JPH01317392A - Production of l-alpha-amino acid - Google Patents

Abstract

PURPOSE:To directly obtain the above compound useful as a raw material for foods, etc., from an alpha-(N-alkylidenamino)nitrile compound of racemic modification and to reduce production cost thereof by using a specific bacterium having nitrile hydrolyzing ability. CONSTITUTION:A nitrile compound (e.g., 2-aminopropanenitrile) shown by formula I or II (R1 and R2 are alkyl or phenyl) is treated with a bacterium [e.g., Nocardia sp. PC-29 (FERM-BP 1561)] belonging to the genus Nocardia, Mycobacterium or Corynebacterium, having hydrolyzing activity to give the aimed compound. The reaction is preferably carried out at 20-70 deg.C at pH8-12 for q-6 days.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention utilizes microorganisms to produce nitrile compounds, particularly racemic α-(N-alkylideneamino)nitrile compounds, and mono-α-amino acids. Regarding the method.

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

Revised by JEED Conventionally, fermentation methods, synthesis methods, enzymatic methods, and extraction methods are known as methods for producing L-α-amino acids. Among these methods, the production of L-α-amino acids by synthetic methods involves synthesizing DL-α-amino acids using the Strecker method or a modified method thereof, and then optically resolving the amino acids to obtain L-α-amino acids.
It produces α-amino acids, and uses an enzymatic method using aminoacylase as the optical resolution method. (Published on the 15th, p. 175).

However, the method for producing L-α-amino acids by the above-mentioned synthetic method is expensive in the optical resolution step, and for economic reasons, the proportion of L-α-amino acids in the production of amino acids has been gradually decreasing17 in recent years.

Further, the following general formula NH is a synthetic intermediate in the synthesis of α-amino acids by the Strecker method.

Attempts to produce α-amino acids from DL-α-aminonitrile represented by R-CH-CN (R represents an alkyl group, phenyl group, etc.) using microorganisms have also been reported [Y,
Fukuda et alo, [Journal of Fermentation Technology (J.

Ferment, Technol, ) J 49.10
11 (+971) ]. However, in this report, Corynebacterium and erium sp. ) to hydrolyze DL-α-aminopropionitrile and DL-α-aminoisovaleronitrile using microorganisms belonging to
L-alanine and DL-valine are produced, and L-α-amino acids cannot be obtained directly. In addition, Brevibacterium spp.
A report on the production of amino acids by hydrolyzing DL-α-aminonitrile using strain R312 of J.
llagas et al., Advances in Biochemical Engineering (Abv, Biochem, E
ngineer, J., Yu, L. (1980)) also showed that DL-α-aminonitrile was converted to DL-α-
Only amino acids are obtained.

Incidentally, the above report is based on Brevibacterium strain R31.
Brevibacterium sp, a mutant strain obtained from 2.
It is disclosed that L-α-amino acid and D-α-amino acid amide can be produced from DL-α-aminonitrile by using A4; Regarding directly obtaining only
No reports yet.

Furthermore, a method has been disclosed in which amino acids are produced by irradiating light before the hydration reaction of a DL-α-aminonitrile compound by microorganisms is completed (Japanese Patent Application Laid-open No. 162191/1982).
However, with this, only DL, -α-amino acids were obtained, and α-
No example has been reported in which an L-α-amino acid was directly obtained from an aminonitrile compound.

Furthermore, the α-(N-alkylideneamino)diI-lyl compound used as a raw material in the present invention is hydrolyzed using microorganisms to obtain α-
There are no reports yet on the production of amino acids.

11 In view of the above-mentioned current situation, the present inventor has conducted intensive research and has determined that microorganisms capable of hydrolyzing nitrile belonging to the genera Nocardia, Mycobacterium, and Corynebacterium have been treated with α-(N It has been found that L-α-amino acids can be selectively produced when treated with -alkylideneamino)nitriles. The present invention was made based on this knowledge, and utilizes a microorganism that has the ability to hydrolyze nitriles selected from a specific genus to directly convert α-(N-alkylideneamino)nitriles into ■, -α- An object of the present invention is to provide a method for producing amino acids.

