JP2670838B2 - Method for producing L-α-amino acids - Google Patents

Description

TECHNICAL FIELD The present invention utilizes microorganisms to produce L-α-amino acids from nitrile compounds, particularly racemic α- (N-alkylideneamino) nitrile compounds. Regarding the method.

L-α-amino acids are important chemical substances used in various fields such as food, feed, medicine and cosmetics.

BACKGROUND ART Conventionally, as a method for producing L-α-amino acids,
Fermentation methods, synthetic methods, enzymatic methods and extraction methods are known. Among these methods, the L-α-amino acid can be produced by a synthetic method by synthesizing DL-α-amino acid using the Strecker method or its modified method, and then the amino acid is optically resolved to give L-α-amino acid.
An α-amino acid is produced, and an enzymatic method using aminoacylase is adopted as a method of optical resolution (“Chemical” magazine special issue “Asymmetric synthesis and progress of optical resolution”, Showa era).
Published October 15, 1957, p. 175).

However, the method for producing an L-α-amino acid by the above-mentioned synthetic method has a high cost in the step of optical resolution, and therefore the proportion of the amino acid in the production has gradually decreased in recent years due to economic reasons.

In addition, the following general formula, which is a synthetic intermediate in the synthesis of α-amino acid by the Strecker method, (R represents an alkyl group, a phenyl group, etc.)
Attempts to produce α-amino acids from DL-α-aminonitrile using microorganisms have also been reported [Y. Fuku.
da et al., “J.Ferment, Technol.” 49 , 1011
(1971)]. However, in this report, we used DLs using microorganisms belonging to Corynebacterium sp.
-Α-aminopropionitrile and DL-α-aminoisovaleronitrile are hydrolyzed to produce DL-alanine and DL
-Production of valine, L-α-amino acid is not directly obtained. In addition, Brevibacterium (Brevib
Amino acid production by hydrolyzing DL-α-aminonitrile using strain R312 of acterium sp. [JC Jall
agas et al. “Advance of Biochemical Engineering (Abv.Biochem.Engineer.)” 14 , 1 (1
980)], the DL-α-aminonitrile was
Only -α-amino acids were obtained. Incidentally, the above report shows that by using Brevibacterium sp.A4, which is a mutant strain obtained from strain R312 of the genus Brevibacterium.
From L-α-aminonitrile to L-α-amino acid and D-α
Amide acid amide, but there is no report yet on directly obtaining only L-α-amino acid from DL-α-aminonitrile.

Further, a method of producing an amino acid by irradiating with light before the hydration reaction of a DL-α-aminonitrile compound by a microorganism has been disclosed (Japanese Patent Laid-Open No. 61-162191). Only an α-amino acid is obtained, and no example of directly obtaining an L-α-amino acid from an α-aminonitrile compound has been reported. Furthermore, no report has yet been found that an α-amino acid was produced by hydrolyzing an α- (N-alkylideneamino) nitrile compound used as a raw material in the present invention using a microorganism.

Problems to be Solved by the Invention In view of the above-mentioned current situation, the present inventors have conducted intensive studies and as a result, have found that the genus Rhodococcus sp., The genus Mycobacterium ( Mycobacterium sp.), And the genus Arthrobacter ( Arthrobacter) It has been found that when a microorganism having a nitrile hydrolyzing ability belonging to sp.) is allowed to act on α- (N-alkylideneamino) nitriles, L-α-amino acids are selectively produced. The present invention has been made based on such findings, and utilizes a microorganism having a hydrolyzing ability of a nitrile selected from a specific genus to directly obtain an L-α-amino acid from α- (N-alkylideneamino) nitriles. It is an object to provide a method for producing a class.

Means for Solving the Problems The present invention provides the following general formula (I) Alternatively, the following general formula (II) (However, in the formula, R ₁ and R ₂ are an alkyl group, a substituted alkyl group, a phenyl group, a substituted phenyl group, an imidazolyl group, a substituted imidazolyl group, an indolyl group, a substituted indolyl group,
A furyl group, a substituted furyl group, a pyridyl group, a substituted pyridyl group, a thiazolyl group, or a substituted thiazolyl group, wherein R ₁ and R ₂ may be the same or different, and one or more kinds of nitriles Compounds include Rhodococcus sp., Mycobacte
rium sp.) or Arthrobacter s
p.) and is converted into L-α-amino acids by the action of a microorganism having a nitrile hydrolysis activity.

