JP3204924B2 - Method for producing L-aspartic acid and fumaric acid and / or L-malic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a microorganism having a long active half-life, which stably and efficiently produces L-aspartic acid from maleic acid and ammonia and fumaric acid and / or L-malic acid from maleic acid by the same microorganism. On how to do it.

[0002]

BACKGROUND ART Microorganisms that produce L-aspartic acid from maleic acid and ammonia include those belonging to the genus Alcaligenes,
Microorganisms belonging to the genera Pseudomonas, Achromobacter, Aerobacterium, Bacillus, and Plevibacterium are known, and some of these microorganisms have already been applied for patents (Japanese Patent Publication No. 38-2793). Otsuka Kenichi et al.), Japanese Patent Publication No. 42-11993 (Sanraku Ocean), Japanese Patent Publication No. 42-11994 (Sanraku Ocean), Japanese Patent Publication No. 43-8710
No. [Sanraku Ocean]).

When ammonium maleate is converted to L-aspartic acid, two types of enzymes, maleate isomerase and aspartase, are involved. Aspartase is relatively stable, while maleate is relatively stable. Acid isomerases are unstable, and have a characteristic that their activity tends to be reduced particularly by heat. In addition, the stability of this maleate isomerase varies depending on the strain, and when the cells are used in an immobilized state, the activity half-life is short and a satisfactory production amount is not obtained at present. .

[0004]

An object of the present invention is to provide a method for producing stable and efficient L-aspartic acid, fumaric acid and / or L-malic acid by microorganisms.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies on microorganisms that produce L-aspartic acid and found that Pseudomonas fluorescens (Pseudomonas fluosens).
orescens) NSM-4 strain, Citrobacter amalanaticus NSM-10 strain, etc., have a longer maleic acid isomerase half-life than known strains and have stable L-aspartic acid-producing ability, The present invention has been completed. That is, the present invention provides a method for treating maleic acid and ammonia with a microbial cell having a maleic acid isomerase activity and a long half-life of the activity, a treated microbial cell, or an immobilized product thereof. A method for producing L-aspartic acid, which comprises producing aspartic acid.

Further, the present invention provides a method of treating maleic acid with a microbial cell having a maleic acid isomerase activity and a long half-life of the activity, a treated product of the microorganism, or an immobilized product thereof to obtain fumaric acid. And / or L-malic acid. A process for producing fumaric acid and / or L-malic acid. And, as the above microorganism, Pseudomonas fluorescens
NSM-4 strain or Citrobacter amalanaticus NSM-10 strain and the like.

[0007] Furthermore, the present invention relates to a microorganism Pseudomonas fluorescens (Pseudomonas fluosens) capable of producing L-aspartic acid from maleic acid and ammonia, or fumaric acid and / or L-malic acid from maleic acid.
orescens) NSM-4 strain itself or Citrobacter amalanaticus NSM-1
0 shares per se. Hereinafter, the present invention will be described in detail. The present inventors have determined that NSM-4 strain and N
The SM-10 strain was isolated. The mycological properties of the NSM-4 and NSM-10 strains are as follows.

[0008]

[0009]

With respect to the above mycological properties,
Manual of Systematic Bacteriology
vol.1-4 (1986)
The M-4 strain is a Pseudomonas fluus from the viewpoint of gram negative, motility +, OF test-, oxidase +, assimilation of sugar, assimilation of organic acid, arginine dehydrolase +, production of fluorescent dye, and the like. It turned out to belong to Olesens.
In addition, NSM-10 strain is gram negative, motility +, OF
Test +, oxidase - catalase +, assimilating citric acid +, lysine decarboxylase -, VP test -, indole product +, assimilation of malonic acid -, generation of H ₂ S -, assimilation of sugars For this reason, it turned out to belong to Citrobacter Amaronaticas. The NSM-4 strain and the NSM-10 strain were transferred to FERM P-15560 (Deposited on April 12, 1996) by the National Institute of Advanced Industrial Science and Technology.
No.) and FERM P-16142 (deposit date: March 19, 1997).

