JPH0417627B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to microbial cells containing S-adenosylmethionion in high concentration, and more specifically,
The present invention relates to a microorganism belonging to the genus Satucharomyces cerevisiae that has a low content of adenine-related substances and ninhydrin-reactive substances and can efficiently obtain highly pure S-adenosylmethionine through simple operations. S-adenosylmethionine (hereinafter abbreviated as SAM) has long been known as a substance that has therapeutic effects on hepatemia, hyperlipidemia, arteriosclerosis, etc., and its mass production is expected in recent years. ing. Therefore, when producing such SAM, a fermentation method using microorganisms is mainly used (for example, Journal of Biological Chemistry 229, p. 1037, published in 1957, Special Publication No. 52).
-17118, etc.), these methods have little SAM content accumulated in the microbial cells, and also contain large amounts of adenine-related substances and ninhydrin reaction-positive substances that are difficult to separate from SAM.
It was difficult to isolate SAM by simple operations. In addition, since SAM is an extremely unstable substance, it is often transported to distant locations while containing SAM inside the bacterial body, but in such cases, the small content of SAM is economically viable. It had become a big problem. Therefore, the present inventors carried out intensive studies to improve this point in the conventional technology, and as a result, we found that SAM in microorganisms belonging to the genus Satucharomyces cerevisiae
When the content is 10% by weight or more based on dry bacterial cells, methylthioadenosine, adenine, and S-adenosylhomocysteine, which are difficult to separate from SAM present in the bacterial cells, will be less than 0.1% and 1%, respectively, based on the amount of accumulated SAM. ×10 ^-2 % or less and 0.5% or less,
As a result, the present inventors discovered that high-purity SAM can be recovered economically and in a high yield, and have completed the present invention. Thus, according to the present invention, 10% by weight or more, preferably 12% by weight or more on a dry bacterial body basis,
A microbial cell containing SAM is provided. The microorganism used in the present invention is a yeast belonging to the genus Satucharomyces cerevisiae that has the ability to produce SAM and is capable of accumulating 10% by weight or more of SAM in the bacterial cells on a dry bacterial cell basis in a methionine-containing liquid medium. Specific examples of such targets include S. cerevisiae IFO 2342, S. cerevisiae IFO 2343, S. cerevisiae IFO 2345, S. cerevisiae IFO 2346, S. cerevisiae IFO 2347, and Yeast No. 9 of the Association. Natural and artificial mutant strains of bacteria can be similarly used as long as they have the above-mentioned properties. The microorganism cells of the present invention contain SAM in an amount of 10% by weight or more, preferably 12% by weight or more, based on dry cells. The method for producing such microbial cells is not particularly limited, but usually involves culturing them under aerobic conditions in a liquid medium containing methionine, a carbon source, a nitrogen source, an inorganic salt, and an organic trace nutrient source. It is done by folding. Methionine is usually added at a rate of 0.2 g/dl or more. Methionine can be added in either the entire amount at once or divided into parts and added sequentially, but if the former method is used when a large amount of methionine is added, the amount of SAM accumulated in the cells tends to decrease. Therefore, in such a case, it is appropriate to adopt the latter method. Glucose is used as a carbon source. Sucrose.
Sugars such as fructose; ethanol. Alcohols such as glycerin; and starch hydrolyzate containing alcohols. Molasses. Soybean whey. Fruit juice waste. Fish processing waste liquid. Fermentation waste liquid. Pulp waste liquid can also be used. Urea is also used as a nitrogen source. Ammonium succinate. Ammonium citrate. Ammonium lactate and the like are preferred. Potassium phosphate is an inorganic salt. Sodium phosphate. Calcium phosphate. Phosphates such as lithium phosphate. Potassium salts such as potassium chloride. Sodium chloride. Sodium salts such as sodium carbonate. Magnesium sulfate. Magnesium salts such as magnesium chloride. Manganese sulfate. Manganese salts such as manganese chloride. Common inorganic salts such as iron salts such as iron sulfate and iron chloride, zinc salts, copper salts, and cobalt salts can be used as appropriate. Vitamins are an organic trace nutrient source. amino acid. Yeast extract containing these. Meat extract. Malt extract. Cornstarch liquor. Casamino acids. Soy flour. Soybean hydrolysis. peptone. Tryptone. Casein decomposition solution etc. can be used as needed. Cultivation is preferably carried out under aerobic conditions, usually at a temperature of 15°C to 45°C, preferably 20°C to 35°C, for 2 days while controlling the pH of the medium to 3 to 8, preferably 3.5 to 7. By culturing for 10 days, SAM is produced and accumulated in the microbial cells. The method for obtaining SAM from the microbial cells thus obtained is not particularly limited, and may be carried out according to conventional methods. That is, the target product is obtained by separating the bacterial cells from the culture medium, extracting the SAM in the bacterial cells, and then isolating the SAM in the extract. Generally, it is stabilized and recovered in the form of a salt or double salt, such as an acid salt or a sulfosalicylate. According to the present invention, a high concentration of
It is convenient for transporting unstable SAM because it contains SAM, and it is easy to purify SAM because the content of impurities that are difficult to separate from SAM is small, and as a result, highly pure SAM can be efficiently recovered. make it possible to The present invention will be explained in more detail with reference to Examples below. Example 1 Glucose 5g/dl, polypeptone 0.5g/dl,
KH ₂ PO ₄ 0.4g/dl, K ₂ HPO ₄ 0.4g/dl,
MgSO ₄・7H ₂ O0.02g/dl, yeast extract 0.2g/
dl, agar slant medium consisting of agar 2g/dl (PH6.0)
One platinum loop of Saccharomyces cerevisiae IFO2346 grown for 2 days was mixed with 10g of Sucrose/
dl, yeast extract 1g/dl, KH ₂ PO ₄ 0.4g/dl,
_MgSO4・_7H2O0.01g /dl, L-methionine 1.0
g/dl, ZnSO ₄・7H ₂ O0.25mg/dl, MnSO ₄ 4~
The cells were inoculated into 10 ml of a heat-sterilized medium containing 1.25 mg/dl of 6H ₂ O, adjusted to pH 6.0, and shaken at 28° C. for 4 days. After collecting bacteria by centrifugation and washing with physiological saline, the bacterial cells were suspended in 1.5N perchloric acid and shaken at room temperature for 1 hour to extract SAM. The extract was analyzed using paper chromatography and high performance liquid chromatography, and SAM, adenine (hereinafter abbreviated as Ad),
S-adenosylhomocysteine (hereinafter abbreviated as SAH), methylthioadenosine (hereinafter abbreviated as MTA)
The results are shown in Table 1.

