JPH09322792A - Production of l-aspartic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a profitable method for producing L-aspartic acid from maleic acid as a raw material by an enzyme-stabilized reaction to enable a stable continuous operation. SOLUTION: This method for producing L-aspartic acid has a characteristic comprising bleeding a solution containing the L-aspartic acid and ammonia at least from a spot in the below-described processes. The process (1): a process for successively or simultaneously executing a reaction for isomerizing maleic acid, etc., in the presence of an enzyme and a reaction for reacting at least a part of the isomerization reaction product with ammonia in the presence of an enzyme in water solvent to produce L-aspartic acid ammonium salt, the process (2): a process for adding maleic acid, etc., to the solution obtained in the process (1) to deposit the L-aspartic acid, and the process (3): a process for recovering the L-aspartic acid obtained in the process (2) and recycling the mother liquor to the process (1).

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing L-aspartic acid. More specifically, the present invention relates to an industrially advantageous process for producing L-aspartic acid by enzymatic action using maleic acid and / or maleic anhydride as a raw material.

[0002]

2. Description of the Related Art L-aspartic acid is in increasing demand as a pharmaceutical and food additive. In addition, although development of new applications is also under consideration, at present, a manufacturing cost is high and an economically excellent industrial process has not been established.

As a method for producing L-aspartic acid, a method is known in which ammonium fumarate is obtained by an enzymatic reaction in the presence of aspartase or a microorganism producing it. Also known is a method of isomerizing maleic acid and ammonia in the presence of isomerase or a microorganism producing the same to obtain fumaric acid, and then obtaining L-aspartic acid by the same enzymatic reaction as described above [Y. Takamura e
t al. , Agric, Biol. Chem. , 3
0 , No. 4, 345-350 (1966)]. However, in the conventional method, since L-aspartic acid ammonium after enzymatic reaction is acid-deposited with sulfuric acid or hydrochloric acid, a large amount of an inorganic acid ammonium salt having a low economical value is produced as a by-product, resulting in the production of L-aspartic acid. It was supposed to raise costs.

As a method for improving this drawback, a mother liquor containing ammonium maleate after ammonium L-aspartate after enzymatic reaction is acid-deposited with maleic acid and / or maleic anhydride to recover L-aspartic acid crystals. Has been proposed as a raw material (US Pat. No. 4,560,653, JP-A-8-33491).
Issue). Since these methods use maleic anhydride as a raw material, which is easily available in large quantities and is inexpensive, it can be an industrially highly desirable method.

However, in the above proposal, the isomerization reaction of ammonium maleate is applied by a conventionally known chemical reaction or enzymatic reaction, and continuous industrially stable operation is continuously performed. -It was still difficult to produce aspartic acid inexpensively and efficiently.
In the case of the method proposed above, a large amount of maleic acid must be added to crystallize L-ammonium aspartate.
-The recovery rate of aspartic acid crystals could not be increased,
The amount of maleic acid added in the crystallization step depends on the L-
The condition exceeds the amount of aspartic acid. Therefore, when the mother liquor containing ammonium maleate after crystallization is recycled and used in the reaction system, the problem that the concentration of L-aspartic acid in the system gradually increases, continuous movement becomes difficult, and an efficient industrial production method is required. Is hard to say.

[0006]

DISCLOSURE OF THE INVENTION The present invention uses maleic acid and / or maleic anhydride as a raw material, isomerizes isomerase or a microorganism producing it in a water solvent, and then produces aspartase or this. Microbes,
L in the presence of ammonia and ammonia in an aqueous solvent
Obtaining ammonium aspartate, L after enzymatic reaction
-A method for producing L-aspartic acid, wherein ammonium aspartate is acid-deposited with maleic acid and / or maleic anhydride, and a mother liquor containing ammonium maleate after recovering L-aspartic acid crystals is recycled to an enzymatic reaction step, It is an object of the present invention to provide a novel L-aspartic acid production process capable of a balanced and stable continuous operation.

[Means for Solving the Problems]

The inventors of the present invention have made diligent studies in view of the above situation,
Upon crystallization of L-aspartic acid ammonium, when a large amount of maleic acid is added, the recovery rate of L-aspartic acid crystals is increased, while the amount of maleic acid added in the crystallization step is different from that of L-aspartic acid to be crystallized. If the mother liquor containing ammonium maleate after crystallization is recycled and used in the reaction system, the amount of L-in the system gradually increases.
There is a problem that the aspartic acid concentration rises, continuous operation becomes difficult, and it is not an efficient industrial manufacturing method.
The present invention was completed by finding that a liquid containing L-aspartic acid and ammonia can be bleed from at least one location after the step in the present recycling process, and stable continuous production of L-aspartic acid can be achieved over a long period of time. Came to do.