4. Measures for carrying out 1) The present invention is based on the following general formula (I) or the following general formula (U) (wherein R1 and R1 are an alkyl group, a substituted alkyl group, a phenyl group, a substituted phenyl group). , imidazolyl group,
Substituted imidazolyl group, indolyl group, substituted indolyl group, furyl group, substituted furyl group, pyridyl group, substituted pyridyl group, thiazolyl group, substituted thiazolyl group, R1, R
, may be the same group or different groups), and L -α-amino acids.

As mentioned above, the microorganisms used in the present invention have the ability to hydrolyze nitriles selected from the group belonging to the genus Nocardia, Mycobacterium, and Corynebacterium, and the microorganisms shown in Table 1 below are I can give an example.

Table 1 These microorganisms have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under the deposit acceptance number shown in Table 1 on November 5, 1988.

Next, Table 2 shows the mycological properties of each microorganism in doffing.

As shown in Table 2 below, the microorganisms used in the present invention are all Durham-positive aerobic bacteria and do not grow at all in anaerobic conditions. In addition, it does not exhibit motility, is positive for catalase, and does not form heat-resistant spores. It also exhibits pleomorphism, with many mycelial cells at the early stage of culture, and branched cells and spherical cells are also observed. Regarding the utilization of sugar, it does not produce gas from sugar, has weak acid-generating ability, and when cultured in lidmus milk, it becomes alkaline and turns the milk blue.

Among the above-mentioned microorganisms, Nocardia sp. and PC-29 form slightly dry wrinkle-like colonies on meat juice agar, and show highly branched mycelial growth in the early stage of culture, at temperatures ranging from 5 to 32°C.
It grows at 37°C, but not at 37°C.

On the other hand, Nocardia sp, PA-34, AB-16
Also, BA-1 does not grow at 5-10℃, but at 37
They grow even at ~42°C, become hyphae-like at the early stage of culture, and then produce a large number of Oidiospore-like spherical cells.

Mycobacterium sp, AB-43 was stained with acid-fast staining (A
cid-fast 5tain), it does not grow at 45°C and forms sticky yellow-orange colonies. In addition, Corynebacterium sp, PA-15, and PC-3 are Durham-positive rods, but their morphology is irregular and when cultured for a long time, granules form in the cells, and Durham staining is negative. As a result, spherical cells also appear. This strain is a psychrophilic bacterium that grows at 5 to 10°C and does not grow above 37°C. The colonies on gravy agar have a yellow-green color, are creamy, and have a strong ability to hydrolyze gelatin and starch, but do not have the ability to decompose cellulose.

In view of Table 2 and the above-mentioned properties, the above microorganisms are
Verge's Manual of Systematics
Bacteriology (Bergey's Manual
of Systematic Bacteriology
Y, 1986).

In the present invention, the general formula (r) is a substrate used to produce L-α-amino acids using each of the above microorganisms.
and α-(N-alkylideneamino)nitrile compound represented by (I[) (hereinafter abbreviated as raw material nitrile)
For example, α- is the first reaction of the Strecker method.
Synthesis of aminonitrile compounds (e.g., Journal of Fermentation Technology (J, Ferme)
nt, Technol, ), 49 Loll (
+971) or Chemistry Letters (Chem
, Lett,), 687 (+987)),
Once the disappearance of the raw material aldehyde is confirmed by analysis using gas chromatography or the like, the inorganic salt can be immediately separated by an operation such as filtration or extraction, and then concentrated. This crude product has the general formula (1)
It may contain α-aminonitrile, which is a partial hydrolyzate of the α-(N-alkylideneamino)nitrile compound represented by 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.

The general formula (
In particular, R1 and R1 in 1) and (II) include , 2
Although not limited, substituents included in each substituted alkyl group include, for example, hydroxy, methoxy, mercapto,
Methyl mercapto, amino, halogen, carboxyl,
Carbofusamide, phenyl, hydroxyphenyl or guanyl are preferred. Furthermore, if raw nitriles having the same group for R1 and R1 are used, a type of 1-α-amino acid can be obtained, and if raw nitriles having different groups are used, a mixture of the two L-α-amino acids can be obtained. Amino acids can be obtained. Moreover, it goes without saying that there is no problem even if two or more of the above raw material nitriles are used as a mixture.

Examples of the α-(N-alkylideneamino)nitrile compound include 2-aminopropanenitrile, 2-aminobutanenitrile, 2-amino-3-methylbutanenitrile, 2-aminobutanenitrile,
Amino-4-methylpentanenitrile, 2-amino-3
-Methylpentanenitrile, 2-amino-3-hydroxypropanenitrile, 2-amino-3-hydroxybutanenitrile, 2-amino-5-g1,7nidinopentanenitrile, 2-amino-3-sulcaptopropanenitrile , 2,7-diamitu-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-cyanopropanamide, 2,6-cyamitsuhexanenitrile, 2,6-diamitsu-5- Hydroxyhexanenitrile, 2-amino-3-(3-indolyl)propanenitrile, 2-amino-3-(4-imidazolyl)propanenitrile, 2-cyanopyrrolidine, 2-cyano-
Examples include Schiff bases of α-aminonitrile compounds such as 4-hydroxypyrrolidine and 2-amino-2-phenylethanenitrile and aldehydes that are raw materials for the synthesis of these α-aminonitriles.

In the present invention, in order to produce L-α-amino acids by causing each of the above-mentioned microorganisms having nitrile hydrolyzing activity belonging to the genus Nocardia, Mycobacterium, and Corynebacterium to act on the raw material nitrile, For example, any of the following methods (a) to (c) may be applied.

That is, (a) a method of culturing and proliferating microorganisms in a medium containing a nitrile compound, for example propionitrile, and bringing the raw material nitrile into contact with the resulting bacterial cells to react;
(b) A method in which a microorganism is cultured in advance, the resulting microbial cells are brought into contact with a nitrile compound, such as propionitrile, and then a raw material nitrile is added to the microorganisms to cause a reaction; and (C) a method in which the microorganism is A method is applied in which the bacterial cells obtained by culturing and multiplying are brought into direct contact with the raw material nitrile and reacted.

In addition, in these reaction methods, crushed bacterial cells after proliferation,
It is also possible to use dried bacterial cells, treated bacterial cells such as isolated and purified nitrile hydrolase, or fixed bacterial cells and treated bacterial cells according to conventional methods.

In addition to propionitrile, the nitrile compounds used in the methods (a) and (b) above include acetonitrile,
Examples include n-butyronitrile, n-capronitrile, methacrylonitrile, isobutyronitrile, geltalonitrile, triacrylonitrile, crotononitrile, lagtonitrile, succinonitrile, acrylonitrile, benzonitrile, and phenylacetonitrile.

In method (a) above, in addition to the nitrile compound, carbohydrates such as glucose, sucrose, molasses, and starch hydrolysates as carbon sources, or substances that have a bacterial growth effect such as acetic acid are added to the medium. Additionally, 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 boric acid,
The inoculum of each of the above microorganisms is inoculated into a medium containing salts such as copper and zinc, that is, so-called trace elements, and, if necessary, growth-promoting substances such as vitamins, yeast extract, and corn staple liquor. The cells are grown by culturing under specific conditions. The raw material nitrile is supplied to the thus obtained bacterial cell culture, a suspension of bacterial cells separated from the culture, or a treated product of the bacterial cells and allowed to react.