Microorganisms used in the present invention, as described above,
Rhodococcus (Rhodococcus sp.), Mycobacterium (Mycobacterium sp.) And Arthrobacter (A
rthrobacter sp.) having a hydrolyzing ability of a nitrile selected from the group belonging to rthrobacter sp.), which can be exemplified by those shown in Table 1 below.

These microorganisms have been deposited at the Institute of Microbial Technology, Institute of Industrial Science and Technology with the deposit accession numbers shown in Table 1 on November 5, 1987.

 Next, Table 2 shows the mycological properties of the above-mentioned microorganisms.

As seen in Table 2, all of the microorganisms used in the present invention are Gram-positive aerobic bacteria and do not grow at all in the anaerobic state. It also shows no motility, is positive for catalase and does not form thermostable spores. In addition, it shows polymorphism, many mycelial cells are present in the early stage of culture, and branched cells and spherical cells are also observed. For sugar availability,
It produces no gas from sugar, has a weak acid-producing ability, and when cultured in litmus milk, it becomes alkaline and turns milk blue.

Among the above-mentioned each microorganism, Rhodococcus sp. (Rhodococcus
sp.) PC-29 forms a slightly dry wrinkle-like colony on broth agar, shows hyphal growth with marked branching in the early stage of culture, and grows at 5 to 32 ° C, but not at 37 ° C. .

Meanwhile, Rhodococcus sp. (Rhodococcus sp.) PA -34,
AB-16 and BA-1 do not grow at 5-10 ° C, but 37
It grows even at ~ 42 ° C, and both become hyphae at the beginning of culture.
It then gives rise to a large number of Oidiospore-like spherical cells.

Mycobacterium sp. AB-43 was treated with acid-fast staining (Acid-f
ast stain) is positive and does not grow at 45 ℃ and forms a viscous yellow-orange colony. Arthrobac sp. ( Arthrobac
ter sp.) PA-15 and PC-3 are Gram-positive bacilli, but their morphology is quite irregular, and when cultured for a long time, granules are formed in the cells, Gram stainability becomes negative, and globular Cells also appear. This strain is a psychrophilic bacterium that grows at 5-10 ° C and does not grow at 37 ° C or higher. The broth agar-like colony has a strong yellow-green, creamy, gelatin and starch hydrolyzing power, but does not have a cellulose decomposing power.

In view of those Table 2 and the above-mentioned properties, the above-mentioned microorganisms are the Bergey's Manual of Systemati
c Bacteriology, 1986).

In the present invention, the α- (N-alkylideneamino) nitrile compounds represented by the general formulas (I) and (II), which are substrates used for producing L-α-amino acids using the above-mentioned microorganisms (hereinafter The raw material nitrile is abbreviated as the first step reaction of the Strecker method, for example, synthesis of an α-aminonitrile compound [eg, Journal of Fermentation Technology (J.
ent.Technol.), 49 1011 (1971) or Chemistry Letters (Chem. Lett.), 687 (1987)],
When the disappearance of the starting aldehyde is confirmed by analysis by gas chromatography or the like, it can be obtained by immediately separating the inorganic salt by an operation such as filtration or extraction and concentrating it. This crude product has the general formula (I)
And α- (N-alkylideneamino) nitrile compound represented by (II), which is a partial hydrolyzate of α-aminonitrile, and aldehyde which is a synthetic raw material may be contained as impurities. After removing with
Although it is used as a substrate, it is also possible to use the crude product as it is as a substrate.

R ₁ and R ₂ in the general formulas (I) and (II) of the nitrile compound used as the substrate of the present invention are not particularly limited, but the substituents contained in each of the substituted alkyl groups and the like are
For example, hydroxy, methoxy, mercapto, methylmercapto, amino, halogen, carboxyl, carboxamide, phenyl, hydroxyphenyl or guanyl are suitable. Further, if R ₁ and R ₂ are used as raw material nitriles of the same group, one kind of L-α-amino acid can be obtained, and if raw material nitriles of different groups are used, respectively, two kinds of L-α-amino acids are mixed. An α-amino acid can be obtained. Further, it goes without saying that there is no problem even if two or more kinds of the above raw material nitriles are mixed and used.

