JPH0724590B2 - Method for producing L-α-amino acids - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a corresponding L-α-amino acid from an aminonitrile compound using a microorganism. More specifically, a racemate and an α-aminonitrile compound are used in the presence of an aldehyde in the presence of a microorganism to obtain L.
This is a method for producing -α-amino acids.

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, a fermentation method, a synthesis method, an enzyme method and an extraction method have been known. Among these methods, the L-α-amino acid can be produced by the synthetic method by synthesizing DL-α-amino acid by using the Strecker method or its modified method, and then the amino acid is optically resolved to give L-α-amino acid.
-For producing amino acids, the enzymatic method using aminoacylase is adopted as a method of optical resolution ("Chemical" magazine special issue "Asymmetric synthesis and progress of optical resolution", Showa 57).
Issued October 15, 2014, page 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, an attempt to produce an α-amino acid using a microorganism from DL-α-aminonitrile, which is a synthetic intermediate in the synthesis of α-amino acid by the Strecker method, has been reported [Y. Fukuda et al. ., “Journal of Fermentation Technology (J.Ferment.Te
chnol.) " 49 , 1011 (1971)]. However, in this report, a microorganism belonging to Corynebacterium (Corynebacterium sp.) Was used to hydrolyze DL-α-aminopropionitrile and DL-α-aminoisovaleronitrile.
It produces DL-alanine and DL-valine, and cannot directly obtain L-α-amino acid. In addition, a report of hydrolyzing DL-α-aminonitrile to produce an amino acid using a strain R312 of the genus Brevibacterium sp. [JC Jallageas et al. “Advance of Biochemical Engineering (Abv. Biochem.
Engineer.) ” 14 , 1 (1980)], DL-α-
Only DL-α-amino acids are obtained from aminonitrile. Incidentally, the above report shows that strains of the genus Brevibacterium
Brevibacterium sp.A, a mutant obtained from R312
From DL-α-aminonitrile to L-
Although it is disclosed that α-amino acids and D-α-amidoamides can be produced, DL-α-aminonitriles can be used to form L-amino acids.
No report has yet been made on directly obtaining only -α-amino acids.

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.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention The present inventor, as a result of intensive research in view of the above-mentioned present situation, has found that Rhodococcus sp., Mycobacterium sp. And Arthrobacter sp. It was found that a microorganism having a nitrile hydrolyzing ability belonging to sp.) selectively produces L-α-amino acids when allowed to act on α-aminonitriles in the presence of aldehyde. The present invention was made on the basis of such findings, and for producing L-α-amino acids directly from α-aminonitriles by utilizing a microorganism having a hydrolyzing ability of nitrile selected from a specific genus. The purpose is to provide a method.

Means for Solving the Problems The present invention has the following general formula (I). Alternatively, the following general formula (II) (In the formula, R is 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, and thiazolyl. Group or a substituted thiazolyl group), one or more α-aminonitrile compounds represented by the following general formula (III) R-CHO (III) or the following general formula (IV) R-CH ₂ CHO (IV) (wherein R is the same as the above general formula (I) or (II)) in the presence of the corresponding aldehyde, Rhodococcus sp., Mycobacterium sp.
ycobacterium sp.) or Arthrobacter (Arthro)
bacter sp.) which has a nitrile-hydrolyzing activity and acts to convert it into L-α-amino acids.

The microorganisms used in the present invention are, as described above, the genus Rhodococcus (Rhodococcus sp.), The genus Mycobacterium (Mycobacterium sp.) And the genus Arthrobacter (Art
hrobacter sp.) having a nitrile hydrolysis ability selected from the group belonging to hrobacter sp.), and examples thereof include 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, the mycological properties of the above-mentioned microorganisms are shown in Table 1-2.

As seen in Table 1-2, all 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. Concerning the utilization of sugar, it does not generate gas from sugar, its acid production capacity is weak, and when it is cultured in litmus milk, it shows alkalinity and turns milk into blue.