The reaction is carried out at a pH of 4 to 13, preferably 8 to 12, for 1 to 6 days. Various buffers can be used for the reaction, but ammonia-based buffers are preferred; mixtures of ammonia water and ammonium chloride aqueous solution, mixtures of ammonia water and ammonium sulfate aqueous solution, mixtures of ammonia water and ammonium phosphate aqueous solution, ammonia water and aqueous ammonium phosphate solution, etc. A mixture with an aqueous ammonium acetate solution is preferred. In addition, an alkaline source for pH adjustment and other components can be appropriately added to maintain and increase the bacterial cell concentration and the nitrile hydrolyzing ability of the bacterial cells. Further, as a method for supplying the raw material nitrile, any of a method of adding it at the start of the reaction, a method of adding it intermittently, and a method of adding it continuously can be adopted.

The L-α-amino acid produced by the above reaction undergoes phase separation,
Separation and collection are performed by applying known means such as J-filtration, extraction, and column chromatography.

Next, in the method (b) above, the nitrile compound is not added during the culture and proliferation of the microbial cells in the method (a) above, but a nitrile compound is added after the microbial cells have grown to increase the nitrile hydrolyzing ability of the microbial cells. After activation, the raw material nitrile is reacted to produce L-α-amino acids.

Further, in the method (c) above, immediately after the bacterial cells grow in the method (b) above, a raw material nitrile is added and reacted to produce L-α-amino acids.

In both methods (b) and (c) above, the culture conditions, reaction conditions, and separation and collection of the produced L-α-amino acid may be the same as those in method (a) above.

The L-α-amino acids obtained according to the present invention in the form of two series are used in various fields such as foods, feeds, medicines, and cosmetics.

The present invention will be specifically explained below using Examples.

Su JLI fresh 1 ■ Preparation of medium 100 ml of the medium with the following composition was placed in a 500 ml flask, sterilized in an autoclave for 120 minutes, and 0.0 ml of medium was placed in a 500 ml flask and sterilized in an autoclave for 120 minutes.
Propionitrile 1 sterilized with a 2μ Millipore filter
ml was added to prepare a medium for bacterial cell preparation.

Medium composition Nigelcose 10 g/9 Yeast extract 0.1 g/Q Na, HPO, 12H, 02,5 g / QKH, P
o, 2.0 g/Q flash gS04 ・
7H, OO, 5g/Q.

FeSO4・7H,00,03g/QCaCl,
・2H,O0006g / QpH7,2 Bacterial strain used: Bacterium-1 Strain name FERM-B
P Fish Nocardia (Nocardia sp,) PC
-291561 Nocardia (Nocardia sp.
) PA-341559 Nocardia (Nocard
ia sp,) AB-161555 Nocardia (N
ocardia sp, ) BA-11557 Mycobacterium (Mycobacterium sp, ) A
B-431556 Corynebacterium (Coryne
bacueri+llTl5p,)PA-151558
Corynebacterium (Corynebacterium)
msp, ) PC-31560 ■ Culture and growth of bacterial cells Three platinum loops of the following seven strains were each inoculated into the above medium, and cultured at a temperature of 30'C and 140 rpm for 48 hours.

The bacterial cells obtained by the above culture were separated by centrifugation, and NH
After washing once with 0.1M buffer (pH 10) of ,C1-N1+, Ni was washed so that the optical density (OD) was 40.
1, CI-NH, in 0.1M buffer.

■Reaction 5 ml of each bacterial cell suspension obtained as described above was placed in a test tube with a diameter of 24 mm, and DL-3-methyl-
Add 100μα of 2-N-(2-methylpropylidene)aminobutanenitrile, seal tightly, and heat to 30℃ for 30 minutes.
Incubation was carried out for 48 hours at 0 rpm.

After the reaction was completed, the reaction solution was extracted twice using 5 ml of hexane, and the resulting aqueous phase was analyzed by high performance liquid chromatography.

The produced L-valine was quantified using As as a column packing material.
Ahipak Inertsil ODS (manufactured by Asahi Kasei Corporation) was used to determine the absolute configuration and optical purity. The results are shown in Table 3.

