JPH0260318B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for efficiently enzymatically synthesizing and collecting S-adenosyl-L-homocysteine from adenosine and homocysteine using cells of specific microorganisms as an enzyme source. S-adenosyl-L-homocysteine (hereinafter referred to as
SAH (abbreviated as SAH) is an important physiologically active substance generated in vivo through a methyl group-donating reaction involving S-adenosylmethionine (hereinafter abbreviated as SAM). Recently, SAH has been found to be effective as a sedative, sleep-inducing agent, etc., and its mass production is expected. Conventionally, the method for producing SAH is to extract it from yeast of the genus Satucharomyces or Candeida [for example, the method described in Archives of Biochemistry and Biophysics (Arch.
Biochem. Biophys) 69, 575 (1962)], enzymatic demethylation methods from SAM [e.g.
Biological Chemistry (J.Biol.Chem)
240, 2512 (1965)], and a chemical demethylation method from SAM (for example, Japanese Patent Publication No. 37536/1983). However, all of these methods are very expensive and cannot be called industrial production methods. On the other hand, a method of enzymatically synthesizing adenosine and homocysteine in the presence of a bacterial cell extract of yeast of the genus Satucharomyces or Candeida [for example, Archives of Biochemistry and Biophysics (Arch, Biochem, Biophys)]
69, 575 (1962)] is also known. However, this method is difficult to operate because it uses extracts from bacterial cells, and is not necessarily practical. In view of the actual situation as described above, the inventors of the present invention
As a result of various studies on the enzymatic synthesis method of
When bacterial cells belonging to the genus Satucharomyces or Candeida are used as an enzyme source, adenosine and homocysteine can be easily and efficiently extracted from adenosine and homocysteine.
We discovered that SAH can be constructed and completed the present invention. Thus, according to the present invention, adenosine and homocysteine are brought into contact with each other in an aqueous medium in the presence of the cells of a microorganism belonging to the genus Satucharomyces or Candeida that has the ability to synthesize SAH from adenosine and homocysteine, and thereby react with SAH. Provided is a method for producing SAH, which comprises synthesizing and collecting SAH. The microorganism used as an enzyme source in the present invention may belong to the above-mentioned genus and may be any microorganism that has the ability to synthesize SAH from adenosine and homocysteine in the form of live or dry cells. is Saccharomyces cerevisiae (Saccharomyces cerevisiae) IFO
1805, Satucharomyces cerevisiae IAM
4175, Saccharomyces chevalieri IFO 1727, Saccharomyces exigus
exiguus) IFO 0215, Saccharomyces uvarum IFO 0565, Saccharomyces fermentati IFO 0422, Saccharomyces rouxii
rouxii) IFO 0487, Saccharomyces bayanus IFO 0206, Saccharomyces rosei
IFO 1172, Saccharomyces delburueckii IFO 0955,
Candida utilis IFO
0396, Candida guilliermondii IFO 0566, Candida sake IFO 1633, Candida tropicalis
IFO 0587, Candida Crucei
krusei) IFO 0013, Candida macedoniensis (Candida macedoniensis) IFO 0706,
Candida seudotropicalis
pseudotropicalis) IFO 1065, Candida maltosa ATCC 38042, Candida intermediaa (Candida
intermedia) IFO 0761, Candida albicans (Candida albicans) IFO 1594, etc. Commercially available baker's yeast, sake yeast, etc. can also be used as long as they belong to the above-mentioned genus. Furthermore, even natural and artificial mutants of these bacteria
As long as it has the ability to synthesize SAH, it can be used in the same way. The method for synthesizing SAH in the present invention utilizes the action of intracellular enzymes in the form of living or dried microbial cells, but this enzyme can be prepared by culturing microorganisms in a conventional manner. can. The medium for culturing the microorganism may be a conventional medium containing a carbon source, a nitrogen source, an inorganic salt, and an organic micronutrient source, and may be appropriately selected and used depending on the type of microorganism. The culture method is usually carried out in a liquid medium, but it can also be carried out by solid surface culture. Culture conditions may be selected appropriately depending on the type of microorganism, and a temperature of 10 to 60℃ and a pH of 3 to 10 are used, but generally, a temperature of 20 to 40℃ and a pH of 4 to 9 and a pH of 10 to 120 are used. All you have to do is cultivate it for a while. The growth of microorganisms can be promoted by aeration and stirring during culturing. In the SAH synthesis reaction of the present invention, the microorganisms cultured as described above are used in the form of live cells or dry cells. For example, the SAH synthesis reaction will proceed even if the cells in the microbial culture solution are used as they are, but if the components in the culture solution become an obstacle or if you want to use a large amount of bacteria, they can be separated from the culture solution. It is preferable to use microbial cells that have been prepared. Bacterial cells can be used as viable cells, but for convenience in storage and handling, air-dried cells, freeze-dried cells,
It can also be used as dried bacterial cells such as acetone bacterial cells. Furthermore, the bacterial cells can be immobilized and used according to a conventional method. The SAH synthesis reaction in the present invention is carried out by bringing adenosine, which is an enzyme reaction substrate, into contact with homocysteine in an aqueous medium in the presence of microbial cells. Homocysteine to be subjected to the reaction can be used in either the L form or the DL form. In addition, the reaction conditions may be selected appropriately.
The substrate concentration may be adenosine of 1 mM or more, preferably 2 to 20 mM, and homocysteine concentration of 1 mM or more, preferably 2 to 200 mM. Note that the term homocysteine in the present invention should be understood to include substances that supply homocysteine in the reaction system (eg, homocysteine, homocysteine thiolactone, etc.). The pH of the system in the reaction is 4-12, preferably 6-12.
It is sufficient to maintain the pH at 10, and the pH may be adjusted by a conventional method, for example, using a potassium phosphate buffer, a sodium phosphate buffer, a Tris buffer, an ammonium chloride buffer, a glycine buffer, etc. The reaction temperature may be any temperature that allows the reaction to proceed well and does not affect enzyme activity, substrates and products, and is usually 15 to 60°C, preferably 20 to 50°C. The reaction time can be set to increase the conversion rate of the substrate to SAH, and in the case of a batch method, it is usually 0.1 to 48
The time is preferably 0.5 to 36 hours. Depending on the purpose, the reaction can also be carried out by adding organic solvents such as acetone or ethanol or various surfactants to the aqueous medium. In this way, a reaction solution containing SAH is obtained, but
A known method may be used to collect SAH from the reaction solution. As a separation method, for example, SAH
By contacting the containing liquid with a strongly acidic cation exchange resin,
After adsorbing SAH, it is eluted with sulfuric acid, and phosphotungstic acid is added to the eluate to precipitate SAH.
After adsorption, it was eluted with ethanol/water/concentrated ammonia water (5:50:1), the eluate was concentrated under reduced pressure, and the pH of the concentrate was adjusted to 7 with acetic acid.
Examples include a method of crystallizing SAH at 0°C. The present invention will be explained in more detail with reference to Examples below. However, the present invention is not limited to these examples. In addition, the quantification of SAH in Examples was performed as follows. That is, after the reaction was completed, the reaction solution was immediately cooled to 0 to 5°C, and perchloric acid was added to stop the reaction. Next, insoluble matters were removed by centrifugation, and then a potassium phosphate buffer (PH7.0) was added to the resulting supernatant, and the generated potassium perchlorate was removed by centrifugation. A predetermined amount of the supernatant thus obtained was collected and SAH was quantified using high performance liquid chromatography (manufactured by Hitachi, Model 638-30, Column: Cosmocil 5C ₁₈ , Detector: UV 260 nm). Example 1 Glucose 5g/dl, peptone 0.5g/dl, yeast extract 0.1g/dl, KH ₂ PO ₄ 0.2g/dl,
K ₂ HPO ₄ 0.1g/dl, MgSO ₄・7H ₂ O0.02g/dl,
In an agar slant medium (PH6.0) consisting of 2 g/dl agar.
A loopful of strain 1 listed in Table 1 cultured at 28°C for 48 hours was treated with glucose 5g/dl, peptone 0.5g/dl,
Yeast extract 0.1g/dl, KH ₂ PO ₄ 0.2g/dl,
K ₂ HPO ₄ 0.1g/dl, Mg SO ₄・7H ₂ O0.02g/dl
A liquid medium sterilized and adjusted to pH 6.5 consisting of 10
ml and cultured with shaking at 28°C for 40 hours. After collecting bacteria by centrifugation and washing with 0.1M sodium phosphate buffer (PH8.5), the bacterial cells were mixed with a substrate consisting of 2mM adenosine, 4mM DL homocysteine, and 100mM sodium phosphate buffer (PH8.5). The mixture was suspended in 1 ml of solution and reacted by shaking at 30°C for 4 hours. The yield of SAH was measured and the results are shown in Table 1.

