JPH0884594A - Method for producing l-aspartic acid - Google Patents

Abstract

PURPOSE: To industrially and advantageously obtain the compound useful for food additives, infusion solutions, etc., by treating maleic acid and ammonia with a microorganism having a maleic acid isomerase activity and a microorganism having an aspartase activity in an aqueous solution. CONSTITUTION: The method for efficiently producing the L-aspartic acid in a high yield comprises treating maleic acid and ammonia with a microorganism (e.g. Alcaligenes faecalis IFO 12669 strain) having a maleic acid isomerase activity or its treated product and a microorganism [e.g. Brevibacterium flavum MJ-233-AB-41 strain (FERM BP-1498)] having an aspartase activity or its treated product in an aqueous solution at 30 deg.C for 24hr to produce the L-aspartic acid from the maleic acid and the ammonia, while the fumaric acid produced by the isomerization of the maleic acid in the reaction solution is maintained in a concentration of <=0.5wt./vol.%, and subsequently collecting the product from the reaction solution.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for efficiently producing L-aspartic acid from maleic acid.

[0002]

2. Description of the Related Art L-Aspartic acid is produced mainly from fumaric acid and ammonia by an enzymatic method and is widely used for food additives, infusions and the like. As a method for producing aspartic acid from fumaric acid by an enzymatic method, a method using Escherichia coli [Kitahara et al .; Amino Acids Fermentation and metabolism; 1
Vol., P102 (1959)], a method using Pseudomonas bacteria [Takahashi et al .; Amino Acids Fermentation and metabolism; Vol. 7, p23 (1963), US Pat. No. 3,31.
0,475], a method using a bacterium of the genus Brevibacterium [Watanabe et al .; Journal of the Japanese Society of Agricultural Chemistry; 38, p434 (1).
964) and Japanese Examined Patent Publication No. 61-29718].

Maleic acid is cheaper than fumaric acid and is more advantageous as a raw material for aspartic acid. As a method for producing aspartic acid from maleic acid, a method for obtaining aspartic acid by using a microbial cell that produces aspartic acid from maleic acid (Japanese Patent Publication Nos. 42-11993, 11994, 11994, 11996, US Pat. Patent No. 3,391,059) is known, but it cannot be said that the yield of aspartic acid with respect to the raw material and the production rate are sufficient. The molar yield based on the theoretical yield of L-aspartic acid described in US Pat. No. 3,391,059 is 92%.

Further, as a method of producing L-aspartic acid from maleic acid, a method of isomerizing maleic acid with a Br catalyst and allowing the aspartic acid-producing microorganism to act on fumaric acid produced (US Pat. No. 2,955,136) No. 3,332,992), but in general, the isomerization with a chemical catalyst has a problem that the yield is not sufficient and impurities are likely to be generated because heating is accompanied during the isomerization. .

[0005]

DISCLOSURE OF THE INVENTION The present invention has been made for the purpose of providing a method for producing L-aspartic acid with high yield and efficiency from a new viewpoint.

[0006]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to establish a method for efficiently producing L-aspartic acid, and as a result, a microorganism having maleate isomerase activity or a treated product thereof, It was found that L-aspartic acid is efficiently produced in high yield by mixing a microorganism having aspartase activity or a treated product thereof in an aqueous solution and simultaneously performing two kinds of enzyme reactions, and further, maleic acid. It was found that the yield and efficiency can be further improved by maintaining the concentration of fumaric acid in the reaction solution produced by isomerization of 0.5% (wt / vol) or less, and completed the present invention. .

Thus, according to the present invention, a microorganism having maleate isomerase activity or a treated product thereof, a microorganism having aspartase activity or a treated product thereof, maleic acid and ammonia are mixed in an aqueous solution to obtain maleic acid. And L-aspartic acid is produced from ammonia by an enzymatic reaction, and L-aspartic acid is collected from the reaction solution.

Further, as a preferred embodiment of the present invention, in the above method, the concentration of fumaric acid in the reaction solution produced by the isomerization of maleic acid is maintained at 0.5% (wt / vol) or less. A method is provided.

