JPH0433439B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing S-adenosylmethionine by a fermentation method, and more specifically, a method for producing S-adenosylmethionine of high purity in high yield and economically by culturing microorganisms belonging to the genus Satucharomyces in a liquid medium containing methionine. It relates to a method of manufacturing. S-adenosylmethionine (hereinafter abbreviated as SAM) is an important physiologically active substance involved in the metabolism of fats, proteins, sugars, etc. in vivo.
Recently, SAM has been associated with hepatemia, hyperlipidemia,
It has been found to have therapeutic effects on arteriosclerosis, depression and psychosis, degenerative arthropathy, neurological pain sensation, insomnia, etc., and mass production is expected. Conventionally, as an industrial method for producing SAM, yeast of the genus Satucharomyces is cultured in a methionine-containing liquid medium, and the SAM accumulated in the microbial cells is extracted.
Purification methods are known [for example, in the Journal of Biological Chemistry (J.
Biol.Chem) 229, p. 1037 (1957)]. However, the SAM content in the bacterial cells obtained by this method is about 3% at most, and the SAM content in the bacterial cell extract is
It was extremely difficult to purify because it contains many adenine-related substances and ninhydrin-reactive substances that are difficult to separate. Especially adenine, S-adenosylhomocysteine and methylthioadenosine (hereinafter referred to as
It is difficult to separate SAM (abbreviated as MTA), and several SAM purification methods are known to overcome this drawback.
No. 21079, No. 53-20998, JP-A-56-145299, etc.), all of these methods were insufficient to economically obtain SAM with high purity and high recovery rate. In addition, there are also known methods of culturing yeast belonging to the genus Candeida, Pichia, Hansenula, Rhodotorula, etc., and producing and accumulating SAM in the bacterial cells or in the culture solution (for example, Japanese Patent Publication No. 52-17118). It is difficult to purify intracellular SAM using this method, and since various metabolites and SAM decomposition products coexist with SAM in the culture medium, it is difficult to purify SAM from the culture medium.
Recovery of SAM was more difficult. Therefore, the present inventors conducted intensive studies on the industrial production method of SAM using the fermentation method, and found that
When the SAM content exceeds a certain value that could not be achieved with conventional technology, the amount of impurities that are difficult to separate from SAM existing within the bacterial cells becomes relatively extremely small.
The present inventors have found that purification is extremely easy by using such microbial cells, and that highly purified SAM can be produced economically with high recovery rate, and have completed the present invention. Thus, according to the present invention, a microorganism capable of producing SAM is cultured in a methionine-containing liquid medium, and 10% by weight or more of SAM on a dry bacterial cell basis and 0.1% by weight or less of MTA are added to the bacterial cells relative to the accumulated amount of SAM. Provided is a method for producing SAM, which is characterized in that after accumulation, the bacterial cells are separated from the culture medium, and then SAM is obtained from the bacterial cells in a stable form. The microorganism used in the present invention belongs to the genus Satucharomyces, has the ability to produce SAM, and has a dry cell basis in the cell body in a methionine-containing liquid medium.
It is capable of accumulating 10% by weight or more of SAM and 0.1% by weight or less of MTA based on the accumulated amount of SAM. A specific example is Satucharomyces cerevisiae.
IFO2342, Satucharomyces cerevisiae
IFO2343, Satucharomyces cerevisiae
IFO2345, Satucharomyces cerevisiae
IFO2346, Satucharomyces cerevisiae
Examples include IFO2347 and Kyokai No. 9 yeast. Natural and artificial mutants of these strains can also be used in the same manner as long as they have the above-mentioned properties. In the present invention, it is essential to culture the microorganism until the intracellular SAM content reaches 10% by weight or more, preferably 12% by weight or more. At this time, if the SAM content is less than 10% by weight, the content of adenine-related substances such as MTA and ninhydrin reaction-positive substances is relatively high, and therefore
Purification of SAM cannot be carried out efficiently. The culture conditions for increasing the SAM content to 10% by weight or more are not particularly limited as long as the MTA content is 0.1% by weight or less based on the SAM content, but include methionine, carbon source, nitrogen source, inorganic salts, and organic Preferably, it is carried out under aerobic conditions in a liquid medium containing micronutrient sources. 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. In such cases, it is appropriate to adopt the latter method. Carbon sources include glucose, sucrose,
Sugars such as fructose; Alcohols such as ethanol and glycerin; Furthermore, starch hydrolyzate containing these, molasses, soybean whey, fruit juice waste liquid,