【table】

[Table] Example 2 Candeida utilis IFO cultured in the same manner as in Example 1 except that the amount of liquid medium was 5 ml.
After adjusting 5 ml of culture solution of 0396 to pH 8.5 with IN NaOH, add 5 μmol of adenosine and DE-homocysteine.
5 μmol was added and the mixture was shaken and reacted at 30° C. for 4 hours. The yield of SAH was 0.24 μmol/ml. Example 3 Example 1 except that the liquid medium volume was 500 ml
Satucharomyces cerevisiae cultured in the same manner as
Bacterial cells were collected from the IFO 1805 culture solution by centrifugation, washed once with physiological saline, and collected again by centrifugation. Next, air-dried cells, freeze-dried cells, and acetone-dried cells were prepared using the washed cells according to the following procedure. That is, the washed bacterial cells were dried at room temperature under ventilation for about 15 hours, and then further dried at 5° C. for 1 day in a vacuum desiccator containing phosphorus pentoxide. Next, the dried bacterial cells were ground into powder in a mortar and dried again in a vacuum desiccator to prepare air-dried bacterial cells. Freeze-dried bacterial cells were prepared by directly vacuum-freezing the washed bacterial cells. In addition, for acetone-dried bacterial cells, the washed bacterial cells were added to 500 times the volume of acetone cooled to 5°C, and after stirring for 5 minutes,
The cells were collected in a vacuum, washed with dehydrated acetone, and then the acetone was distilled off under reduced pressure. Next, 50 mg of each of these three types of dried bacterial cells were suspended in 1 ml of a substrate solution consisting of 2 mM adenosine, 4 mM DL-homocysteine, and 100 mM potassium phosphate buffer (PH6.5), and the mixture was shaken at 37°C for 4 hours to react. Ta. The amount of SAH synthesized for each is shown in Table 2.

