JPH0455677B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing S-adenosylmethionine by a fermentation method.
- A method for producing microbial cells containing a high amount of adenosylmethionine. 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 neurological psychosis, degenerative arthropathy, neurological pain sensation, insomnia, etc., and its mass production is expected. Conventionally, SAM production methods involve cultivating yeast belonging to the genera Satucharomyces, Pichia, Candeida, Hansenula, and Rhodotorula, and filamentous fungi belonging to the genera Mucor, Rhizopus, Aspergillus, and Penicillium in a methionine-containing medium. A method of culturing SAM and producing and accumulating SAM in the bacterial body or culture solution has been reported [e.g.
AminoAcids No. 4, page 95 (1966), Tokko Sho
No. 52-17118, Special Publication No. 47-37038, Journal of
Bacteriology, Vol. 121, p. 267 (1975), etc.). However, with these methods, the generated
Since a considerable amount of SAM flows into the medium,
The SAM content was low, and as a result, a large amount of medium had to be treated in the purification process, and SAM accumulated in the medium was unstable to heat, making it difficult to purify. Therefore, the present inventors conducted intensive studies on the method of obtaining microbial cells that contain a high amount of SAM, and found that
The present invention was completed by discovering that when culturing microorganisms capable of producing SAM in a methionine-containing medium, it is extremely effective to add a specific substance to the medium. That is, the purpose of the present invention is to provide a method for producing microbial cells containing a high amount of SAM,
The object of the present invention is to provide a method for producing SAM by culturing yeast belonging to Saccharomyces cerevisiae having SAM-producing ability in a methionine-containing medium, in which 0.2% of an organic acid ammonium salt is added to the medium.
This is achieved by its presence at a concentration of ~2 g/dl. A method for producing SAM by culturing Satucharomyces cerevisiae in a methionine-containing medium has been conventionally known. In such known techniques, inorganic salts such as ammonium sulfate and ammonium chloride are generally used as the nitrogen source, but in the present invention, it is essential to use a predetermined amount or more of an organic acid ammonium salt as the nitrogen source. This makes it possible to accumulate SAM at a high concentration within the bacterial body, and at the same time, promote the growth of the bacteria. Specific examples of organic acid ammonium salts used in the present invention include mono-, di-, An example is ammonium tricarboxylate. Such organic acid ammonium salts are present in the culture medium at a concentration of 0.2 to 2 g/dl. The microorganism used in the present invention may be any microorganism that belongs to Saccharomyces cerevisiae and has the ability to accumulate SAM in a methionine-containing medium. Specific examples include IFO 2342,
IFO 2343, IFO 2345, IFO 2346, IFO 2347,
Examples include IFO 2376. Natural and artificial mutants of these strains can also be used in the same manner as long as they have the ability to produce SAM. In the present invention, the culture medium is prepared according to conventional methods, except for the presence of the above-mentioned specific compounds in the culture medium. For example, carbon sources include sugars such as glucose, sucrose, and fructose;
Alcohols such as ethanol and glycerin; and starch hydrolyzate, molasses, soybean whey, fruit juice waste liquid, fish processing waste liquid, fermentation waste liquid, pulp waste liquid, etc. containing these can also be used. Furthermore, inorganic salts, organic micronutrients, etc. are used as necessary. The culture in the present invention is preferably carried out under aerobic conditions, and the pH of the normal medium is 3 to 8, preferably 4.
15°C to 45°C, preferably 20°C while controlling the temperature to 7°C
By culturing at a temperature of ~35°C for 2 to 10 days, a significant amount of SAM is produced and accumulated in the microbial cells. SAM that has been produced and accumulated can be collected using conventional methods. For example, SAM-containing bacterial cells are extracted with perchloric acid, the extract is neutralized with potassium bicarbonate under ice cooling, and then extracted with strong acidic cations. Contact with exchange resin, then adsorb SAM, then elute with sulfuric acid,
SAM can be isolated by adding phosphotungstic acid to the eluate to precipitate it. The present invention will be explained in more detail with reference to Examples below. In addition, the quantitative determination of SAM and the measurement of dry bacterial weight in Examples were performed as follows. (1) Quantification of SAM: After culturing, the bacterial cells and the culture solution were separated by centrifugation, and the bacterial cells were extracted with approximately 5 times the amount of 1.5N perchloric acid. The resulting extraction supernatant was then subjected to paper chromatography (developing solvent: ethanol/1-butanol/water/acetic acid/1% aqueous sodium pyrophosphate solution: 35/30/30/1/
1), detect spots corresponding to SAM with an ultraviolet detector, extract this with 0.1N hydrochloric acid,
The amount of SAM in the sample was calculated from the absorbance at 260 nm. (2) Measurement of dry bacterial weight After culturing, measure the turbidity of the culture solution using a Hitachi photoelectric colorimeter in the wavelength range of 610 nm, and calculate the turbidity using a calibration curve of dry bacterial weight and turbidity determined in advance. The dry weight of bacteria in the culture solution was determined. 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 Satucharomyces cerevisiae IFO 2346 grown for 2 days at 28°C was treated with sucrose.
10g/dl, casamino acid 1g/dl, tryptone 1g/dl, KH ₂ PO ₄ 0.4g/
dl, K ₂ HPO ₄ 1g/dl, MgSO ₄・7H ₂ O0.04g/
dl, 0.75 g/dl of L-methionine and 0.5 g/dl of various additives shown in Table 1, adjusted to pH 6.0, heat sterilized and inoculated into 5 ml of a medium, and shaken at 28°C for 5 days. Next, the accumulated amount of SAM and dry bacterial weight were measured, and the results are shown in Table 1.

[Table] From these results, the addition of organic acid ammonium salt increases the amount of SAM accumulated in the bacterial cells,
Moreover, it is clear that the amount of bacterial growth also increases. Example 2 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 IFO 2346 grown for 2 days was mixed with 10 g 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 5 ml of a heat-sterilized medium containing 1.25 mg/dl of 6H ₂ O and predetermined amounts of various additives shown in Table 2, adjusted to pH 6.0, and shaken at 28° C. for 5 days. then
The accumulated amount of SAM and dry bacterial weight were measured, and the results are shown in Table 2.

【table】

Claims (1)

[Claims] 1 S- belonging to Satucharomyces cerevisiae
A method for preparing S-adenosylmethionine by culturing a microorganism capable of producing adenosylmethionine in a methionine-containing liquid medium, characterized in that an organic acid ammonium salt is present in the medium at a concentration of 0.2 to 2 g/dl. Method for producing S-adenosylmethionine.