The microorganism used in the present invention can be cultured by the same method as known microorganisms belonging to the genus Pseudomonas and the like. As the medium, any synthetic medium or natural medium can be used as long as the medium appropriately contains assimilable carbon sources, nitrogen sources, inorganic substances, and necessary growth promoting substances. Organic acids such as maleic acid, fumaric acid, malic acid, glucose, molasses, etc. can be used as the carbon source, and ammonia, ammonium sulfate, peptone, yeast extract, corn steep liquor, etc. are used as the nitrogen source. Can be. In addition, if necessary, an enzyme-inducing substance such as malonic acid such as a phosphate, magnesium sulfate, a trace amount of an inorganic salt such as a heavy metal salt or the like can be added. Since microorganisms cultured in a medium containing malonic acid have higher maleic acid isomerization activity than microorganisms cultured in a medium containing no malonic acid,
It is preferred that malonic acid be added to the medium for culture growth. The culture temperature is preferably 25 to 40 ° C., and the pH of the medium is 5 to 5 with a mineral acid such as hydrochloric acid or an organic acid such as maleic acid.
It is preferable to maintain at 9. When culturing is performed under such conditions, a sufficient amount of microorganisms can be obtained within 6 to 48 hours from the start of culturing.

[0012] The microorganism cells obtained as described above,
L-aspartic acid is produced by treating maleic acid and ammonia with the treated cells and their immobilized products. Alternatively, fumaric acid and / or L-malic acid is produced by treating maleic acid with the same cells, treated cells, or their immobilized products in the absence of ammonia. In the present invention, "treating maleic acid" means a substance derived from maleic acid, for example, treating maleic acid ions, salts containing maleic acid, and the like.
Similarly, “treat ammonia” means treating a substance derived from ammonia. Therefore, the case where ammonium maleate is added to the medium for treatment is also included in the scope of the present invention.

In the present invention, the term "microbial cells" refers to the cells themselves which are obtained by culturing the microorganisms in a culture medium and recovered from the culture, and the treated cells are referred to as crushed cells or cultured cells. (Including cells and culture supernatant). The term "immobilized product" refers to a product obtained by immobilizing the cells or the treated cells on a carrier or the like. Examples of the method for producing L-aspartic acid, fumaric acid, and / or L-malic acid using the cells of a microorganism or a treated product of the cells include the following methods. First, the microorganism of the present invention is cultured in a medium, and the cultured cells are collected from the culture.

The following method can be exemplified as a treatment method using microbial cells or processed cells. First, the microorganism of the present invention is cultured in a medium, and the cells are collected from the culture. Since the culture here is for the purpose of growing microorganisms, the above-described medium is used,
Culture is performed at the above-described temperature, pH, and the like.

Next, the recovered cells or processed cells are
When producing L-aspartic acid, it is inoculated and cultured in a medium containing maleic acid and ammonia. The medium used here is prepared by adjusting the concentration of maleic acid to 0.5 to 5% by weight, the concentration of ammonia to 0.1 to 1.5% by weight, and adding phosphate, magnesium sulfate, yeast extract and the like. After culturing in such a medium for about 6 to 24 hours, maleic acid is added to the medium so that the concentration becomes 5 to 30% and ammonia is 1.5 to 10%. Aspartic acid accumulates. The method for collecting L-aspartic acid from the culture is not particularly limited, but is preferably a precipitation method in the present invention. That is, the culture is separated into cells and supernatant by centrifugation or the like, L-aspartic acid is precipitated as crystals by adding a large amount of water-soluble solute to the supernatant, and this is filtered by suction filtration or the like. Collect. As a solute soluble in water, it is preferable to use maleic acid. This is because, when maleic acid is used, after collecting L-aspartic acid, the culture supernatant can be used again as a medium for producing L-aspartic acid of the microorganism. In addition to maleic acid, a mineral acid such as sulfuric acid can be used as a solute.

