JP2864277B2 - Method for producing optically active amino acids - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to the use of microorganisms to produce optically active amino acids from nitrile compounds, particularly racemic α-aminonitrile compounds and α- (N-alkylideneamino) nitrile compounds. It relates to a method of manufacturing.

Optically active amino acids are important chemical substances used in the fields of food, feed, medicine, cosmetics and the like.

Conventional technology Conventionally, methods for producing optically active amino acids include:
Fermentation, synthesis, enzymatic and extraction methods are known. Of these methods, the production of an optically active amino acid by a synthetic method involves synthesizing a DL-α-amino acid using the Strecker method or a modification thereof, and then optically resolving the amino acid to produce an optically active amino acid. This method employs an enzymatic method using aminoacylase as a method for optical resolution (see “Chemicals” extra edition “Asymmetric synthesis and optical resolution”, Showa
Published October 15, 1957, p. 175).

However, the method for producing an optically active amino acid by the above synthesis method involves a high cost in the optical resolution stage, and the ratio of the amino acid to the production of the amino acid has been gradually decreasing in recent years for economic reasons.

Also, attempts have been reported to produce α-amino acids from microorganisms, DL-α-aminonitrile, which is a synthetic intermediate in the synthesis of α-amino acids by the above Strecker method using a microorganism [Y. Fukuda et al. ., "Journal
Of Technology "(J. Ferm
ent.Tehnol.) 49 , 1011 (1971)]. In this report, however, DL-α-aminopropionitrile and DL-α-aminoisovaleronitrile are hydrolyzed using a microorganism belonging to Corynebacterium sp. To convert DL-alanine and DL-valine. As it is produced, L-α-amino acid cannot be obtained directly. Also, a report was made on the production of amino acids by hydrolyzing DL-α-aminonitrile using strain R312 of the genus Brevibacterim sp. [JC Jallagas et al. “Advance of Biochemical Engineering” (Adv. Bioche
m.Engineer.,) 14 , 1 (1980)]
Only DL-α-amino acids can be obtained from aminonitrile. By the way, the above report is based on strains of the genus Brevibacterium.
Brevibacterium sp.A, a mutant strain obtained from R312
4 to convert L-α-aminonitrile into L-
It discloses that α-amino acids and D-α-amino acid amides can be produced,
No direct report of -α-amino acids alone is reported.

Also disclosed is a method for preparing an optically active amino acid and an amino acid amide by treating a solution of an enantiomeric mixture of DL-α-aminonitrile with an enantioselective nitrilase (Japanese Translation of PCT Application No. 63-500004). The above report reported that DL-α was obtained by using strain 311, which produces nitrilase.
Discloses that a 40% ee L-amino acid amide can be obtained from aminonitrile. On the other hand, in order to obtain an L-amino acid, the enzyme is removed from the reaction solution, and a 2M sodium hydroxide solution is added to adjust the pH. To 11 was repeated 5 times during 6 hours of the reaction, which had the problem of having to chemically hydrolyze L-amino acid amide.

On the other hand, the present inventors have proposed an α-amino nitrile compound and α
A method for directly obtaining an optically active amino acid from a-(N-alkylideneamino) nitrile compound using a specific microorganism has been proposed (JP-A-1-317392, JP-A-1-317393 and JP-A-1-317394). ).

However, a method for producing an optically active α-amino acid by hydrolysis using a microorganism of the genus Nocardiopsis or Bacillus has not yet been known.

Problem to be Solved by the Invention The present invention provides a method for producing optically active amino acids directly from DL-α-aminonitrile using a microorganism having a hydrolytic ability of nitrile selected from a specific genus. The task is to provide.

Means for Solving the Problems The present invention relates to a method for selectively producing optically active amino acids when a microorganism having nitrile hydrolytic ability belonging to the genus Nocardiopsis or Bacillus is allowed to act on DL-α-amino acids. This is based on knowledge.

Accordingly, the present invention provides a method of reacting one or more nitrile compounds represented by the following general formulas (I) to (IV) with a microorganism having a nitrile hydrolytic ability belonging to the genus Nocardiopsis or the genus Bacillus. A method for producing optically active amino acids, characterized by producing optically active amino acids by (Where R in the formulas (I) and (II) 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) Group, substituted pyridyl group, thiazolyl group, substituted thiazolyl group) (Wherein R ₁ and R in the formulas (III) and (IV) are an alkyl group, a substituted alkyl group, a phenyl group, a substituted phenyl group,
Imidazolyl group, substituted imidarizolyl group, indolyl group, substituted indolyl group, furyl group, substituted furyl group, pyridyl group, substituted pyridyl group, thiazolyl group, substituted thiazolyl group, R ₁ and R ₂ are the same groups, respectively, differ Group may be used).

In the present invention, when one or more α-aminonitrile compounds represented by the above general formula (I) or (II) are used as a starting material, the following general formula (V) or (VI) Is characterized by producing the optically active amino acid by allowing the microorganism to act in the presence of the aldehyde represented by

R-CHO (V) or the following general formula (VI) R-CH ₂ CHO (VI) (where R is the same as in the above general formula (I) or (II)).

The microorganism used in the present invention is, as described above,
Those having a nitrile hydrolytic ability selected from the group belonging to the genus Nocardiopsis or the genus Bacillus, and those shown in Table 1 below can be exemplified.

As shown in Table 1, these microorganisms have been commissioned to the Microbiological Laboratory of the National Institute of Advanced Industrial Science and Technology.