The present invention will be described in detail below. Among the microorganisms used in the present invention, the microorganism having maleate isomerase activity is not particularly limited as long as it is a microorganism having the ability to isomerize maleic acid to produce fumaric acid, for example, maleic acid isomerase activity. Examples of microorganisms belonging to the genera Alcaligenes, Pseudomonas, and Xanthomonas. Specifically, Alcaligenes fae
calis) IFO 12669, IFO 1311
1, the same IAM 1473, Alcaligenes eutrophus,
Pseudomonas fluorescens (Pseudomo
NAS Fluorescens) ATCC 2372
8. Xanthomonas maltophilia
onas maltophilia) ATCC 132
70 and the like can be illustrated.

The microorganism having aspartase activity is not particularly limited as long as it is a microorganism capable of producing L-aspartic acid from fumaric acid and ammonia.
Examples include microorganisms having aspartase activity, such as Brevibacterium, Escherichia, Pseudomonas, and Bacillus. Specifically, Brevibacterium flavum (Brevibacterium fl)
avum) MJ-233 (FERM BP-149
7), the same MJ-233-AB-41 (FERM BP-
1498), Brevibacterium ammoniagenes
ATCC 6872, Escherichia coli (Esche
richia coli) ATCC 11303, same A
TCC 27325 etc. can be illustrated.

Cultivation of a microorganism having maleate isomerase activity can be carried out in a medium known per se which is generally used and to which a natural nutrient source such as meat extract, yeast extract or peptone has been added. In the medium, if necessary, as a nitrogen source, ammonium salts such as ammonium chloride, ammonium sulfate and ammonium phosphate, sodium nitrate,
It is possible to add nitrates such as potassium nitrate and ammonium nitrate, ammonia, and potassium phosphate, magnesium sulfate, iron, manganese, zinc, copper and the like as inorganic substances.

Further, in order to increase the maleate isomerase activity, it is desirable to add maleic acid or malonic acid to the medium. The added concentration of maleic acid and malonic acid is appropriately 10 to 200 mM, preferably 50 to 100 mM.

Cultivation of microorganisms having aspartase activity is likewise commonly used per se known meat extract,
It can be performed using a medium to which a natural nutrient source such as yeast extract or peptone is added. Glucose or ethanol can be added as a carbon source. In the medium, as necessary, ammonium salts such as ammonium chloride, ammonium sulfate and ammonium phosphate, nitrates such as sodium nitrate, potassium nitrate and ammonium nitrate, ammonia as a nitrogen source; potassium phosphate, magnesium sulfate and iron as inorganic substances; Manganese, zinc, copper, etc. can be added.

Cultivation of the above-mentioned microorganism having maleate isomerase activity and microorganism having aspartase activity is carried out under aerobic conditions such as aeration and stirring, and the culture temperature is 25 to 40 ° C., preferably 28 to 35 ° C. Is appropriate. The pH during the culture can be around 6 to 9, and the pH during the culture can be adjusted by adding an acid or an alkali.

The method of the present invention comprises a microorganism having maleate isomerase activity or a treated product thereof obtained by culturing two types of microorganisms as described above, a microorganism having an aspartase activity or a treated product thereof, and malein. This is a method in which an acid and ammonia are mixed in an aqueous solution, L-aspartic acid is produced from maleic acid and ammonia by an enzymatic reaction, and L-aspartic acid is collected from this reaction solution.

The bacterial cells are washed bacterial cells as they are recovered from the culture or washed with an appropriate buffer solution, for example, a phosphate buffer solution (pH 6-9) of about 0.05 to 0.2 M. Good. Furthermore, the culture of the above-mentioned microorganism and the enzymatic reaction can be carried out in parallel.

The above-mentioned "treated product" means an immobilized product obtained by immobilizing a culture or a microbial cell recovered therefrom, a pulverized product obtained by pulverizing the microbial cell by a means such as ultrasonic waves or rubbing, and the pulverized product. An extract obtained by extracting the extract with water or the like, an enzyme component obtained by further treating the extract with ammonium sulfate salting out, column chromatography, or the like, and an immobilized product obtained by immobilizing the pulverized product, extract, enzyme component, or the like. It is meant. Immobilization of bacterial cells, etc., according to a commonly used method known per se,
It can be carried out by a method of immobilizing on an appropriate carrier such as acrylamide monomer, alginic acid or carrageenan.