That is, the gist of the present invention is to provide the following steps:
In the method for producing L-aspartic acid, the method for producing L-aspartic acid comprises bleeding a liquid containing L-aspartic acid and ammonia from at least one location after the step. Step: a reaction for isomerizing maleic acid and / or maleic anhydride in the presence of isomerase or a microorganism producing it, and the presence of at least a part of the isomerization reaction product and ammonia aspartase or a microorganism producing it. A step of sequentially or simultaneously carrying out a reaction of reacting in a sewage solvent to produce L-aspartic acid ammonium, and step: adding maleic acid and / or maleic anhydride to the solution obtained in the step to form L-aspartic acid crystals And a step of recovering the L-aspartic acid crystals obtained in the step and recycling the mother liquor to the step.

[0009]

The present invention will be described in more detail below. (Step) In the present invention, maleic acid and / or maleic anhydride is isomerized in the presence of isomerase or a microorganism producing it, and then at least a part of an isomerization reaction product and ammonia are produced aspartase or this. In the presence of the microorganisms mentioned above, the reaction is carried out in a water solvent to produce L-ammonium aspartate. The reaction is carried out by isomerizing ammonium maleate to ammonium fumarate, and then using this as an enzyme in the presence of aspartase or a microorganism that produces this, a two-step reaction method of reacting to produce ammonium aspartate, or maleic acid. Any one-step reaction method in which isomerization of ammonium and formation of ammonium aspartate are simultaneously performed may be used.

The reaction of isomerizing ammonium maleate into ammonium fumarate and the reaction of converting ammonium fumarate into ammonium aspartate are known, and the present invention can be carried out according to known methods. .

The microorganism having maleate isomerase activity used in the present invention is not particularly limited as long as it has this activity, but microorganisms belonging to the genera Alcaligenes, Pseudomonas, Xanthomonas and Patylus are mentioned. It is preferably used. Especially Alcaligenes faec
alis) IFO 12669, IFO 1311
1, IAM 1473, Alcaligenes eutrophus,
Pseudomonas fluorescens
nas fluorescens) ATCC 2372
8. Xanthomonas Xanthomomos
nas marutomonas) ATCC 132
70. Bacillus stearothermophilus (Bacil
lus stearothermophilus) MI
-101 (FERM P-14801), Bacillus brevis MI-10
3 (FERM P-14803) and the like are preferably used. Furthermore, there is no problem even if the above-mentioned genetically modified microorganism in which the gene is recombined is used.

Each cultured bacterial cell can be used after washing with a buffer solution (pH 7) such as a phosphate buffer solution in advance. The concentration of the phosphate buffer for washing is 0.05M to 0.2.
M is preferably used. The microbial cells can be used as they are, and if necessary, the microbial cell crushed product obtained by subjecting the microbial cell to ultrasonic crushing or the like, a cell-free extract obtained by centrifuging the crushed product, or the cell-free extract is ammonium sulfate. It is also possible to use a partially purified enzyme purified by a fractionation method, an ion exchange column, a gel filtration column or the like, or an immobilized product obtained by immobilizing the microbial cell or the disrupted cell with a carrier such as acrylamide monomer or alginic acid. When using bacterial cells, freeze the bacterial cells in advance,
Triton X-100, Tween in the above buffer
After increasing the permeability of the bacterial cells by treating the bacterial cells for 10 to 120 minutes at a temperature of 15 to 40 ° C. in a liquid containing 0.01 to 0.2% by weight of a surfactant such as 20 It can also be used.

In the present invention, when a microorganism having maleate isomerase activity or a treated product thereof is subjected to an enzymatic reaction in an aqueous solution containing maleic acid, the dissolved oxygen concentration in the aqueous solution containing maleic acid (hereinafter abbreviated as DO) 4 ppm or less, preferably 1 ppm or less,
It is more preferable to maintain the reaction at 0.5 ppm or less.

However, even if the dissolved oxygen concentration level may momentarily exceed the above range, the dissolved oxygen concentration level is substantially below the above range during the isomerization reaction, for example, 90% or more of the isomerization reaction time,
It may be maintained preferably for 95% or more, more preferably 98% or more. For example, even if the above range is temporarily exceeded immediately after starting stirring or immediately after preparing the maleic acid-containing aqueous solution, it may be maintained below the range thereafter. In addition, even if the number of agitation is increased to exceed the above range for a very short time, it is sufficient that the reaction time is maintained within the above range for most of the entire reaction time.