Next, the bacteriological and chemical properties of each of the above microorganisms are shown below. The Nocardiopsis strain was mainly identified by Shirling, EB and Gottlieb, D.
nal Journal of Systematic Bacteriology 16 , 313-340,
Performed according to the method described in 1966.

Nocardiopsis A10-12 1. Form A10-12 elongates a well-branched reticulated mycelium under a light microscope. The aerial hyphae elongating from the basal hyphae exhibit a straight-bend shape (Rectiflexibiles) or a fishhook-loop shape (Retinaculiaperti), and often Zig-z
It grows in ag form and breaks down like bacilli with the cultivation time.
No special organs such as transgenic branches, sclerotium, and sporangia are found. Mature spores usually link 10 or more spores, the size is about 0.5-0.8 × 1.0-2.0 microns, and the surface is smooth. No spore motility is observed.

2. Growth conditions on various media The color tone is described in "Color Standards" of the Japan Color Research Institute, 1951, and the symbols and colors in brackets are used in Container Corporation of America.
oration of America) Color harmony manual.

1) Lime malate agar medium (cultured at 27 ° C) The growth is slow and forms colorless growth two weeks after inoculation.
At the third week, white aerial hyphae are slightly formed on the growth of pale yellow [2ca, Lt Ivory], and no soluble pigment is observed.

2) Glucose / nitrate agar medium (cultured at 27 ° C) Light yellow tea [2ec, Biscuit]
1), and no soluble pigment is observed.

3) Glucose-asparagine agar medium (cultured at 27 ° C) A slightly white aerial mycelium grows on colorless growth, and no soluble pigment is observed.

4) Starch / synthetic agar medium (cultured at 27 ° C.) The growth of colorless to pale yellow [2ca, Lt Ivory] produces a slightly white aerial mycelium, and no production of soluble pigment is observed.

5) Sucrose / nitrate agar medium (cultured at 27 ° C) Light yellow tea [2ca ~ 2gc, Lt Ivory ~ Bamboo] grows, and aerial mycelia of tea ash [2ba, Pearl] grow abundantly on the growth of No soluble dye is found.

6) Yeast-malt agar medium (cultured at 27 ° C) White aerial hyphae grow on the growth of light yellow tea [2ic, Honey Gold], and no soluble pigment is observed.

7) Oatmeal agar medium (ISP3) (cultured at 27 ° C) White aerial hyphae grow on the growth of pale yellow [2gc, Bamboo], and no soluble pigment is observed.

8) Starch inorganic salt agar medium (ISP4) (cultured at 27 ° C) Aerial hyphae of white to light yellow [2ca, Lt Ivory] were formed on the growth of light yellow [2ea-2gc, Lt Wheat-Bamboo] and soluble. No pigment is observed.

9) Glycerin / asparagine agar medium (ISP5) (27 ° C)
Culture) White aerial hyphae grow on the growth of pale yellow [2gc, Bamboo], and no soluble pigment is observed.

10) Tyrosine agar medium (ISP7) (cultured at 27 ° C) Aerial hyphae of white to yellowish ash [2ec, Biscuit] formed on the growth of light yellow [2ea-2gc, Lt Wheat-Bamboo]. unacceptable.

11) Nutrient agar medium (cultured at 27 ° C) White aerial hyphae grow abundantly on the growth of pale yellow [2ca, Lt Ivory], and no production of soluble pigment is observed.

3. Physiological properties 1) Growth temperature range Using yeast extract / starch agar medium
As a result of culturing at 10 ° C, 20 ° C, 25 ° C, 31 ° C, 36 ° C, 40 ° C, 45 ° C, and 50 ° C, the A 10-12 strain grew at any temperature from 10 ° C to 36 ° C. , 40 ° C, 45 ° C and 50 ° C did not grow. The optimum growth temperature seems to be around 25 ° C to 31 ° C.

2) Liquefaction of gelatin (15% simple gelatin at 20 ° C, glucose / peptone / gelatin at 27 ° C) No liquefaction was observed in any medium.

3) Starch hydrolyzability (starch inorganic salt agar medium 27)
Cultivation at about 14 ° C) Slightly hydrolyzable from about 14 days after cultivation, the effect is weaker.

4) Coagulation of defatted milk and peptone formation (cultured at 37 ° C defatted milk) Coagulation starts around 10 days after culture and is completed around 21 days.
Peptone conversion began around day 14 and was almost complete on day 21. Its effect is weaker.

5) Utilization of malate lime (cultivation at 27 ° C of lime malate agar medium) No dissolution of lime malate was observed.

6) Nitrate reduction (peptone water containing 1.0% sodium nitrate ISP8 at 27 ° C) Positive.

7) Cellulose degradation (cultured at 27 ° C) Negative.

8) Melanin-like pigment formation (trypton yeast broth ISP1, peptone yeast iron agar medium ISP6,
Tyrosine agar medium ISP7 is cultured at 27 ° C.) No melanin-like pigment is produced in any medium.

9) Utilization of carbon source (Pleedham Gottlieb agar medium ISP9, cultivation at 27 ° C) D-glucose, D-xylose, D-fructose, L-arabinose, D-mannitol and L-rhamnose grow well. Sucrose is also utilized. Inositol is probably not assimilated,
Raffinose does not assimilate.

4. Cell wall components Lechevalier, MP, Lechevalier, HA, Actinomycete T
The cell wall type of all cells was examined according to the method described in the text of axonomy workshop, Soc. Ind. Microbiol. In addition, the sugar component type of all strains was C type in which only ribose was detected, and the acyl type of muramic acid was acetyl type.