Of the above microorganisms, Rhodococcus sp.
p.) PC-29 forms a slightly dry wrinkled 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. PA-34, AB
-16 and BA-1 do not grow at 5-10 ° C, but 37-
It grows even at 42 ° C, and all become hyphae in the early stage of culture, and thereafter, many Oidiospore-like spherical cells are formed.

Mycobacterium sp. AB-43 was treated with acid-fast staining (Acid-fas
T stain) is positive and does not grow at 45 ℃ and forms a viscous yellow-orange colony. In addition, Arthrobacter sp.
r 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. Villages on broth agar have a strong yellow-green, creamy, gelatin and starch hydrolyzing power but no cellulose degrading power.

In view of Table 1-2 and the above-mentioned properties, the above-mentioned microorganisms are categorized by Bergey's Manual of Systematic.
Bacteriology, 1986).

In the present invention, a DL-α-aminonitrile compound (hereinafter abbreviated as a raw material nitrile) that is a substrate used for producing L-α-amino acids using each of the above microorganisms is, for example, “organic synthesis collectible volume ( Org.Syn.Col.Vol.) I, p21 and III, p84 ”or“ Journal of Fermentation Technology (J.Ferment.Tachnol.), 49 1011 (1971) ”and the like. Can be synthesized.

R in the general formulas (I) and (II) of the α-aminonitrile compound used as the substrate of the present invention is not particularly limited, but the substituents contained in each of the substituted alkyl groups and the like are, for example, hydroxy, methoxy, Preferred are mercapto, methylmercapto, amino, halogeno, carboxyl, carboxamide, phenyl, hydroxyphenyl and guanyl.

Examples of the α-aminonitrile compound include 2-aminopropanenitrile, 2-aminobutanenitrile, 2-amino-3-methylbutanenitrile, 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) propanenitrile, 2-cyanopyrrolidine, 2-cyano-4-hydroxypyrrolidine,
2-amino-2-phenylethane nitrile etc. can be illustrated.

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.

In the present invention, the above raw material nitrile is replaced by the above general formula (II
The compound is reacted with a microorganism in the presence of the aldehyde represented by I) or (IV). As R of this aldehyde, a compound having the same type of substituent as the starting nitrile can be used. In this case, a compound having the same substituent as the starting nitrile can be used to obtain a single L-α-amino acid, and a compound having different substituents can be used to obtain a mixture of different types of La-amino acids. .

In the present invention, the raw material nitrile is added to the genus Rhodococcus sp. And the genus Mycobacte.
rium sp.) and Arthrobacter s
p.) to produce L-α-amino acid by acting each of the above microorganisms having a nitrile hydrolysis activity,
For example, any one 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, and contacting the raw material nitrile with the bacterial cell.
(B) a method of pre-culturing a microorganism and contacting the obtained bacterial cell with a nitrile compound, for example, propionitrile, and then reacting by adding a raw material nitrile to the bacterial cell, and (c) a microorganism. A method of reacting by directly contacting the raw material nitrile with the bacterial cells obtained by culturing and proliferating in advance is applied.

Further, in these reaction methods, crushed cells of the bacterial cells after growth,
It is also possible to use dried bacterial cells, treated bacterial cells such as separated and purified nitrile hydrolase, or immobilized bacterial cells and treated bacterial cells according to a conventional method.

As the nitrile compound used in the above methods (a) and (b), in addition to propionitrile, acetonitrile,
Examples thereof include n-butyronitrile, n-capronitrile, methacrylonitrile, isobutyronitrile, glutaronitrile, triacrylonitrile, 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 sulfate, Inorganic salts such as manganese sulfate, ferrous sulfate, ferric chloride, calcium chloride, manganese chloride, and salts such as boric acid, copper, zinc, that is, so-called trace elements,
Further, if necessary, a seed culture of each of the above-mentioned microorganisms is inoculated into a medium to which a growth promoting substance such as vitamins, enzyme extracts and corn stapler is added, and cultured under aerobic conditions to grow the bacterial cells. The bacterial 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 at pH 4 to 13, preferably pH 8 to 12 for 1 to 6 days. Although various buffers may be used in the reaction, ammonia-based buffers are preferred, such as a mixture of ammonia water and ammonium chloride aqueous solution, a mixture of ammonia water and ammonium sulfate aqueous solution, a mixture of ammonium water and ammonium phosphate aqueous solution, and ammonia water. A mixture with an aqueous solution of ammonium acetate and the like are preferable. Ammonia water is preferable as the alkali for pH adjustment.