When the concentration of dissolved oxygen in the reaction solution in the step is high, the stability of the enzyme isomerase is lowered and it becomes difficult to stably continue the isomerization reaction. In particular, ammonium L-aspartate after the enzymatic reaction is treated with maleic acid and / or
Alternatively, in the method of reusing the mother liquor containing ammonium maleate after the L-aspartic acid crystals have been recovered by acid precipitation with maleic anhydride as a raw material, isomerase or the enzyme of the microorganism producing the isomerase when a long-term recycling operation is carried out. The action gradually decreases, and it is very difficult to maintain continuous operation.

In the present invention, an inert gas is used for the reaction,
The inside of the maleic acid-containing aqueous solution tank may be sealed. The gas inert to the reaction is not particularly limited as long as it is a gas containing oxygen. A gas selected from so-called rare gases such as Ar and He, nitrogen gas, carbon dioxide gas, etc. may be used,
You may combine 1 type of gas or 2 or more types of gas.
As a method of sealing the inside of the maleic acid-containing aqueous solution tank with these gases, for example, stirring while blowing the above gas into the maleic acid aqueous solution, a method of introducing the above gas after degassing the maleic acid-containing aqueous solution, etc. Can be adopted. In the method of the present invention, the inside of the maleic acid-containing aqueous solution tank is preferably sealed with the above gas, and it is not always necessary to continuously blow the above gas into the maleic acid containing aqueous solution tank. In addition, as a method of sealing the inside of the reaction tank with the gas, a method of continuously blowing the gas into the reaction tank, a method of blowing the gas into a reaction solution in the reaction tank, and then sealing the reaction liquid are performed.

In the present invention, DO may be lowered by adding an oxygen scavenger inert to the reaction to the reaction solution. Examples of the oxygen scavenger include ascorbic acid, protocatechuic acid, sulfite and the like. The concentration of the oxygen absorber added is 1 ppm to 1000 ppm, preferably 10 ppm to 500 ppm. The addition position is not particularly limited to the steps, and may be any step or between any steps. You may divide and add in several places.

Further, the reaction solution containing ammonium maleate used in this step may be degassed to reduce the dissolved oxygen concentration and then subjected to an enzymatic reaction. In particular, in a method of reusing the mother liquor containing ammonium maleate after the L-aspartic acid ammonium after the enzymatic reaction is acidified with maleic acid or maleic anhydride and the L-aspartic acid crystals are recovered as a raw material, It is preferable to degas the recycled mother liquor in advance to reduce DO, and then to carry out an enzymatic reaction.

In the present invention, the conversion of ammonium fumarate into ammonium L-aspartate is carried out in the presence of aspartase or a microorganism producing aspartase. This method can be carried out by a known method. I can. The aspartase-producing microorganism is not particularly limited as long as it is a microorganism capable of producing L-aspartic acid from fumaric acid and ammonia, and examples thereof include Brevibacterium, Escherichia,
Examples thereof include microorganisms of the genus Pseudomonas and the genus Bacillus. Specifically, Brevibacterium flavum (Br
Evibacterium flavum) MJ-23
3 (FERM BP-1497), the same MJ-233-A
B-41 (FERM BP-1498), Brevibacterium ammoniagenes ATCC 6872, Escherichia coli
ATCC 11303 and ATCC 27325 can be exemplified.

The concentration of the raw material aqueous solution for the isomerization reaction and the reaction for producing L-aspartic acid is usually determined by the concentration of the monoammonium maleate aqueous solution recovered from the step described below. The concentration of the raw material aqueous solution in the step of the present invention is usually 45 to 700 g / l, preferably 90 to 450 in terms of monoammonium maleate.
g / l.

Isomerization reaction and L in the process of the present invention
-The reaction for producing aspartic acid can also be carried out under pH adjustment with an alkaline agent other than ammonia. The pH in the reaction system at this time is usually
It is preferably 7.5 to 10, and both reactions may be carried out simultaneously in the same reaction tank. When they are carried out at the same time, the bacteria having the respective properties can be used together, and the conditions suitable for both reactions can be selected and carried out. It is not always necessary to use them in combination as long as they have both properties.