To summarize the above properties, the A10-12 strain has a well-branched mycelium on the agar medium and elongates like a mesh. An aerial mycelium growing in a zigzag shape is observed at the beginning of the culture, and the hypha is divided along with the growth. Aerial hyphae are straight to curved (Rectifle
xibiles) or fish hook to loop (Retinaculiape)
rti). The spores have a size of 0.5 to 0.8 × 1.0 to 2.0 microns, usually 10 or more spores are linked, and the surface is smooth.

No special organs such as sporangia, axle branching and sclerotia are found on various agar media. The A10-12 strain develops white aerial hyphae on the growth of light yellow tea on various agar media, and production of a soluble pigment is not recognized.

A The reduction of nitrate of strain 10-12, coagulation of milk, peptization of milk, and utilization of lime malate were positive, but starch hydrolysis, gelatin liquefaction, cellulose degradation,
Production of melanin-like pigments (ISP1, ISP6, ISP7) was negative. The strain is a neutral salt actinomycete exhibiting a salt tolerance of 7.5% to 10%.

A Cell wall type using all the cells of the 10-12 strain is type III,
The sugar component type was C type, and the acyl type of muramic acid was acetyl type.

Bergey's manual of determinative b
acteriology (8th edition) and "Experimental method for identification of actinomycetes"
When searched in 1985, edited by the Japanese Society of Actinomycetes, A 10
The −12 strain has Actinomadura, Excellospora, and Actinosynnem because it has a cell wall type III and links 20 or more spores.
a, Nocardiopusis.
However, Actinomadura and Excellospora differ from the genus A10-12 in that they do not contain madurose in the cell wall.
The genus nnema differs from the A strain in that motile cells are formed in the aerial hyphae.

The A10-12 strain (1) forms aerial hyphae and links 10 or more spores, (2) aerial hyphae grows in a Zig-zag form, and breaks down over time, (3) cell wall type III Type,
(4) Type C in which the only sugar contained in the cell wall is ribose, (5) Aerial mycelia of white to yellowish ash on the growth of light yellow tea,
Nocardiopus proposed by J. Meyer
is genus (Nocardiopsis, a new genus of the order Actin
omycetes.Int.J.Syst.Bacteriol., 26 (4), 487-493,1
976) is considered appropriate.

Nocardiopusis spp.N.alba, N.atra, N.coeruleofusc
a, N.flava, N.longispora, N.mutabilis, N.syringae, Nd
assonvillei, N.antarticus, N.trehalosei, N.africane,
"Seeds" such as N. streptosporus are known, but A 10
Strain -12 is probably N. dassonvillei in terms of carbon source assimilation and the fact that only ribose is detected as a sugar component in the cells.
Is considered to be a species similar to

The A10-12 strain is determined to be an actinomycete belonging to the genus Nocardiopusis based on characteristics such as morphology, physiology, and cell wall components.

B 9-47 Strain 1. Morphology The B 9-47 strain elongates a well-branched reticulated basal hypha under a light microscope. The aerial hyphae elongating from the basal hyphae exhibit a straight-bend shape (Rectiflexibiles) or a fishhook-loop shape (Retinaculiaperti), and often Zig-z
It grows in ag form and breaks down like bacilli with the cultivation time.
No special organs such as transgenic branches, sclerotium, and sporangia are found. Mature spores usually link 10 or more spores, the size is about 0.5-0.8 × 1.0-2.0 microns, and the surface is smooth. No spore motility is observed.

2. Growth conditions on various media The color tone is described in "Color Standards" of the Japan Color Research Institute, 1951, and the symbols and colors in brackets are used in Container Corporation of America.
oration of America) Color harmony manual.

1) Lime malate agar medium (cultured at 27 ° C) White aerial hyphae grow on colorless growth, and no soluble pigment is observed.

2) Glucose / nitrate agar medium (cultured at 27 ° C) White aerial hyphae grow on the growth of light yellow tea [2ec, Biscuit], and the soluble pigment takes on a tea taste.

3) Glucose-asparagine agar medium (cultured at 27 ° C) Almost no growth was observed.

4) Starch / synthetic agar medium (cultured at 27 ° C) White aerial mycelia form on the growth of light yellow tea [2gc, Bamboo], and the soluble pigment takes on a yellowish tinge.

5) Sucrose / nitrate agar medium (cultured at 27 ° C) White aerial hyphae grow on colorless growth, and no soluble pigment is observed.

6) Yeast-malt agar medium (cultured at 27 ° C) White aerial hyphae grow on colorless growth, and no soluble pigment is observed.

7) Oatmeal agar medium (ISP3) (cultured at 27 ° C) White aerial hyphae grow on colorless growth, and no soluble pigment is observed.

8) Starch inorganic salt agar medium (ISP4) (cultured at 27 ° C) Aerial mycelia of light yellowish brown [3ca, Shell] were formed on the growth of light yellow tea [2ea, Lt Wheat], and no soluble pigment was observed.

9) Glycerin / asparagine agar medium (ISP5) (27 ° C)
Culture) White aerial hyphae grow on the growth of pale yellow [2gc, Bamboo], and no soluble pigment is observed.

10) Tyrosine agar medium (ISP7) (cultured at 27 ° C) White aerial mycelia form on the growth of pale yellow [3ec, Bisque], and a reddish soluble pigment is observed.

11) Nutrient agar medium (cultured at 27 ° C) White aerial hyphae grow abundantly on the growth of pale yellow [3ec, Bisque], and no production of soluble pigment is observed.