The addition ratio of the aldehyde to the raw material nitrile is appropriately selected in the range of 0.1 to 10 moles of aldehyde, particularly preferably 0.5 to 3 moles, relative to 1 mole of the raw material nitrile.

The reaction temperature is preferably in the range of 20 to 70 ° C., and the carbon source used for cell growth during the reaction, the nitrogen source, and other components are appropriately added to increase the cell concentration and the nitrile hydrolysis of cells. It can be maintained and enhanced. Further, as a method for supplying the raw material nitrile and aldehyde, 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 purified by the above reaction is phase-separated,
Separation and collection are carried out by applying known means such as filtration, extraction, column chromatography and the like.

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, raw material nitrile and aldehyde are added and reacted to produce L-α-amino acid.

In the method (c), the raw material nitrile and aldehyde are added immediately after the growth of the cells in the method (b) to react them to produce L-α-amino acids.

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.

The present invention will be specifically described below with reference to examples.

Example 1 100 ml of a medium having the following composition was placed in a 500 ml volumetric flask.
After sterilizing in an autoclave for 1 minute, 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 0.5g / FeSO 4 · 7H 2 O 0.03g / CaCl 2・ 2H ₂ O 0.06g / pH /7.2 Rhodococcus sp. (Rhodococcus.sp) PA-34 was added to the above medium.
Three platinum loops of each strain were inoculated and shake culture was carried out at 30 ° C. for 48 hours.

The cells obtained by the above culture were separated by centrifugation, and NH ₄ C
After two washes with 0.1M buffer l-NH ₃ (pH10), and resuspended in 0.1M buffer NH ₄ Cl-NH ₃ as optical density (OD) is 40.

500 ml of the bacterial cell suspension obtained as described above was placed in a 1.2 fermenter, and 4.9 g (50 mmol) of DL-2-amino-3-methylbutanenitrile was added thereto, and the reaction temperature was 30 ° C.
The reaction was carried out for 48 hours while keeping the pH at 10.0 with 4N ammonia water and 10.0N at a stirring speed of 700 rpm and an air flow rate of 0.2 vvm.

After the reaction is complete, centrifuge the reaction mixture to obtain 0.45
The mixture was filtered through a μ millipore filter, and the obtained aqueous layer was analyzed by high performance liquid chromatography. The accumulated concentration of L-valine was 1.3 mg / ml, and the optical purity was 96% ee.

The amount of L-valine produced was determined 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.).

Example 2 5 ml of the cell suspension prepared by the method described in Example 1 except that Mycobacterium sp. AB-43 was used was placed in a φ24 mm test tube, and DL-2-amino-3- Methylbutanenitrile (49 mg) and isobutyraldehyde (72 mg) were added and the mixture was tightly stoppered, and the reaction was carried out at a reaction temperature of 30 ° C and a shaking frequency of 300 rpm for 48 hours. When analyzed by the method described in Example 1, the accumulated concentration of L-valine was 0.6 mg / ml and the optical purity was 97% ee.

Example 3 3 platinum loops of Arthrobacter sp. PA-15 strain were treated with NBG medium (“Labrengo” powder from Oxoid Co., 10 g of code L29, 10 g of bacteriological peptone, code L37, 10 g of glucose, Sodium chloride 5g
Deionized water was added to 1000 ml to adjust the pH to 7.5 with a 1N aqueous solution of caustic soda, and the mixture was inoculated into a 500 ml Sakaguchi flask containing 100 ml of a liquid medium sterilized by heating at 120 ° C for 15 minutes in an autoclave at 30 ° C. The cells were shake-cultured (150 times / min) for 48 hours. The cells produced by this culture were collected by the method described in Example 1, washed, and resuspended in 0.1 M NH ₄ Cl-NH ₃ buffer to an optical density (OD) of 40. . The bacterial suspension was treated with DL-2-amino-3- by the reaction method described in Example 2.
Reacted with methylbutanenitrile, L-valine 0.3 mg / m
l, optical purity of 95% ee was obtained.