As the alkaline agent, sodium hydroxide,
Although not particularly limited to potassium hydroxide, L
-It is preferable in the present invention to use at least ammonia, which is a raw material for the reaction for producing aspartic acid, and does not have any adverse effect on the isomerization reaction.
When ammonia is used alone, the amount of ammonia used is 1.0 with respect to the raw material monoammonium maleate.
It is 5 to 2.0 molar times, and preferably 1.1 to 1.5 molar times. When the above alkaline agent is used in combination with ammonia, the amounts of ammonia and alkaline agent are adjusted so as to be within the pH range in the reaction system. The temperature for the isomerization reaction and the reaction for producing L-aspartic acid is selected at a temperature at which the enzymatic reaction is efficiently carried out, and is usually 10
-80 degreeC, Preferably it is 15-60 degreeC.

The reaction operation of the enzyme reaction is not limited, but continuous operation is more preferable than batch operation. Usually, as a reaction method, a method of passing a raw material aqueous solution through a packed bed in which cells are immobilized, or while supplying the raw material aqueous solution into a reactor in which the cells themselves or the immobilized cells are suspended, the reaction solution A method of extracting the cells, separating the cells using a separation membrane or a centrifuge, and returning the cells to the reactor can be mentioned.

The reaction liquid obtained by the reaction for producing L-aspartic acid mainly contains ammonium L-aspartate, and the ammonium salt is a mixture of monoammonium salt and diammonium salt. The proportion of diammonium salt is usually 10 to 60% mol. The reaction solution may also contain unreacted ammonium fumarate and ammonium maleate, but the content in this case is 5 g / l or less, preferably 2 g / l or less, particularly 1
It is desirable to control to g / l or less. The reaction solution after the enzymatic reaction in the step is separated into bacterial cells by a conventional method and then sent to the next step. Usually, cells are separated using a separation membrane or a centrifuge.

(Step) Maleic acid and / or maleic anhydride is added to the solution obtained in the above step to crystallize L-aspartic acid. The maleic acid or maleic anhydride to be added may be a powder, a melt, an aqueous solution, or a slurry. Mixing the two acids in any ratio is not subject to any restrictions.

The amount of maleic acid and / or maleic anhydride added in the step is such that the molar ratio of (maleic acid + maleic anhydride) / L-ammonium aspartate is
It is added so as to be 0.5 to 1.4, preferably 0.6 to 1.3. If this molar ratio is too small, the recovery rate of L-aspartic acid in the crystallization recovery will be insufficient, and the aspartic acid concentration in the recycle system will be high. If it is too large, the added maleic acid and maleic anhydride will be Not efficiently used for crystallization.

The crystallization temperature is usually from 0 to 90 ° C, preferably from 10 to 80 ° C. If the temperature is too low, the resulting crystals will be too fine, making the solid-liquid separation operation cumbersome,
Rinse efficiency also deteriorates. That is, since the mother liquor retention amount (water-containing liquid amount) of the wet cake obtained by solid-liquid separation is large and a sufficient rinsing effect cannot be obtained, the crystal purity is lowered or the rinsing amount is increased to increase L-aspartic acid. Will be the situation to reduce the recovery rate of. On the other hand, if the temperature is too high,
Not only is the recovery rate of L-aspartic acid low, but thermal degradation of maleic acid may occur, which is not preferable.

The processing time of the crystallization step is usually 0.5 to 5
It's about time. The crystallization is usually carried out using a stirring tank type crystallization tank. The maleic acid and / or maleic anhydride may be added at any position in the crystallization tank or in the transfer pipe from the step leading to the crystallization tank. In addition, crystallization is performed by continuously supplying the solution obtained in the step, or optionally the solution obtained in the step described below, and maleic acid and / or maleic anhydride, while continuously producing the produced slurry. A continuous type for withdrawing is desirable, but it may be partially performed by an intermittent operation or a batch type. (Step) The slurry obtained in the above step is subjected to solid-liquid separation, and if necessary, the obtained crystals are rinsed with water to recover L-aspartic acid crystals. The obtained crystals can be dried by a conventional method and recovered as a product having a purity of 99% or more.

The mother liquor obtained by the solid-liquid separation contains monoammonium maleate, and further contains L-ammonium aspartate for the solubility. This mother liquor is recycled to the above process and reused as a raw material for the process. Solid-liquid separation of the slurry is not particularly limited, but is in the temperature range of 0 to 80 ° C., preferably 10 to 5
Perform at 0 ° C. At low temperatures, the viscosity of the slurry is high and handling becomes difficult. At high temperatures, the solubility of L-aspartic acid increases, and the recovery rate decreases. The amount of water used for the rinsing operation performed as necessary is not particularly limited, but is performed at 5 times or less, preferably 3 times or less with respect to the wet cake. If the amount of rinsing is too small, the rinsing effect is not sufficient, and if it is too large, the recovery of L-aspartic acid decreases. The temperature of the rinsing water is not particularly limited.

