JP2798886B2 - Method for producing L-aspartic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for preventing the discharge of wastewater containing a large amount of ammonium ions when producing L-aspartic acid from fumaric acid and ammonia.

[0002]

2. Description of the Related Art Conventionally, as a method for producing L-aspartic acid from ammonium fumarate using a microorganism having aspartase activity, a microorganism having resistance to α-aminobutyric acid is aerobically cultured and then subjected to a reaction. (JP-B-61-29718), a method using microbial cells cultured in a fumaric acid-added medium (Japanese Patent Application Laid-Open No. 60-120983).
Publication), Escherichia coli (Esche
Various methods are known, such as a method using an immobilized microorganism-filled column on which R. richia coli is immobilized (JP-A-53-6483).

[0003] Usually, in order to recover L-aspartic acid from a reaction solution of fumaric acid and ammonia, a mineral acid such as sulfuric acid is used to adjust the pH of the reaction solution to pH2, which is the isoelectric point of L-aspartic acid. After adjusting to about 7, a crystal of L-aspartic acid is precipitated by cooling, and the crystal is filtered off.

This method is very economically advantageous because it uses an inexpensive mineral acid, has a high L-aspartic acid product recovery rate as crystals, and has a high purity of the L-aspartic acid obtained. It is a way.

However, from the viewpoint of industrial waste, there is a problem that a large amount of wastewater containing a high concentration of ammonium salt such as ammonium sulfate is discharged. Removal of ammonium ions in an aqueous solution is also extremely difficult in terms of wastewater treatment, and problems such as water pollution due to an increase in the concentration of nitrogen containing ammonium ions have arisen in lakes and inland bays such as the Seto Inland Sea. Also recently
It seems that various ministries are also examining the regulation of nitrogen concentration in industrial wastewater. Therefore, it is desired to develop a system that does not generate a large amount of by-products such as ammonium sulfate even in the production of L-aspartic acid.

[0006] In US Pat. No. 4,560,563, during the production of L-aspartic acid, aspartase or an aspartase-producing bacterium is allowed to act on fumaric acid and ammonia, and the resulting aqueous solution of ammonium aspartate is made acidic by adding maleic acid. A method has been proposed in which L-aspartic acid is precipitated and the reaction solution is recycled by isomerizing the filtrate. This method does not generate by-products such as ammonium sulfate. However, in this method, the maleic acid used for the precipitation of L-aspartic acid is
The process includes the step of isomerizing to fumaric acid capable of acting aspartase using a catalyst containing bromine ions and removing the catalyst after the isomerization, which has the disadvantage of complicating the production process of L-aspartic acid. doing.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to produce L-aspartic acid from fumaric acid and ammonia.
An object of the present invention is to provide a method for producing L-aspartic acid, which does not discharge a large amount of ammonium salt.

The present inventors have conducted intensive studies on a simple method for producing L-aspartic acid in which wastewater containing such a high concentration of ammonium ions is not discharged in large amounts. If the alkali metal ion is used in combination with ammonia alone to neutralize the acid, the conversion and selectivity of the reaction are comparable to those of the conventional method. It was found that the present invention was completed.

[0009]

According to the present invention, L-aspartic acid is produced by allowing an enzyme-containing substance having aspartase activity to act on a substrate medium containing fumaric acid, ammonia and an alkali metal ion. -Adding a mineral acid to the reacted medium containing aspartic acid, filtering and recovering precipitated L-aspartic acid crystals, and discharging a waste liquid containing an alkali metal salt of the mineral acid as a main component. And a method for producing L-aspartic acid.

The fumaric acid used in the present invention is selected from fumaric acid or a fumarate, and may be a mixture thereof. The concentration of fumaric acid at the time of the reaction is usually preferably 5 to 30% by weight, but particularly preferably 10 to 25% by weight in view of the solubility and productivity of the fumarate.

As the ammonia used in the present invention, liquid ammonia, an aqueous ammonia solution and the like can be used, but an ammonia aqueous solution is advantageous in handling.

The concentration of the aqueous ammonia is not particularly limited, but is preferably 10 to 35% by weight industrially.

The amount of the alkali metal ion used for neutralizing the fumaric acid used in the present invention is 0.5 to 1.5 times mol of fumaric acid, preferably 0.9 to 1.
It is good to use 3 times mole, more preferably 1.10 to 1.25 times mole.

As the alkali metal ion used in the present invention, various alkali metal ions can be used in addition to sodium ion and potassium ion, but it is economically preferable to use sodium hydroxide or potassium hydroxide as the alkali metal ion. These alkali metal hydroxides may be used in combination of two or more. Further, instead of neutralizing fumaric acid with an alkali metal ion, an alkali metal salt of fumaric acid may be used as it is.