Examples of the α- (N-alkylideneamino) nitrile compound include 2-aminopropanenitrile, 2-aminobutanenitrile, 2-amino-3-methylbutanenitrile and 2
-Amino-4-methylpentanenitrile, 2-amino-
3-methylpentanenitrile, 2-amino-3-hydroxypropanenitrile, 2-amino-3-hydroxybutanenitrile, 2-amino-5-guanidinopentanenitrile, 2-amino-3-mercaptopropanenitrile, 2,7- Diamino-4,5-dithiaoctanenitrile, 2
-Amino-4-methylthiobutanenitrile, 2-amino-3-phenylpropanenitrile, 3- (4-hydroxyphenyl) propanenitrile, 3-amino-3-cyanopropanoic acid, 4-amino-4-cyanobutanoic acid, 3 −
Amino-3-cyanopropanamide, 4-amino-4-
Cyanobutanamide, 2,6-diaminohexanenitrile, 2,6-diamino-5-hydroxyhexanenitrile, 2-amino-3- (3-indolyl) propanenitrile, 2-amino-3- (4-imidazolyl) propane Α-aminonitrile compounds such as nitrile, 2-cyanopyrrolidine, 2-cyano-4-hydroxypyrrolidine and 2-amino-2-phenylethanenitrile;
A Schiff base body with an aldehyde which is a synthetic raw material of amino nitrile can be exemplified.

In the present invention, the above raw material nitrile is added to the genus Rhodococcus sp. And Mycobacterium sp.
acterium sp.) and Arthrobacter
In order to produce the L-α-amino acid by causing each of the above microorganisms having a nitrile hydrolysis activity belonging to sp.) to produce L-α-amino acid, for example, any of the following methods (a) to (c) may be applied.

That is, (a) a method of reacting a microorganism obtained by culturing a microorganism by culturing the microorganism in a medium containing a nitrile compound, for example, propionitrile, with a raw material nitrile to react, and (b) culturing the microorganism in advance Then, the bacterial cells obtained by growing are contacted with a nitrile compound, for example, propionitrile, and then a method of reacting by adding a raw material nitrile to the bacterial cells,
And (c) a method of culturing a microorganism in advance and directly contacting the raw material nitrile with the bacterial cells obtained by the growth and reacting them.

In addition, in these reaction methods, crushed cells after growth, dried cells, or treated cells such as separated and purified nitrile hydrolase, or cells and cells immobilized according to a conventional method A processed product can also be used.

Examples of the nitrile compound used in the above methods (a) and (b) include, in addition to propionitrile, acetonitrile, n-butyronitrile, n-capronitrile, methacrylonitrile, isobutyronitrile, glutaronitrile, triacrylonitrile, Examples thereof include crotononitrile, lactonitrile, succinonitrile, acrylonitrile, benzonitrile and phenylacetonitrile.

In the method (a) above, in addition to the nitrile compound, glucose, sucrose, molasses, a sugar such as a starch hydrolyzate as a carbon source, or a substance having a cell growth action such as acetic acid is added to the medium. In addition, nitrogen sources such as ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, aqueous ammonia, sodium nitrate, amino acids and other assimilable organic nitrogen compounds, potassium phosphate, sodium phosphate, magnesium nitrate, Inorganic salts such as manganese sulfate, ferrous nitrate, ferric chloride, calcium chloride and manganese chloride, and salts such as boric acid, copper and zinc, that is, so-called trace elements,
Further, if necessary, a seed medium of each of the above-mentioned microorganisms is inoculated into a medium to which a growth promoting substance such as vitamins, yeast extract and corn stapler is added, and cultured under aerobic conditions to grow the cells. The cell culture thus obtained,
Alternatively, the raw material nitrile is supplied to the suspension of the bacterial cells separated from the culture or a treated product of the bacterial cells to react them.