The separating operation is not limited,
For example, it can be performed by a conventional method such as Nutsche or centrifugal separation. The mother liquor obtained by solid-liquid separation is an acidic aqueous solution mainly containing monoammonium maleate and having a pH of about 3-6. If necessary, remove water by a concentration step,
Ammonia can be further added and recycled to the process. In the present invention, it is preferable that the aqueous solution containing maleic acid to be recycled to the step is degassed in advance to reduce the dissolved oxygen concentration before use. The concentration level of dissolved oxygen is 4 ppm or less, preferably 1 ppm or less,
More preferably, the liquid deaerated to 0.5 ppm or less is recycled. Deaeration is not particularly limited as long as it is an operation capable of reducing DO by expelling dissolved oxygen in the recycled liquid.

Examples of the method of expelling dissolved oxygen include a method of heating the recycle liquid under reduced pressure, a method of vibrating by ultrasonic waves, and the like. In the degassing operation, the operation may be carried out while supplying an inert gas to the enzyme reaction. The inert gas is not particularly limited as long as it is a gas containing oxygen. A gas selected from so-called rare gases such as Ar and He, nitrogen gas, carbon dioxide gas, etc. may be used, and one kind of gas or two or more kinds of gas may be combined. These gases are preferably fed into the degassed recycle liquid. On the other hand, by supplying the above inert gas into the tank for storing the recycled liquid, the DO
May be reduced. Preferably, the inert gas is continuously supplied and the gas containing oxygen is continuously discharged. For example, methods such as stirring while blowing the gas into the recycle liquid and sealing the inside of the tank with the gas are performed.

Further, a method using a general evaporator (concentrator) such as a natural circulation type using saturated vapor, a forced circulation type, a thin film centrifugal type, or a method of obtaining a degassed liquid from the bottom by a distillation operation may be used. Absent. These deaeration operations may be carried out under normal pressure or reduced pressure, and are carried out within the range of 10 to 100 ° C, preferably 20 to 70 ° C. In order to perform the degassing operation at a low temperature, the effect cannot be obtained unless the degree of pressure reduction is increased, and the operational restriction becomes large. High temperatures are not preferred because they cause thermal degradation of the liquid composition.

(Step) In the method of the present invention, preferably, the reaction liquid obtained in the step is distilled or stripped before the step is carried out to remove a part of ammonia, whereby ammonium L-aspartate is obtained. Can be substantially an L-aspartic acid monoammonium salt, which can provide an even more advantageous process. The proportion of L-aspartic acid monoammonium salt obtained in this step is 90 mol% or more, preferably 95 mol% or more, and more preferably 98 mol% or more with respect to the entire ammonium salt of L-aspartic acid.

By this deammonification treatment, the amount of maleic acid and / or maleic anhydride used in the step can be suppressed, and a well-balanced industrial process capable of continuous operation for a longer period can be realized. In the process in which the step is carried out as a preferred embodiment, the liquid obtained in the step is sent to the step and subjected to acid precipitation. In that case, the added amount of maleic acid and / or maleic anhydride is (maleic acid). The molar ratio of (acid + maleic anhydride) / L-monoammonium aspartate is 0.5 to 1.1, preferably 0.6 to 1.0. If this molar ratio is too small, the recovery rate of L-aspartic acid in the crystallization recovery will be insufficient, and the aspartic acid concentration in the recycle system will be high, which is not efficient, and if it is too large, the added maleic acid and Maleic anhydride is not efficiently used for crystallization.

Regarding the other crystallization conditions of the step in the process for carrying out the step, the method described in the above step may be used. The distillation operation or stripping operation (hereinafter sometimes referred to as ammonia removal operation) may be carried out under normal pressure or reduced pressure, and can be carried out in the range of 30 to 100 ° C, preferably 40 to 80 ° C. In order to perform the ammonia removing operation at a low temperature, the degree of pressure reduction must be increased, and the operational restrictions are increased.

