JP2950896B2 - Method for producing D-α-phenylglycine - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing D-α-phenylglycine from racemic phenylglycinonitrile by the action of a microorganism.

D-α-phenylglycine is important as a raw material for medicinal and agricultural chemicals such as penicillin antibiotics and cephem antibiotics.

[Conventional technology and problems]

The method for producing D-α-phenylglycine is as follows: (1) A synthesis method in which a DL-5-substituted hydantoin is converted into an N-carbamyl-D-amino acid by a D-form-specific dihydropyrimidinase and then decarbamylated [S. Takahashi e
t.al.J.Ferment.Technol., 56 492 (1978);
(2) A method of converting DL-5-substituted hydantoin into D-amino acid in one step by a microorganism containing D-hydantoinase and N-carbamoyl-D-amino acid hydrolase [JP-A-54-89088] (3) Method of converting DL-amino acid amide to D-amino acid by hydrolyzing it with a D-form specific amidase [Japanese Patent Publication No. Sho 63]
-87998] is known.

However, in the method for hydrolyzing DL amino acid amide, an L-form amino acid amide remains as an unreacted component, and a step of separating, collecting, racemizing, and reusing it is further required. In addition, the method using a hydantoin derivative as a raw material is economical because the reaction involves a racemization of the raw material, but is disadvantageous in that aliphatic hydantoin is generally less likely to racemize than aromatic hydantoin. Is limited.

On the other hand, methods for specifically producing amino acids from aminonitrile include: (1) Production of L-methionine, L-alanine, L-phenylalanine, etc. from the corresponding aminonitrile by Brevibacterium [JC Jallageas., Et al. .al, Advan
ces reference in Biochemical Engineering 14 1], (2) the genus Acinetobacter L- alanine production from DL-alpha-aminopropionitrile by [Anne M., et.a
1, Biotechnology Letters 7 865 (1985)], (3) A method for producing L-valine and L-leucine from the corresponding DL aminonitrile by Nocardia, Mycobacterium, and Corynebacterium [Japanese Unexamined Patent Publication No. −3173
No. 93].

However, these methods all relate to a method for producing L-amino acids from aminonitrile, and do not directly produce D-amino acids from aminonitrile. In addition, DL-α-phenylglycinonitrile is converted to D-α
There is no finding that phenylglycine was produced by microbial action.

[Problems to be solved by the invention]

The present inventors have obtained inexpensively chemical synthesis in order to develop an industrially advantageous production method of D-α-phenylglycine.
As a result of diligent research on a novel technique for specifically converting DL-α-phenylglycinonitrile to D-α-phenylglycine, nitrile hydrolysis activity that is optically specific for DL-α-phenylglycinonitrile DL-α-phenylglycinonitrile by the action of a microorganism having
-Specific conversion to α-phenylglycine, and D-specific hydrolysis reaction by this microorganism and racemization reaction of phenylglycinonitrile occurring in a neutral or basic aqueous medium in the presence of ammonia It has been found that substantially all of DL-α-phenylglycinonitrile can be converted to D-α-phenylglycine by a reaction conjugate with the above. The present invention is based on these findings.

That is, the present invention provides a method for converting D-α into an aqueous medium by the action of a microorganism having an optically specific nitrile hydrolysis activity for DL-α-phenylglycinonitrile.
-D-α-phenylglycine, characterized in that it is converted to -phenylglycine or that this hydrolysis reaction is carried out under neutral or basic conditions in the presence of ammonia.

According to the present invention, DL-α- is obtained by chemical racemization of a substrate aminonitrile and D-specific nitrile hydrolysis.
The target product can be theoretically obtained at a yield of 100% from phenylglycinonitrile, which is economically advantageous.

In the present invention, the microorganism used is a genus Acinetobacter and a caseobacter.
er), a microorganism belonging to the genus Alcaligenes or the genus Pseudomonas, or a mutant derived therefrom, and more specifically, the Caseobacter sp. BC4 newly isolated from the soil by the present inventors.
(Pierce No. 3316), Caseobacter sp. BC23 (Pierce No. 11261), Pseudomonas sp. BC13-2 (Pierce No. 3319), Pseudomonas sp. BC15-2 ( Microorganisms, Inc. No. 3320), Alcaligenes sp. BC16-2 (Microtechnical Co., Ltd. No. 3321), and Acinetobacter sp. BC9-2 (Microtechnical Research, Inc. No. 3317). . All of these microorganisms have been deposited under the above numbers with the Research Institute of Microbial Industry and Technology (MIC) at the National Institute of Advanced Industrial Science and Technology, and their mycological properties are as follows.

The above mycological properties are described in the
Systematic Bacteriology (Bergey's Manual
According to the classification of BC4 and BC23, the strains BC4 and BC23 belong to the genus Caseoobacter, BC13-2 and BC15-2 belong to the genus Pseudomonas, BC16-2 belong to the genus Alcaligenes and BC9-2 belong to the genus Acinetobacter. Identified.

 Next, embodiments of the present invention will be described.

For cultivation of the microorganism used in the present invention, a medium containing a carbon source, a nitrogen source, and inorganic nutrients necessary for the growth of the microorganism is used. For example, glucose, glycerol, sucrose, molasses, etc. as a carbon source, yeast extract, ammonium sulfate, ammonium chloride, etc. as a nitrogen source, and sodium sulfate, magnesium chloride, calcium chloride, manganese sulfate, ferric chloride as an inorganic nutrient source , Zinc sulfate and the like. Also, nitriles (such as nitrile cinnamate, benzyl cyanide, benzonitrile, 2-cyanopyridine, propionitrile, isobutyronitrile, etc.) and amides (phenyl) at concentrations that do not significantly inhibit growth in the early or middle stages of culture Acetamide, 4
-Pyridinecarboxylic acid amide, isobutylamide) and lactams (ε-caprolactam, γ-butyrolactam, etc.) are preferable because higher enzyme activity can be obtained.

