JPH0665313B2 - Method for producing trans-4-cyanocyclohexane-1-carboxylic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention provides trans-4-cyanocyclohexane-1-
It relates to a novel method for producing a carboxylic acid.

The object of the present invention is trans-4-aminomethylcyclohexane-1-, which is extremely effective as a medicine such as a hemostatic agent.
The present invention is to industrially produce trans-4-cyanocyclohexane-1-carboxylic acid, which is a carboxylic acid and an intermediate for producing cetraxate hydrochloride, which is highly effective as an anti-ulcer agent, by a novel method.

(Prior Art) As a conventional method for producing 4-cyanocyclohexane-1-carboxylic acid, for example, a method of contacting hexaterephthalic acid with ammonia gas (Japanese Patent Publication No. 47-23).
535), 1,4-dicyanocyclohexane is passed through hydrochloric acid gas in methanol to form its imidate, and then hydrolyzed to produce methyl 4-cyanocyclohexane-1-carboxylate (JP-A-55-15451), 1. A method of contacting 1,4-dicyanocyclohexane with ammonia gas (Japanese Patent Laid-Open No. 48-34144) is known.

(Problems to be Solved by the Invention) Since all conventional production methods use synthetic techniques, high-temperature, high-pressure reaction conditions are required, and the production of by-products makes purification of the target product complicated. is there. Moreover, in the conventional manufacturing method, from the raw material of the transformer body, trans-4 is used.
It is difficult to produce -cyanocyclohexane-1-carboxylic acid while maintaining the configuration.

(Means for Solving the Problems) In order to solve the above problems, the present inventors utilize a novel biochemical action of a microorganism capable of maintaining the configuration at room temperature and normal pressure. The manufacturing method was found. That is, the formula (I) From trans-1,4-dicyanocyclohexane represented by the formula (II) using the action of microorganisms. Trans-4-cyanocyclohexane-1-
It is a method for producing a carboxylic acid.

Generally, the biochemical action of converting a nitrile group to a carboxyl group, that is, the enzyme is known as nitrilase [Arch. Biochem. Biophys.
Vol. 62-68 (1964), or Fermentation & Kogaku, Vol. 41, 382-388 (1983)].
However, the reaction of converting a nitrile compound containing a cyclohexane ring into a corresponding carboxylic acid as a substrate has never been known. Furthermore, it has not been known at all for converting only one of the two nitrile groups of (I) into a carboxyl group while maintaining the configuration.

Therefore, the present inventors searched for a microorganism having the ability to convert (I) into (II) from soil isolates and publicly preserved strains, discovered a microorganism having the conversion ability, and have already found Japanese Patent Application No. -1
Patent application for 64823 was made, and further, the present invention by another microorganism was completed.

Next, a method for implementing the present invention will be described.

(i) Reaction method The reaction method of converting (I) to (II) in the present invention, specifically, a method of culturing the microorganism in the presence of (I), a microbial culture, and There are two methods, namely, a method of contacting the cells (I) with the cells or treated cells (for example, crushed cells or enzymes separated and extracted from the cells). In addition, after the cells, the treated cells, or the enzyme extracted from the cells is immobilized on a carrier by an appropriate method,
It may be reacted with (I).

(ii) Culturing method Cultivation of the microorganism used in the present invention can be carried out according to a known method. As the medium to be used, those known as nutrient sources for general microorganisms can be used, glucose, glycerin, ethanol, carbon sources such as sucrose or dextrin, nitrogen sources such as ammonium sulfate or ammonium chloride, yeast extract, malt extract, peptone or meat. Organic nutrient sources such as extracts and inorganic nutrient sources such as phosphoric acid, magnesium, potassium, iron and manganese can be used in appropriate combination. In addition, as a substance that promotes the conversion activity of microorganisms from (I) to (II), vitamins or trans-1,4
-A cyano compound such as dicyanocyclohexane may be added.

The pH of the medium may be selected in the range of 6 to 10, and the culture temperature is 1
The temperature is 8 to 37 ° C, preferably 25 to 32 ° C. The number of days of culture may be 1 to 10 days until the activity becomes maximum.

(iii) Reaction conditions As the reaction medium, an aqueous medium such as water, buffer solution or culture solution can be used. To the reaction medium, (I) is added as a powder as it is or after being dissolved in an appropriate solvent. The addition concentration of (I) is preferably about 0.01 to 5% by weight, and does not need to be completely dissolved in the reaction medium. When the bacterial cells are used in the reaction, the bacterial cell concentration is usually in the range of 0.05 to 5% by weight. Reaction temperature is 5 to 50 ° C, preferably 15 to 37 ° C, reaction pH is 4
-11, and preferably 6-9. Reaction is usually 1-10
A suitable time may be selected within the range of 0 hours. Consumed
(I) may be added continuously or intermittently and added so that the concentration in the reaction solution is maintained within the above range.