Examples of the method for allowing a microorganism having maleate isomerase activity or a treated product thereof and a microorganism having aspartase activity or a treated product thereof to act on an aqueous solution containing maleic acid and ammonia include a fermentation method and an enzymatic method. To be Here, the "fermentation method" means that at least, in an aqueous medium containing a component in which the microorganism to be used is proliferative, while the microorganism is proliferating under conditions (temperature, pH) in which the microorganism can be proliferated, This is the method to generate. The "enzymatic method" means that the microorganism used is cultivated by an appropriate culturing method, and the obtained bacterial cell or a treated product thereof is used to produce a target substance in an aqueous solution that does not necessarily contain components necessary for growth. It is a method to let.

In the case of the fermentation method, the above-mentioned culture or cells are used as the microorganism having maleate isomerase activity and the microorganism having aspartase activity, and a medium obtained by adding maleic acid and ammonia to the medium is used as an aqueous solution. Used.

The concentration of maleic acid and ammonia in the medium is not particularly limited as long as the two types of microorganisms can produce L-aspartic acid, but the maleic acid concentration is usually 1 to 40% (wt / vol). ), Preferably 5-2
In the range of 0% (wt / vol), the ammonia concentration is usually 0.2 to 8M, preferably 1 to 4M. Maleic acid and ammonia can be added to the medium all at once or sequentially. Further, in the continuous reaction, the maleic acid concentration may be maintained at about 0.01 to 1% for the reaction.

The culture temperature is 25 to 40 ° C., preferably 2
A temperature of 8 to 35 ° C. is suitable, and the pH of the medium during culture is 6 to 9
The pH can be adjusted to the vicinity, and the pH can be adjusted by adding an acid or an alkali. Culturing can be normally performed for about 10 to about 72 hours under aerobic conditions such as aeration and stirring.

By culturing as described above, a large amount of L-aspartic acid can be produced and accumulated in the medium. In the case of the enzymatic method, as a microorganism having maleate isomerase activity or a treated product thereof and a microorganism having aspartase activity or a treated product thereof, the bacterial cells or a treated product thereof is used, and as an aqueous solution, at least maleic acid and An aqueous solution containing ammonia or a suitable buffer solution, for example, a phosphate buffer solution of about 0.05 to 0.2 M is used.

The amount of the bacterial cells or treated product thereof prepared as described above is not particularly limited, but is 0.5 to 30% (wt / vo) each based on the volume of the aqueous solution.
l) is suitable.

The concentration of maleic acid and ammonia in the aqueous solution is not particularly limited as long as maleic acid and ammonia can be converted by an enzymatic reaction to produce L-aspartic acid, but the maleic acid concentration is usually 1 to. 40
% (Wt / vol), preferably 5 to 20% (wt / v)
ol), the ammonia concentration is usually 0.2 to 8 M,
A range of 1 to 4M is suitable. Maleic acid and ammonia can be added all at once or sequentially to the aqueous solution.

If desired, a divalent metal salt such as a calcium salt, a magnesium salt or a manganese salt can be added to the aqueous solution. The enzyme reaction temperature in the above-mentioned aqueous solution is usually in the range of 20 to 50 ° C., preferably 25 to 37 ° C., and the pH of the aqueous solution during the reaction is 6 to 1
The pH can be adjusted to 0, preferably 7 to 9, and the pH can be adjusted by adding an acid or an alkali to the aqueous solution. The enzymatic reaction can be carried out usually under aerobic conditions such as aeration and stirring and shaking for 10 to 72 hours.

