JP3065175B2 - Method for producing 3-cyclohexyl-L-alanine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to 3-cyclohexyl-L
It relates to a method for producing alanine. 3-cyclohexyl-
L-alanine is useful as a pharmaceutical synthesis intermediate.

[0002]

BACKGROUND OF THE INVENTION 3-Cyclohexyl-L-alanine is
Since it is an unnatural amino acid, it cannot be produced by fermentation or extraction. On the other hand, as a method for producing an optically active amino acid, a method of optically resolving a racemic amino acid produced by organic chemical synthesis is well known (Bull.
Agr. Chem. Soc. Jap., 21 , 304 (1957), J. Am. Chem.
Soc., 76 , 6045 (1954), J. Biol. Chem., 188 , 657 (1
951)), however, this method has the disadvantage that the optical resolution operation is complicated and the yield is low.

[0003]

An object of the present invention is to provide a process for producing 3-cyclohexyl-L-alanine which is advantageous for industrial implementation.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies on a method for producing 3-cyclohexyl-L-alanine, and as a result, have found that DL-5 obtained by a chemical synthesis method.
By allowing the cells of a microorganism belonging to the genus Bacillus or Flavobacterium to act on cyclohexylmethylhydantoin, the hydantoin ring is optically specifically cleaved and hydrolyzed to convert only the L-form of 3-cyclohexylalanine. They have found that they can be obtained in good yield, and have completed the present invention.

That is, the present invention relates to a microorganism or a treated product of a microorganism belonging to the genus Bacillus or Flavobacterium and having the ability to convert DL-5-cyclohexylmethylhydantoin into 3-cyclohexyl-L-alanine. It is intended to provide a method for producing 3-cyclohexyl-L-alanine, which comprises reacting DL-5-cyclohexylmethylhydantoin and collecting the produced 3-cyclohexyl-L-alanine.

The microorganism used in the present invention belongs to the genus Bacillus or the genus Flavobacterium, and DL-5-cyclohexylmethylhydantoin is replaced with 3-cyclohexyl-L-.
Any microorganism can be used as long as it has the ability to convert to alanine. Specific examples thereof include Bacillus brevis AJ12299 (FERM P-8837; JP-A-63-24895) and Flavobacterium aminogenes AJ3940 ( FERM P-31
35; Japanese Patent Publication No. 54-8749). Further, it is a matter of course that a mutant strain derived from these microorganisms by a natural mutation or a mutation treatment such as a chemical mutagen or ultraviolet irradiation may be used.

[0007] In order to obtain cells of such a microorganism, the microorganism is preferably cultured and grown in an appropriate medium. Such a medium is not particularly limited, and may be a normal medium containing a normal carbon source, a nitrogen source, inorganic ions, and if necessary, an organic nutrient source.

As the carbon source, carbohydrates such as glucose, alcohols such as glycerol, organic acids and the like are appropriately used. As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salt or the like is used. As the inorganic ion, a magnesium ion, a phosphate ion, a potassium ion, an iron ion, a manganese ion and others are appropriately used as needed. As organic nutrients, vitamins, amino acids and the like and yeast extracts, peptone, meat extracts, corn steep liquor, casein hydrolyzate and the like containing these are appropriately used.

When a small amount of a 5-substituted hydantoin such as 5-isopropylhydantoin or 5-indolylmethylhydantoin is further added to the medium, the activity of converting DL-5-cyclohexylmethylhydantoin to 3-cyclohexyl-L-alanine is increased. High cells may be obtained (acclimation).

There are no particular restrictions on the culture conditions.
Culture may be carried out under aerobic conditions for about 12 to 48 hours at pH 5 to 8 and temperature of 25 to 40 ° C. while appropriately controlling the pH and temperature.

As the cells to act on DL-5-cyclohexylmethylhydantoin, a culture solution containing the cells obtained as described above may be used as it is.
Alternatively, the cells may be used after separating and washing the cells from the culture solution. As the treated cells, freeze-dried cells, acetone-treated cells, surfactants, cells treated with toluene, or the like are appropriately used. Furthermore, the cells can be used as a treated product of cells immobilized by entrapping the cells in a polymer such as carrageenan. The amount of the cells or the processed product of the cells may be an amount (effective amount) that exhibits a desired effect in the case of a given reaction, and this effective amount can be easily determined by those skilled in the art by a simple preliminary experiment. For example, in the case of washed wet cells, the amount is 1 to 40 g per 1 dl of the reaction solution.

The concentration of DL-5-cyclohexylmethylhydantoin is 0.1 to 30% of the total weight of the reaction mixture.
%, Preferably 0.1-10%, but if necessary
For example, if the reaction is inhibited at a high concentration, DL
-5-Cyclohexylmethylhydantoin can be supplemented during the reaction.

