JPH0153038B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing S-adenosylmethionine (hereinafter abbreviated as SAM) by a fermentation method,
More specifically, the present invention relates to a method for efficiently producing SAM by culturing yeast in a methionine-containing liquid medium supplemented with a specific compound. SAM is an important substance involved in the metabolism of fats, proteins, sugars, etc. in vivo. Recently, it has been discovered that SAM has a therapeutic effect on hepatemia, hyperlipidemia, arteriosclerosis, depression and neurological manifestations of psychosis, degenerative joint disease, neurological pain sensation, insomnia, etc. mass production is expected. Conventionally, cultivation methods using various microorganisms have been known as methods for producing SAM, but among these methods, methods using yeast are considered preferable, and a specific example is a method using microorganisms of the genus Satucharomyces or Candeida ( For example, Journal of
Bacteriology, vol 121, p. 267 (1975), etc.)
Pichia spp., Rhodotorula spp., Cryptococcus spp.
Hansenula spp., Trichosporon spp., Crecera spp.
Methods using microorganisms of the genus Hanseniaspora, Sporobolomyces, Lipomyces, or Debaryomyces (Japanese Patent Publication No. 17118/1982) are known. However, even in these methods
The accumulated amount of SAM was not necessarily fully satisfactory, and further improvements were required. Therefore, the present inventors conducted intensive studies on improving the accumulation of SAM by fermentation, and found that it is effective to use a medium to which methionine, a precursor of SAM, and a specific compound were added. completed. That is, the purpose of the present invention is to provide a method for producing SAM using a fermentation method with excellent productivity.
The object of the present invention is to provide a method for producing SAM by culturing yeast capable of producing SAM in a methionine-containing medium, in which the medium contains (A) at least one base selected from the group consisting of uracil, thymine, and purine; (B) at least one nucleoside selected from the group consisting of inosine, guanosine, xanthosine, uridine, cytidine, thymidine, and deoxyadenosine, (C) inosine-5'-monophosphate, guanosine-5'-monophosphate , xanthosine-5'-monophosphate, uridine-5'-monophosphate, cytidine-5'-monophosphate, adenosine-
2'-monophosphoric acid, adenosine-3'-monophosphoric acid,
At least one nucleotide selected from the group consisting of deoxyadenosine-5'-monophosphate and deoxythymidine-5'-monophosphate, (D) ribonucleic acid or (E) deoxyribonucleic acid, preferably at least 0.01 g/dl. This is achieved by making it exist at a concentration of 0.02 g/dl or more. The yeast used in the present invention may be any yeast as long as it has the ability to accumulate SAM in a methionine-containing medium.
Genus, Hansenula, Pichia, Rhodotorula,
Belongs to the genus Cryptococcus, Trichosporon, Kloeckera, Hanseniaspora, Sporobolomyces, Lipomyces, Debaryomyces, etc. Yeasts include Satucharomyces cerevisiae IFO2346 and Candeida macedoniensis as specific examples.
IFO0960, Hanzenula Huabianii IFO1370,
Pihia falinosa IFO0193, Rhodotorula glutinis IFO0389, Cryptococcus albidus
IFO0385, Trichosporon capitatum
IFO0743, Crechera apiquyulata IFO0151, Hanseniaspora barbaiensis IFO0115, Sporobolomyces salmonicolor IFO0374, Lipomyces lipofuel IFO0673, Debaryomyces
Examples include Hanzenii IFO0026. Furthermore, these natural and artificial mutant bacteria can be similarly used as long as they have the ability to produce SAM. In the present invention, the culture medium is prepared according to a conventional method except that the specific compounds mentioned above are present alone or in combination of two or more kinds. For example, sugars such as glucose, sucrose, and fructose, organic acids such as acetic acid, hydrocarbons, alcohols such as methanol and ethanol, etc. can be used as carbon sources, and in addition, ordinary nitrogen sources, inorganic salts, and organic micronutrients can be used. Used as needed. Cultivation is performed under good aerobic conditions, and the culture temperature is from 20℃.
A range of 40°C is preferred. During culture, adjust the pH of the medium to 3.
Adjusting to a range of 8 to 8 usually yields the most desirable results. Thus, after 2 to 10 days of culture, SAM is produced and accumulated in the bacterial cells and/or the culture solution, and this can be treated using conventional methods.
Able to obtain SAM. For example, SAM-containing bacterial cells are extracted with perchloric acid, the extract is neutralized with potassium bicarbonate under ice-cooling, and if necessary, it is brought into contact with a strongly acidic cation exchange resin, then SAM is adsorbed, and then sulfuric acid is added. Elute,
SAM can be isolated by adding phosphotungstic acid to the eluate to precipitate it. The present invention will be described in more detail with reference to Examples below, but the present invention is not limited thereto. In addition, the quantification of SAM in Examples was performed as follows. That is, after the cultivation was completed, the bacterial cells and the culture solution were separated by centrifugation, and the bacterial cells were extracted with about 5 times the amount of 1.5N perchloric acid. Next, the obtained extraction supernatant was mixed with the culture solution, and then subjected to paper chromatography (developing solvent: ethanol/1-butanol/water/acetic acid/1% aqueous sodium pyrophosphate solution = 35/30/30/1/1 ), a UV detector detects spots equivalent to SAM, and
The sample was extracted with 0.1N hydrochloric acid, and the amount of SAM in the sample was detected from its absorbance at 260 nm. 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 IFO2346 grown for 2 days was added to Satucharose 10g/dl.
Casamino acids 1g/dl, tryptone (Bacto
tryptone) 1g/dl, KH ₂ PO ₄ , 0.4g/dl,
K ₂ HPO ₄ 1g/dl, MgSO ₄・7H ₂ O0.04g/dl, L
- 5 ml of a heat-sterilized medium containing 0.75 g/dl of methionine and a predetermined amount of purine, adjusted to pH 6.0, was inoculated and shaken at 28°C for 5 days. Next, the amount of accumulated SAM was measured, and the results are shown in Table 1.

