JPH0720432B2 - 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 a DL-α-aminonitrile compound using a microorganism. 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 a synthetic method by synthesizing a DL-α-amino acid by using the Strecker method or a modified method thereof, and then optically resolving the amino acid to obtain L-α-amino acid.
-For producing amino acids, an enzymatic method using aminoacylase is adopted as a method for 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 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
"Farmentation Technology" (J.Ferment.Te
hnol.) 49 , 1011 (1971)]. But in this report,
Using microorganisms belonging to Corynebacterium sp., DL-α-aminopropionitrile and
DL-α-aminoisovaleronitrile is hydrolyzed to DL-
A method for producing alanine and DL-valine, comprising:
-Α-amino acids are not directly available. In addition, using strain R312 of the genus Brevibacterrium sp.
Report of producing amino acid by hydrolyzing L-α-aminonitrile [J-C Jallagas et al. "Advance of Biochemical Engineering" (Adv. Biochem.
Engineering,) 14 , 1 (1980), only DL-α-amino acid was obtained from DL-α-aminonitrile. Incidentally, the above report indicates that the strain R3 of the genus Brevibacterium is R3.
Brevibacterium sp. A4, a mutant obtained from 12.
From DL-α-aminonitrile to L-α
-Amino acid and D-α-amino acid amide are disclosed, but L-
No report has yet been made on directly obtaining only α-amino acids.

Also, as disclosed in JP-A-61-162191, it is disclosed that DL-amino acids are produced from α-aminonitrile compounds, and α-aminonitrile compounds are used to produce L-α-amino acids.
There are no reports of directly obtaining amino acids.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention The present invention provides a method for producing L-α-amino acids directly from DL-α-aminonitriles by utilizing a microorganism having a hydrolyzing ability of a nitrile selected from a specific genus. The challenge is to provide a method.

The present invention is a microorganism belonging to the genus Rhodococcus or the genus Mycobacterium having a nitrile hydrolyzing ability, under the conditions of pH 8 to 12, when acting on DL-α-aminonitriles mainly in an Ammonia buffer, the L-α-amino acid. It was made based on the knowledge of selectively producing a kind.

The present invention will be described in detail below.

Structure of the invention The structural feature of the present invention, microorganisms having a nitrile hydrolyzing ability selected from the group belonging to the genus Rhodococcus or Mycobacterium, pH8-12 conditions, DL- in mainly Ammonia buffers It is to act on an α-aminonitrile compound to selectively produce L-α-amino acid.

Means for Solving the Problems As described above, the microorganisms used in the present invention have hydrolysis ability of nitrile selected from the group belonging to the genus Rhodococcus or Mycobacterium and are shown in Table 1 below. The thing can be illustrated.

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

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

As seen in Table 2, all the microorganisms used in the present invention are Gram-positive aerobic bacteria and do not grow at all in the anaerobic state. In addition, it 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.

Among the above microorganisms, Rhodococcus sp. PC-29 forms a slightly dry wrinkle-like colony on broth agar, and shows markedly branched mycelial growth in the early stage of culture, and grows at 5 to 32 ° C. , Does not grow at 37 ℃.

On the other hand, Rhodococcus sp. PA-34, AB-16 and BA-1
Does not grow at 5-10 ° C, but grows at 37-42 ° C,
Both of them became hyphae at the beginning of culture, and then many Oidi
It produces ospore-like spherical cells.

Mycobacterium sp. AB-43 was treated with acid-fast staining (Acid-f
aststain) is positive and does not grow at 45 ℃ and forms a viscous yellow-orange colony.

In view of the Table 2 and the properties described above, the above-mentioned microorganisms are
Bergey's Manual of Systematic Bacte
riology, 8th edition.

In the present invention, a DL-α-aminonitrile compound (hereinafter abbreviated as a raw material nitrile) that is a substrate used to produce L-α-amino acids using each of the above microorganisms is, for example, [[Organic Synthesis Collective Volume ( Org.Syu.Col.Vol.) I, p, 21 and III, p.84 ”] and [Y.Fukuda et al.,“ Journal of Future Technology Technology ”(J.Ferment.Techno.
l.,) 49 , 1011 (1971)] and the like.