By carrying out the enzymatic reaction as described above, a considerable amount of L-aspartic acid can be produced and accumulated in the aqueous solution. In the present invention, the isomerization reaction of maleic acid to fumaric acid and the reaction of producing fumaric acid and ammonia to form L-aspartic acid proceed in parallel, so that the fumaric acid concentration in the reaction solution is within a certain range. And L-aspartic acid is efficiently produced. Particularly, the concentration of fumaric acid in the reaction solution is 0.5% (wt / v
ol) It is preferable to keep below. The concentration of fumaric acid is 0.5% for most of the time during the reaction.
It is only necessary to maintain the concentration below (wt / vol), and it is possible to exceed this concentration momentarily. In order to maintain the fumaric acid concentration at 0.5% (wt / vol) or less, the activity of aspartase should be kept relatively low with respect to the activity of maleate isomerase. Examples of the method include maintaining on the side, increasing the ammonia concentration, and excessively adding a microorganism having an aspartase activity to a microorganism having a maleate isomerase activity.

After L-aspartic acid is produced and accumulated in the aqueous solution as described above, L-aspartic acid can be collected from the aqueous solution by a commonly used separation and purification method known per se. For example, L-aspartic acid can be collected as crystals by precipitating and separating by a method such as sulfuric acid isoelectric precipitation, washing with water and drying.

[0028]

EXAMPLES Next, the present invention will be described more specifically by way of examples. However, the following examples are given only as an aid to gain a specific recognition of the present invention,
This in no way limits the scope of the invention.

Example 1 (1) Cultivation of Microorganism Having Maleate Isomerase Activity 10 g of meat extract, 10 g of peptone, 5 g of NaCl, 10 g of maleic acid and 1000 ml of distilled water (adjusted to pH 7.0 with caustic soda) were added to 100 ml of a medium. The mixture was dispensed into a 500 ml Erlenmeyer flask and sterilized at 120 ° C. for 20 minutes. In the medium, Alcaligenes faecalis IFO 1
The 2669 strain was inoculated and cultured with shaking at 30 ° C. for 24 hours.

In addition, 1500 ml of the same medium as above was used for 3 times.
The mixture was placed in an L-volume jar fermenter and sterilized at 120 ° C. for 20 minutes, and 30 ml of the shaking culture solution was inoculated, and this was 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 washed once with 0.1 M phosphate buffer (pH 7.0) and subjected to the following reaction. did.

(2) Cultivation of microorganism having aspartase activity 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.
_{5g, FeSO 4 · 7H 2 O} 20mg, MnSO 4 · nH
20 mg of ₂ O, 200 μg of D-biotin, 100 μg of thiamine hydrochloride, 1 g of yeast extract and 1 g of casamino acid, and distilled water was added to make a total volume of 1000 ml (pH 6.
6) 100 ml was dispensed into a 500 ml Erlenmeyer flask and sterilized at 120 ° C. for 15 minutes. To this medium was added 4 ml of a sterilized 50% glucose aqueous solution, and Brevibacterium flavum MJ-233-AB-41 strain (F
ERM 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 ml.
20 ml of the shaking culture solution and 200 ml of sterilized 50% glucose aqueous solution were added to the sterilized material at 120 ° C. for 20 minutes in an L jar fermenter, and the mixture was cultured at 33 ° C. for 24 hours. The obtained culture solution was centrifuged (8000 rpm, 15 minutes, 4 ° C.) to collect the cells. Next, 100 g of aspartic acid, 180 ml of ammonia,
2.2 g of calcium chloride and polyoxyethylene sorbitan monolaurate (trade name: Tween 20) 0.8
Water was added to g to make the total volume 1000 ml, and the collected bacterial cells were suspended, shaken at 45 ° C for 3 hours, and then centrifuged (8000 rpm, 15 minutes, 4 ° C) to remove the bacterial cells. By collecting, the by-product of malic acid contaminating the bacterial cells was removed.

(3) L- from maleic acid and ammonia
Production of aspartic acid Both bacterial cells (IFO 1 recovered in the above (1) and (2)
2669 strain 40 g, MJ-233-AB-41 strain 1
20 g) was added to the reaction solution [an aqueous solution (pH 8) in which water was added to 100 g of maleic acid and 132 ml of 25% ammonia water to make the total amount 500 ml], and the mixture was reacted at 30 ° C. for 24 hours. At this time, the concentration of fumaric acid in the reaction solution was maintained at approximately 0.2% (wt / vol).