3. Physiological properties 1) Growth temperature range Using yeast extract starter agar medium
As a result of culturing at 10 ° C, 20 ° C, 25 ° C, 31 ° C, 36 ° C, 40 ° C, 45 ° C, and 50 ° C, the B9-47 strain grew at any temperature from 10 ° C to 36 ° C. , 40 ° C, 45 ° C and 50 ° C did not grow. The optimum growth temperature seems to be around 25 ° C to 31 ° C.

2) Liquefaction of gelatin (cultured at 15% simple gelatin at 20 ° C, glucose / peptone / gelatin at 27 ° C) Liquefaction was observed in any medium, and the effect was moderate.

3) Hydrolysis of starter (starch inorganic salt agar medium 27)
Cultivation at about 14 ° C) Slightly hydrolyzable from about 14 days after cultivation, the effect is weaker.

4) Coagulation of defatted milk and peptone formation (cultured at 37 ° C defatted milk) Coagulation starts around 10 days after culture and is completed around 21 days.
Peptone conversion began around day 14 and was almost complete on day 21. Its effect is weaker.

5) Utilization of malate lime (cultivation at 27 ° C of lime malate agar medium) No dissolution of lime malate was observed.

6) Nitrate reduction (peptone water containing 1.0% sodium nitrate ISP8 at 27 ° C) Positive.

7) Cellulose degradation (cultured at 27 ° C) Negative.

8) Melanin-like pigment formation (trypton yeast broth ISP1, peptone yeast iron agar medium ISP6,
Tyrosine agar medium ISP7 is cultured at 27 ° C.) No melanin-like pigment is produced in any medium.

9) Utilization of carbon source (Pleedham Gottlieb agar medium ISP9, cultivation at 27 ° C.) D-glucose, D-xylose, D-fructose, L-arabinose, and D-mannitol were used to grow well, and inositol, Sucrose, L-rhamnose and raffinose do not assimilate.

4. Cell wall components Lechevalier, MP, Lechevalier, HA, Actinomycete T
The cell wall type of all cells was examined according to the method described in the text of axonomy workshop, Soc. Ind. Microbiol. In addition, the sugar component type of all the cells was C type in which ribose and some glucose were detected, and the acyl type of muramic acid was acetyl type.

To summarize the above properties, in the B9-47 strain, the mycelium in the basal medium is well branched on the agar medium and elongates like a mesh. An aerial mycelium growing in a zigzag shape is observed at the beginning of the culture, and the hypha is divided along with the growth. Aerial hyphae are straight to curved (Rectifle
xibiles) or fish hook to loop (Retinaculiape)
rti). The spores have a size of 0.5 to 0.8 × 1.0 to 2.0 microns, usually 20 or more spores, and the surface is smooth.

No special organs such as sporangia, axle branching and sclerotia are found on various agar media. The B9-47 strain develops white aerial mycelia on the growth of light yellow to light yellow tea on various agar media, and the soluble pigment has a reddish to brown taste.

B 9-47 strain showed reduction of nitrate, coagulation of milk, peptonization of milk, and liquefaction of gelatin, but the utilization of lime malate, starch hydrolysis, cellulose degradation, melanin-like pigment production (ISP1, ISP6, ISP7) were negative. The strain is a neutral salt actinomycete exhibiting a salt tolerance of 7.5% to 10%.

The cell wall type using all the cells of the B9-47 strain is type III, the sugar component type is type C, and contains ribose and some glucose. Acyl type of muramic acid showed acetyl type.

Bergey's manual of determinative b
acteriology (8th edition) and "Experimental method for identification of actinomycetes"
When searched in 1985, edited by the Japanese Society of Actinomycetes, B 9
The -47 strain has Actinomadura, Excellospora, and Actinosynnem because it has a cell wall type III and links 20 or more spores.
a, Nocardiopusis.
However, Actinomadura and Excellospora are different from the genus Actinosy in that the B9-47 strain does not contain madurose in the cell wall.
The genus nnema is B 9-47 in that motile cells are formed in the aerial hyphae.
Different from strains.

The B9-47 strain is (1) aerial mycelium grows in a zigzag shape and breaks down with the lapse of time, (2) cell wall type III
(3) Genus Nocardiopusis proposed by J. Meyer (Nocardiopsis, a new genus of the order Actinomyc)
etes. Int. J. Syst. Bacteriol., 26 (4), 487-493,197.
I think it is appropriate to belong to 6).

Nocardiopusis spp.N.alba, N.atra, N.coeruleofusc
a, N.flava, N.longispora, N.mutabilis, N.syringae, Nd
assonvillei, N.antarticus, N.trehalosei, N.africane,
"Seeds" such as N. streptosporus are known, but B 9-
The 47 strains are probably species similar to N. dassonvillei, N. antarticus, or N. trehalosei in that they utilize carbon sources and detect ribose and some glucose as sugar components in bacterial cells. I believe that.

The B9-47 strain is determined to be an actinomycete belonging to the genus Nocardiopusis from the characteristics such as morphology, physiology, and cell wall components.

Next, the bacteriological and chemical properties of the Bacillus strain will be described.

In the present invention, a DL-α-aminonitrile compound (hereinafter abbreviated as “raw nitrile”), which is a substrate used for producing an optically active amino acid using each of the microorganisms described above, may be, for example, [“Organic Synthesis Collective Volume (Org. Syn.Col.Vol.) I, p. 21, and III, P. 84 "]
And [Y. Fukuda et al., "Journal of Technology Technology" (J. Ferment.
l.,) 49 , 1011 (1971)] and the like.