(iv) Recovery The produced (II) can be easily recovered by a known method (solvent extraction, crystallization, etc.) from the reaction-completed liquid in which insoluble matters such as bacterial cells have been removed.

(v) Strains to be used In carrying out the present invention, Corynebacterium sp. C5 (Cornerobacter sp. No. 8931) belonging to the genus Corynebacterium isolated from soil in Kyoto city, And as a publicly preserved strain, Corynebacterium nitrilophilus ATCC2 belonging to the genus Corynebacterium
1419, Rhodococcus sp. ATCC19070 belonging to the genus Rhodococcus, Rhodococcus erythropolis J
A strain of microorganism such as CM3132 may be used. However, Corynebacterium sp. C5 and Rhodococcus sp. ATCC19070 are preferable because they have strong target reactions. The soil isolate C5 strain has been deposited at the Micro Incorporated with the above number, and its mycological properties are as shown below.

(a) Morphology 1. Cell shape and size; bacilli, 0.4-0.6 x 2.5-6.0μ
m.

2. Presence or absence of cell polymorphism; long rod-shaped (1st day), develops with snapping, and later (3rd day) ruptures into short rod-shaped (0.4-0.6 × 0.6-1.2μm) .

3. Presence or absence of motility; None 4. Presence or absence of spores; None 5. Gram stainability; + 6. Anti-acidity ;-( b) Growth condition in each medium 1. Meat agar plate culture; Diameter 1.0 to 2.2 mm, circle , Smooth and dry, hemispherical, white to pale pink.

2. Meat broth agar slope culture; medium growth, smooth, dry, white to light pink.

3. Meat broth liquid culture; vigorous growth, moderate turbidity, sedimentation with growth.

4. Meat broth gelatin puncture culture; grows well on the surface, grows along the puncture site, and does not liquefy.

5. Litmus milk; no change.

(c) Physiological properties 1. Reduction of nitrate; -2. Denitrification reaction; -3. MR test; -4. VP test; -5. Indole formation; -6. Hydrogen sulfide formation; -7. Starch Hydrolysis of water; -8. Utilization of citric acid (a) Medium of Coser ;-( b) Medium of christensene; + 9. Utilization of inorganic nitrogen source (a) Nitrate; + (b) Ammonium salt; + 10. Dye (A) King A medium ;-( b) King B medium;-11. Urease; + 12. Oxidase;-13. Catalase; + 14. Growth range (a) pH; 6-10 (b) Temperature 10-37 ° C 15. Attitude toward oxygen; Aerobic 16. OF test; -17. Production of acid and gas from sugar and cosmetic makeup 18. Heat resistance in skim milk at 72 ° C for 15 minutes in 10% skim; No 19. Ornithine decarboxylation; + 20. Lysine decarboxylation; − The above mycological properties are based on the Bergy's Ma
nual of Determinative Bacteriology 8th Edition) and “Manual of Clinical Microbiology 4th Edition (19
1985) ".

The C5 strain is an aerobic, gram-positive, and catalase-positive, endospore-free bacillus that does not grow flagella, develops a long rod-like shape at the early stage of development with snapping, and then ruptures into a short rod-like shape. By doing so, it is clear that they belong to coryneform bacteria. In addition, it has no cellulose decomposing ability, Gram dyeing is not variable, 10% skim milk, 72 ° C, no heat resistance for 15 minutes, further, it is not absolutely aerobic, and the OF test is negative. From this, it is clear that the C5 strain belongs to the genus Corynebacterium.

The C5 strain grows at 37 ° C. and is aerobic as regards its attitude toward oxygen, but it can be said to be facultative anaerobic, and OF
The test was low, the assimilation of glucose was weak, and D-fructose, sucrose, D-mannitol and ethanol were assimilated, there was no nitric acid reduction ability, hydrogen sulfide was not produced, and citric acid was assimilated by Coser medium However, since it has urease activity and catalase activity, there is no applicable species judging from the classification documents mentioned above. Therefore, the C5 strain is considered to be a new species of the genus Corynebacterium.

(Examples) Next, the present invention will be described with examples. Unless otherwise specified,% in the examples means% by weight.

