JP3043511B2 - Method for separating and recovering D-malic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for separating and recovering D-malic acid with high efficiency. According to the present invention, D-malic acid in an aqueous solution can be easily collected with high purity and high yield.

[0002] D-malic acid is expected as an industrially important compound as an intermediate material for pharmaceuticals, agricultural chemicals and the like.

[0003]

2. Description of the Related Art An industrial production method of D-malic acid has been scarcely known. The present inventors have previously proposed a method for producing D-malic acid by an enzymatic method (Japanese Patent Application No. Hei.
136331).

[0004]

DISCLOSURE OF THE INVENTION In order to collect D-malic acid with higher efficiency, the present inventors have intensively studied methods for separating and recovering D-malic acid, As a result, the present inventors have found a suitable separation and recovery method, and have completed the present invention.

[0005]

SUMMARY OF THE INVENTION The present invention provides a highly pure D-malic acid by treating a mixed aqueous solution of D-malic acid and L-aspartic acid with a strongly acidic cation exchange resin to collect D-malic acid. It is intended to provide a method for separating and recovering D-malic acid with high yield.

[0006] The method for preparing the aqueous solution containing D-malic acid and L-aspartic acid used in the present invention is not particularly limited, but usually, inexpensive DL-malic acid is used as a raw material, and fumarase activity and aspartase are used. An active microbial cell or a treated product thereof is allowed to act in an aqueous solution. L-malic acid is converted to fumaric acid by the action of fumarase, and further converted to L-aspartic acid by aspartase. Thus, a mixed aqueous solution of L-aspartic acid and unreacted D-malic acid is prepared.

A mixed aqueous solution of D-malic acid and L-aspartic acid is passed through a strongly acidic cation exchange resin, but the strongly acidic cation exchange resin is not particularly limited. SK-1B "H type (Mitsubishi Kasei)," Dowex 50WX "
(Manufactured by Dow Chemical), "Amberlite (Amberl)
item) IR120, IR112, IR118 ”(Rohm
and Hass), "AGMP-50" (Bio-Rad), "Duolite ES-26" (Diammond-
Shamrock Chemical), "Ionac"
CFZ "(manufactured by Ionac Chemical) and the like are preferably used.
The liquid passing conditions are space velocity (ml / ml resin · hr) 0.5
-5, preferably 1-3. The temperature of the treatment liquid is not particularly limited, but is usually from room temperature to 50 ° C.

According to the method of the present invention, D-malic acid can be recovered in a yield of 80% or more of the theoretical value.

[0009]

EXAMPLES The present invention will be described in detail below with reference examples and examples. Reference Example 1 Preparation of cells containing fumarase and aspartase Medium (0.4% urea, 1.4% ammonium sulfate,
KH ₂ PO ₄ 0.05%, K ₂ HPO ₄ 0.05
%, MgSO ₄ .7H ₂ O 0.05%, CaCl _{2.
2H 2 O 2ppm, FeSO 4 ·} 7H 2 O 2ppm, M
nSO ₄ .4 to 6H ₂ O 2 ppm, ZnSO ₄ .7H ₂
O 2 ppm, NaCl 2 ppm, biotin 200 μg /
Liter, thiamine.HCl 100 μg / liter,
Casamino acid 0.1% and yeast extract 0.1%) 1
After dispensing 00 ml into a 500 ml Erlenmeyer flask, sterilizing (pH 7.0 after sterilization), Brevibacterium flavum MJ-233-AB-41.
(FERM BP-1498), and aseptically remove 5
2 ml of 0 (W / V)% glucose was added, followed by shaking culture at 30 ° C. for 2 days.

Next, the medium (ammonium sulfate 2.3)
%, KH ₂ PO ₄ 0.05%, K ₂ HPO ₄ 0.0
_{5%, MgSO 4 · 7H 2} O 0.05%, FeSO 4
・ 7H ₂ O 20ppm, MnSO ₄・₄ ～ 6H ₂ O 2
0 ppm, biotin 200 μg / liter, thiamine
HCl 100 μg / liter, casamino acid 0.3%
And yeast extract 0.3%) 1 liter was charged into a 2 liter aeration stirred tank and sterilized (120 ° C, 20 minutes)
Thereafter, 20 ml of 50 (W / V)% glucose and 20 ml of the above culture were added aseptically, and the mixture was cultured for 15 hours at 1,000 rpm, aeration of 1 vvm, temperature of 33 ° C. and pH 7.6.

[0011] Glucose is 50% (W / V)% so that the concentration of the medium during the culture does not exceed 1 (W / V)%.
Glucose was added intermittently about every 1-2 hours.

After completion of the culture, the cells were collected from 1 liter of the culture by centrifugation.

Reference Example 2 Preparation of D-malic acid-containing reaction solution The collected cells obtained in Reference Example 1 were used as a reaction solution [DL-malic acid].
50 g, polyoxyethylene sorbitan monolaurate 0.8 g / distilled water 1 liter (the pH was adjusted to 8.0 with 25% aqueous ammonia)] and suspended at 45 ° C. for 24 hours. After the reaction, centrifugation (8
The supernatant was separated from the cells at 2,000 rpm for 40 minutes at 2 ° C.).

Example D The measurement of the amount of malic acid was carried out using high performance liquid chromatography (Shimadzu LC-5A). Further, the optical purity of D-malic acid was confirmed by a specific rotation meter.

[0015] 100 ml of the reaction solution prepared in Reference Example 2
(D-malic acid 25 mg / ml, L-aspartic acid 50
mg / ml) into a column packed with 100 ml of a strongly acidic cation exchange resin ("Diaion SK-1B" H type).
After passing the solution at a space velocity of 2.0, the treatment liquid was concentrated and dried by a vacuum evaporator. The amount of crystals of the recovered D-malic acid was 2300 mg, and the yield from the reaction solution was 90%.
That was all. Further, when the specific rotation of the crystal was measured, it was [α] _D ²⁰ = + 2.20 (c = 8, H ₂ O).

[0016]

According to the present invention, D-malic acid and L-malic acid
From the mixed aqueous solution of aspartic acid, D-malic acid can be easily collected with high purity and high yield.

Continuation of front page (72) Inventor Hideaki Yukawa 8-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Pref. Tsukuba Research Laboratory, Mitsubishi Yuka Corporation (56) References JP-A-58-170488 (JP, A) JP-A-59-45895 (JP, A) JP-A-60-126092 (JP, A) JP-A-57-56439 (JP, A) JP-A-63-130555 (JP, A) JP-A-1-285193 ( JP, A) JP-A-1-129991 (JP, A) JP-A-3-183487 (JP, A) JP-A-6-72945 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C07C 59/245 C07C 51/487 C12P 13/20 C12P 7/46 CAPLUS (STN) REGISTRY (STN) WPIDS (STN)

Claims (1)

(57) [Claims] An aqueous solution of a mixture of D-malic acid and L-aspartic acid is treated with a strongly acidic cation exchange resin to obtain D-malic acid.
A method for separating and recovering D-malic acid, comprising collecting malic acid.