In the α-aminonitrile compound used as the substrate of the present invention, R in the general formulas (I) and (II) is not particularly limited, but the substituent contained in each of the substituted alkyl group and the like is, for example, hydroxy, methoxy, mercapto , Methyl mercapto, amino, halogen, carboxyl, carboxamide, phenyl, hydroxyphenyl or 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-methylcaptopropanenitrile, 2,7-diamino-4,5-dithiaoctanedinitrile, 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-cyanobutanamide, 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,
Examples thereof include 2-amino-2-phenylethanenitrile and the like.

Needless to say, there is no problem even if two or more of the above-mentioned raw material nitriles are used in combination.

In the present invention, the general formula (II)
The α- (N-alkylideneamino) nitrile compounds represented by I) and (IV) can be used, for example, in the synthesis of α-aminonitrile compounds in the first reaction of the Strecker method [eg, Journal of Fermentation]
Technology (J.Ferment.Technol.), 49 , 1011 (197
1) Or Chemistry Letters (Chem. Lett.), 687
(1987)], if the disappearance of the starting aldehyde is confirmed by analysis by gas chromatography or the like, the inorganic salt can be obtained by immediately separating and concentrating the inorganic salt by an operation such as filtration or extraction.

The crude product thus obtained is represented by the general formula (II)
The α-aminonitrile, which is a partial hydrolyzate of the α- (N-alkylideneamino) nitrile compounds represented by I) and (IV), and aldehyde, which is a raw material for its synthesis, may be contained as impurities. After removal by such operations as above, it is used as a substrate. However, it is also possible to use the crude product as it is as a substrate. Note that α-
Examples of the (N-alkylideneamino) nitrile compound include a Schiff base compound of each of the above nitrile compounds and an aldehyde which is a raw material for synthesizing these α-amino nitriles.

In the present invention, the above-mentioned nitrile compound (hereinafter referred to as a raw material nitrile) may be a microorganism having a hydrolytic ability of nitrile belonging to the genus Nocardiopsis or the genus Bacillus,
For example, Nocardiopsis sp.A10-12, Nocardiopsis sp.B9-47, Bacillus sp.B9-40 or Bacillus sp.A9
In order to produce an optically active amino acid by acting -1, any of the methods exemplified as the following (a) to (c) may be applied.

That is, (a) a method in which a microorganism obtained by culturing and growing a microorganism in a medium containing a nitrile compound, for example, propionitrile, is brought into contact with a raw material nitrile in the presence of a corresponding aldehyde, and reacted. (B) a method in which a microorganism obtained by preculturing and growing a microorganism is brought into contact with a nitrile compound, for example, propionitrile, and then the raw material nitrile and a corresponding aldehyde are added to the microorganism to cause a reaction;
And (c) a method of preliminarily cultivating and growing microorganisms and reacting them by directly contacting the starting nitrile and aldehyde with the cells obtained. In these reaction methods, crushed bacterial cells after growth as microorganisms, dried bacterial cells, or treated cells such as separated and purified nitrile hydrolase,
Alternatively, cells immobilized according to a conventional method and processed cells 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, isobutylnitrile, glutaronitrile, triacrylonitrile, crotononitrile Examples include nitrile, lactonitrile, succinonitrile, acrylonitrile, benzonitrile, phenylacetonitrile, and the like.

In the above method (a), in addition to the nitrile compound, as a carbon source, glucose, sucrose, molasses, a saccharide such as starch hydrolyzate, or a substance having a cell growth activity such as acetic acid is added to the medium. And nitrogen sources such as ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, aqueous ammonia, sodium nitrate, amino acids and other assimilating 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 boron, copper, and zinc, that is, so-called trace elements;
Further, if necessary, a seed medium of each of the above microorganisms is inoculated into a medium to which a growth promoting substance such as vitamins, yeast extract and corn stap liquor has been added, and cultured under aerobic conditions to proliferate the cells. The cell culture thus obtained,
Alternatively, a raw material nitrile and an aldehyde are supplied and reacted with a suspension of bacterial cells or a treated bacterial cell separated from the culture.

The reaction is carried out at pH 5-12, preferably at pH 8-12, from 1-6.
Perform for days. A variety of buffers may be used for the reaction, but ammonia-based buffers are preferred, and mixtures of aqueous ammonia and aqueous ammonium chloride, mixtures of aqueous ammonia and aqueous ammonium sulfate, mixtures of aqueous ammonia and aqueous ammonium acetate, aqueous ammonia And the like of an ammonium phosphate aqueous solution. Further, it is preferable to use aqueous ammonia as an alkali for pH adjustment.

When the hydrolysis of the starting nitrile is performed in the presence of the aldehyde, the addition ratio of the aldehyde to the starting nitrile may be 0.1 mol to 10 mol per 1 mol of the starting nitrile, but may be 0.5 mol to 3 mol. It is preferable to use The reaction temperature is preferably in the range of 20 to 70 ° C. In addition, 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 hydrolysis ability of the cells. And increase. 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 intermittent addition, and a method of continuous addition may be used.

The optically active amino acid produced by the above reaction is separated and collected by applying a known means such as phase separation, filtration, extraction, column chromatography and the like.

Next, in the method (b), the nitrile compound is added after the cells are grown without adding the nitrile compound during the culture and growth of the cells in the method (a), and the nitrile hydrolysis ability of the cell microorganisms is increased. After activation, the starting nitrile and the aldehyde are added and reacted to produce an optically active amino acid.

In the method (c), the starting nitrile and the aldehyde are added and reacted immediately after the growth of the cells in the method (b) to produce an optically active amino acid.

In any of the above methods (b) and (c), the method in the above method (a) can be applied to culture conditions, reaction conditions, and separation and collection of the generated optically active amino acids.