On the other hand, high temperature is not preferable because it causes thermal deterioration of the liquid composition. From the viewpoint, the temperature conditions of this step, which may result in the highest temperature among all steps in the method of the present invention, especially the upper limit temperature, should be defined as described above from this viewpoint. As the type of the ammonia distillation column, a usual tray column or packed column can be used. The L-ammonium aspartate aqueous solution obtained by the enzymatic reaction of the step is subjected to ammonia removal operation by the above method,
In the distillation still, a residual liquid having a molar ratio of ammonia to L-aspartic acid of about 1.0, that is, containing L-aspartic acid monoammonium can be obtained.

Only ammonia and water are separated as vapor in the ammonia removing operation, and ammonia water can be obtained by recovering this vapor as a liquid by using a cooling pipe or the like. The temperature of the obtained ammonia water is affected by the temperature, pressure, vapor recovery temperature, etc. of the ammonia removal operation. The aqueous solution after the steam operation is sent to the process to recover L-aspartic acid crystals, but the concentration of ammonium L-aspartate in the aqueous solution supplied to the crystallization process is usually 50.
８００800 g / l, preferably 100-500 g / l. If this temperature is too low, the crystal recovery rate will be low, and conversely, if it is too high, the concentration of the recovered slurry will be too high, which is not preferable in operation.

The ammonia water separated as vapor in the ammonia removing operation in the step and recovered in a cooling pipe or the like is reused in the above step or steps as necessary.
It is preferably added to the process, preferably degassed with the recycling liquid of the process, and then recycled to the process. The supply temperature in this case is not particularly limited, but in consideration of the reaction or operating temperature of each step, 5 to 80 ° C.,
Preferably, 10 to 50 ° C can be adopted. At a high temperature, the vapor pressure of ammonia is undesirably high. In addition, there is no problem even if it is supplied at a temperature lower than the temperature reaction.

(Bleed) In the present invention, in order to provide a stable and continuous operation for a long period of time and to provide an efficient new industrial production method of L-aspartic acid, after the step in the recycling process, L from at least one place
Characterized by bleeding a liquid containing ammonium aspartate.

In summary, in the process of the present invention,
Naturally, L-ammonium aspartate produced was present in the reaction liquid of the step, but ammonium L-aspartate which was not crystallized in the process was always present in the recycled mother liquor of the process. Therefore, the bleeding position may be bleeding from any position after each step. It is also possible to bleed at a plurality of locations. Above all, it is preferable to bleed from the aqueous solution supplied for fixation, that is, between the steps or between the steps.

The amount of bleed is usually from 1 to 20%, preferably 3% by volume of the mixture of interest at the bleed position.
~ 17%. If the bleed rate is low, the effect is small and meaningless, and if the bleed rate is high, the bleed system has the same equipment volume as the main process, which gives an economically disadvantageous process. The bleeding method may be continuous or intermittent as long as the bleeding rate is satisfied.

It is preferable to recover L-aspartic acid as crystals from the bleed solution, and it is usually preferable to add an inorganic acid such as sulfuric acid or hydrochloric acid. The addition amount of the inorganic acid is equivalent to or more than the amount of ammonium L-aspartate. That is, it is desirable to add an inorganic acid to the bleed solution so that the isoelectric point of L-aspartic acid becomes 2.8 to improve the recovery rate.

The crystallization method and conditions are the same as those of the main line crystallization described above. For example, the crystallization temperature is 10 to 100 ° C.
Preferably, it is 20 to 80 ° C. The slurry obtained by crystallizing the bleed solution is also subjected to solid-liquid separation in the same manner as in the main line, and then the obtained crystals are rinsed with water and then dried to obtain a product. Therefore, the crystals after solid-liquid separation may be rinsed and then mixed with the crystals in the main line and dried together.

In the present invention, during crystallization of the bleeding liquid, it is preferable that L-malic acid is present in the crystallization system in an amount of 0.5 g / l or more, preferably 2.0 to 50 g / l. In short, in the crystallization of ammonium L-aspartate by the addition of an inorganic acid, the precipitated crystals tend to be fine, but the presence of a specific amount of L-malic acid causes
A columnar crystal with good handleability can be obtained.

As a method for supplying L-malic acid, L-malic acid may be added to the crystallization tank, but it is preferable to add L-malic acid in a necessary amount to the ammonium aspartate aqueous solution. In addition, when an aqueous solution of ammonium aspartate is obtained from an aqueous solution of ammonium fumarate or ammonium maleate, L-malic acid may be by-produced depending on the reaction conditions. The coexistence amount does not reach the above range. However, this by-product L
-The concentration of L-malic acid in the crystallization system may be adjusted by adjusting malic acid or concentrating the by-product L-malic acid.

