JPS6314957B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a novel method for producing S-adenosyl-L-methionine. (Prior art) S-adenosyl-L-methionine (hereinafter referred to as S-
AM (abbreviated as AM) exists widely in living organisms, and is highly expected to be used as a methyl group donor for methylation reactions by various transferases, as well as for medical use in treating various metabolic disorders. ing. However, although it has such important biochemical meaning and is expected to be widely used for medical and research purposes, there are currently no industrial methods for producing S-AM. Only complicated extraction methods from yeast and filamentous fungi are known. Regarding the above, the present inventors have conducted various studies and found that adenosine-5'-triphosphate (hereinafter abbreviated as ATP)
We have now established a new method for efficiently producing S-AM from L-methionine and L-methionine. (Structure and Effects of the Invention) That is, according to the present invention, the S-AM is composed of a bacterial cell of a microorganism that belongs to the genus Satucharomyces or a processed product thereof that generates S-AM from ATP and L-methionine, and a product that belongs to the genus Escherichia. It can be produced by reacting ATP or its precursor with L-methionine in the presence of a microorganism that produces ATP from the ATP precursor or a processed product thereof. The above reaction of the present invention can be carried out in the following manner. (However, in the formula, L-Met is L-methionine, Pi is inorganic phosphoric acid, E _A is the cells of a microorganism of the genus Satucharomyces that generates S-AM from ATP and L-methionine, or a processed product thereof, and E _B is Each represents a bacterial cell of a microorganism belonging to the genus Escherichia or a processed product thereof.) The present invention will be described in detail below. In the present invention, microorganisms of the genus Satucharomyces that produce S-AM from ATP and L-methionine include microorganisms that contain enzymes involved in the biosynthesis of S-AM (for example, methionine adenosyltransferase, etc.). Examples of such microorganisms include Satucharomyces cerevisiae IFO2044). Examples of processed bacterial cells include dried bacterial cells, organic solvent-treated bacterial cells, surfactant-treated bacterial cells, cell-free extracts from bacterial cells, and methionine adenosyltransferase purified from the extracts. It will be done. In addition, microorganisms of the genus Escherichia that generate ATP from ATP precursors catalyze reactions that generate ATP from ATP precursors such as adenosine and adenosine-5'-diphosphate (hereinafter abbreviated as ADP). from enzymes, especially adenosine and phosphate donors (e.g. acetyl phosphate)
glycolytic enzymes that catalyze reactions that produce ATP;
Examples include microorganisms that contain acetate kinase or pyruvate kinase, which catalyze the reaction that generates ATP from ADP and a phosphate donor. Examples of such microorganisms include Escherichia coli B strain ATCC23226. In addition, as mentioned above, examples of processed bacterial cells include dried bacterial cells, organic solvent-treated bacterial cells, surfactant-treated bacterial cells, cell-free extracts from bacterial cells, and enzymes purified from the extracts. . The reaction is from the above ATP and L-methionine to S
- A microorganism of the genus Satucharomyces or a processed product thereof that produces AM, a microorganism of the genus Escherichia or a processed product thereof that produces ATP from an ATP precursor, ATP or its precursor, a phosphate donor, L-methionine, and magnesium chloride It can be carried out in a buffer solution containing an appropriate amount of. Examples of the buffer used in the reaction include phosphate buffer, Tris-HCl buffer, etc., and the concentration thereof is preferably about 25 to 200 mM, particularly 50 to 100 mM. The ATP concentration in the reaction solution is approximately 5-60mM,
Especially 10-20mM, ATP precursor concentration is about 5-20mM, especially about 10-15mM for ADP, about 10-60mM, especially about 20-20mM for adenosine.
Preferably it is 40mM. Further, it is appropriate to use a phosphate donor concentration of about 1 to 2 times, preferably about 1.5 times, the ATP concentration. Preferably, the L-methionine concentration is about 5-60mM, especially 10-40mM. Furthermore, the concentration of magnesium chloride is about 5~
Preferably it is 20mM, especially 10mM. The reaction takes place at a pH of about 6 to 9, especially 7 to 8, and a temperature of about 20.
to 50°C, particularly preferably 30 to 40°C. Thus, according to the invention, the