The reaction is carried out in the range of pH 4 to 13, preferably pH 8 to 12 in the range of 1 to 6
Perform for days. Although various buffers may be used in the reaction, ammonia-based buffers are preferable, such as a mixture of ammonia water and an ammonium chloride aqueous solution, a mixture of ammonia water and an ammonium sulfate aqueous solution, a mixture of ammonia water and an ammonium phosphate aqueous solution, and an ammonia water. A mixture with an aqueous solution of ammonium acetate is preferred. Ammonia water is preferred as the alkali for pH adjustment.

The reaction temperature is preferably in the range of 20 to 70 ° C, and the above-mentioned carbon source, nitrogen source, and other components used for cell growth during the reaction are appropriately added to maintain the cell concentration and the nitrile hydrolyzing ability of the cell. And can be enhanced. Further, as a method of supplying the raw material nitrile, any of a method of adding at the start of the reaction, a method of intermittently adding, and a method of continuously adding can be adopted.

The L-α-amino acid produced by the above reaction is separated and collected by applying known means such as phase separation, filtration, extraction and column chromatography.

Next, in the method (b), the nitrile compound is added after the growth of the bacterial cells without adding the nitrile compound during the culture and growth of the bacterial cells in the method (a), so that the nitrile hydrolyzing ability of the bacterial cells can be improved. After activation, it is reacted with the raw material nitrile to produce L-α-amino acid.

In the method (c), the raw material nitrile is added immediately after the growth of the cells in the method (b) to cause the reaction to produce the L-α-amino acid.

In any of the above methods (b) and (c), the method in the above method (a) can be applied to the culture conditions, the reaction conditions, and the separation and collection of the produced L-α-amino acid.

The L-α-amino acid obtained according to the present invention as described above is used in various fields such as food, feed, medicine and cosmetics.

 Hereinafter, the present invention will be described specifically with reference to examples.

Example 1 Preparation of medium 100 ml of medium having the following composition was placed in a 500 ml flask.
After sterilizing in an autoclave for 20 minutes, 1 ml of propionitrile that had been sterilized with a 0.2 μm Millipore filter was added to obtain a medium for cell preparation.

Medium composition: Glucose 10 g / yeast extract _{0.1 g / Na 2 HPO 4 ·} 12H 2 O 2.5 g / KH 2 PO 4 2.0 g / MgSO 4 · 7H 2 O 0.5 g / FeSO 4 · 7H 2 O 0.03g / CaCl 2・ 2H ₂ O 0.06g / pH 7.2 Strains used: Strain name FERM - BP No. Rhodococcus sp. PC-29 1561 Rhodococcus sp. PA-34 1559 Rhodococcus sp. AB-16 1555 Rhodococcus (Rhodococcus sp.) BA-1 1557 Mycobacterium (Mycobacteriumu sp.) AB-43 1556 Arthrobacter sp. (Arthrobacter sp.) PA -15 1558 Arthrobacter sp. (Arthrobacter sp.) PC -3 1560 Cultivation and Proliferation of Bacterial Cells The above medium was inoculated with 3 platinum loops of the following 7 strains, and shaking culture was performed at a temperature of 30 ° C. and 140 rpm for 48 hours.

The cells obtained by the above culture were separated by centrifugation and
After washing once with 0.1 M buffer of ₄ Cl-NH ₃ (pH 10), it was resuspended in 0.1 M buffer of NH ₄ Cl-NH ₃ so that the optical density (OD) was 40.

Reaction 5 mL of each cell suspension obtained as described above was
Store in a 4 mm test tube and add DL-3-methyl-2-
(N-2-methylpropylideneamino) butanenitrile
After 100 μl was added and the container was tightly closed, shaking culture was performed at a temperature of 30 ° C. and 300 rpm for 48 hours.

After completion of the reaction, the reaction solution was extracted twice with 5 ml of hexane, and the obtained aqueous phase was analyzed by high performance liquid chromatography.

The amount of L-valine produced was determined by using Asah as a column packing material.
ipak Inert Sill ODS (manufactured by Asahi Kasei) was used to determine the absolute configuration and optical purity of CHIRALPAK WH.
(Manufactured by Daicel Chemical Industries, Ltd.). Table 3 shows the results
As shown in FIG.