The amount of L-aspartic acid in the reaction solution was quantified by high performance liquid chromatography. As a result, 100 g of maleic acid was used to give L-aspartic acid at a molar yield of 99% or more of the theoretical yield.
Aspartic acid was obtained. After the reaction, sulfuric acid was added to ammonium L-aspartate to precipitate aspartic acid, which was washed with water and dried to obtain L-aspartic acid crystals. The amount of crystals obtained was 112 g. [Reference Example 1] (1) Cultivation of a microorganism having maleic acid isomerization ability In the same manner as in Example 1, Alcaligenes faecalis I.
The FO 12669 strain was cultured to obtain bacterial cells.

(2) Cultivation of microorganism having aspartase activity 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.
_{5g, FeSO 4 · 7H 2 O} 20mg, MnSO 4 · nH
20 mg of ₂ O, 200 μg of D-biotin, 100 μg of thiamine hydrochloride, 1 g of yeast extract and 1 g of casamino acid, and distilled water was added to make a total volume of 1000 ml (pH 6.
6) 100 ml was dispensed into a 500 ml Erlenmeyer flask and sterilized at 120 ° C. for 15 minutes. To this medium was added 4 ml of a sterilized 50% glucose aqueous solution, and Brevibacterium flavum MJ-233-AB-41 strain (F
ERM 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 ml.
20 ml of the above shaking culture solution and 100 ml of sterilized 50% aqueous glucose solution were added to the sterilized material at 120 ° C. for 20 minutes in an L jar fermenter, and this was cultured at 33 ° C. for 24 hours. The obtained culture solution was centrifuged (8000 rpm, 15 minutes, 4 ° C.) to collect the cells. Next, 100 g of aspartic acid, 180 ml of ammonia,
2.2 g of calcium chloride and polyoxyethylene sorbitan monolaurate (trade name: Tween 20) 0.8
Water was added to g to make the total volume 1000 ml, and the collected bacterial cells were suspended, shaken at 45 ° C for 3 hours, and then centrifuged (8000 rpm, 15 minutes, 4 ° C) to remove the bacterial cells. By collecting, malic acid by-productivity contaminating the bacterial cells was removed.

(3) L- from maleic acid and ammonia
Production of aspartic acid Both bacterial cells (IFO 1 recovered in the above (1) and (2)
2669 strain 40 g, MJ-233-AB-41 strain 6
0 g) was added to the reaction solution [an aqueous solution (pH 8) in which water was added to 100 g of maleic acid and 132 ml of 25% ammonia water to make the total amount 500 ml], and the mixture was reacted at 30 ° C. for 24 hours. At this time, the concentration of fumaric acid in the reaction solution was maintained at about 0.6% (wt / vol).

The amount of L-aspartic acid in the reaction solution was quantified by high performance liquid chromatography. As a result, L-aspartic acid was obtained from 100 g of maleic acid in a molar yield of 95% of the theoretical yield. After the reaction, sulfuric acid was added to ammonium L-aspartate to precipitate aspartic acid, which was washed with water and dried to obtain L-aspartic acid crystals.
The crystals obtained were 105 g.

[0039]

According to the method of the present invention, L-aspartic acid can be efficiently produced from maleic acid and ammonia in a high yield.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Masato Terasawa 8-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Prefecture Tsukuba Research Center, Mitsubishi Chemical Co., Ltd. (72) Hideaki Yukawa 8-cho, Ami-cho, Inashiki-gun, Ibaraki Prefecture 3-1 Mitsubishi Chemical Co., Ltd. Tsukuba Research Center

Claims (2)

[Claims] 1. A microorganism having maleate isomerase activity or a treated product thereof, a microorganism having aspartase activity or a treated product thereof, and maleic acid and ammonia are mixed in an aqueous solution, and an enzymatic reaction is performed from maleic acid and ammonia. L-aspartic acid is produced by the method described above, and L-aspartic acid is collected from this reaction solution. 2. The fumaric acid concentration in the reaction solution produced by isomerization of maleic acid according to claim 1, is 0.5% (w).
t / vol) or less, the manufacturing method of L-aspartic acid characterized by maintaining below.