[Table] Example 4 Using 50 mg of air-dried bacterial cells of each strain shown in Table 3 prepared using the same culture method and drying method as in Example 3,
The reaction was carried out in the same manner as in Example 3. The yield of SAH is shown in Table 3.

[Table] Example 5 50 mg of air-dried bacterial cells of Satucharomyces cerevisiae IFO 1805 prepared using the same culture and drying methods as in Example 3 were mixed with 10 mM adenosine, 10 mM L-homocystine, and potassium phosphate buffer (PH8.0).
It was suspended in 1 ml of a 100 mM substrate solution and reacted at 37°C for 4 hours. SAH yield is 3.96μmo/ml
It was hot. Example 6 200 mg of air-dried bacterial cells of Satucharomyces cerevisiae IFO 1805 prepared in the same manner as in Example 3 were placed in a 10 m
Add to 2 ml of M potassium phosphate buffer (PH7.0), suspend, cool on ice, and add 375 mg of acrylamide.
N,N'-methylenebisacrylamide 20mg, 5
Add and dissolve 0.25 ml of a 2.5% aqueous solution of N,N,N',N'-tetramethylenediamine, degas under reduced pressure, and add 0.25 ml of a 2.5% aqueous solution of ammonium persulfate.
ml was added and left to stand under ice-cooling. The bacterial cell-containing gel produced after 1 hour was finely ground and washed with 10 mM potassium phosphate buffer (PH7.0) to prepare a bacterial cell immobilized product. Add 1.9 g of this immobilized material to adenosine 10
The mixture was added to 2 ml of a substrate solution consisting of mM, DL-homocysteine, and 100 mM sodium phosphate buffer (PH9.0), and the mixture was reacted at 30°C for 8 hours.
The yield of SAH was 1.10 μmol/ml. Example 7 2.5 g of air-dried cells of Saccharomyces cerevisiae IFO 1805 prepared in the same manner as in Example 3 were added to 50 ml of a substrate solution consisting of 10 mM adenosine, 10 mM DL-homocysteine, and 100 mM sodium phosphate buffer (PH9.5). When suspended and reacted at 37°C for 6 hours, 238 μmol (90 mg) of SAH was found in the reaction solution.
was synthesized. Thereafter, the reaction was stopped by adding 5 ml of a 30% perchloric acid aqueous solution under ice cooling, and then insoluble matter such as bacterial cell residue was removed by centrifugation. Then,
Add IMKHCO ₃ aqueous solution to the obtained supernatant to adjust the pH.
6.0, and the generated potassium perchlorate precipitate was removed by centrifugation. The supernatant liquid obtained in this way was transferred to a strongly acidic cation exchange resin Dowex 50×8.
After passing the liquid through the (H ⁺ type) column, the column was 0.025
After washing with % thiodiglycol aqueous solution and 2N sulfuric acid containing 0.025% thiodiglycol, 0.025%
The fraction eluted with 6N sulfuric acid containing thiodiglycol was collected, and a 20% aqueous phosphotungstic acid solution was added thereto. The resulting precipitate was collected by centrifugation and washed with cold water, followed by 5 times the volume of acetone/ After dissolving in water (50/50 V/V) and extracting with isoamyl alcohol/ether (1/1 V/V), BaCO ₃ was added to the resulting aqueous layer to adjust the pH to 3.9. When the supernatant liquid from which BaSO ₄ was removed was freeze-dried, 53 mg of a white solid was obtained. When silica gel thin layer chromatography, high performance liquid chromatography, paper chromatography, infrared absorption spectrum, and specific rotation were measured, this white solid was confirmed to be SAH.

Claims (1)

[Claims] 1. Adenosine and homocysteine are synthesized in an aqueous medium in the presence of a microorganism belonging to the genus Satucharomyces or Candeida that has the ability to synthesize S-adenosyl-L-homocysteine from adenosine and homocysteine. Contact and react, S
- A method for producing S-adenosyl-L-homocysteine, which comprises synthesizing and collecting adenosyl-L-homocysteine. 2. The production method according to claim 1, wherein the microorganism cells are live cells or dry cells.