[0013] The reaction yield may be increased by adding an energy source substance such as glucose to the reaction mixture and further supplying oxygen by shaking or aeration and stirring.

As the energy source substance to be added, carbohydrates, alcohols and the like can be used as long as the microorganism can be used as an energy source and ATP consumed during the reaction can be regenerated in the reaction solution including the inside of the cells. , Organic acids and the like, and examples thereof include glucose, glycerol, fructose, galactose, xylose, mannitol, trehalose, succinic acid, fumaric acid, citric acid, acetic acid, ethanol, and mixtures thereof.
The amount of the energy source substance used may be an amount (effective amount) that exhibits the intended effect in the case of a given reaction,
This effective amount can be easily determined by those skilled in the art by simple preliminary experiments, and for example, in the case of glucose, it is 0.1 to 10 g per dl of the reaction solution. The energy source substance may be added during the reaction or during the reaction.

The reaction yield may be improved by adding a surfactant, an organic solvent, a coenzyme, hydroxylamine, metal ions such as cobalt ions and magnesium ions to the reaction mixture.

The reaction temperature is 10 to 70 ° C., preferably 20
５０50 ° C., reaction pH 5-11, preferably 6.5-9
Thus, by performing the reaction for about 5 to 50 hours, DL-5-cyclohexylmethylhydantoin is hydrolyzed optically specifically, and a significant amount of 3-cyclohexyl-L-alanine is produced and accumulated in the reaction mixture.

According to the method of the present invention, 3-cyclohexyl-L-alanine thus produced and accumulated is separated and collected from the reaction-completed mixture.
Since alanine is not produced as a by-product, ordinary methods for collecting L-amino acids such as a method using a synthetic adsorption resin and a method of precipitating at an isoelectric point can be adopted.

[0018]

The present invention will be further described with reference to the following examples.
The amount of 3-cyclohexyl-L-alanine was determined by high performance liquid chromatography (column: CROW manufactured by Daicel Corporation).
NPAK CR (+), Eluent: HClO ₄ aqueous solution (pH 1.5)
+ 15% (v / v) methanol, flow rate 1.0 ml / min,
Temperature 50 ° C., detection 210 nm).

Example 1 Maltose 0.2 g / dl, (NH ₄ ) ₂ SO ₄ 0.5 g / d
l, KH ₂ PO ₄ 0.1 g / dl, K ₂ HPO ₄ 0.3 g / dl,
_{MgSO 4 · 7H 2 O0.05g / dl} , FeSO 4 · 7H 2
_{O1mg / dl, MnSO 4 · 4H} 2 O1mg / dl, medium (pH 7 containing yeast extract 1.0 g / dl, peptone 1.0 g / dl, a DL-5-isopropyl hydantoin 0.2 g / dl.
50) into a 500 ml flask,
Heat sterilization for 5 minutes.

Bacillus brevis AJ12299 which had been cultured at 30 ° C. for 24 hours on a bouillon agar medium in advance.
Was inoculated with one platinum loop and shake-cultured at 30 ° C. for 16 hours. The cells were centrifuged from this culture, washed once with the same amount of physiological saline as the culture, and centrifuged again to collect the cells. This cell was treated with 0.5 g / dl of DL-5-cyclohexylmethylhydantoin and 0.5 g / d of glucose.
l, 0.1 M containing 0.001 g / dl of MgSO ₄ · 7H ₂ O
The solution was added to 50 ml of Tris-HCl buffer (pH 7.0) at a concentration of 5 g / dl, and shaken at 30 ° C. for 24 hours.
Meanwhile, after 12 hours, 0.5 g / dl of glucose was further added.

After completion of the reaction, the cells are removed by centrifugation,
The resulting 3-cyclohexyl-L-alanine was measured by high performance liquid chromatography to find that it was 0.41 g / dl.
Of 3-cyclohexyl-L-alanine.
The 3-cyclohexyl-L-alanine was separated and purified from the reaction solution by hydrophobic adsorption chromatography using a synthetic adsorbent (SP207 manufactured by Mitsubishi Kasei Co., Ltd.), and was analyzed by NMR spectrum, high performance liquid chromatography using an optically active column, and specific rotation. As a result of analysis by a method such as a degree measurement, it was confirmed that each of them was in agreement with the standard (manufactured by Signa).

Example 2 Glucose 0.5 g / dl, (NH ₄ ) ₂ SO ₄ 0.5 g / d
l, KH ₂ PO ₄ 0.1 g / dl, K ₂ HPO ₄ 0.3 g / dl,
_{MgSO 4 · 7H 2 O0.05g / dl} , FeSO 4 · 7H 2
_{O1mg / dl, MnSO 4 · 4H} 2 O1mg / dl, CaCl 2
1 mg / dl, yeast extract 1.0 g / dl, peptone 1.0 g /
dl, DL-5-Indolylmethylhydantoin 0.35
A 500 ml flask containing 50 g of a medium (pH 7.0) containing g / dl was sterilized by heating at 120 ° C. for 15 minutes.