[Table] Example 2 Add 0.05 of the compound shown in Table 2 instead of purine.
Cultivation was carried out under the same conditions as in Example 1, except that it was added to the medium at a concentration of g/dl. The results are shown in Table 2.

[Table] Example 3 Culture was carried out under the same conditions as in Example 1, except that the strains shown in Table 3 were used instead of S. cerevisiae IFO2346, and 0.05 g/dl of various compounds were added to the medium. The results are shown in Table 3.

【table】

[Table] Example 4 1 of Satucharomyces cerevisiae IFO2346 grown for 2 days on the same agar slant medium as in Example 1
Platinum looper with 10 g/dl of sucrose, 1 yeast extract
g/dl, KH ₂ PO ₄ 0.4g/dl, MgSO ₄・7H ₂ O0.01
g/dl, Urea (sterilized separately) 1.8g/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, purine 0.05g/dl and xanthosin-
Contains 0.05g/dl of 5′-monophosphoric acid, adjusted to pH 6.0,
The cells were inoculated into 5 ml of heat-sterilized medium. After shaking at 28℃ for 5 days, the amount of SAM accumulated was 0.36.
g/dl. Furthermore, when similar culture was carried out without adding purine and xanthosine-5'-monophosphate, the amount of SAM accumulated was 0.19 g/dl. Example 5 Culture was carried out under the same conditions as in Example 1, except that the strains shown in Table 5 were used instead of Saccharomyces cerevisiae IFO2346, and 0.05 g/dl of purine was added to the medium. The results are shown in Table 5.

【table】

Claims (1)

[Claims] 1. A method for producing S-adenosylmethionine by culturing yeast capable of producing S-adenosylmethionine in a methionine-containing medium, which comprises
(A) at least one base selected from the group consisting of uracil, thymine, and purine; (B) inosine;
At least one nucleoside selected from the group consisting of guanosine, xanthosine, uridine, cytidine, thymidine and deoxyadenosine, (C) inosine-5'-monophosphate, guanosine-
5'-monophosphoric acid, xanthosine-5'-monophosphoric acid, uridine-5'-monophosphoric acid, cytidine-5'-
Monophosphoric acid, adenosine-2'-monophosphoric acid, adenosine-3'-monophosphoric acid, deoxyadenosine-
Presence of at least one nucleotide selected from the group consisting of 5'-monophosphoric acid and deoxythymidine-5'-monophosphoric acid, (D) ribonucleic acid, or (E) deoxyribonucleic acid at a concentration of 0.01 g/dl or more. A method for producing S-adenosylmethionine, characterized by: 2. The method according to claim 1, wherein the yeast belongs to the genus Satucharomyces, Candeida, or Hansenula.