(Margins below) Table 3 Nocardia sp prepared by the method described in Example 1,
Add 4-methyl-2-N to 5 ml of PA-34 bacterial suspension.
-(3-methylbutylideneamino)pentanenitrile 5
When the reaction was carried out in the same manner as described in Example 1 except that 0 mg was added, 2.5 g/A of L-leucine was produced, and its optical purity was only 93%. In addition, high performance liquid chromatography was used to determine the absolute configuration, and C) IIRALPAK WE (manufactured by Daicel Chemical Industries, Ltd.) was used as a packing material.

1 Toyogoyu DL was added to the bacterial suspension 5d prepared by the method described in Example 1.
-2-N-(butylidene)aminopentanenitrile 50
The reaction was carried out as described in Example 1 except for adding μα. Table 4 shows the results of analyzing the produced L-norvaline using the method described in Example 2.

Table 4 Three platinum loops of each of the microorganisms shown in Table 5 were placed in NBG medium ("Labrengo" powder manufactured by Oxoid, code L29) for 10 minutes.
g, bacteriological peptone, code L37 lo
g, glucose log, IQ by adding deionized water to 5 g of sodium chloride, 500 nl1I containing 100 dl of +J゛liquid medium) of l standard.
inoculated into a flask of 100 ml, and cultured with shaking at 30°C for 48 hours (1
50 times/min). The bacterial cells produced by this culture were collected and washed by the method described in Example 1, and the optical density (OD
) of NH2Cl-NH, is 40. Resuspend in IM buffer. The bacterial suspension was reacted with 50 μα of DL-2-N-(butylidene)aminopentanenitrile using the reaction method described in Example 3, and analyzed using the method described in Example 2, and the results are shown in Table 5. Obtained.

Table 5 The reaction was carried out by the method described in Example 1 except that 50μ0 of DL-2-N-(pentylidene)aminohexanenitrile was added to 511 sides of the bacterial suspension prepared by the method described in Example 1. Table 6 shows the results of analyzing L-norleucine using the method described in Example 3.

Table 6 DL was added to the bacterial suspension 5d prepared by the method described in Example 4.
-2-N-(pentylidene)aminohexanenitrile 5
The reaction was carried out by the method described in Example 1 except that 0 μα was added, and the produced mononorleucine was analyzed by the method described in Example 3. The results are shown in Table 7.

Table 7 Nocardia SP, PA-34 prepared by the method described in Example 1, except that other nitrile compounds were used in place of propionitrile.
50 μQ of DL-3-methyl-2-N-(2-methylpropylidene)aminobutanenitrile was added to 5d of the bacterial suspension, and reaction analysis was performed using the method described in Example 1. The results are shown in Table 8. Ta.

Table 8: Culture, reaction, and analysis were performed as described in Example 2 except for using Mycobacterium sp, AB-43, and the amount of L-leucine produced was 0.6 μ/lll1! , optical purity 83%e
, e, were obtained.

The reaction analysis was carried out by the method described in Example 2, except that the bacterial suspension 5 prepared by the method described in Example 4 was used, and the results shown in Table 9 were obtained.

Table 9 As mentioned above, according to the present invention, it is possible to selectively produce L-α-amino acids directly from racemic α-alkylidene aminonitrile compounds using microorganisms, so it can be used in the above-mentioned fields. It is useful in the production of monoα-amino acids.

Claims (1)

[Claims] (1) The following general formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) Or the following general formula (II) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(II) (However, R_1 and R_2 is an alkyl group, substituted alkyl group, phenyl group, substituted phenyl group, imidazolyl group, substituted imidazolyl group, indolyl group, substituted indolyl group, furyl group, substituted furyl group, pyridyl group, substituted pyridyl group, thiazolyl group, substituted thiazolyl group and R_
1, R_2 may be the same group or different groups)
One or more nitrile compounds represented by the above are converted into L-α-amino acids by the action of a microorganism having nitrile hydrolyzing activity belonging to the genus Nocardia, Mycobacterium, or Corynebacterium. Characteristic method for producing L-α-amino acids.