.F. et al. [Journal of Biological Chemistry (J.
Biol.Chem.) vol. 229, p. 1037 (1957)]
10g of SAM-containing bacterial cells obtained by culturing IFO2044
(Wet amount) is washed twice with distilled water. After that, the bacterial body
After thoroughly suspending in 50g of distilled water, store at -20℃ for 5 minutes.
Leave for some time to freeze. The frozen product is then heated indirectly with water at 20°C to completely thaw it. The molten liquid was centrifuged to remove bacterial cell residue, and the SAM content and relative purity of SAM in the OD ₂₆₀ substance were measured, and the SAM content was 1.72 g.
A value of 42.8% SAM relative purity was obtained.

The purity of SAM is determined by taking a portion of the test solution and developing it with two-dimensional paper chromatography.
A spot of SAM was detected, the SAM concentration of the test solution was detected using an ultraviolet detector, and it was calculated from the measurement of OD ₂₆₀ of the test solution using the following formula.

SAM purity (%) = SAM OD ₂₆₀ / OD ₂₆₀ × 100 Comparative example 2 SAM-containing bacterial cells identical to those used in Comparative example 1
10 g (wet weight) was suspended in 50 ml of 1.5N perchloric acid and extracted with shaking at room temperature for 1 hour. Next, potassium hydrogen carbonate was added to the extract from which bacterial cell residue had been removed by centrifugation to adjust the pH to 5.0, and the resulting precipitate of potassium perchlorate was removed by suction, using the same method as in Example 1. When the content and relative purity of SAM were measured, the SAM content was 1.73g, SAM
The relative purity was 15.6%.

Example 1 10 SAM-containing bacteria used in Comparative Example 1
After washing g (wet amount) twice with distilled water, the pH was 6.4.
The mixture was suspended in 100 parts of tris(hydroxymethyl)aminoethane-2-[N-morpholino]ethanesulfonic acid buffer (buffer concentration: 10 mmolar).

Next, cupric chloride was added to the solution to a concentration of 0.1 millimolar, and the mixture was gently stirred at 30° C. for 1 hour to expel low-molecular impurities from the cells, and then the cells were collected and washed twice with distilled water. Thereafter, it was frozen and thawed in the same manner as in Example 1, and the resulting suspension was centrifuged to remove insoluble substances, and the content and purity of SAM contained in the supernatant was measured.
1.70 g, SAM relative purity 90.9%.

Claims (1)

[Claims] 1. Extraction of S-adenosyl-L-methionine, which is characterized in that yeast cells that have accumulated S-adenosyl-L-methionine in their bodies are brought into contact with an aqueous divalent copper ion solution in advance, and then frozen in water and then thawed. Separation method.