ATP is broken down by ATP degrading enzyme and ADP is generated, but in this reaction, ATP precursor is
ADP can be reduced by the presence of microorganisms of the genus Escherichia or their bacterial cell products that produce
It can be regenerated into ATP and the S-AM production reaction can be carried out efficiently. In addition, even when its precursor is used instead of ATP, the presence of the above-mentioned Escherichia microorganisms or their bacterial cell products can quickly convert ATP.
ATP is generated from the precursor, and S-AM can be efficiently generated. Furthermore, according to the present invention, a microorganism of the genus Satucharomyces or a processed product thereof that produces S-AM from ATP and L-methionine, and a microorganism of the genus Escherichia or a processed product thereof that produces ATP from an ATP precursor are each They can also be used in the above enzymatic reaction even when they are encapsulated or immobilized in a gel lattice of a high molecular weight polymer, either singly or both at the same time. The immobilization of these microorganisms or their bacterial cell products has been adopted for the preparation of immobilized microorganisms and immobilized enzymes, such as polyacrylamide gel entrapment method, carrageenan gel entrapment method, agar gel entrapment method, polyvinyl alcohol gel entrapment method, This can be carried out by a method such as a collagen gel envelopment method or an alginate gel entrapment method, and thus an immobilized product of the bacterial cells or the treated bacterial cells can be obtained. For example, ATP and L-
When immobilizing a microorganism of the genus Satucharomyces that produces S-AM from methionine or a processed product of the microorganism, an acrylamide monomer, a crosslinking agent (e.g. N,N -methylenebisacrylamide), a polymerization accelerator (for example, β-(dimethylamino)propionitrile), and a polymerization initiator (for example, sodium persulfate) to form a gel, thereby obtaining the desired immobilized product. In addition, in the case of the carrageenan gel entrapment method, the microbial cells or the processed material of the cells are suspended in an aqueous carrageenan solution, and this suspension is brought into contact with a gelling agent (for example, potassium chloride) to gel it. An immobilized product can be obtained. These immobilized substances can be treated with surfactants or organic solvents to improve the gel permeability of raw materials or products, so S-AM
It is advantageous for the production of In addition, the above-mentioned immobilized microorganism or its processed product can not only be used repeatedly in enzyme reactions, but also the enzyme reaction using the immobilized product can be carried out not only by the batch method but also by the column method. In particular, when using a column method, S-AM can be produced continuously. Example 1 (1) Preparation of air-dried yeast: 5% glucose, 1.5% casamino acids, 0.1% potassium phosphate, 0.1 potassium phosphate
%, 0.03% magnesium sulfate heptahydrate, and 1.5% yeast extract (PH 5.0) to culture Satucharomyces cerevisiae IFO2044, and collect the yeast cells from the culture. The cells are washed once with physiological saline, dried under ventilation at room temperature for 20 hours, and then dried under reduced pressure in a desiccator. (2) Preparation of air-dried E. coli Escherichia coli B strain in a medium (PH7.0) containing 0.5% glycol, 1.0% peptone, 1.3% yeast extract, 0.5% meat extract, and 0.5% salt.
ATCC23226 is cultured, and the E. coli cells are collected from the culture. After washing the bacterial cells once with physiological saline, they are dried at room temperature under ventilation for 20 hours, and further dried under reduced pressure in a desiccator. (3) S-AM production and results: The air-dried yeast and air-dried Escherichia coli obtained in (1) and (2) were used in the amounts shown in Table 1 below (wet weight), and 20mM of ATP and acetyl phosphate were added. 30m
M, L-methionine 20mM, magnesium chloride
Add 50mM Tris-HCl buffer (PH7.5) to make a total volume of 2.0ml, and react at 37°C for 180 minutes with shaking. Add perchloric acid to the reaction solution at a final concentration of 0.5
% and stop the reaction. The reaction stop solution was centrifuged to remove bacterial cell residue, and S-AM in the supernatant was quantified. S-AM was quantified by passing a predetermined amount of the supernatant through a Dowex 50W (Na ⁺ ) column to adsorb it, eluting it with 4M sulfuric acid, and measuring the ultraviolet absorption of the eluate at 260 nm ( Same hereafter). The amount of S-AM produced was as shown in Table 1 below.

[Table] Example 2 In Example 1, the reaction using 100 mg/ml of air-dried yeast and 10 mg/ml of air-dried Escherichia coli (both as wet weight) was carried out by varying the pH as shown in Table 2 below.
Furthermore, the experiments were carried out in the presence or absence of potassium ions or ammonium ions. S- at that time
The amount of AM produced was as shown in Table 2.

[Table] Example 3 In Example 1, when carrying out the reaction using 100 mg/ml of air-dried yeast and 10 mg/ml of air-dried E. coli (both as wet weight), the same amount as in Example 1 was added every 1.5 hours after the start of the reaction. of acetyl phosphate was added. The amount of S-AM generated at that time was 1.12mg/ml
It was hot. Example 4 The yeast cells and Escherichia coli cells obtained in Example 1 are separately entrapping immobilized in a polyacrylamide gel.
Immobilization of both bacterial cells was carried out as follows. wet bacteria
Suspend 20g in 30ml of 30% potassium chloride aqueous solution,
To this, 5 g of acrylamide monomer and N,
0.25g of N'-methylene-bis-acrylamide in water
Add the solution to 15ml. Add to this 5% β-
Add 6 ml of (dimethylamino)propionitrile,
Furthermore, a solution of 0.4 g of potassium persulfate dissolved in 6 ml of water is added to form a gel. 50 g (wet weight) of the resulting gel is suspended in Tris-HCl buffer (PH 7.5) containing L-methionine, toluene is added to the suspension at a concentration of 10% W/V, and the suspension is stirred at room temperature for 30 to 120 minutes.
The obtained immobilized yeast and immobilized E. coli were
Using the amounts shown in the table, the reaction was carried out under the same conditions as in Example 1 (however, the reaction liquid volume was 4 ml). The amount of S-AM produced at that time was as shown in Table 3.

[Table] Example 5 After mixing the yeast cells and Escherichia coli cells obtained in Example 1 in the ratio shown in Table 4 below, 20 g of the mixed cells were fixed on polyacrylamide gel in the same manner as in Example 4. and treated with toluene. This immobilized bacterial cell 1
g under the same conditions as in Example 1.
The amount of S-AM produced at that time was as shown in Table 4.

[Table] Example 6 Using the immobilized bacterial cells prepared in Example 5 and shown in parentheses in Table 4, the reaction was repeated under the same conditions as in Example 5. The amount of S-AM generated at that time was the fifth
As shown in the table, it was confirmed that the immobilized bacterial cells could be used continuously for the production of S-AM.

【table】

Claims (1)

[Scope of Claims] 1 S-adenosyl-L from adenosine-5'-triphosphate and L-methionine belonging to the genus Satucharomyces
- Cells of a microorganism that produces methionine or a processed product thereof, and cells of a microorganism belonging to the genus Escherichia that generate adenosine-5'-triphosphate from an adenosine-5'-triphosphate precursor or a processed product thereof. 1. A method for producing S-adenosyl-L-methionine, which comprises reacting adenosine-5'-triphosphate or its precursor with L-methionine in the presence of L-methionine. 2 From adenosine-5'-triphosphate and L-methionine belonging to the genus Satucharomyces, S-adenosyl-L
- Cells of a microorganism that produces methionine or a processed product thereof, and cells of a microorganism belonging to the genus Escherichia that generates adenosine-5'-triphosphate from an adenosine-5'-triphosphate precursor, or a processed product thereof. , either alone or in combination, are encapsulated and immobilized in a gel lattice of a polymer, and an enzymatic reaction is carried out in the presence of the immobilized product.