JPH0438398B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-2-amino-4-phenylbutyric acid. The target compound of the present invention, L-2-amino-4-phenylbutyric acid, is an important substance as a pharmaceutical raw material,
It is also a useful substance that has various uses in the biochemical field as a similar compound to the essential amino acid L-phenylalanine. Conventionally, L-2-amino-4-phenylbutyric acid has been produced by optically resolving chemically synthesized DL-2-amino-4-phenylbutyric acid (V.
Du Vigneaud and OJIrish.J.Biol.Chem. 122 349
(1938)), but these methods have problems in terms of yield and operation, and cannot be said to be industrially advantageous. On the other hand, typical methods for directly biochemically converting α-keto acids into α-amino acids include reductive amination using enzymes or transfer of the amino group of other α-amino acids. However, when such a biochemical reaction is used, for example, the enzyme that converts pyrupic acid to L-alanine converts oxalacetic acid into L-alanine in most cases.
The enzyme used usually has high substrate specificity, such as not converting it to aspartic acid. Therefore, even though there are enzymes that catalyze chemical changes in biological substances, it is not possible to use such biochemical methods to treat 2-oxo-4-phenylbutyric acid, which is not a biological substance. Whether or not the same compound is L
This was completely unpredictable since no enzyme that converts -2-amino-4-phenylbutyric acid has been discovered so far. As a result of extensive research in view of this situation, the present inventors discovered that certain microorganisms contain an enzyme that converts 2-oxo-4-phenylbutyric acid to L-2-amino-4-phenylbutyric acid. , we have completed the present invention. That is, according to the present invention, L-2-amino-4-
Phenylbutyric acid is derived from the genus Achromobacter, genus Acinetobacter, genus Bacillus,
Belongs to the genus Paracoccus, and produces L-2-amino-4 from 2-oxo-4-phenylbutyric acid.
- It can be produced by allowing a culture solution of a microorganism capable of producing phenylbutyric acid, bacterial cells obtained from the culture solution, or a processed product of the bacterial cells to act on 2-oxo-4-phenylbutyric acid in the presence of L-amino acids. can. The microorganisms used in the present invention belong to the genus Achromobacter, Acinetobacter, Bacillus, and Paracoccus, and have the ability to produce L-2-amino-4-phenylbutyric acid from 2-oxo-4-phenylbutyric acid. Any of them can be used, such as Achromobacter, Achromobacter liquidum OUT 8012 (Feikoken Bibori No. 12684), Acinetobacter, Acinetobacter calcoacetics IFO
12552, Bacillus cereus
Preferred examples include IFO 3001 and Paracoccus denitrificans IFO 12442. The above microorganisms can be cultured under normal conditions. 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 glycerose and sorbitol, and fumaric acid. Organic acids such as , citric acid and the like can be used. The amount added to the medium is usually preferably about 0.1 to 10%. Examples of nitrogen sources include ammonium salts of inorganic acids such as ammonium chloride, ammonium sulfate, and ammonium phosphate, ammonium salts of organic acids such as ammonium fumarate, and natural sources such as meat extract, yeast extract, corn staple liquor, and casein hydrolyzate. An organic nitrogen source can be used, and in many cases, the organic nitrogen source can also be used as a carbon source. The amount of nitrogen source added is usually preferably in the range of 0.1 to 10%.
Preferred examples of inorganic salts include alkali metal phosphates such as potassium phosphate and sodium phosphate, alkali metal chlorides such as potassium chloride and sodium chloride, metal sulfates such as magnesium sulfate, and ferrous sulfate. can be used,
The amount used is usually preferably in the range of 0.001 to 1%. For culturing microorganisms, pH is approximately 5-8, 20-40℃
In particular, it is preferably carried out under aerobic conditions at a pH of about 6-7 and 30-37°C. Next, the culture fluid obtained as described above, the bacterial cells obtained from the culture fluid, or the processed product of the bacterial cells are used as an enzyme source, and this is used as an enzyme source in the presence of L-amino acids.
L-2-amino-4-phenylbutyric acid can be produced by acting on oxo-4-phenylbutyric acid. Examples of bacterial cells obtained from a culture solution include bacterial cells separated by centrifugation, filtration, etc., and processed bacterial cells include freeze-dried bacterial cells, acetone-dried bacterial cells, washed bacterial cells, and bacterial cell polishing. Examples include crushed materials, autolysed bacterial cells, ultrasonicated bacterial cells, bacterial cell extracts, and those obtained by immobilizing these by a known immobilization method such as a gel entrapment method or an adsorption method. Specific examples of immobilized materials include culture solutions, bacterial cells, or treated bacterial cells, such as polyacrylamide gels, sulfur-containing polysaccharide gels (carrageenan, fur-cerelan, etc.), collagen gels, alginate gels,
Examples include those fixed with polyvinyl alcohol gel, agar gel, etc. Furthermore, as the enzyme source of the present invention, an enzyme purified and collected from a bacterial cell extract by a combination of known methods can also be used. The reaction may be carried out by adding a substrate to a culture solution containing microbial cells, or the microbial cells obtained from the culture solution or the processed product of the microbial cells may be added to an aqueous solution of the substrate and the reaction may be carried out. . Examples of the L-amino acid used in the above-mentioned main reaction include L-aspartic acid, L-glutamic acid, and L-alanine, and it is preferable to use at least an equivalent molar amount to the substrate. In addition, shortening of reaction time or L-2-amino-
In order to increase the amount of 4-phenylbutyric acid accumulated, it is preferable to carry out the reaction in the presence of a surfactant and/or a coenzyme. Examples of surfactants that can be used for this purpose include cetyltrimethylammonium bromide, polyethylene glycol, and p-isooctyl phenyl ether (trade name: Triton X-100, manufactured by Rohm and Haas). The amount used is 0.0001~ relative to the reaction solution.
It is preferably about 0.1%. In addition, as a coenzyme that can be used for the above purpose, for example, pyridoxal phosphate can be mentioned, and it is generally
It is preferable to use it at a concentration of about 0.001 to 1 mM. The L-2-amino-4-phenylbutyric acid accumulated in the reaction solution in this way is almost insoluble in water and can be easily separated and collected from the reaction solution using conventional means such as filtration or centrifugation. can do. In addition, since L-2-amino-4-phenylbutyric acid becomes soluble in water when made into an acid or alkaline salt, an acid or alkali is added to the reaction solution to remove impurities and the solution is neutralized. -amino-4-
After phenylbutyric acid crystals are precipitated, they can be separated and collected from the reaction solution by conventional methods such as filtration or centrifugation. Hereinafter, the present invention will be specifically explained with reference to Examples. In addition, the quantitative determination of 2-oxo-4-phenylbutyric acid and L-2-amino-4-phenylbutyric acid in the examples was carried out by liquid chromatography.
-Amino-4-phenylbutyric acid was confirmed using the elemental analysis values of the obtained crystals and the pre-synthesized N-acetyl-
L-2-amino-4-phenylbutyric acid produced by reacting commercially available aminoacylase with DL-2-amino-4-phenylbutyric acid and specific rotation, NMR and IR
This was done by comparing spectra. In this specification, "%" means "weight/volume (g/ml)". Example 1 (1) 1% glucose, 1% casein hydrolyzate,
Yeast extract 1%, diammonium hydrogen phosphate
0.2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, ferrous sulfate 0.001%, calcium chloride 0.01%, and Calalin 102 (trade name manufactured by Sanyo Chemical Industries, Ltd.) 0.003%.
(PH7.0) was sterilized at 120°C for 10 minutes. One platinum loop of Paracoccus denitrificans IFO 12442 was inoculated into the medium, and cultured with shaking at 30°C for 18 hours. After culturing, the culture solution is centrifuged to collect bacteria, and the cells are freeze-dried to obtain 0.5 g of freeze-dried cells.
was prepared. (2) 4g of 2-oxo-4-phenylbutyric acid, 3.2g of L-aspartic acid and pyridoxal phosphate
Dissolve 0.003g in water, adjust the pH to 8.5 with aqueous ammonia, add water to make a total of 100ml, and add 1g of the freeze-dried bacterial cells prepared in (1) above to the substrate solution.
The reaction was carried out at 30°C for 2 days. Hydrochloric acid was added to the reaction solution to dissolve the product, 2 g of activated carbon was added, and the mixture was filtered under suction. By neutralizing the liquid with sodium hydroxide and collecting the precipitated crystals, L-2
3.1 g of -amino-4-phenylbutyric acid was obtained. Yield 78% Melting point: 286-288°C (decomposition) [α] ²² _D +47°C (C = 1, 1N-HCl) Examples 2 to 4 In the same manner as Example 1-(1), the results are shown in Table 1 below. The microorganisms shown were cultured, and the bacterial cells collected from 100 ml of the culture solution by centrifugation were added to a substrate solution (2-oxo-4
-4 g of phenylbutyric acid, 3.2 g of L-aspartic acid,
Cetyltrimethylammonium bromide 10mg to 100ml
Dissolved in water, pH8.5 with sodium hydroxide aqueous solution
(prepared as ). This suspension was heated at 30℃ for 2 hours.
By reacting for several days, L-2-amino-4
-Phenylbutyric acid was obtained. The results are shown in the table below.

