JPH05268991A - Production of d-malic acid - Google Patents

Abstract

PURPOSE:To obtain D-malic acid in a good yield by treating a DL-malic acid- containing reaction solution with the cells of a microorganismic strain having a fumarase activity and an aspartase activity or its treated material under a specific condition. CONSTITUTION:A reaction solution containing DL-malic acid is treated with the cells of a microorganismic strain having a fumarase activity and an aspartase activity or its treated material. DL-Malic acid is added to the reaction solution so that the concentration of L-malic acid does not exceed 100mM in the reaction solution, and the reaction is not inhibited with D-malic acid. The reaction finishes usually after approximately 3-48hrs.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a highly efficient method for producing D-malic acid by an enzymatic method. According to the invention, the DL
-The conversion of L-malic acid in malic acid to L-aspartic acid facilitates the separation of D-malic acid,
-It is possible to collect malic acid with high purity and high yield.

D-malic acid is expected as an industrially important compound as an intermediate raw material for medicines, agricultural chemicals and the like.

[0003]

The industrial production method of D-malic acid is hardly known until now. The present inventors have diligently studied the development of a process for producing D-malic acid by an enzymatic method, and have proposed a method for producing D-malic acid more efficiently than inexpensive DL-malic acid (Japanese Patent Application No. Hei 3). -136331).

However, in this method, if the concentration of DL-malic acid charged is increased in order to increase the final concentration of D-malic acid and improve the efficiency of separation and purification, the high concentration of D-male remaining in the reaction solution is obtained. The acid has a drawback that the enzymatic conversion process for producing L-aspartic acid from L-malic acid is inhibited, the reaction rate is reduced, and the reaction time is prolonged.

[0005]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies to solve the above problems, and found that L
-By adding DL-malic acid as a reaction substrate successively or continuously so that the concentration of malic acid does not exceed 100 mM,
The inventors have found that the reaction can be completed in a short time without being inhibited by D-malic acid and the final concentration of D-malic acid is not lowered, and have completed the present invention.

That is, according to the present invention, a microbial cell having fumarase activity and aspartase activity or a treated product thereof is allowed to act on a reaction solution containing DL-malic acid to give L-malate.
In the method of converting malic acid into L-aspartic acid by an enzymatic method and collecting D-malic acid from this, DL-malic acid is successively added so that the concentration of L-malic acid in the reaction solution does not exceed 100 mM. Alternatively, it is a method for producing D-malic acid, which comprises continuously adding to the reaction solution.

According to this method, D-malic acid can be efficiently produced.

The microbial cells used in the present invention include those having fumarase activity and aspartase activity,
For example, Brevibacterium flavum
um flavum) MJ-233 (FERM BP-149
7), the same MJ-233-AB-41 (FERM BP-
1498), Escherichia coli
K-12 ATCC 27325, Escherichia coli BATCC 11303 and the like are preferably used.

The above-mentioned microorganism used in the present invention can be used as it is as a microbial cell, and as "a treated product of a microbial cell", a crushed cell, a microbial cell or a microbial cell crushed by a treatment such as ultrasonic crushing. The crushed material is polyacrylamide, alginic acid,
A suitable immobilizing agent such as κ-carrageenan immobilized by a method known per se is included.

The medium used for preparing the above-mentioned microbial cells used in the method of the present invention is not particularly limited and may be one used for general microorganisms. For example, in a microorganism belonging to the genus Brevibacterium,
As the carbon source of the medium, for example, glucose, ethanol, acetic acid or the like can be used. As the nitrogen source of the medium, inorganic salts such as ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea can be used,
Also, peptone, yeast extract, corn steep liquor,
Organic nutrient sources such as casamino acids can also be used.
As the inorganic salt, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and the like are used.

The culture is carried out under aerobic conditions such as aeration and stirring, and the culture temperature is 20 ° C to 40 ° C, preferably 28 ° C to
37 ° C. The pH during the culture is 5 to 10, preferably around 7 to 8. The pH during the culture is adjusted by adding an acid or an alkali. The concentration of the carbon source in the medium at the start of culture is 0.05 to 10% by weight, for example, when glucose is used, the glucose concentration is preferably
0.05-1.0% by weight, more preferably 0.1-
0.3 wt% is suitable. Culture period is 10 hours to 4 days,
The optimum period is 15 hours to 3 days.

The cells are collected from the culture thus obtained, washed with water or an appropriate buffer and used in the enzymatic reaction of the present invention.

In the method of the present invention, an aqueous solution containing DL-malic acid is subjected to an enzymatic reaction in the presence of the above-prepared microbial cell, its crushed product, or its immobilized product.

Here, the concentration of DL-malic acid contained in the aqueous solution is 0.3 as the final concentration added at the end of the reaction.
-4 M, preferably 0.5-2 M, which is added to the reaction solution either continuously or continuously. A powder or an aqueous solution is used as the form of addition, but it is preferable to use a powder or a high-concentration aqueous solution in order to prevent an increase in the amount of the reaction liquid.