Example 2 Rhodococcus sp. Prepared by the method described in Example 1.
The reaction was carried out by the method described in Example 1 except that 50 mg of 4-methyl-2- (N-3-methylbutylideneamino) pentanenitrile was added to 5 ml of the bacterial suspension of ( Rhodococcus sp.) PA-34. As a result, L-leucine (2.5 g /) was produced, and its optical purity was 93% ee. High-performance liquid chromatography was used to determine the absolute configuration, and CH was used as the packing material.
IRALPAK WE (manufactured by Daicel Chemical Industries, Ltd.) was used.

Example 3 DL was added to 5 ml of the bacterial suspension prepared by the method described in Example 1.
-2- (N-butylideneamino) pentanenitrile 50μ
The reaction was carried out by the method described in Example 1 except that was added. The results of analyzing the produced L-norvaline by the method described in Example 2 are shown in Table 4.

Example 4 3 platinum loops of each of the microorganisms shown in Table 5 were deionized into NBG medium (Oxoid "Labrengo" powder, 10 g of code L29, 10 g of bacteriological bepton, code L37, glucose 10 g, sodium chloride 5 g). After adding water to 1 to adjust the pH to 7.5 with 1N sodium hydroxide aqueous solution, inoculate into a 500 ml flask containing 100 ml of liquid medium sterilized by heating in an autoclave at 120 ° C for 15 minutes.
Shaking culture (150 times / min) was performed at 48 ° C for 48 hours. The cells produced by this culturing were collected and washed by the method described in Example 1, and the optical density (OD) of NH ₄ Cl-NH ₃ was adjusted to 0.4.
Resuspended in 1M buffer. The bacterial suspension was reacted with 50 μL of DL-2- (N-butylideneamino) pentanenitrile by the reaction method described in Example 3, analyzed by the method described in Example 2, and the results shown in Table 5 were obtained. Got

Example 5 DL was added to 5 ml of the bacterial suspension prepared by the method described in Example 1.
-2- (N-pentylideneamino) hexanenitrile 50
The reaction was performed by the method described in Example 1 except that μ was added, and the produced L-norleucine was analyzed by the method described in Example 3. The results are shown in Table 6.

Example 6 DL was added to 5 ml of the bacterial suspension prepared by the method described in Example 4.
-2- (N-pentylideneamino) hexanenitrile 50
The reaction was carried out by the method described in Example 1 except that μ was added, and the produced L-norleucine was analyzed by the method described in Example 3 and the results are shown in Table 7.

Example 7 Example 1 except that among the methods described in Example 1, other nitrile compounds are used instead of propionitrile.
Rhodococcus sp. ( Rhodococc
us sp.) DL-3-methyl-2-to 5 ml of bacterial suspension of PA-34
(N-2-methylpropylideneamino) butanenitrile
Table 8 shows the results of reaction analysis by the method described in Example 1 after adding 50 μm.

Example 8 Example 2 except that Mycobacterium sp. AB-43 is used.
Culture, reaction, and analysis were carried out by the method described in 1., and the result was that the amount of L-leucine produced was 0.6 mg / ml and the optical purity was 83% ee.

Example 9 Reaction analysis was carried out by the method described in Example 2 except that 5 ml of the bacterial suspension prepared by the method described in Example 4 was used.
Was obtained.

Effects of the Invention As described above, according to the present invention, microorganisms can be utilized to selectively produce L-α-amino acids directly from a racemic α- (N-alkylideneamino) nitrile compound. It is useful in the production of L-α-amino acids used in the field.

Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display area C12R 1:06) (C12P 13/04 C12R 1:32)

Claims (1)

(57) [Claims] 1. A compound represented by the following general formula (I) Alternatively, the following general formula (II) (However, in the formula, R ₁ and R ₂ are an alkyl group, a substituted alkyl group, a phenyl group, a substituted phenyl group, an imidazolyl group, a substituted imidazolyl group, an indolyl group, a substituted indolyl group,
A furyl group, a substituted furyl group, a pyridyl group, a substituted pyridyl group, a thiazolyl group, or a substituted thiazolyl group, and R ₁ and R ₂ may be the same or different groups) 1
Rhodococcus sp., Mycobacterium spp.
rium sp.) or Arthrobacter s
A method for producing L-α-amino acids, which is characterized in that a microorganism having a nitrile hydrolysis activity belonging to p.) is allowed to act to convert it into L-α-amino acids.