The pH of the reaction solution is in the range of 5 to 10, preferably in the range of 7.5 to 9.0, and more preferably about 8.0 to 8.5, which is the optimum pH of aspartase. It may be adjusted by adding ammonia.

The enzyme-containing substance having aspartase activity used in the present invention can be obtained by physically or microbial cells having high aspartase activity or by physical treatment such as ultrasonication, milling, freeze-thawing, enzymatic treatment and surfactant treatment. Any of the crushed bacterial cells obtained by biochemical treatment and crushing, and any of the enzymes obtained by conventional methods such as ammonium sulfate salting out and acetone precipitation can be used. Examples of the microorganism having aspartase activity include microorganisms belonging to the genus Escherichia (Escherichia col.
i) ATCC 11303, ATCC 9637, ATCC
27325), Brevibacterium
The microorganism is not particularly limited as long as it is a microorganism such as a microorganism belonging to the genus and has a feature of producing L-aspartic acid from fumaric acid in a high yield. These enzyme-containing substances having aspartase activity can also be used by immobilizing them on a carrier. As a carrier for immobilization, a suitable natural polymer such as cellulose, alginic acid, carrageena, or nanman gel, or a suitable synthetic polymer such as an ion exchange resin or polyvinyl alcohol or polyacrylamide can be used in a conventional manner.

It is also possible to use, in the reaction, an enzyme in which an enzyme other than aspartase activity that may interfere with the reaction, such as a fumalase activity, contained in the enzyme-containing substance having aspartase activity used in the reaction is previously inactivated. It is. For example, the enzyme-containing substance is previously heated in the presence of L-aspartic acid and ammonium ions in an alkaline region at 40 to 60 ° C.
The fumarase activity can be previously inactivated by heat treatment.

The reaction between fumaric acid and ammonia is carried out in an aqueous medium in which they are dissolved, for example water or a buffer.
The concentration of fumaric acid as a raw material at the time of the reaction is preferably 5 to 30% by weight, but considering the solubility of the fumarate and the reactivity of the biocatalyst, it is particularly effective to react with an aqueous solution in the range of 10 to 25% by weight. It is a target.

The reaction solution further contains a manganese salt such as manganese chloride and manganese sulfate, or magnesium chloride,
0.1 to 50 magnesium salts such as magnesium sulfate
It is preferable to add at a concentration of mM, especially 1 to 10 mM.

The mode of the reaction tank in the present invention is not particularly limited. For example, the reaction can be performed in a conventionally known reaction tank such as a batch type reaction apparatus or a column type reaction apparatus. The number of reaction vessels may be one or more. In the case of a column-type reaction device, it is also possible to carry out the reaction by changing the flow rate depending on the amount of the enzyme packed in the column.

The lower limit of the reaction temperature is usually about 20 ° C. because the reaction rate is low at a low temperature, and the upper limit is preferably about 50 ° C. at a high temperature to inactivate aspartase at a high temperature. It is good to carry out in the range of 25 to 40 ° C.

L-Aspartic acid in the solution after the reaction can be easily recovered by an isoelectric point precipitation method or the like as usual. For example, a mineral acid such as sulfuric acid may be added to the reaction solution to lower the pH to about 2.77, which is the isoelectric point of L-aspartic acid, and the crystals may be precipitated by cooling.

The mineral acid used in the present invention is sulfuric acid, hydrochloric acid,
Phosphoric acid and the like can be used.

The precipitated L-aspartic acid crystals are separated from the liquid by a conventional method such as filtration, centrifugation, decantation and the like, and dried according to a conventional method. The concentration of ammonium ions in the liquid from which the crystals have been separated in this way is a few to several tenths of that in the usual industrially practiced method. It is a metal salt.

[0025]

According to the present invention, from fumaric acid and ammonia, L
-In the production of aspartic acid, the main component of wastewater can be changed from conventional ammonium sulfate to alkali metal salt of sulfuric acid, and it is possible to comply with recent regulations on nitrogen in industrial wastewater.

[0026]

Next, the method of the present invention will be described with reference to examples, but the present invention is not limited to these examples.