Example 4 Culture, reaction and analysis were carried out by the method described in Example 2 except that the microorganisms listed in Table 2 were used as the microorganisms. The results are shown in Table 2.

Example 5 Rhodococcus sp. BA-as a microorganism
Culture, reaction, and analysis were carried out by the method described in Example 2 except that 1 was used, L-valine accumulation was 0.4 mg / ml, and optical purity was
100% result was obtained.

Example 6 DL- was added to 5 ml of the bacterial suspension prepared by the method described in Example 1.
The reaction was carried out by the method described in Example 1 except that 50 μm of 2-aminopentanenitrile and 60 μm of n-butyl aldecht were added, and the produced L-norvaline was used as a column with Asahipak Inertosyl ODS as a packing material and CHIRALPAK WE as a packing material. Table 3 shows the results of high-performance liquid chromatography analysis using the column.

Example 7 DL- was added to 5 ml of the bacterial suspension prepared by the method described in Example 3.
The reaction was carried out by the method described in Example 3 except that 50 μm of 2-aminopentanenitrile and 60 μm of n-butyl aldecht were added, and the produced L-norvaline was used as a column having Asahipak Inertosyl ODS as the packing material and CHIRALPAK WE as the packing material. The results of high performance liquid chromatography analysis using the column are shown in Table 4.

Example 8 DL- was added to 5 ml of the bacterial suspension prepared by the method described in Example 1.
4-methyl-2-aminopentanenitrile 50μ and 3
-The reaction was carried out by the method described in Example 1 except that 60 μl of methylbutyraldehyde was added, and the L-leucine produced was packed with a column containing Asahipak Inertosyl ODS as a packing material and C
Table 5 shows the results of high performance liquid chromatography analysis using a column having HIRALPAK WE as a packing material.

Example 9 DL- was added to 5 ml of the bacterial suspension prepared by the method described in Example 3.
4-methyl-2-aminopentanenitrile 50μ and 3
-The reaction was carried out by the method described in Example 3 except that 60 µl of methylbutyl aldecht was added, and the L-leucine produced was packed with a column containing Asahipak Inertosyl ODS as a packing material and C
Table 6 shows the results of high performance liquid chromatography analysis using a column having HIRALPAK WE as a packing material.

Example 10 DL- was added to 5 ml of the bacterial suspension prepared by the method described in Example 1.
The reaction was carried out by the method described in Example 1 except that 50 μm of 2-aminohexanenitrile and 60 μm of n-pentylaldecht were added, and the produced L-norleucine was used as a column with Asahipak Inertosyl ODS as a packing material and CHIRALPAK WE.
Table 7 shows the results of high-performance liquid chromatography analysis using a column with the above as a packing material.

Example 11 DL- was added to 5 ml of the bacterial suspension prepared by the method described in Example 3.
The reaction was carried out by the method described in Example 3 except that 50 μm of 2-aminohexanenitrile and 60 μm of n-pentylaldecht were added, and the produced L-norleucine was used as a column with Asahipak Inertosyl ODS as a packing material and CHIRALPAK WE.
Table 8 shows the results of analysis by high performance liquid chromatography using a column having the above as a packing material.

EFFECTS OF THE INVENTION As described above, according to the present invention, it is possible to selectively produce L-α-amino acids directly from a racemic α-aminonitrile compound using a microorganism. It is useful in producing the L-α-amino acid used.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication C12R 1:32) (C12P 13/04 C12R 1:06) (C12P 41/00 C12R 1: 365) (C12P 41/00 C12R 1:32) (C12P 41/00 C12R 1:06)

Claims (1)

[Claims] 1. The following general formula (I): Alternatively, the following general formula (II) (In the formula, R is 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, and thiazolyl. Group or a substituted thiazolyl group), one or more α-aminonitrile compounds represented by the following general formula (III) R-CHO (III) or the following general formula (IV) R-CH ₂ CHO (IV) (wherein R is the same as the above general formula (I) or (II)) in the presence of 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.