[Table] From this result, it is clear that the amount of
It can be seen that when the SAM content is 12% or more, there are fewer impurities that are difficult to separate from SAM. Example 2 Same culture medium as Example 1, culture time, medium PH,
Satucharomyces cerevisiae IFO2346 was cultured at different culture temperatures to prepare bacterial cells with different SAM contents per dry bacterial cell. The bacterial cells were extracted and analyzed using the same method as in Example 1, and the results are shown in Table 2.

【table】

[Table] Example 3 Glucose 5g/dl, polypeptone 0.5g/dl,
KH ₂ PO ₄ 0.4g/dl, K ₂ HPO ₄ 0.4g/dl,
MgSO ₄・7H ₂ O0.02g/dl, yeast extract 0.2g/
dl, adjusted to PH6.0, heat sterilized medium
Inoculate one platinum loop of the various bacterial strains shown in Table 3 into 10 ml,
Shaking culture was performed at 28°C for 24 hours. On the other hand, sucrose 10g/dl, yeast extract 1
g/dl, KH ₂ PO ₄ 0.4g/dl, MgSO ₄・7H ₂ O0.01
g/dl, urea (sterilized separately) 1.5g/dl, L-methionine 0.75g/dl, CaCl ₂ 2H _{2 O} 0.02g/dl,
ZnSO ₄ 7H ₂ O0.25mg/dl, FeSO ₄ 7H ₂ O0.25mg/
dl, MnSO ₄ 4~6H ₂ O125mg/dl, CuSO ₄・
5H ₂ O2 μg/dl, H ₃ BO ₃ 2 μg/dl, CoCl ₂・
Medium 1 containing 0.2 μg/dl of 6H ₂ O and 1 μg/dl of KI and adjusted to pH 6.0 was placed in a 2-volume fermenter, and after sterilization,
5 ml of the above seed culture solution was inoculated and cultured with aeration and stirring at 28°C for 72 hours. After culturing, collect the bacteria by centrifugation, wash once with physiological saline, and add 100ml of the bacteria.
Suspend in 1.5N perchloric acid and shake at room temperature for 1 hour.
SAM was extracted. Next, after removing bacterial cell residue by centrifugation, potassium hydrogen carbonate was added to adjust the pH to 4.5.
The resulting potassium perchlorate precipitate was removed by centrifugation to obtain an extract containing SAM. SAM in the extract was quantified and the results are shown in Table 3 as the amount of SAM per dry bacterial cell. Weakly acidic cation exchange resin Amberlite IRC-50 was added to the extract so that the SAM amount was 0.2g
(H ⁺ form) was passed through a column packed with 50 ml to adsorb SAM. Pass the eluate through 0.005N acetic acid into the column.
Impurities were removed by washing until the absorbance at 260 nm became 0.1 or less. The amount of 0.2N acetic acid required at this time is shown in Table 3. Next, pass 0.1N sulfuric acid through the column until the absorbance of the eluate at 260nm is 0.05.
SAM was eluted until: This eluate was treated with Amberlite IRA900 resin (OH ^- type) to adjust the pH to 3.0, and then freeze-dried to obtain SAM/sulfate. The recovery rate of SAM at this time is shown in Table 3. The purity of SAM was measured by cellulose thin layer chromatography, paper chromatography, and high performance liquid chromatography and is shown in Table 3.

【table】

[Table] As is clear from Table 3, in the examples of the present invention, the amount of eluate required to remove impurities is small, and it is clear that both the SAM recovery rate and the purity of SAM are extremely good.

Claims (1)

[Claims] 1. More than 10% by weight of S- in the bacterial cells based on dry bacterial cells.
Contains adenosylmethionine, and contains methylthioadenosine, adenine, and S-adenosylhomocysteine at 0.1% or less, 1×10 ^-2 % or less, and 0.5% or less relative to the accumulated amount of S-adenosylmethionine, respectively.
Microorganisms belonging to Satucharomyces cerevisiae containing less than %.