Example 1 20 g of fumaric acid and phosphoric acid 1 were placed in a 2 L jar fermenter.
1 g of potassium, 0.5 g of magnesium sulfate heptahydrate, 20 g of yeast extract, and 20 g of corn steep liquor are dissolved in water, 1 L of a medium whose pH is adjusted to 6.8 with ammonia is charged and sterilized, and then separately placed in a 500 ml shake flask. Was inoculated with Escherichia coli ATCC 11303, which had been cultivated in 50 ml of the above medium.
The culture was carried out by aeration and stirring at ℃. At the time when the fumaric acid in the medium disappeared, acetic acid was added to the bacterial cell culture to adjust the pH to about 5,
After standing at 45 ° C. for 1 hour, the culture was centrifuged,
The cells were separated. The cells were divided into three equal parts, frozen at -80 ° C and refrigerated.

200 g of fumaric acid and 0.25 g of magnesium sulfate heptahydrate were placed in 600 ml of water, and 82.8 g of sodium hydroxide (1.2 times mol of fumaric acid) was added.
The pH was adjusted to 8.3 with 25% aqueous ammonia, and water was added to make 1 L, which was used as a reaction substrate solution.

[0029] One of the frozen microbial cells previously divided into three equal parts was added to the substrate solution and reacted at 37 ° C for 5 hours while gently shaking. L-Aspartic acid in this reaction solution contained 221.
3. g. After the reaction solution was centrifuged to remove the cells, sulfuric acid was added to adjust the pH to 2.77. This was heated to 60 ° C. and then cooled. After cooling, the mixture was filtered by suction with a suction filter, and the crystals in the filter were washed while suctioning with about 150 ml of water. The crystals were dried and checked for weight and purity. The weight was 216.4 g and the purity was 99.6. % L-aspartic acid was obtained. When the ammonia concentration in 1 L of the filtrate was measured, it was about 1.0 g / L as NH ₃ .

Example 2 The same operation as in Example 1 was carried out except that the amount of sodium hydroxide added to the reaction substrate was changed to 100 g (1.45 mol of fumaric acid). L-aspartic acid in the reaction solution after the reaction was 178.1 g. L-aspartic acid was crystallized in the same manner as in Example 1 to obtain L-aspartic acid having a weight of 219 g and a purity of 80%.
When the ammonia concentration in the filtrate was measured, it was about 0.85 g / L as NH ₃ .

Example 3 In Example 1, sodium hydroxide was changed to potassium hydroxide as the reaction substrate, and 96.7 g of this was added (to 1.0% of fumaric acid).
The reaction was performed in the same manner as in Example 1 except that the compound was added (double mole). L-aspartic acid in the reaction solution after the reaction was 225.
9 g. L-aspartic acid was crystallized in the same manner as in Example 1 to obtain a weight of 222.0 g and a purity of 99.4%.
Of L-aspartic acid was obtained. When the ammonia concentration in the filtrate was measured, it was about 5.7 g / L as NH ₃ .

Comparative Example 1 The same operation as in Example 1 was performed, except that 25% aqueous ammonia was added without adding sodium hydroxide to the reaction substrate, and the pH was adjusted to 8.3. L-aspartic acid in the reaction solution after the reaction was 227.3 g. This reaction solution was treated in the same manner as in Example 1 to obtain a crystal and a furnace solution. When the crystals were dried and examined for weight and purity,
L-aspartic acid having a weight of 223.3 g and a purity of 99.6% was obtained. When the ammonia concentration in the filtrate was measured,
It was about 35.2 g / L as NH ₃ .

[0033]

According to the present invention, L-aspartic acid can be efficiently produced from fumaric acid without involving environmentally undesirable by-products such as a high-concentration aqueous solution of ammonium sulfate.

────────────────────────────────────────────────── (5) Continuation of the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:13) (56) References JP-A-63-102693 (JP, A) JP-A-59-113895 (JP, A) JP-A-49-25189 (JP, A) U.S. Pat. No. 4,606,653 (US, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12P 13/00-13/24 CA (STN)

Claims (4)

(57) [Claims] An L-aspartic acid is produced by allowing an enzyme-containing substance having aspartase activity to act on a substrate medium containing fumaric acid, ammonia and an alkali metal ion, followed by a reaction containing L-aspartic acid. A method for producing L-aspartic acid, comprising adding a mineral acid to a waste medium, filtering and recovering L-aspartic acid crystals, and discharging a waste liquid containing an alkali metal salt of the mineral acid as a main component. 2. The method according to claim 1, wherein the enzyme-containing substance having aspartase activity is a microbial cell having enzymatic activity, a crushed cell, a partially purified enzyme or a purified enzyme, or an immobilized substance containing these enzymes. Method. 3. The method according to claim 1, wherein the alkali metal ion is contained in an amount of 0.5 to 1.5 times mol of fumaric acid. 4. The method according to claim 1, wherein the alkali metal ion is a sodium ion and / or a potassium ion.