JP3266635B2 - Method for producing 1-pipecolic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for industrially producing 1-pipecolic acid. 1-Pipecolic acid is a compound useful as a raw material for synthesizing drugs such as the immunosuppressant FK506 and related compounds. In addition, it is sometimes used after being converted to a useful compound α-aminoadipic acid. 1-Pipecolic acid is not a normal amino acid present in natural proteins, but is an analog of 1-proline and occurs in plants and animals, and its production in the brain suggests a role as a neurotransmitter. (HP Broquist, Ann. Re.
v. Nutr. 1991, No. 11,435-488
page). It is also used as an analog of proline in the synthesis of ACE inhibitors (AA Patch S, N
aure, 288, 280-283, 1980.
Year).

[0002]

2. Description of the Related Art 1-Pipecolic acid is a plant (J. Ame).
r. Chem. Soc. 74, 2949, 1952
), The isolation of Neurospora from cultures has been reported. (JP Greens
tein and M.E. Winitz: Chemistr
y of The Amino Acids, John
Wiley and Sons Inc. New Yo
rk (1966) Volume 1, p29 and Volume 2 p141
2). Aspergillus nislans (Asperg)
production in lysine-requiring strains of H. illus nidulans has been reported (Biochemistry No. 1).
Vol., 606-612, 1962). Production method by a synthesis method from L-lysine (J. Chem. Soc. Chem.
Commun. 1985, 633-635). It was also made by optical resolution of DL-pipecoline made by synthetic method from pyrophosphate (Method
of Enzymol. 17B, pages 174 to 188, 1
971). As a method of optical resolution, a diastereomer salt method using d-tartaric acid is used, and D-tartrate from pig liver is used.
A method is known in which the d-form is decomposed with amino acid oxidase to leave one form.

[0003] Also, L-lysine, ε.
Reduction of delta-1-piperidein-6-carboxylic acid generated by natural conversion from α-aminoadipate semialdehyde generated by oxidation with dehydrogenase or L-lysine-α-ketoglutarate aminotransferabi with hydrogen Is known to produce 1-pipecolic acid (Biochemistry, 7).
Vol., 4102, 1968). In addition, α of L-lysine
-A metabolic pathway for producing 1-piperidein-2-carboxylic acid from the decarboxylation reaction to produce pipecolic acid has been estimated in Pseudomonas bacteria (J.ge.
n. Microbiol. , 99, 139-155
Pp. 1977).

[0004]

In the above-mentioned known method, the amount of 1-pipecolic acid produced is small, the optical resolving agent used is expensive, the operation is complicated, and the purified enzyme cannot be used. It is not efficient as an industrial process due to drawbacks such as the use and use of expensive intermediates.

[0005]

SUMMARY OF THE INVENTION The inventor of the present invention has conducted research on a method for producing 1-pipecolic acid which is industrially advantageous by overcoming the drawbacks of the conventional method, and as a result, the method has been inexpensively supplied to the market. The present inventors have discovered a method of directly producing 1-pipecolic acid in a reaction solution by allowing a microorganism or a processed product thereof to act on existing L-lysine, and have completed the present invention based on this discovery.

[0006]

DETAILED DESCRIPTION OF THE INVENTION The microorganisms used in the present invention include the genus Alcaligenes, the genus Providencia, and the genus Proteus (Pr).
oteus), Bacillus, Agrobacterium,
It is a microorganism belonging to the genus Morganella and the genus Planococcus. More specific examples of the bacterial species and strains include Alcaligenes faecalis.
calis) ATCC 8750, Providencia alkaciens (Providencia alk)
alfaciens) ATCC 9886, Providencia retteri (Providencia rett)
geri) ATCC 9918, Proteus mirabilis ATCC 1
5290, Proteus mirabilis
mirabilis) IFO 3549, Proteus
Mitagiri (Proteus mitajiri) ATC
C 21136, Providencia Stuarchy (Pr
Ovidencia Sturti) ATCC 25
825, Bacillus sphaericus
sphaericus) IFO 3525, Agrobacterium tumefaciens
um tumefaciens) IFO 3058, Morganellamorga
nii) ATCC 25830, Planococcus citreus A
TCC 14404.