Flavobacterium aminogenes which had been cultured for 24 hours at 30 ° C. on a bouillon agar medium
One platinum loop of AJ3940 cells was inoculated into the cells, and the cells were incubated at 30 ° C.
The cells were cultured with shaking for 6 hours. The cells were centrifuged from this culture, washed once with the same amount of physiological saline as the culture, and centrifuged again to collect the cells. The cells were 0.1 M containing 0.5 g / dl of DL-5-cyclohexylmethylhydantoin.
5 g / dl in 50 ml of Tris-HCl buffer (pH 8.0)
And reacted at 40 ° C. for 24 hours.

After completion of the reaction, the cells are removed by centrifugation.
When the produced 3-cyclohexyl-L-alanine was measured by high performance liquid chromatography, it was found to be 0.21 g / dl.
Of 3-cyclohexyl-L-alanine.
In the same manner as in Example 1, this 3-cyclohexyl-
L-alanine was separated and purified, and analyzed by NMR spectrum, high-performance liquid chromatography using an optically active column, specific rotation measurement, and the like. As a result, it was confirmed that all of the results agreed with the standard (manufactured by Signa Corporation).

Example 3 Bacillus brevis AJ12299 was cultured with shaking at 30 ° C. for 16 hours in the same manner as in Example 1. Add DL to this culture
0.5 g / d of 5-cyclohexylmethylhydantoin
l, added glucose _{0.5g / dl, MgSO 4 · 7H} 2 O aseptically so that 0.001 g / dl, and incubated for 24 hours at further 30 ° C.. As a result, 0.39 g / dl of 3-cyclohexyl-L-alanine was produced in the culture solution.

Example 4 Flavobacterium aminogenes AJ3940 was cultured with shaking at 30 ° C. for 16 hours in the same manner as in Example 2. DL-5-Cyclohexylmethylhydantoin was aseptically added to this culture solution to a concentration of 0.5 g / dl, and further added for 3 hours.
The cells were cultured at 0 ° C. for 24 hours. As a result, 0.20 g / dl of 3-cyclohexyl-L-alanine was produced in the culture solution.

Example 5 Bacillus brevis AJ12299 was cultured with shaking at 30 ° C. for 16 hours in the same manner as in Example 1. The cells were centrifuged from this culture, washed once with the same amount of physiological saline as the culture, and centrifuged again to collect the cells. 5 g / d of these cells
The mixture was added to a phosphate buffer solution (pH 7.5) containing 100 ppm of cobalt ions so that the concentration became 1 l, and the mixture was treated with 10 Kc ultrasonic waves for 10 minutes. The processed product was centrifuged to obtain a supernatant. To 5 ml of the supernatant, 0.5 g / dl of DL-5-cyclohexylmethylhydantoin and 2 g of ATP were added.
dl, was added MgSO ₄ · 7H ₂ O to 0.001 g / dl, the KCl becomes 1.5 g / dl, and reacted for 24 hours at 30 ° C. after pH correction to 7.0. As a result, 0.35 g / dl of 3-cyclohexyl-L-alanine was formed in the reaction solution.

Example 6 Flavobacterium aminogenes AJ3940 was cultured with shaking at 30 ° C. for 16 hours in the same manner as in Example 2. The cultured cells were subjected to ultrasonic treatment in the same manner as in Example 5, and the treated product was centrifuged to obtain a supernatant. DL-5 was added to 5 ml of the supernatant.
Cyclohexylmethylhydantoin was added at a concentration of 0.5 g / dl, and the pH was adjusted to 8.0.
The reaction was performed for 4 hours. As a result, 0.15 g was contained in the reaction solution.
/ Dl of 3-cyclohexyl-L-alanine was produced.

[0029]

According to the method of the present invention, 3-cyclohexyl-L-alanine, an unnatural amino acid, can be efficiently produced from racemic 5-cyclohexylmethylhydantoin obtained by a chemical synthesis method, Suitable for industrial production of 3-cyclohexyl-L-alanine.

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12P 13/06 BIOSIS (DIALOG) WPI (DIALOG) CA (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] 1. A microorganism or a treated product of a microorganism belonging to the genus Bacillus or Flavobacterium and capable of converting DL-5-cyclohexylmethylhydantoin into 3-cyclohexyl-L-alanine. Act on cyclohexylmethylhydantoin to form 3
-A method for producing 3-cyclohexyl-L-alanine, which comprises collecting cyclohexyl-L-alanine.