The optically active amino acids obtained according to the present invention as described above are 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 25 g of bouillon [Nutrient Broth (OXOID)] and 10 g of glucose were added to 900 ml of ion-exchanged water and stirred, and this was used as solution A. Solution A is added to the Sakaguchi flask in 90 ml portions, and
Sterilize in an autoclave for 0 minutes. 27 g of NaCO ₃ .12H ₂ O is added to 100 ml of ion-exchanged water, and sterilized in an autoclave at 120 ° C. for 20 minutes to obtain a solution B. In a clean bench, add 10 ml of Solution B to a Sakaguchi flask, and use this as a culture medium.

Culture and Propagation of Microorganisms Three platinum loops of the following two strains were ingested into the above medium, and shaking culture was performed at 30 ° C. and 140 rpm for 48 hours.

Strain used: strain name FERM-BP No. Nocardiopsis A 10-12 2422 (Nocardiopsis sp.) Nocardiopsis B 9-47 2423 (Nocardiopsis sp.) The mycelium obtained by the above culture was separated by centrifugation, NH
After washing twice with a 0.1 M buffer of ₄ Cl-NH ₃ (pH 10), the cells were resuspended in a 0.1 M buffer of NH ₄ Cl-NH ₃ so that the optical density (OD) became 10.

Reaction 5 ml of each bacterial cell suspension obtained as described above was
The tube was housed in a test tube having a length of 50 m, and DL-2-amino-3-methylbutanenitrile (50 μm) was added thereto, the tube was sealed, and shaking culture was performed at a temperature of 30 ° C. at 300 rpm for 48 hours.

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

The amount of L-valine produced was determined by Asah as a column packing material.
The measurement was performed using ipak inert sill ODS (manufactured by Asahi Kasei Corporation), and the absolute configuration and optical purity were determined using the same CHIRALPAK WH.
(Manufactured by Daicel Chemical Industries, Ltd.). Table 2 shows the results
As shown in FIG.

Example 2 5 ml of the suspension prepared by the method described in Example 1 was
The tube was housed in a test tube having a diameter of 50 m, and DL-2-amino-4-methylpentanenitrile (50 μl) was added thereto. The tube was sealed, and shaking culture was performed at a temperature of 30 ° C. at 300 rpm for 48 hours.

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

The amount of L-leucine produced was determined using As
Performed using ahipak inert sill ODS (manufactured by Asahi Kasei Corporation)
CHIRALPAK WE was also used to determine its absolute configuration and optical purity.
(Manufactured by Daicel Chemical Industries, Ltd.). Table 3 shows the results
As shown in FIG.

Example 3 5 ml of the bacterial suspension prepared by the method described in
The tube was housed in a 4 mm test tube, 50 μL of DL-2-amino-methylpentanenitrile was added to the tube, the tube was sealed, and shaking culture was performed at a temperature of 30 ° C. at 300 rpm for 48 hours.

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

The amount of L-norvaline produced was determined as column packing.
Asahipak Inertsill ODS (Asahi Kasei Co., Ltd.) was used to determine its absolute configuration and optical purity.
This was performed using KWE (manufactured by Daicel Chemical Industries, Ltd.). The results are as shown in Table 4.

Example 4 5 ml of the bacterial suspension prepared by the method described in
It was housed in a 4 mm test tube, 50 μL of DL-2-amino-4-hexanenitrile was added thereto, and the tube was sealed and brought to a temperature of 30 ° C.
Shaking culture was performed at 300 rpm for 48 hours.

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

The L-norleucine produced was quantified using Asahipak Inertosyl ODS (manufactured by Asahi Kasei Corporation) as a column packing material, and its absolute configuration and optical purity were also determined using CHIRALPA.
This was performed using KWE (manufactured by Daicel Chemical Industries, Ltd.). The results are as shown in Table 5.

Example 5 5 ml of the bacterial suspension prepared by the method described in
It was placed in a 4 mm test tube, and DL-2-amino-3-methylbutanenitrile (50 μl) and isobutyraldehyde were added thereto.
After adding 60 μl, the mixture was sealed, and shaking culture was performed at a temperature of 30 ° C. at 300 rpm for 48 hours.

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

The amount of L-valine produced was determined by Asah as a column packing material.
The measurement was performed using ipak inert sill ODS (manufactured by Asahi Kasei Corporation), and the absolute configuration and optical purity were determined using the same CHIRALPAK WH.
(Manufactured by Daicel Chemical Industries, Ltd.). Table 6 shows the results
As shown in FIG.

Example 6 5 ml of the bacterial suspension prepared by the method described in
It was placed in a 4 mm test tube, and DL-2-amino-4-methylpentanenitrile (50 µm) and isovaleraldehyde (60 µm) were added to the tube, the tube was sealed, and shaking culture was performed at 30 ° C at 300 rpm for 48 hours.

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

The amount of L-leucine produced was determined using As
Performed using ahipak inert sill ODS (manufactured by Asahi Kasei Corporation)
CHIRALPAK WE was also used to determine its absolute configuration and optical purity.
(Manufactured by Daicel Chemical Industries, Ltd.). Table 7 shows the results
As shown in FIG.

Example 7 5 ml of the bacterial suspension prepared by the method described in
The reaction was carried out in the same manner as in Example 1 except that the reaction solution was accommodated in a 4 mm test tube, and 50 μL of DL-2-aminopentanenitrile and 60 μm of n-butyraldehyde were added thereto.

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

The amount of L-norvaline produced was determined as column packing.
Asahipak Inertsill ODS (Asahi Kasei Co., Ltd.) was used to determine its absolute configuration and optical purity.
This was performed using KWE (manufactured by Daicel Chemical Industries, Ltd.). The results are shown in Table 8.