The pH of the reaction solution is adjusted with aqueous ammonia. This is to generate L-aspartic acid from fumaric acid and ammonia generated in the reaction solution. If necessary, ammonium salts such as ammonium chloride and ammonium sulfate can be added.

The pH during the reaction is 6 to 10, preferably 8 to
9 and the reaction temperature is 30 to 50 ° C., preferably 3
5 to 46 ° C. The reaction time is usually about 3 to 48 hours.

When microbial cells are used as they are in the reaction, a nonionic surfactant or the like can be added in order to improve the membrane permeability of the cells.

The D-malic acid and L-aspartic acid produced in the reaction solution obtained by the above reaction method are sterilized from the reaction solution by centrifugation to remove L-asparagine present in the supernatant. The acid is precipitated and separated by an isoelectric focusing method as usual. D-malic acid can be recovered from the residual liquid by an ordinary ion exchange method or the like.

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.

[0020]

EXAMPLES The present invention will be described in detail below with reference to examples and examples. Reference Example Preparation of Fumarase- and Aspartase-Containing Bacteria Medium (urea 0.4%, ammonium sulfate 1.4%,
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・ 4-6H ₂ O 2ppm, ZnSO ₄・ 7H ₂
O 2ppm, NaCl 2ppm, biotin 200μg /
Liter, thiamine / HCl 100 μg / liter,
Casamino acid 0.1% and yeast extract 0.1%) 1
00 ml was dispensed into a 500 ml Erlenmeyer flask and sterilized (pH 7.0 after sterilization), and then Brevibacterium flavum MJ-233-AB-41.
(FERM BP-1498) was inoculated and aseptically 50
2 ml of (W / V)% glucose was added and shake culture was carried out 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 1% of yeast extract (0.3%) were sterilized (120 ° C, 20 minutes) in a 2 liter aeration and stirring tank.
Thereafter, 6 ml of 50 (W / V)% glucose and 20 ml of the culture were aseptically added, and the culture was carried out for 15 hours at a rotation speed of 1000 rpm, an aeration rate of 1 vvm, a temperature of 33 ° C. and a pH of 7.6.

Glucose contains 50 (W / V) so that the concentration of the medium in the culture does not exceed 0.3 (W / V)%.
V)% glucose was added intermittently about every 1-2 hours.

After completion of the culture, 1 liter of the culture was collected by centrifugation.

Example The quantification of D-malic acid and L-malic acid in this example was carried out by a known method such as high performance liquid chromatography and derivative gas chromatography.

From the collected cells obtained in the above Reference Example, DL-malic acid 10 g / liter, polyoxyethylene sorbitan monolaurate 0.8 g / liter (pH adjusted to 8.0 with 25% ammonia water)] The reaction solution was suspended in 1 liter of the reaction solution and the reaction was started at 45 ° C. under stirring conditions. While quantifying the L-malic acid concentration on the way, the L-malic acid concentration was 1
5 g each of DL-malic acid (L
-About 18 mmol as malic acid) was added sequentially to the reaction solution.
Regarding the decrease in pH accompanying the addition of DL-malic acid, pH8 was maintained by the addition of aqueous ammonia. When the total amount of DL-malic acid reached 200 g, the successive addition was stopped, and the reaction completion time (L-in the added DL-malic acid was
The reaction completion time is defined as the time at which L-aspartic acid corresponding to malic acid is produced.
It was 5 hours (experimental zone 1).

On the other hand, when the DL-malic acid concentration at the start of the reaction was set to 200 g / liter and no addition was made thereafter, the reaction completion time was 3 when the reaction was conducted under the same conditions as above.
It was 0.6 hours (experimental section 2).

When the concentration of DL-malic acid at the start of the reaction is 100 g / liter and 50 g of DL-malic acid is added 5 hours and 10 hours after the start of the reaction, the reaction end time is 29.4 hours. Was (Experimental Zone 3).

From the above experiment, the L-malic acid concentration at the start of the reaction was 10 compared with the case where the reaction was carried out so that the L-malic acid concentration in the reaction solution did not exceed 100 mM (Experimental group 1).
When it greatly exceeds 0 mM (Experimental group 2), when the amount of DL-malic acid added in the middle greatly exceeds 100 mM as the L-malic acid concentration in the reaction solution (Experimental group 3), the reaction end time is It is significantly extended to 25% and 20%, respectively, and it is understood that it is important to control the addition amount of the substrate as in the method of the present application in order to shorten the reaction time.

[0029]

According to the present invention, DL-malic acid
D-malic acid can be collected efficiently.

Front Page Continuation (72) Inventor Hideaki Yukawa 8-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Mitsubishi Petrochemical Co., Ltd. Tsukuba Research Institute

Claims (1)

[Claims] 1. A microbial cell having fumarase activity and aspartase activity or a treated product thereof is allowed to act on a reaction solution containing DL-malic acid to convert L-malic acid into L-aspartic acid by an enzymatic method. In the method for collecting D-malic acid from this, DL-malic acid is added to the reaction solution sequentially or continuously so that the concentration of L-malic acid in the reaction solution does not exceed 100 mM. D-
Method for producing malic acid.