By the treatment of the bleeding solution described above, the contained L-ammonium aspartate can be recovered without loss, and the impurities accumulated in the system are purged. The present invention, by connecting the respective steps of the ammonia recovery from the process main line and preferably the deammonification process, and preferably the bleed solution treatment process,
It is possible to provide an industrially advantageous process capable of a balanced and stable continuous operation, and to obtain a high-purity L-
It enables the production of aspartic acid.

In the present invention, the processing operations of the above steps (1) to (3) are sequentially carried out, but each of the steps (1) to (3) may be a batch method or a continuous method. For example, even when all the steps are carried out by a batch method, according to the present invention, even if the reaction is repeatedly carried out, each step can be operated under the same processing conditions with the same throughput. Of course, when all the steps are performed by a continuous method, a balanced and stable process results.

[0048]

EXAMPLES The present invention will be described in more detail by way of examples, but the present invention is not limited to the description of the examples as long as the gist thereof is not exceeded. The analysis of L-aspartic acid (hereinafter abbreviated as ASP), maleic acid (hereinafter abbreviated as MA) and fumaric acid (hereinafter abbreviated as FA) was performed by high performance liquid chromatography, and ammonia in ASP crystals was analyzed. Analysis of the content (hereinafter abbreviated as NH ₃ ) was quantified by ion chromatography.

[Reference Example] Preparation of enzyme (1) Cultivation of microorganism having maleate isomerase activity Meat extract: 10 g, peptone: 10 g, NaCl: 5
g, maleic acid 10 g and distilled water: 1000 ml (adjusted to pH 7.0 with caustic soda).
aliquots into an Erlenmeyer flask having a volume of 120 ml and sterilized at 120 ° C. for 20 minutes.
The O.12669 strain was inoculated and cultured with shaking at 30 ° C. for 24 hours. Moreover, 1000 ml of the same medium as above is used for 3
30 ml of the above shaking culture solution was inoculated into a jar fermenter having an L volume and sterilized at 120 ° C. for 20 minutes, and cultured at 30 ° C. for 24 hours. The obtained culture solution was centrifuged (8000 rpm, 15 minutes, 4 ° C.), and the collected bacterial cells were subjected to 0.1 M phosphate buffer (pH 7.0).
, And subjected to the following reaction.

(2) Cultivation of a microorganism capable of producing aspartic acid from fumaric acid Urea: 4 g, (NH ₄ ) ₂ SO ₄ : 14 g, KH ₂ PO
₄ : 0.5 g, K ₂ HPO ₄ : 0.5 g, MgSO _{4.
7H 2 O: 0.5mg, FeSO 4} · 7H 2 O: 20m
g, MnSO ₄ .nH ₂ O: 20 mg, D-biotin:
200 μg, thiamine hydrochloride: 100 μg, yeast extract 1
g, casamino acid 1 g and distilled water: 1000 ml (pH
6.6) 100 ml of medium was dispensed into a 500 ml Erlenmeyer flask and sterilized at 120 ° C. for 15 minutes, and 4 ml of sterilized 50% glucose aqueous solution was added to the mixture, and Brevibacterium flavum AB-41 strain (FERM) was added.
BP-1498) was inoculated and shake-cultured at 33 ° C. for 24 hours.

Further, 1000 ml of the same medium as described above was added to 2
To a L-jar fermenter, which had been sterilized at 120 ° C. for 20 minutes, 20 ml of the above-mentioned shaking culture solution and 200 ml of sterilized 50% glucose aqueous solution were added, and cultured at 33 ° C. for 24 hours. The obtained culture was centrifuged (8000 rpm, 15 minutes, 4 ° C.) to collect bacteria.
In this cell, the contaminating malic acid side-life was removed by the following method. That is, aspartic acid: 100 g, ammonia: 180 ml, calcium chloride: 2.2 g, Twee
n20: Suspend the collected cells in a composition liquid consisting of 0.8 g (total volume is 1 L with water), shake at 45 ° C. for 3 hours, and centrifuge (8000 rpm, 15 minutes, 4 ° C.) to remove the cells. Recovered.