After culturing these microorganisms to obtain cells,
The cells are suspended in a reaction solution containing L-lysine, and L-lysine is suspended.
The reaction is allowed to proceed until most of the lysine is converted to 1-pipecolic acid. The cells may be crushed to extract the enzyme activity for converting 1-lysine to 1-pipecolic acid and then act on L-lysine, or the extract may be further purified to act on L-lysine. Alternatively, L-lysine can be directly added to the growth culture for reaction.

[0008] As components of a medium for culturing microorganisms to obtain cells, those commonly used for culturing microorganisms such as a carbon source, a nitrogen source, and inorganic salts are used. Examples of the carbon source include sugars such as glucose, organic acids such as citric acid, and natural products such as molasses that can be used by microorganisms. As the nitrogen source, natural products such as ammonium sulfate, ammonium chloride, peptone, meat extract and yeast extract are usually used. As the inorganic salt, potassium phosphate, magnesium sulfate, or a trace amount of a so-called trace element but effective for growth is used. L-Lysine as a reaction substrate may be added to the medium.

The aqueous reaction solution used for the reaction is
A phosphate buffer, an acetate buffer, or the like is usually used so as to reduce the fluctuation of the pH of the reaction solution. A reaction solution containing an organic solvent can be used as long as it contains water. Reaction pH 4
9 at a temperature of 10 to 60 ° C. If the reaction solution is gently shaken or stirred, the reaction progresses quickly, but the objective can be achieved even by standing reaction. When the reaction is carried out under aerobic conditions such as shaking, and when the reaction is carried out in the presence of carbohydrates such as glucose and fructose that can be metabolized by microorganisms, the amount of pipecolic acid produced is significantly increased. When the amount of L-lysine in the reaction solution decreases and the production of 1-pipecolic acid reaches a substantial amount, the cells are removed from the reaction solution by an operation such as centrifugation, or the ion exchange resin is used as it is. 1-Pipecolic acid can be isolated and recovered from the reaction solution by applying a known method to separation and purification of pipecolic acid, such as treatment, concentration, and crystallization. A method for collecting pipecolic acid is described in the literature (Methods of Enz).
ymology 17B, pp. 174-188, 1971
Year) and that method can be used.

[0010]

The present invention will be described more specifically with reference to the following examples. In the examples, the quantification of 1-pipecolic acid was by high performance chromatography. The condition is column: SU
MICHIRAL OA-5000, mobile phase: 1 mM copper sulfate, speed: 1 ml / min, temperature: 30 ° C., detection: UV2
50 nm. Under these conditions, 1-pipecolic acid and d-
Pipecolic acid can be separated and quantified. In addition, in order to follow the progress of the reaction qualitatively and semi-quantitatively, as a simpler method, observe the color intensity of the purple spot of pipecolic acid by heating after spraying ninhydrin by thin layer chromatography. To do was also used.

Example 1 10 g of glucose, 10 g of peptone, 5 g of yeast extract,
10 g of sterile medium prepared by dissolving 5 g of sodium chloride, 0.8 g of monopotassium phosphate, 0.4 g of dipotassium phosphate, and 0.2 g of magnesium sulfate in 1 liter (pH 7.2) by dissolving in water.
l in a large test tube containing Alcaligenes faecalis A
After inoculation with TCC 8750 and shaking culture at 20 ° C. and 295 reciprocations per minute for 2 days, the bacteria were centrifuged and the pH was adjusted to 5 at pH 7.0.
After the suspension was suspended twice in 0 mM phosphate buffer and centrifuged twice, the cells were placed in a test tube containing 10 ml of 50 mM phosphate buffer (pH 7.0) containing 1% L-lysine hydrochloride and 5% glucose. In addition, a shaking reaction was performed at 26 ° C. at 295 reciprocations per minute. After 4 days, 0.300% reaction 7
0.417% of L-pipecolic acid was produced in one day. This concentration is about 47% and 59% relative to L-lysine in molar yield, with some errors based on the cell volume. Note that d-pipecolic acid was not detected in the reaction solution.