[Table] Example 5 100g of glucose, 150g of casein hydrolyzate,
50g corn staple liquor, 100g yeast extract,
Diammonium hydrogen phosphate 20g, dipotassium hydrogenphosphate 10g, magnesium sulfate 5g, ferrous sulfate
A medium (PH7.0) consisting of 0.1 g, 1 g of calcium chloride, 0.3 g of Calalin 102 (trade name manufactured by Sanyo Chemical Industries, Ltd.) and 10 g of water was added to MSJ-u manufactured by Marubishi Corporation.
Pour into a 20 volume jar fermentor and heat at 120℃ for 10 minutes.
Sterilized for minutes. Cell fluid of Paracoccus denitrificans IFO 12442 cultured in a flask under shaking at 30°C for 18 hours in this medium.
Inoculate 100ml and inoculate at 1/2vwm, 400rpm, 30℃ for 20
Cultured for hours. Next, 2-oxo-
4-phenylbutyric acid 400g, L-aspartic acid 320g
After adding 1 g of cetyltrimethylammonium bromide and adjusting the pH to 8.5 with aqueous ammonia, the mixture was reacted at 37°C for 2 days. 2-oxo-4-phenylbutyric acid was not observed in the culture solution after 2 days, and L-2-
360 g of amino-4-phenylbutyric acid was produced and accumulated. Impurities such as bacterial cells and culture fluid were removed from the reaction solution using a centrifuge, and the remaining crystals were suspended in pure water and washed to obtain 320 g of crystals of L-2-amino-4-phenylbutyric acid. Yield 79.5%.

Claims (1)

[Claims] 1 2-oxo-4-phenylbutyric acid to L-2-
A culture solution of a microorganism of the genus Achromobacter, Acinetobacter, Bacillus or Paracoccus that has the ability to produce amino-4-phenylbutyric acid, cells obtained from the culture solution, or a processed product of the cells are used to produce L-amino acids. 2-oxo-4-phenylbutyric acid in the presence of L-2-amino-4-
L-2 characterized by collecting phenylbutyric acid
-Production method of amino-4-phenylbutyric acid. 2. The production method according to claim 1, wherein the L-amino acid is L-aspartic acid, L-glutamic acid, or L-alanine.