[Example 1] Enzymatic reaction (1) Nitrogen gas was blown for 30 minutes while stirring, and the reaction solution was nitrogen-substituted <maleic acid 175 g, 25% ammonia water 257 ml (water makes the total volume 1000 ml)>
Was transferred to a 3 L jar fermenter, and both bacterial cells (20 g of isomerase bacteria, 120 of aspartase bacteria) were collected.
g) was added, nitrogen gas was supplied at a rate of 0.02 vvm to seal the inside of the tank with nitrogen gas, and the dissolved oxygen concentration was maintained at 0.5 ppm or less during the reaction. When reacted at 30 ° C. for 48 hours, 199 L-aspartic acid was found in the reaction solution.
g / l was obtained.

[Example 2] Recycling operation (A) The enzyme reaction solution obtained by the same method as in Example 1 (1) was sealed with nitrogen gas so as not to reduce DO, and the ultrafiltration membrane ( Asahi Kasei-AVC-3050) was used to remove the cells. The concentrated liquid of the bacterial cells was transferred to a container sealed with nitrogen gas and stored refrigerated at 4 ° C. The enzyme reaction solution from which the bacterial cells had been removed gave 995 ml of which the ASP was 199 g /
l, NH ₃ was the composition of 32.4g / l (NH ₃ / ASP molar ratio 1.27).

(B) Except for 100 ml (0.1 as a bleed rate) of the enzyme reaction solution obtained in (A) above, the rest was heated to 60 ° C. by flowing warm water into a 1000 ml jacketable separable flask. MA 140g (MA / ASP molar ratio 0.9
0) was added and crystallized. After adding MA, keep the temperature at 60 ° C for 30 minutes while stirring, and then at 20 ° C for 1 hour.
Until it was cooled and kept warm for another 30 minutes.

(C) The obtained slurry was subjected to Nutsche solid-liquid separation, further rinsed with 400 g of distilled water, and dried under reduced pressure at about 60 ° C. to obtain 139.3 g of a white solid. The obtained solid had 99.3 wt% ASP and MA
It contained 0.6% by weight of ammonium and 0.1% by weight of FA ammonium. The ASP recovery rate was 78.0%. On the other hand, the mother liquor obtained by solid-liquid separation is ASP 3
4.5g / l, MA122.7g / l, NH 3 25.
8g / l composition, pH about 4.5, volume 1125
It was ml. In addition, substantially all of the obtained ammonium ammonium was a monoammonium salt in view of its NH ₃ balance.

(D) The mother liquor obtained in (C) above is the mother liquor.
1125 ml was depressurized at 80 ° C. (300 to 400 mmH
g) Underneath, water was removed and concentrated. 25% in the resulting concentrate
NH _Three Add about 128 ml of water and distilled water
When 1 L of liquid was prepared, ASP 38.8 g / l,
FA 0.9 g / l, MA 139.8 g / l, NH _Three 
 The composition was 57.5 g / l.

(E) The reaction solution obtained in (D) above was subjected to an enzymatic reaction in the same manner as in Example 1 (1) using the cells refrigerated in (A) above. A in 48 hours
The SP was 198 g / l. (F) Furthermore, the same operation as above was repeated three times without changing the conditions. The results are shown in Table 1.

[0058]

[Table 1]

[0059]

INDUSTRIAL APPLICABILITY According to the present invention, when maleic acid and / or maleic anhydride is used as a raw material to produce L-aspartic acid by an enzymatic method, the components of the raw material can be effectively utilized, It is possible to set up a stable continuous process.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Miyuki Miura 1 Tohocho, Yokkaichi-shi, Mie Mitsubishi Chemical Co., Ltd. Yokkaichi Research Institute

Claims (4)

[Claims] 1. A method for producing L-aspartic acid by carrying out the following steps (1) to (3), wherein a liquid containing ammonium L-aspartic acid is bleeding from at least one location after the step. Manufacturing method. Step: a reaction for isomerizing maleic acid and / or maleic anhydride in the presence of isomerase or a microorganism producing it, and at least a part of the isomerization reaction product and ammonia for aspartase or a microorganism producing it. Step of sequentially or simultaneously carrying out a reaction of reacting in a water solvent in the presence of water to produce ammonium L-aspartate, step: L-aspartic acid by adding maleic acid and / or maleic anhydride to the solution obtained in the step A step of precipitating crystals, a step: a step of recovering the L-aspartic acid crystals obtained in the step and recycling the mother liquor to the step. 2. The method according to claim 1, wherein the amount of bleeding is 1 to 20 wt%. 3. The method according to claim 1, wherein the amount of bleeding is 3 to 17 wt%. 4. The method according to claim 1, wherein the following steps are added between the steps. Step: A deammonification step of removing ammonia from the reaction solution obtained in the step to obtain a solution substantially containing L-monoammonium aspartate.