General formula (1) of the DL-α-aminonitrile compound of the present invention
Alternatively, R in (2) is not particularly limited, but the substituent contained in each of the substituted alkyl groups and the like is, for example, hydroxy, methoxy, mercapto, methylmercapto, amino, halogen, carboxyl, carboxamide, phenyl, hydroxyphenyl or Such as guanyl.

As the DL-α-aminonitrile compound, 2-aminopropanenitrile, 2-aminobutanenitrile, 2-amino-3-methylbutanenitrile, 2-amino-4-methylpentanenitrile, 2-amino-3-methylpentane Nitrile, 2-amino-3-hydroxypropanenitrile, 2-amino-3-hydroxybutanenitrile, 2-
Amino-5-guanidinopentanenitrile, 2-amino-3-methylcaptopropanenitrile, 2,7-diamino-4,5-dithiaoctanenitrile, 2-amino-4-methylthiobutanenitrile, 2-amino-3- Phenyl propane nitrile, 3- (4-hydroxyphenyl) propane nitrile, 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-imidazoyl) propanenitrile, 2-cyanopyrrolidine, 2-cyano-4-hydroxypyrrolidine,
2-amino-2-phenyl ethane nitrile etc. can be illustrated.

In the present invention, each of the above-mentioned raw material nitriles is allowed to act with each of the above-mentioned microorganisms having a nitrile hydrolysis activity belonging to the genus Rhodococcus or Mycobacterium to produce L-α-
To produce an amino acid, for example, the following (a) to (c)
It is good to apply any of the above methods.

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.

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

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 above method (a), in addition to the nitrile compound, glucose, sucrose, molasses, sugar such as 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 sulfate, amino acids and other assimilable organic nitrogen compounds, potassium phosphate, sodium phosphate, magnesium sulfate, sulfuric acid. Inorganic salts such as manganese, ferrous sulfate, ferric chloride, calcium chloride and manganese chloride, and salts such as boron, 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 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 in the pH range of 8 to 12 for 1 to 6 days. At pH values outside this range, α-amino acids rich in L-form are produced, but the optical purity is low and not practical. Although various buffers may be used in the reaction, an ammonia-based buffer is preferable, 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 an ammonia water and an ammonium acetate aqueous solution, and an ammonia water. A mixture with an aqueous solution of ammonium phosphate is preferable. Ammonia water is preferable as the alkali used as the pH adjuster.

The reaction temperature is preferably in the range of 20 to 70 ° C. In addition, the carbon source, the nitrogen source, and other components used for cell growth during the reaction can be appropriately added to maintain and increase the cell concentration and the nitrile hydrolyzing ability of the cell.

Further, as a method of supplying the raw material nitrile, either a method of adding at the start of the reaction or a method of continuously adding can be used.

The L-α-amino acid produced by the above reaction is phase-separated,
Separation and collection are carried out by applying known means such as filtration, extraction and column chromatography.

Next, in the method (b), the nitrile compound is not added during the culture and growth of the cells in the method (a), but the nitrile compound is added after the growth of the cells to improve the nitrile hydrolysis of the cell microorganism. 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.

As described above, according to the present invention, the use of microorganisms
Since the L-α-amino acids can be selectively produced directly from the DL-α-aminonitrile compound, it is useful for producing the L-α-amino acids used in the above various fields.

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

Example 1 Preparation of medium 100 ml of a medium having the following composition was placed in a 500 ml flask.
After sterilizing in an autoclave at 0 ° C. for 20 minutes, 1 ml of propionitrile sterilized with 0.2 μm Millipore filter was added to prepare a cell culture medium.