Example 8 5 ml of the bacterial suspension prepared by the method described in
The reaction was carried out in the same manner as in Example 1 except that the reaction solution was placed in a 4 mm test tube, and 50 µL of DL-2-aminohexanenitrile and 60 µL of valeraldehyde were added thereto.

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

The L-norleucine produced was quantified using Asahipak Inertosyl ODS (manufactured by Asahi Kasei Corporation) as a column packing material, and its absolute configuration and optical purity were also determined using CHIRALPA.
This was performed using KWE (manufactured by Daicel Chemical Industries, Ltd.). The results are shown in Table 9.

Example 9 5 ml of the bacterial suspension prepared by the method described in
It was placed in a 4 mm test tube, and DL-3-methyl-2-
(N-2-Methylpropylidene) aminopentanenitrile (50 μ) was added, the mixture was sealed, and shaking culture was performed at a temperature of 30 ° C. at 300 rpm for 48 hours.

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

The amount of L-valine produced was determined by Asah as a column packing material.
The measurement was performed using ipak inert sill ODS (manufactured by Asahi Kasei Corporation), and the absolute configuration and optical purity were determined using the same CHIRALPAK WH.
(Manufactured by Daicel Chemical Industries, Ltd.). The results are in Table 10
As shown in FIG.

Example 10 To 5 ml of the bacterial suspension prepared by the method described in Example 1,
4-methyl-2- (N-3-methylbutylideneamino)
The reaction was carried out as described in Example 9 except that 50 μ of pentanenitrile was added.

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

The amount of L-leucine produced was determined using As
Performed using ahipak inert sill ODS (manufactured by Asahi Kasei Corporation)
CHIRALPAK WE was also used to determine its absolute configuration and optical purity.
(Manufactured by Daicel Chemical Industries, Ltd.). The results are in Table 11
As shown in FIG.

Example 11 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

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

The amount of L-norvaline produced was determined as column packing.
Asahipak Inertsill ODS (Asahi Kasei Co., Ltd.) was used to determine its absolute configuration and optical purity.
This was performed using KWE (manufactured by Daicel Chemical Industries, Ltd.). The results are shown in Table 12.

Example 12 To 5 ml of the bacterial suspension prepared by the method described in Example 1,
2- (N-pentylideneamino) pentanenitrile 50μ
The reaction was carried out by the method described in Example 1 except that

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

The amount of L-norleucine was determined using column
Asahipak Inertsill ODS (Asahi Kasei Co., Ltd.) was used to determine its absolute configuration and optical purity.
This was performed using KWE (manufactured by Daicel Chemical Industries, Ltd.). The results are shown in Table 13.

Example 13 Preparation of Medium 25 g of bouillon [Nutrient Broth (OXOID)] and 10 g of glucose were added to 900 ml of ion-exchanged water, and the mixture was stirred to obtain a liquid A. Solution A is added to the Sakaguchi flask in 90 ml portions, and
Sterilize in an autoclave for 0 minutes. Na ₂ CO ₃・ 12H ₂ O27g
Was added to 100 ml of ion-exchanged water, and sterilized in an autoclave at 120 ° C. for 20 minutes to obtain a solution B. In a clean bench, add 10 ml of Solution B to a Sakaguchi flask, and use this as a culture medium.

Culture and Propagation of Medium Three platinum loops of the following two strains were respectively collected in the above medium, and cultured at 30 ° C. with shaking at 140 rpm for 48 hours.

Bacterial strain used: Bacterial name FERM-P No. Bacillus B9-40 (Bacillus sp.) P-11478 Bacillus A9-1 (Bacillus sp.) P-11477 The mycelium obtained by the above culture was separated by centrifugation, and NH
After washing twice with a ₄ Cl-NH ₃ 0.1 M buffer (pH 10), the cells were resuspended in a 0.1 M buffer of NH ₄ Cl-NH ₃ so that the optical density (OD) became 10.

Reaction 5 ml of each bacterial cell suspension obtained as described above was
Then, 50 μl of DL-2-amino-4-methylpentanenitrile was added thereto, sealed tightly, and heated at 30 ° C. and shake-cultured at 300 rpm for 48 hours.

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

The amount of L-leucine produced was determined using As
Performed using ahipak inert sill ODS (manufactured by Asahi Kasei Corporation)
CHIRALPAK WE was also used to determine its absolute configuration and optical purity.
(Manufactured by Daicel Chemical Industries, Ltd.). The results are in Table 14
As shown in FIG.

Example 14 5 ml of the suspension prepared by the method described in Example 13 was φ24 m
m, and sealed with 50 μL of DL-2-amino-methylpentanenitrile.
Shaking culture was performed at 300 rpm for 48 hours.

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

The amount of L-norvaline produced was determined as column packing.
Asahipak Inertsill ODS (Asahi Kasei Co., Ltd.) was used to determine its absolute configuration and optical purity.
This was performed using KWE (manufactured by Daicel Chemical Industries, Ltd.). The results are as shown in Table 15.

Example 15 5 ml of the cell suspension prepared by the method described in Example 13 was placed in a test tube having a diameter of 24 mm, and DL-2-amino-4- was added thereto.
Hexanenitrile (50 μ) was added and the mixture was sealed, followed by shaking culture at 30 ° C. at 300 rpm for 48 hours.

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

The L-norleucine produced was quantified using Asahipak Inertosyl ODS (manufactured by Asahi Kasei Corporation) as a column packing material, and its absolute configuration and optical purity were also determined using CHIRALPA.
This was performed using KWE (manufactured by Daicel Chemical Industries, Ltd.). The results are as shown in Table 16.