The culture was carried out under aerobic conditions at pH 4-10, temperature 20-50 ° C, 24
The control may be performed in a range suitable for each microorganism in about 96 hours.

In the hydrolysis reaction, the microorganism is cultured as described above, and the culture solution, the bacterial cells separated from the culture solution, the treated bacterial cells (crushed bacterial cells, extracted enzymes) or the bacterial cells or enzymes immobilized by a conventional method are subjected to DL. It may be mixed with -α-phenylglycinonitrile. At this time, ammonia was allowed to coexist in the reaction system, and the system was adjusted to a neutral to basic pH value of 7 to 11 to obtain a theoretically 100% yield from DL-α-phenylglycinonitrile. To obtain the desired product. Further addition of ammonia to make cyan ions present can further improve the yield.

The amount of ammonia to be added is 1 to 500 mol, preferably 10 to 100 mol, of ammonia to 1 mol of the raw nitrile. As the ammonia source, any commonly used aqueous ammonia or ammonium salt may be used. It is effective to use an ammonia-based buffer such as ammonium. A cyanide source such as potassium cyanide and sodium cyanide can be used as a cyanide ion source.
It is 0.1 to 100 mol, preferably 1 to 50 mol.

DL-α-phenylglycinonitrile in the reaction solution is usually 0.01 to 5.0% by weight, and the amount of microorganisms used for DL-α-phenylglycinonitrile is 0.01 to
5.0% by weight, reaction temperature is freezing point ~ 60 ° C, preferably 10 ~ 5
The reaction may be performed at 0 ° C. for 0.5 to 72 hours.

The raw materials DL-α-phenylglycinonitrile, ammonia and cyanide may be added at the same time at the start of the reaction, or sequentially or continuously after the start of the reaction.

D-α from a reaction solution containing D-α-phenylglycine
-Isolation of phenylglycine is to remove the cells by centrifugation or the like, and then obtain D-α-phenylglycine as a target substance by using a known method such as concentration, ion exchange, extraction, or crystallization. Can be.

〔The invention's effect〕

According to the present invention, DL-α-phenylglycinonitrile can be hydrolyzed D-specifically, and furthermore, a D-specific hydrolysis reaction by this microorganism and near neutral or basic in the presence of ammonia. Substantially all of the DL-α-phenylglycinonitrile can be converted to D-α-phenylglucine by a conjugate reaction with the racemization reaction of phenylglycinonitrile that occurs in an aqueous medium.

〔Example〕

Hereinafter, the present invention will be described specifically with reference to Examples, but these Examples do not limit the present invention.

Example 1 (1) Culture Each microorganism shown in Table 1 was inoculated into the following medium and cultured.

Medium composition Glycerol 30 g / cyanide 0.2 g / yeast extract _{3 g / K 2 HPO 4 3} g / Na 2 SO 4 0.3 g / MgCl 2 0.2 g / CaCl 2 40 mg / MnSO 2 · 4H 2 O 4 mg / _{FeCl 3 · 7H 2 O 0.7mg /} ZnSO 4 30 ℃ in 500ml Erlenmeyer flask containing 0.1 mg / pH 7.2 culture the medium 100 ml, 2
Shaking culture was performed for one day.

(3) Hydrolysis reaction The cells were collected from the resulting culture by centrifugation, washed with 20 mM phosphate buffer (pH 7.5), and the precipitated cells were resuspended in 10 ml of the same phosphate buffer. . 50 mM phosphate buffer (pH 7.) with a final concentration of 10 mM DL-α-phenylglycinonitrile.
5) Add 1 ml of the above cell suspension to a total of 2 ml, and add 15 ° C
For 9 hours. After completion of the reaction, the reaction solution was centrifuged to remove bacterial cells, and the supernatant was subjected to Daicel Chemical Industries, Ltd.
Analysis was performed by high performance liquid chromatography using a column containing IR-ALPAK CR (+) as a packing material, and D-α-
The amount of phenylglycine was measured.

 The results are shown in Table 1.

Example 2 Microorganisms of each genus were cultured as in Example 1, collected by centrifugation, washed, and suspended in a 20 mM phosphate buffer. Next, 10 ml of DL-α-phenylglycinonitrile containing 1 ml of the above cells
M, ammonia water 0.4M, and potassium cyanide 02M. 2m
l of the reaction solution was prepared. This solution was reacted at 35 ° C. for 4 hours. After completion of the reaction, the reaction solution was centrifuged to remove cells, and the supernatant was analyzed by high performance liquid chromatography as in Example 1.

 The results are shown in Table 2.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:05) (C12P 13/04 C12R 1:01) (C12P 13/04 C12R 1:05) (58) Fields surveyed ( Int.Cl. ⁶ , DB name) C12P 41/00 C12P 13/04 CA (STN) REGISTRY (STN) BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A genus of Acinetobacter, which has an optically specific nitrile hydrolysis activity for DL-α-phenylglycinonitrile,
cter) by the action of a microorganism belonging to the genus Alcaligenes or to the nitrile in aqueous medium.
-A method for producing D-α-phenylglycine, which is converted into α-phenylglycine. 2. The process according to claim 1, wherein the hydrolysis reaction is carried out in the presence of ammonia under neutral or basic conditions.