Fish processing waste liquid, fermentation waste liquid, pulp waste liquid, etc. can also be used. Preferable nitrogen sources include urea, ammonium succinate, ammonium citrate, and ammonium lactate. Inorganic salts include phosphates such as potassium phosphate, sodium phosphate, calcium phosphate, and lithium phosphate; potassium salts such as potassium chloride; sodium salts such as sodium chloride and sodium carbonate; magnesium salts such as magnesium sulfate and magnesium chloride; manganese sulfate; Common inorganic salts such as manganese salts such as manganese chloride, iron salts such as iron sulfate and iron chloride, zinc salts, copper salts, and cobalt salts can be used as appropriate. Organic micronutrient sources include vitamins, amino acids, yeast extract containing these, meat extract, malt extract, corn staple liquor, casamino acids, soybean flour, soybean hydrolyzate, peptone, tryptone, casein decomposition solution, etc. as required. It can be used as Cultivation is preferably carried out under aerobic conditions, usually at a temperature of 15°C to 45°C, preferably 20°C to 35°C, 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. In the present invention, after culturing, the bacterial cells are separated from the medium, and then SAM is extracted and purified from the bacterial cells. The methods used in these steps are not particularly limited, and any known method may be used. In other words, when separating bacterial cells from a culture medium, methods such as centrifugal separation and filtration are exemplified, and when obtaining SAM from bacterial cells, perchloric acid, hydrochloric acid, sulfuric acid, After extracting SAM with extractants such as formic acid, acetic acid, formate ester, acetate ester, ethanol, etc.
Highly purified stabilized SAM can be obtained by purifying the SAM in the extract according to a conventional method. The purification method for SAM is not particularly limited, and examples include chromatography using activated carbon, strongly acidic cation exchange resin, weakly acidic cation exchange resin, chelate resin, Reinetske's salt, picric acid, phosphotungstic acid, picloronic acid, etc. There are methods of purifying SAM by precipitating it using, for example, acetone, ethanol, etc., and methods of precipitating SAM using an organic solvent such as acetone or ethanol. These methods can be used in combination as appropriate. At this time, in order to stabilize and recover SAM, it is common to add acids such as sulfuric acid, para-toluenesulfonic acid, and sulfosalicylic acid to recover SAM in the form of a salt or double salt. 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 cells were suspended in 1.5N perchlorine fermentation 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) and methylthioadenosine were analyzed, and the results are shown in Table 1.

[Table] From this result, it is clear that the amount of
When the SAM content is 10% by weight or more, the MTA content is 0.1% by weight or less based on the accumulated amount of SAM, indicating that there are few impurities that are difficult to separate from SAM. Example 2 Using the same medium as in Example 1, culturing time, medium PH, and culturing temperature were changed to cultivate S. cerevisiae.
IFO2346 was cultured, and cells with different SAM contents per dry cell were prepared. The bacterial cells were extracted and analyzed using the same method as in Example 1, and the results are shown in Table 2.

[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
10ml of medium, adjusted to pH 6.0 and heat sterilized.
One platinum loopful of the various bacterial strains shown in Table 3 was inoculated into the water at 28°C.
The cells were cultured with shaking for 24 hours. On the other hand, seuucrose 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 O0.25
mg/dl, MnSO ₄ 4-6H ₂ O1.25mg/dl, CuSO ₄・
5H ₂ O2μg/dl, H ₃ BO ₃ 2μg/dl, CoCl ₂・6H ₂
Medium 1 containing 0.2 μg/dl of 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 at 28°C for 72 hours. Ivy. After culturing, the bacteria were collected by centrifugation and diluted with physiological saline.
The washed bacterial cells were suspended in 100 ml of 1.5N perchloric acid and shaken at room temperature for 1 hour to extract SAM. 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, and SAM
An extract containing the following was obtained. 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 acetic acid through the column until the absorbance at 260 nm of the eluate is 0.05.
SAM was eluted until below. This eluate was treated with Amberlite IRA900 resin (OH ^- type) to adjust the pH to 3.0, and then lyophilized 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] 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 A microorganism belonging to Satucharomyces cerevisiae that has the ability to produce S-adenosylmethionine, with an amount of S-adenosylmethionine of 10% by weight or more and 0.1% by weight or less based on the accumulated amount of S-adenosylmethionine on a dry cell basis. A microorganism capable of accumulating methylthioadenosine in its bacterial cells was cultured in a methionine-containing liquid medium, and 10% by weight or more of S-adenosylmethionine and 0.1% by weight or less of S-adenosylmethionine accumulated on a dry bacterial cell basis were cultured. A method for producing S-adenosylmethionine, which comprises accumulating methylthioadenosine in the cells, separating the cells from the culture medium, and then obtaining S-adenosylmethionine from the cells in a stable form.