Example 16 5 ml of the bacterial suspension prepared by the method described in Example 13 was
It was placed in a 4 mm test tube, and DL-2-amino-4-methylpentanenitrile (50 µm) and isovaleraldehyde (60 µm) were added to the tube, the tube was sealed, and shaking culture was performed at 30 ° C at 300 rpm for 48 hours.

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

The amount of L-leucine produced was determined using As
Performed using ahipak inert sill ODS (manufactured by Asahi Kasei Corporation)
CHIRALPAK WE was also used to determine its absolute configuration and optical purity.
(Manufactured by Daicel Chemical Industries, Ltd.). The results are in Table 17
As shown in FIG.

Example 17 Example 5 was repeated except that 5 ml of the cell suspension prepared by the method described in Example 13 was placed in a φ24 mm test tube, and 50 μL of DL-2-aminopentanenitrile and 60 μl of n-butyraldehyde were added thereto. The reaction was carried out according to the method described in 13.

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

The amount of L-norvaline produced was determined as column packing.
Asahipak Inertsill ODS (Asahi Kasei Co., Ltd.) was used to determine its absolute configuration and optical purity.
This was performed using KWE (manufactured by Daicel Chemical Industries, Ltd.). The results are shown in Table 18.

Example 18 5 ml of the bacterial cell suspension prepared by the method described in Example 13 was placed in a φ24 mm test tube, and DL-2-aminohexanenitrile 50 μ and valeraldehyde 60 μ were added thereto. The reaction was performed in the manner described.

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

The L-norleucine produced was quantified using Asahipak Inertosyl ODS (manufactured by Asahi Kasei Corporation) as a column packing material, and its absolute configuration and optical purity were also determined using CHIRALPA.
This was performed using KWE (manufactured by Daicel Chemical Industries, Ltd.). The results are shown in Table 19.

Example 19 5 ml of the bacterial suspension prepared by the method described in Example 13 was
The sample was placed in a 4 mm test tube, and 4-methyl-2- (N-
3-methylbutylideneamino) pentanenitrile 50μ
The reaction was carried out by the method described in Example 13 except that was added.

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

The amount of L-leucine produced was determined using As
Performed using ahipak inert sill ODS (manufactured by Asahi Kasei Corporation)
CHIRALPAK WE was also used to determine its absolute configuration and optical purity.
(Manufactured by Daicel Chemical Industries, Ltd.). Results are in Table 20
As shown in FIG.

Example 20 To 5 ml of the bacterial suspension prepared by the method described in Example 13,
2- (N-3-butylideneamino) pentanenitrile 50
The reaction was performed by the method described in Example 13 except that μ was added.

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

The amount of L-norvaline produced was determined as column packing.
Asahipak Inertsill ODS (Asahi Kasei Co., Ltd.) was used to determine its absolute configuration and optical purity.
This was performed using KWE (manufactured by Daicel Chemical Industries, Ltd.). Table 21 shows the results.

Example 21 5 ml of the bacterial cell suspension prepared by the method described in Example 13
2- (N-pentylideneamino) pentanenitrile
The reaction was performed by the method described in Example 13 except that 50 μ was added.

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

The amount of L-leucine was determined by Asah as a column packing material.
The measurement was performed using ipak inert sill ODS (manufactured by Asahi Kasei Corporation).
(Manufactured by Daicel Chemical Industries, Ltd.). Table 22 shows the results.

Effect of the Invention As described above, according to the present invention, optically active amino acids can be selectively produced directly from a DL-α-aminonitrile compound using a microorganism, so that the optically active amino acids used in the above various fields are used. It is beneficial in the production of amino acids.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12R 1:07) (C12P 13/08 C12R 1:01) (C12P 13/08 C12R 1:07) (72) Inventor Keizo Furuhashi Nihon Mining Co., Ltd., 3-17-35 Niisominami, Toda City, Saitama Prefecture (56) References JP-A-4-166091 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12P 13/06

Claims (2)

(57) [Claims] 1. A method comprising reacting one or more nitrile compounds represented by the following general formulas (I) to (IV) with a microorganism having a nitrile hydrolytic ability belonging to the genus Nocardiopsis or the genus Bacillus. A method for producing optically active amino acids, characterized by producing optically active amino acids, (Where R in the formulas (I) and (II) 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) Group, substituted pyridyl group, thiazolyl group, substituted thiazolyl group) (Wherein R ₁ and R ₂ in the formulas (III) and (IV) are an alkyl group, a substituted alkyl group, a phenyl group, a substituted phenyl group,
Imidazolyl group, substituted imidazolyl group, indolyl group,
A substituted indolyl group, a furyl group, a substituted furyl group, a pyridyl group, a substituted pyridyl group, a thiazolyl group, and a substituted thiazolyl group, and R ₁ and R ₂ may be the same or different. 2. The following general formula (I) or (II) (Where R in the formulas (I) and (II) 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) Group, substituted pyridyl group, thiazolyl group, substituted thiazolyl group)
One or more α-aminonitrile compounds represented by the following general formula (V) R-CHO (V) or the following general formula (VI) R-CH ₂ CHO (VI) (wherein R Is the same as the above-mentioned general formula (I) or (II)) to produce an optically active amino acid by reacting a microorganism having a nitrile hydrolytic ability belonging to the genus Nocardiopsis or Bacillus in the presence of the corresponding aldehyde represented by the general formula (I) or (II). A process for producing optically active amino acids.