Example 2 As a microorganism, Providencia stuartii ATCC
In the same manner as in Example 1 except that 25825 was used, 0.159% of 1-pipecolic acid was produced in the reaction solution on the 2nd reaction day and 0.184% in the 7th reaction day. Example 3 As a microorganism, Proteus mitaziri ATCC2113
Reaction 7 was carried out in the same manner as in Example 1 except that Compound 6 was used.
In the day, 0.04% of 1-pipecolic acid was produced in the reaction solution.

Example 4 When the reaction conditions for reacting the cells cultured in the same manner as in Example 1 with L-lysine were allowed to stand or shake without glucose, the shake was performed in the presence of 1 to 5% of glucose. In this case, the amount of 1-pipecolic acid produced on days 1 to 7 of the reaction is 1st.
As shown in the table. As shown in Table 1, the production rate of 1-pipecolic acid was slow in the standing reaction, and the production rate was faster in the shaking reaction. In the shaking reaction, the production of 1-pipecolic acid was promoted by the presence of glucose, and the decrease of 1-pipecolic acid which would occur when the reaction time was prolonged was suppressed.

[Table 1]

Example 5 The same strain, medium and reaction solution composition as in Example 1 were used, and an Erlenmeyer flask was used instead of a test tube. That is,
The cells obtained by inoculating and culturing the cells into a 300 ml Erlenmeyer flask containing 50 ml of the medium were placed in a 300 ml Erlenmeyer flask containing 50 ml of the reaction solution.
The mixture was added to a ml Erlenmeyer flask and shaken for 4 days. 1-Pipecolic acid was produced in the reaction solution at a concentration of 0.35%. A liquid obtained by removing cells by centrifugation from 200 ml of the reaction solution was passed through a column of a strongly ionized cation exchange resin Dowex 50 W × 8 (200 to 400 mesh) in the form of hydrogen ion, and the solution was diluted with a 1N ammonium hydroxide solution. Eluted. The eluate fractions containing 1-pipecolic acid were combined and concentrated under reduced pressure. The residue was dissolved in 100 ml of water, passed through a column of strongly basic anion exchange resin Dowex 1 (acetic acid type), and treated with 1N acetic acid. Eluted. The 1-pipecolic acid fractions were combined and concentrated to give 1-pipecolic acid crystals 310 m
g. [Α] ²⁵ _D = −34.7 ° (c = 5,
water).

Example 6 The same procedure was performed as in Example 1 except that the various strains shown in Table 2 were used. After the reaction for 6 days, the amount of 1-pipecolic acid produced was as shown in Table 2.

[Table 2]

Example 7 Example 1 was repeated except that the strains shown in Table 3 were allowed to stand and reacted in a reaction solution from which glucose in the reaction solution composition had been removed.
The amount of 1-pipecolic acid produced during the 7 days of the reaction in the same manner as in Example 1 was as shown in Table 3.

[Table 3]

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12P 17/12 (C12P 17/12 C12R 1:37) C12R 1:37) (C12P 17/12 (C12P 17/12 C12R 1 : 07) C12R 1:07)

Claims (2)

(57) [Claims] 1. A microorganism belonging to any of the genera Alcaligenes, Prodensia, Proteus, Bacillus, Agrobacterium, Morganella, and Planococcus or a treated product thereof is allowed to act on L-lysine and cultured.
A method for producing 1-pipecolic acid, wherein 1-pipecolic acid is produced without adding hydrogen to the liquid . 2. The genus Alcaligenes, the genus Prodensia and the genus
Roteus, Bacillus, Agrobacterium, mol
Microbes belonging to any of the genus Ganela or Planococcus or a processed product thereof are allowed to act on L-lysine and cultured.
In the method for producing l-pipecolic acid which produces l-pipecolic acid without adding hydrogen to the liquid, the reaction is carried out aerobically by the presence of a carbohydrate that can be metabolized by the bacteria used in addition to L-lysine. A method for producing 1-pipecolic acid, which is characterized in that: