JPH0738794B2 - Method for producing L-malic acid - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing L-malic acid,
More specifically, for microorganisms having fumarase activity,
The present invention relates to a method for efficiently producing L-malic acid by suppressing the enzyme activity involved in the production of succinic acid which is a side reaction while maintaining its activity.

Conventional technology and its problems Conventionally, L from fumaric acid or its salt by fumarase
-For the production of malic acid, a method is known in which a microbial cell having a fumarase activity is used as it is or after being subjected to treatment such as immobilization (Japanese Patent Publication No. 37-4511).
No. 44-1191, etc.). The present inventors have also established a production method using Brevibacterium bacteria (Japanese Patent Laid-Open No. 61-26).
5096).

However, in these methods, succinic acid is often found as a by-product of fumaric acid due to the use of microbial cells, and it is difficult to efficiently separate this succinic acid by-produced from L-malic acid. Therefore, it is important as an industrial production method to suppress the by-production of succinic acid from fumaric acid.

As a method for suppressing the by-production of succinic acid, a method of contacting an immobilized microorganism with an organic solvent (Japanese Patent Publication No. 52-8396) and a method of contacting the immobilized microorganism with bile acid (Japanese Patent Publication No.
No. 52-31952) is known.

However, these methods require special equipment when using an organic solvent due to the nature of the solvent, and therefore require special equipment.When using bile acid, bile acid is used. Is expensive, the processing time is long, etc.
Industrially, there are still problems, and a simpler processing method has been desired.

SUMMARY OF THE INVENTION The present invention provides a method for producing L-malic acid by enzymatic reaction from fumaric acid or a salt thereof in the presence of a fumarase-containing bacterium or a treated product thereof, wherein the bacterium or the treated product is a surfactant. The present invention provides a method for producing L-malic acid, which is characterized by being heat-treated in an aqueous solution of L-malic acid or a salt thereof containing.

EFFECTS OF THE INVENTION According to the method of the present invention, a succinic acid by-product is produced when L-malic acid is produced from fumaric acid or a salt thereof by enzymatic reaction in the presence of fumarase-containing cells or a treated product thereof. Can be significantly reduced.

The heat treatment for suppressing the succinic acid by-product can be performed in a short time and at a relatively low temperature by using a surfactant and an aqueous solution of L-malic acid or a salt thereof together. It becomes advantageous as a target method.

Detailed Description of the Invention Examples of the bacterium having a fumarase activity used in the present invention include Brevibacterium flavum MJ-233 (FERM BP-1497) (Japanese Patent Publication No.
-26755) and MJ-233-AB-41 (FER, which was obtained from this fungus as an α-amino-n-butyric acid resistant strain.
M BP-1498) (see Japanese Examined Patent Publication No. 59-28398).

Although these cells can be used as they are,
Furthermore, the bacterial cell or the enzyme (fumarase) contained therein
What was fixed by the well-known method may be used for reaction.
The heat treatment, which is a side reaction suppressing treatment for the immobilization product, may be performed before or after the immobilization. As the immobilization, a method usually used for immobilizing this type of bacterial cell or enzyme can be used,
For example, it is used after being immobilized on polyacrylamide gel, carrageenan gel, membrane polymer, or the like.

As a condition for suppressing the succinic acid byproduct, the heating temperature is 30 to 60.
C. is used, but preferably more than 35.degree. C. to 55.degree. C., pH is preferably 4-10, more preferably pH 5-9, L-malic acid concentration is preferably 0.01-5 mol, more preferably 0.
The reaction time is usually from 0.1 to 0.3 mol, and the reaction time varies depending on the cell concentration, but is usually 10 minutes to 3 hours.

Examples of the surfactant used in the present invention include dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, cetyltrimethylammonium bromide, dodecylpyridinium bromide, cetylpyridinium chloride, and cationic surfactants such as tetradecylammonium bromide. Sodium dodecyl sulfate, sodium tetradecyl sulfate, sodium dodecyl sulfonate, sodium tetradecyl sulfonate,
Sodium dodecylbenzenesulfonate, dodecyl-N
An anionic surfactant such as sodium sarcosinate,
Polyoxyethylene (n) decyl ether, polyoxyethylene (n) dodecyl ether, polyoxyethylene (n) tridecyl ether, polyoxyethylene (n)
Tetradecyl ether, polyoxyethylene (n) cetyl ether, polyoxyethylene (n) stearyl ether, polyoxyethylene (n) oleyl ether, polyoxyethylene (n) cetyl stearyl ether, polyoxyethylene (n) para-tert- Octyl phenyl ether, polyoxyethylene (n) para-octyl phenyl ether, polyoxyethylene (n) para-nonyl phenyl ether, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, polyoxy Ethylene (n) sorbitan monolaurate, polyoxyethylene (n) sorbitan monopalmitate, polyoxyethylene (n) sorbitan monostearate, polyoxyethylene (n) monoolein Nonionic surfactants such as sorbitan acid, palmitoyl lysolecithin, dodecyl-N-
Betaine, stearyl-N-betaine, dodecyl-β-
Examples thereof include amphoteric surfactants such as alanine, and surfactants having a steroid skeleton such as sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium taurocholate, sodium taurodeoxycholate, and digitonin. However, (n) after polyoxyethylene means that the exemplified substances include a group of compounds having different numbers (n) of oxyethylene (—CH ₂ CH ₂ O—) units.

Among these surfactants, cationic surfactants,
Nonionic surfactants and surfactants having a steroid skeleton are preferable, and particularly cetyltrimethylammonium bromide, cetylpyridinium chloride, polyoxyethylene (n) para-tert-octylphenyl ether, sorbitan monolaurate, sorbitan monopalmitate. , Sorbitan monostearate, sorbitan monooleate, polyoxyethylene (n) sorbitan monolaurate, polyoxyethylene (n) sorbitan monopalmitate, polyoxyethylene (n) sorbitan monostearate, polyoxyethylene (n) mono Preference is given to sorbitan oleate, sodium cholate, sodium deoxycholate and the like.

The addition concentration of these surfactants is 0.005% (wt / vol) or more, though it depends on the kind of the surfactant, but it is preferably 0.01 to 2%, particularly preferably 0.02 to 1%.

When the heating temperature for the succinic acid by-product suppression treatment is higher than the above range, the fumarase activity of the desired reaction is inactivated, and at low temperatures, the side reaction suppression effect is reduced, which is unsuitable. Regarding the L-malic acid concentration, if the concentration is higher than the above, the effect is saturated and it is uneconomical, and if the concentration is low, the effect of suppressing only side reactions cannot be obtained.

Since the succinic acid by-product of the bacterial cell or its immobilized product obtained as described above is remarkably reduced, when producing L-malic acid using the bacterial cell or the immobilized product, a by-product of succinic acid is produced. It has become clear that this can be suppressed. Therefore, in the presence of the treated cells or immobilized product, fumaric acid or its fumaric acid salt such as sodium or ammonium can be efficiently converted to L-.
Can produce malic acid.

This enzymatic reaction can be carried out in the temperature range of about 15 to 60 ° C, but considering the stability of fumarase, it is 20 to 55 ° C.
It is desirable to carry out. This enzymatic reaction is carried out in a water solvent, but it is also possible to use a solvent such as a phosphate buffer or Tris-HCl buffer at a concentration of 10 to 500 mM in addition to water.
Further, in order to adjust the pH of the reaction solution to 4 to 10, alkalis such as potassium hydroxide, sodium hydroxide and ammonium hydroxide, and inorganic acids such as hydrochloric acid and sulfuric acid can be added.

The amount of fumaric acid or a salt thereof used during the reaction is not particularly limited, but it is generally suitable to use it in the range of 0.5 to 30% (wt / vol). In addition, the amount of the microbial cells containing fumarase that has been subjected to a succinic acid by-product suppression treatment or a treated product thereof is not particularly limited, but is generally 0.5 to 10%.
It can be used in the range of (wt / vol).

Separation and purification of L-malic acid produced in the reaction solution obtained by enzymatically reacting fumaric acid with water using the treated product should be carried out by a known method such as adsorption or desorption with an ion exchange resin, activated carbon or the like. You can

According to the method of the present invention, since the microbial cells obtained by removing only the by-product of succinic acid or its immobilized product are used without impairing the fumarase activity contained in the microbial cells, this treated microbial cell is
When used for the production of malic acid, the yield of fumaric acid is significantly improved. Therefore, the present invention greatly contributes to the industrial production of L-malic acid.

Hereinafter, the present invention will be described in more detail by showing Examples of the present invention, but the present invention is not limited thereto.

Experimental Example Reference Example 1 50 ml of a medium having the composition shown in Table 1 was dispensed into a 500 ml Erlenmeyer flask and autoclaved at 120 ° C. for 15 minutes, and then 2% by volume of ethanol was added thereto. Flavum MJ-233 (FERM BP-1498) was inoculated with one platinum loop, and
Shaking culture was carried out at ℃ for 24 hours.

20 ml of this culture solution was added to the second volume in a 2-volume jar fermenter.
Inoculated to the medium 1 shown in the table, 33 ℃, pH7.6, aeration 1vv
The cells were cultured under the condition of m, ethanol was intermittently added so that the ethanol concentration was maintained at 1 to 1.5% by volume, and the cells were cultured for 30 hours.

After completion of the culture, the culture solution was centrifuged (6,000 rpm, 15 minutes) to obtain the cells as test cells.

0.2% (wt / vol) of each surfactant shown in Table 3 was added to an aqueous solution containing 0.2 mol / L-malic acid adjusted to pH 7.0 with caustic soda. Take 50 ml of each of the thus obtained aqueous solutions and obtain the bacterial cells obtained above.
2.5 g each was suspended in each. Then these are 45 ℃
Heat treatment was carried out for 2 hours, and the cells were collected by centrifugation.

2.5 g of each of the heat-treated cells thus obtained was suspended in 50 ml of a 1.0 mol / fumaric acid aqueous solution which had been adjusted to pH 7.0 with caustic soda, and enzymatically reacted at 45 ° C. for 2 hours.

The amount of produced L-malic acid and by-product succinic acid in each reaction solution is
It was quantified by high performance liquid chromatography. As a result, the amount of L-malic acid and succinic acid produced when the same enzymatic reaction as the above was carried out by heat-treating the cells in the same manner as above except that the surfactant was not added, respectively. Ten
The ratio when 0 is shown in Table 3.

Table 1 Urea 4.0 g Ammonium sulfate 14.0 g KH ₂ PO ₄ 0.5 g K ₂ HPO ₄ 0.5 g MgSO ₄ / 7H ₂ O 0.5 g FeSO ₄ / 7H ₂ O 6.0 mg MnSO ₄ / 4-6H ₂ O 6.0 mg Yeast extract 1.0 g Casamino acid 1.0 g Biotin 200 μg Thiamine hydrochloride 100 μg Distilled water 1000 ml pH 7.6 Table 2 Ammonium sulfate 23.0 g KH ₂ PO ₄ 0.5 g K ₂ HPO ₄ 0.5 g MgSO ₄ / 7H ₂ O 0.5 g FeSO ₄ / 7H ₂ O 20 mg MnSO ₄・ 4-6H ₂ O 20 mg Yeast extract 3 g Casamino acid 3 g Biotin 200 μg Thiamine hydrochloride 100 μg Distilled water 1000 ml Reference Example 2 Test cells were obtained in the same manner as in Reference Example 1. "Tween 20" whose pH has been adjusted to 7.0 with caustic soda is the fourth
2.5 g of each of the above-mentioned test cells was added to 50 ml of a 0.2 mol / concentration L-malic acid aqueous solution contained at the concentration shown in the table, and each was heat-treated at 45 ° C. for 2 hours. Then, the cells were collected by centrifugation.

The fumaric acid aqueous solution was enzymatically reacted in the same manner as in Reference Example 1 except that the heat-treated bacterial cells thus obtained were used, and L-malic acid and by-product succinic acid in the obtained reaction solution were used in Reference Example 1. The measurement was conducted in the same manner as above, and the results are shown in Table 4.

Reference Example 3 Test cells were obtained in the same manner as in Reference Example 1. 2.5 mol each of these cells contained 0.2% (wt / vol) of "Tween 20" 0.2 mol /
Add 50 ml of malic acid aqueous solution (pH 7.0) of
The heat treatment was performed for 2 hours at each treatment temperature shown in the table.

L-malic acid and by-product succinic acid in the reaction solution obtained by enzymatically reacting an aqueous solution of fumaric acid in the same manner as in Reference Example 1 except that the heat-treated cells were collected and used.
Measurement was carried out in the same manner as above, and the results are shown in Table 5.

Reference Example 4 2.5 g each of the test cells obtained in the same manner as in Reference Example 1 was added to "Twee
n 20 "containing 0.2% (wt / vol) of 0.2 mol / concentration of L
-Suspended in 50 ml of an aqueous solution of malic acid (pH 7.0), and heat-treated at 45 ° C for the time shown in Table 6.

The obtained heat-treated bacterial cells were collected by centrifugation, and the fumaric acid aqueous solution was enzymatically reacted in the same manner as in Reference Example 1 except that these bacteria were used to obtain L-malic acid and L-malic acid in the reaction solution. The succinic acid by-product was measured in the same manner as in Reference Example 1, and the results are shown in Table 6.

Example 1 2.5 g of the test cells obtained in the same manner as in Reference Example 1 was added to "Tween 2
Suspended in 50 ml of 0.2 mol / concentration L-malic acid aqueous solution (pH 7.0 adjusted with caustic soda) containing 0.2% (wt / vol) of "0", heat treated at 45 ° C for 2 hours, and then collected by centrifugation. did.

50 ml of 1 mol / concentration fumaric acid aqueous solution
(PH 7.0 was adjusted with caustic soda), and the enzyme reaction was carried out at 45 ° C. for 2 hours. L- generated in the reaction solution after completion of the reaction
The amounts of malic acid and succinic acid were quantified by high performance liquid chromatography, and the molar yield was 78% (39 mmol) of L-malic acid and 0.05% of succinic acid based on the starting fumaric acid.
Less than (2.5 mmol) was formed.

The pH of this solution was adjusted to about 2 with a 6N HCl solution, and the solution was passed through a column filled with a strong base resin (Amberlite IRA-400, manufactured by Rohm and Haas Co., R ₂ CO ₃ type). L-malic acid was adsorbed on the resin. Next, it was eluted with 1N ammonium carbonate and then concentrated to precipitate crude crystals of L-malic acid. After washing this with acetone and drying, L-
2.8 g of malic acid crystals were obtained.

Claims (1)

[Claims] 1. A method for producing L-malic acid from fumaric acid or a salt thereof by an oxygen reaction in the presence of a fumarase-containing bacterium or a treated product thereof, wherein the bacterium or the treated product contains a surfactant. The method for producing L-malic acid is characterized by being heat-treated in an aqueous solution of L-malic acid or a salt thereof.