Medium composition: Glucose Mml 10 g / l Yeast extract 0.1 g / l NaHPO ₄・ 12H ₂ O 2.5 g / l KH ₂ PO ₄ 2.0 g / l MgSO ₄・ 7H ₂ O 0.5 g / l FeSO ₄・ 7H ₂ O _{0.03g / l CaCl 2 · 2H 2} O 0.06g / l pH 7.2 strains used: strain name FERM-BPNo Rhodococcus (Rhodococcus) sp./PC-29 1561 Rhodococcus (Rhodococcus) sp./PA-34 1559 Rhodococcus (Rhodococcus) sp./AB-16 1555 Rhodococcus sp./BA-1 1557 Mycobacterium sp. AB-43 1556
Cultivation and Proliferation of Bacterial Cells The above medium was inoculated with 3 platinum loops of the following 7 strains, and shake 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 NH ₄ C
After washing once with 0.1M buffer of 1-NH ₃ (pH 10), it was resuspended in 0.1M buffer of NH ₄ C 1 -NH ₃ so that the optical density (OD) was 40.

Reaction 5 ml of each cell suspension obtained as described above,
In a test tube, DL-50-amino-2-phenylethanenitrile (50 mg) was added, and the container was tightly sealed.
Shaking culture was performed at 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-phenylglycine produced was quantified using an amino acid analyzer (Model 7300 manufactured by Beckmann), and its absolute configuration and optical purity were determined by high performance liquid chromatography using CHIRALPAK WM (manufactured by Daicel Chemical Industries) as a column. I went with. The results are shown in Table 3.

Example 2 Using Rhodococcus sp. PA-34, a bacterial suspension was prepared by the same procedure as described in Example 1. Prepared bacterial suspension 50
0 ml was stored in a 1.2 l fermenter and DL-2-amino-
3-Methylbutanenitrile (4.9 g, 50 mmol) was added, and the reaction was carried out at a reaction temperature of 30 ° C. and a stirring rotation speed of 700 rpm for 48 hours while maintaining PH at 10.0 ± 0.1 with 4N aqueous ammonia.

After the reaction is complete, centrifuge the reaction mixture to obtain 0.45
The mixture was filtered through a μ pore filter and the obtained aqueous layer was analyzed by high performance liquid chromatography.

The storage concentration of L-valine was 0.5 mg / ml, and the optical purity was 67% ee. The L-valine was quantified using Asahipak Inertosil ODS (manufactured by Asahi Kasei) as a column packing material, and the absolute configuration and the optical purity were determined in the same manner.
It was performed using CHIRALPAK WH (manufactured by Daicel Chemical Industries).

Next, a comparative example will be shown as a control.

Comparative Example 1 PH was 7.0 ± 0.1 with 4N ammonia water and 1N HC1.
The reaction was carried out by the method described in Example 2, except that
analyzed. The storage concentration of L-valine is 3.5 mg / ml, and the optical purity is
32% ee.

Comparative Example 2 After the cells obtained by culturing in the same procedure as described in Example 1 were centrifuged, 0.1 M Na ₂ HPO ₄ —KH ₂ PO ₄ buffer solution (P
After washing twice with H7.0), the cells were resuspended in 0.1 M Na ₂ HPO ₄ —KH ₂ PO ₄ buffer so that the optical density (OD) was 40.
Using the obtained bacterial suspension, the reaction was carried out in the same procedure as described in Comparative Example 1. The storage concentration of L-valine is 4.3mg / ml,
The optical purity was 17% ee.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12R 1: 365) (C12P 41/00 C12R 1:32) (C12P 41/00 C12R 1: 365)

Claims (2)

[Claims] 1. A general formula (1) or (2) (Wherein 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, thiazolyl Group or a substituted thiazolyl group), a nitrile compound represented by Rhodococcus (Rhod
ococcus sp.) or Mycobacteria (Mycobacteri
um sp.) and has a nitrile-hydrolyzing activity in the pH range of 8-12.
-A method for producing α-amino acids. 2. A microorganism having nitrile hydrolysis activity, Rhodococcus sp. PC-29 (Microtechnology Research Institute No. 1561, FER
M BP-1561), Rhodococcus sp. PA-34 (Mikken Kenjoyori No. 1559, FERM BP-1559), Rhodococcus sp. AB-16
(Microtechnical Research Institute No. 1555, FERM BP-1555), Rhodococcus sp. BA-1 (Microtechnical Research Article No. 1557, FERM BP-1557)
And Mycobacterium sp. AB-43
L-α-according to claim (1), wherein at least one kind of microorganism selected from the group consisting of No. FERM BP-1556) is used.
A method for producing amino acids.