Example 1 Ethanol 0.4%, trans-1,4-dicyanocyclohexane 0.2%, dipotassium phosphate 0.2%, sodium chloride.
1%, magnesium sulfate 0.02%, biotin 0.01 μg / m
Corynebacterium sp. C5, which had been cultivated in the same medium in advance, was inoculated in 50 ml of a sterilizing medium containing 1 and 0.01 µg / ml of thiamine hydrochloride and having a pH of 8.0, and cultured by shaking at 32 ° C for 2 days. After culturing, centrifuge the cells (wet cells 0.34
g) is collected, and 0.01M phosphate buffer (pH 7.0) is collected.
After suspension in a test tube containing 10 ml, trans-1,4
-0.3 g of dicyanocyclohexane was added and reacted at 32 ° C with shaking. After 20 hours, the reaction was completed, the bacterial cells were removed by centrifugation, concentrated hydrochloric acid was added to the supernatant to adjust the pH to 1.0 to 2.0, and the mixture was left in a refrigerator at 5 ° C. 0.33 g of cyanocyclohexane-1-carboxylic acid was obtained as white crystals. This product showed a single peak in high performance liquid chromatography analysis, and the cis-peak was not noticeable.

The elemental analysis values are as follows.

Calculated CH value 62.75% 7.19% Measured value 61.5% 7.2% Melting point was 150 to 152 ° C, and absorption of 2235 cm ^-1 (cyano group) was observed in the infrared absorption spectrum (KBr).

The high performance liquid chromatographic analysis was performed as follows. Analyzer; Waters 6000A type pump,
Toyo Soda RI-8 type RI detector, JASCO UVI
DEC-100 type UV detector. Column; Unisil C-
18. Solvent: Water / acetonitrile = 77/23 (volume ratio) + PIC B-7 (Waters) 1.5 volume%.

Example 2 Glycose 0.4%, trans-1,4-dicyanocyclohexane 0.2%, dipotassium phosphate 0.2%, sodium chloride 0.
1%, magnesium sulfate 0.02%, biotin 0.01 μg / m
l, thiamine hydrochloride 0.01 μg / ml, pH 9.0 in 10 ml of sterilized medium, Corynebacterium microorganisms were cultivated in broth agar medium and Rhodococcus microorganisms in nutrient agar medium, and 1 platinum loop was inoculated respectively. The cells were cultured at 30 ° C with shaking.

After removing the cells from the culture solution by centrifugation, trans-4-cyanocyclohexane-1 was prepared in the same manner as in Example 1.
-Carboxylic acid was obtained and confirmed by high performance liquid chromatography analysis.
Table 1 shows the amount of trans-4-cyanocyclohexane-1-carboxylic acid produced at this time.

Example 3 Glucose 0.8%, yeast extract 0.05%, peptone 0.1%,
Sterilization medium 100 ml containing ammonium sulfate 0.1%, sodium chloride 0.1%, magnesium sulfate 0.02%, trans-1,4-dicyanocyclohexane 0.2% and pH adjusted to 9.0
Rhodococcus sp. ATCC 19070, which had been cultivated in the same medium in advance, was inoculated into the cells and cultured at 30 ° C. for 4 days with shaking. After culturing, the cells (wet cells 1.8 g) were collected by centrifugation and reacted under the same conditions as in Example 1. Four
After 8 hours, the reaction was terminated, and the cells were removed by centrifugation. In the same manner as in Example 1, trans-4-cyanocyclohexane-1-carboxylic acid 0.30 g was obtained as white crystals. This product showed a single peak in high performance liquid chromatography analysis, and no cis peak was observed.

(Effects of the Invention) By utilizing the present invention, trans-4-cyanocyclohexane-1-carboxylic acid can be produced at a high concentration under the reaction conditions of normal temperature and normal pressure, and the configuration can be maintained. Therefore, it is economically very advantageous.

Claims (3)

[Claims] 1. Corynebacterium
Genus or Rhodococcus genus, trans-1,4-dicyanocyclohexane trans-4
Trans-4, characterized in that trans-4-cyanocyclohexane-1-carboxylic acid is produced from trans-1,4-dicyanocyclohexane by the action of a microorganism having the ability to convert it to cyanocyclohexane-1-carboxylic acid. -Method for producing cyanocyclohexane-1-carboxylic acid. 2. Corynebacterium
Microorganisms belonging to the genus Corynebacterium sp
The method according to claim 1, which is rynebacterium sp.) C5 (Microtechnology Research Institute, Microbiology Co., Ltd. 8931). 3. A microorganism belonging to the genus Rhodococcus is Rhodococcus sp.
The method of claim 1 which is ATCC 19070.