JPH025397B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing N-acetyl-L-methionine, and more particularly to a method for producing N-acetyl-L-methionine from N-acetyl-DL-methionine amide using an enzyme. N-acetyl-L- which is the object compound of the present invention
Methionine is a compound useful as a pharmaceutical compound such as an infusion component or a food additive. Conventionally, the method for producing N-acetyl-L-methionine has been as follows: (1) After reacting optically active lysine with N-acetyl-DL-methionine to form a salt,
A method of obtaining the desired L-form by fractional crystallization (Japanese Patent Application Laid-open No. 48-22418) and (2) a method of acetylating L-methionine with acetic anhydride (Japanese Patent Application Laid-Open No. 55-42986) are known. However, production method (1) requires repeated recrystallization to obtain a compound with high optical purity, resulting in poor yields, while production method (2) tends to cause racemization during the reaction. Therefore, it has the disadvantage that reaction conditions must be strictly controlled. In recent years, there have been reports of a method for producing N-acetyl-L-methionine by using the enantiomer-distinguishing hydrolysis ability of enzymes and allowing serine protease or sulfhydryl protease to act on the ester of N-acetyl-DL-methionine. Yes (Unexamined Japanese Patent Publication No. 51-142595
issue). However, as the enzymatic reaction progresses, N-acetyl-L-methionine ester is hydrolyzed and the target product N-acetyl-L-methionine is produced.
Due to methionine, the pH value of the reaction solution decreases. Therefore, in order to maintain and continue the enzymatic reaction, there is a problem in that the pH of the reaction solution must be constantly adjusted to an optimum range. Under such circumstances, the inventors of the present invention have carried out intensive research to overcome the technical and economical difficulties described above, and as a result, it was completely unexpected that Erwinia (Erwinia ) Enantiodifferential hydrolysis catalyzed by the enzymes contained in the culture fluid obtained by culturing microorganisms belonging to the genus genus, bacterial cells collected from the culture fluid, or processed products of the bacterial cells. Through the reaction, only the 1-position amide bond of N-acetyl-L-methionine amide is specifically hydrolyzed to form N-
It was discovered that acetyl-L-methionine was produced. The present invention relates to N-acetyl-DL-methionine amide, which belongs to the genus Erwinia, and is derived from N-acetyl-DL-methionine amide.
The N-acetyl-L produced by allowing a culture of a microorganism capable of producing L-methionine, bacterial cells collected from the culture, or a processed product of the bacterial cells to act on N-acetyl-DL-methionine amide.
- A new method for producing N-acetyl-L-methionine that collects methionine. The method of the present invention will be explained in detail below. The microorganism used in the present invention belongs to the genus Erwinia and has the ability to produce N-acetyl-L-methionine from N-acetyl-DL-methionine amide.
carotovora) 3057 (Feikoken Bibori No. 6176). The enzyme-containing culture solution that catalyzes the enantiomer-differentiated hydrolysis reaction of the present invention can be prepared by culturing the above-mentioned microorganisms in a conventional manner. That is, as a carbon source for the nutrient medium, any carbon source that can be used by the above-mentioned microorganisms can be used, such as sugars such as glucose, fructose, sucrose, maltose, and dextrin, sugar alcohols such as glycerol and sorbitol, and fumar. Acids, organic acids such as citric acid, etc. can be suitably used. The amount added to the medium is usually 0.1 to 10
% is sufficient. Nitrogen sources include various inorganic acids such as ammonium chloride, ammonium phosphate, and ammonium fumarate, ammonium salts of organic acids, and natural organic nitrogen sources such as meat extract, yeast extract, cornstarch liquor, and casein hydrolyzate. In particular, natural organic nitrogen sources can also be used as carbon sources in many cases. Further, as inorganic salts, for example, potassium phosphate, sodium phosphate, potassium chloride, sodium chloride, magnesium sulfate, ferrous sulfate, etc. can be used. The culture conditions for the microorganism may be a temperature of about 20 to 40°C, a pH of about 5 to 8, and more preferably a temperature of about 30 to 35°C, a pH of about 6 to 7, and a pH of about 6 to 7.
It is convenient to carry out the cultivation under aerobic conditions for 16 to 72 hours. The culture fluid thus obtained may be used as is in the method of the present invention, or if components in the culture fluid interfere with the method of the present invention or if a large amount of bacterial cells is desired to be used, the culture fluid may be centrifuged. The bacterial cells may be once separated by a method such as the following, and then the isolated bacterial cells may be used.
Furthermore, processed products of the bacterial cells, such as freeze-dried bacterial cells,
Acetone-dried microbial cells, ground microbial cells, microbial extracts, and microbial cells or processed products thereof immobilized by known methods can also be used. The enantiomer-differentiated hydrolysis reaction of the present invention is usually carried out by mixing N-acetyl-DL-methionine amide, which is a substrate, with the above-mentioned culture solution, bacterial cells collected from the culture solution, or a treated product thereof in an aqueous medium. It is preferable to do so. The substrate concentration in the reaction solution is approximately
It can be used up to a high concentration of about 0.1 to 20%,
When reacting with a high concentration of substrate, even if the substrate remains insoluble in the reaction solution, it will dissolve as the reaction progresses, so it will not interfere with the progress of the reaction.
The mixing method of substrate and culture solution etc. can be batch method or
A continuous method using a column is also possible. In the reaction, the pH of the reaction solution is about 5 to 9, preferably about 5.5 to 7.5, and the reaction temperature is usually about 20 to 60°C.
More preferably, the process proceeds smoothly when the temperature is about 30 to 40°C. Target compound N of the present invention obtained as described above
- Confirmation and identification of acetyl-L-methionine can be carried out, for example, by extracting the target product from the reaction solution by treatment with an ion exchange resin, recrystallizing it from isopropanol, elemental analysis of the obtained crystals, and thin layer chromatography using various developing solvents. The R value, melting point, optical rotation, NMR spectrum, IR spectrum, etc. of the sample were compared with those of the standard substance. For quantitative determination of the target product, for example, take a portion of the reaction solution, add one part as is, and add commercially available mold acylase (which specifically acts only on N-acetyl-L-methionine) to the other at 37°C. to react with N-
Acetyl-L-methionine was completely converted to L-methionine, and L-methionine in both samples was quantified by a microbial assay using Leuconostoc mesenteroides P-60. The amount of acetyl-L-methionine produced was calculated. As detailed above, according to the method of the present invention, N-
N-acetyl-L-methionine is produced by hydrolysis of the amide moiety at position 1 of acetyl-L-methionine amide, and at the same time, the same mole of ammonia is produced as a by-product, so there is essentially no PH fluctuation in the reaction solution. The operation is extremely simple as there is no need to adjust the pH of the reaction solution throughout the reaction period, and since the reaction is an enzymatic reaction, the specificity for optical isomers is extremely high, and the resulting N-acetyl-L-methionine It has many advantages such as extremely high optical purity. Incidentally, it has not been known until now that the enzyme that catalyzes the above-mentioned new enzymatic reaction is present in the culture solution of Erwinia carotovora, the cells collected from the culture solution, and the bacterial cell products. There has also been no report on an enzymatic reaction that acts on acetyl-DL-methionine amide to produce N-acetyl-L-methionine. Furthermore, known enzymes such as acylamidase or enzymes that act on DL-amino acid amide to produce L-amino acids, such as leucine aminopeptidase and aminoacylamidase, are completely N-
It has no effect on acetyl-DL-methionine amide. Therefore, the enzyme that catalyzes the enzymatic reaction of the present invention, which acts on N-acetyl-DL-methionine amide to produce N-acetyl-L-methionine, is a completely new enzyme. Example 1 Dibasic sodium phosphate/decahydrate 0.875%, monobasic potassium phosphate 0.34%, ammonium sulfate 0.1%,
Magnesium sulfate heptahydrate 0.058%, camsium chloride dihydrate 0.006%, ferrous sulfate heptahydrate 0.002
%, manganese sulfate hexahydrate 0.0002%, L-asparagine 0.2%, and yeast extract 1.0%. Erwinia cultured on agar medium at 30℃ for 20 hours.
One platinum loop of Carotovora 3057 (Feikoken Bacteria No. 6176) was inoculated and cultured at 30℃ for 24 hours with shaking (rotation speed
140 γ pm, amplitude 8 cm) and use the culture solution obtained as the seed culture solution. 2% glycerol, 0.1% ammonium chloride,
Monobasic potassium phosphate 0.1%, magnesium sulfate
Nutrient liquid medium (PH
7.0) Pour 50ml into a 500ml Sakaguchi flask and make 120
After sterilizing at 30°C for 10 minutes, 1 ml of the above seed culture was inoculated and cultured with shaking at 30°C for 2 hours. Collect bacteria from 50 ml of culture solution by centrifugation, and
After washing with 1 ml of physiological saline, suspend in 25 ml of 0.2 M acetate buffer (PH5.5), and add 100 ml of the same buffer separately.
N-acetyl-DL dissolved to mg/ml
- Add 25 ml of methionine amide solution and carry out the enzyme reaction at 30°C. Take a 0.5ml sample from the reaction solution and incubate at 100℃.
After stopping the reaction by heating for 5 minutes, water 4.5
ml and remove bacteria by centrifugation. Appropriately dilute a portion of the supernatant to obtain standard N-acetyl-L.
- Thin layer chromatography simultaneously with methionine (solvent; chloroform: methanol: acetic acid = 85:
15:3) and confirm the production of N-acetyl-L-methionine by iodine coloring. In addition, a portion of the supernatant was diluted 50 times with 50 mM phosphate buffer at pH 7.5, and 10 mg of commercially available Aspergillus oryzae acylase was added to 1 ml and reacted at 37°C for 1 hour. - Hydrolyze methionine to L-methionine. This solution was diluted 50 to 100 times and Leuconostoc mesenteroides P-60 was added.
L-methionine was quantified by a microbial assay using a microbial assay, and the amount of N-acetyl-L-methionine was determined. The amount of N-acetyl-L-methionine produced with respect to the reaction time was 100 mg/ml of N-acetyl-DL-
50 mg/ml N after 20 hours from methionine amide.
-Acetyl-L-methionine was produced, and its time course is shown in Table 1.

[Table] Example 2 Erwinia carotovora 3057 (Feikoken Bibori No. 6176), which was seed cultured in the same manner as in Example 1, was mixed with 2% glycerol, 0.1% ammonium chloride, 2% polypeptone, and 0.1% monobasic potassium phosphate. , 1% was inoculated into a nutrient liquid medium (PH7.0) consisting of 0.05% magnesium sulfate/heptahydrate, and cultured with shaking at 30°C for 28 hours. Bacteria were collected from 200 ml of culture solution by centrifugation, washed once with physiological saline, and suspended in pure water to make 100 ml.
There, N-acetyl-DL-methionine amide
Add 100ml of an aqueous solution containing 15g and adjust the pH to 6.0 with acetic acid.
It was prepared as follows. After reacting at 30°C for 24 hours, the amount of N-acetyl-L-methionine contained in the supernatant after removing the bacterial cells by centrifugation was determined using the same method as in Example 1. The amount was 7.2 g (conversion rate 98%). ). After adjusting the pH of this supernatant to 2 with hydrochloric acid, it is passed through a weakly basic ion exchange resin WA-10 (OH ^- type, trade name manufactured by Mitsubishi Kasei) column to adsorb N-acetyl-L-methionine. After thoroughly washing the column with water, N-acetyl-L-methionine is eluted with 3% aqueous ammonia. Excess ammonia was removed under reduced pressure, and the eluate was passed through a strongly acidic ion exchange resin IR-120 (H ⁺ type, trade name manufactured by Rohm and Haas) column, and the passed liquid was concentrated to obtain N-acetyl-L- Obtain crude crystals of methionine. By recrystallizing this from a small amount of isopropanol, 5.8 g of N-acetyl-L-methionine was obtained. The melting point of this product is 104°C, and the specific optical rotation is [α] _D -20.2° (C=4, water). Example 3 2 g of live cells of Erwinia carotovora 3057 (Feikoken Bacterium No. 6176) prepared in the same manner as in Example 2
Add 2 ml of physiological saline to the solution, stir, and add 8.5 ml of 5% carrageenan sol prepared in advance.
Cool on ice to gel, then add to 2% potassium chloride aqueous solution.
After soaking for 30 minutes, shape into a cube of 3 x 3 x 3 mm. 6 ml of this molded gel was packed into a column with a jacket tube in which water at 30°C was circulated, and a 50 mg/ml N-acetyl-DL-methioninamide aqueous solution (coexisting with 2% ammonium acetate, prepared with acetic acid to pH 6.0) SV=
Flow down at 0.2. N-acetyl-L in flowing liquid
- Methionine was quantified in the same manner as in Example 1 and found to be 24.9 mg/ml (conversion rate 99%).

Claims (1)

[Scope of Claims] 1. A culture solution of a microorganism that belongs to the genus Erwinia and has the ability to produce N-acetyl-L-methionine from N-acetyl-DL-methionine amide, and bacterial cells collected from the culture solution. Or N-acetyl-L-methionine is collected by allowing the processed product of the bacterial cells to act on N-acetyl-DL-methionine amide and collecting the produced N-acetyl-L-methionine.
- Method for producing acetyl-L-methionine. 2. The production method according to claim 1, wherein the microorganism is Erwinia carotovora 3057, which has the ability to produce N-acetyl-L-methionine from N-acetyl-DL-methionine amide.