On the other hand, when producing fumaric acid and / or L-malic acid, the above collected cells are inoculated into a medium containing maleic acid and cultured. An appropriate concentration of maleic acid in the medium is 5 to 30%. The medium components other than maleic acid may be the same as the above-described medium used for the growth of microorganisms in principle, but if ammonia is present in the medium, L-aspartic acid is generated, so ammonium sulfate or the like is used as the nitrogen source. When adding an alkaline substance to the medium to correct the pH lowered by maleic acid, an alkali metal salt such as sodium hydroxide or an alkaline earth metal salt is used instead of ammonia. The culture temperature is preferably 5 to 40 ° C, and the pH is preferably 5 to 9. After culturing for a predetermined time under the above conditions, fumaric acid and L-malic acid are recovered from the culture.
Since maleic acid changes to L-malic acid via fumaric acid, the ratio of L-malic acid increases if the culture time is long, and the ratio of fumaric acid increases if the culture time is short. Therefore, if you mainly want to obtain fumaric acid,
The culturing time is shortened, and the culturing time is increased if L-malic acid is mainly intended to be obtained. The method for recovering fumaric acid or L-malic acid from the culture is not particularly limited. Fumaric acid can be recovered, for example, by the following method. The culture is separated into cells and supernatant by centrifugation, a large amount of water-soluble solute is added to the supernatant, fumaric acid is precipitated,
This is collected by suction filtration or the like. As a solute soluble in water, a mineral acid such as sulfuric acid or maleic acid is preferably used. When maleic acid is used, there is an advantage that after collecting fumaric acid, the culture supernatant can be used again as a medium for producing fumaric acid and malic acid of a microorganism.

Further, L-malic acid can be recovered by the following method. The culture is separated into cells and supernatant by centrifugation, and fumaric acid is precipitated by adding a mineral acid to the supernatant to make it acidic. After concentrating the filtrate from which fumaric acid has been removed by suction filtration, malic acid is extracted by adding a solvent capable of dissolving malic acid well, and malic acid can be obtained by concentrating the solvent phase. It is preferable to use inexpensive sulfuric acid as the mineral acid. As the solvent used for the extraction, ether or the like is suitably used. Further, a purification method using an ion exchange resin instead of the extraction can also be adopted.

In the case of producing L-aspartic acid, the cells obtained by culturing the cells in the same manner as described above, or a processed product of the cells, are mixed with a solution containing maleic acid and ammonia to obtain maleic acid and ammonia. It may be reacted with ammonia.
Preferably, the concentration of maleic acid is 5 to 30% and the concentration of ammonia is 1.5 to 10%. The reaction time is 2
It is preferably set to 120 hours. The method of recovering L-aspartic acid from the reaction solution can be performed in the same manner as described above.

On the other hand, when producing fumaric acid and / or L-malic acid, the obtained cultured cells or the treated cells may be mixed with a maleic acid solution and reacted with maleic acid. The concentration of maleic acid is preferably 5 to 30%.
This reaction converts maleic acid to fumaric acid or malic acid. The reaction time is shortened when mainly obtaining fumaric acid and long when mainly obtaining L-malic acid, as in the case of treatment using bacterial cells. The method for recovering fumaric acid and L-malic acid from the reaction solution can be performed in the same manner as described above.

On the other hand, as a production method using the immobilized product, the following method can be exemplified. First,
The microorganisms are immobilized by entrapping in a gel, or an enzyme-containing substance prepared from the microorganisms is supported on an ion exchanger to obtain an immobilized product. Here, as the gel for entrapping and immobilizing microorganisms, for example, carrageenan gel, agar gel, mannan gel, P
VA gel, polyacrylamide gel and the like can be mentioned. The size of the gel sphere depends on the type of gel,
A diameter of about 1 to 10 mm is appropriate. In addition, examples of the ion exchanger supporting the enzyme-containing substance include a cellulose-based, styrene-divinylbenzene-based, and phenol-formalin-based ion exchanger. next,
A column containing the above-mentioned immobilized substance and L-aspartic acid is used to prepare a solution containing maleic acid and ammonia, while fumaric acid and / or L-malic acid is used to prepare maleic acid. The solution containing is circulated.
Preferably, the maleic acid concentration in the solution is 5 to 30% by weight and the ammonia concentration is 1.5 to 10% by weight. In addition, an SH compound such as mercaptoethanol, cysteine and glutathione, a reducing agent such as a sulfite, and an enzyme activator such as a magnesium ion and a manganese ion can also be added to the solution. The speed of the solution flowing through the column varies depending on the size of the column and the amount of the immobilized substance, but an SV of about 0.05 to 10 is appropriate. The solution that has passed through the column contains a large amount of L-aspartic acid or fumaric acid and / or L-malic acid due to the action of microorganisms or enzymes. The method of collecting L-aspartic acid or fumaric acid and / or L-malic acid from the solution can be performed in the same manner as the above-described method using the cells.

[0021]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. Note that the present invention is not limited to the embodiments. Example 1 A 3 L medium having the composition shown in Table 1 was charged into a 5 L jar fermenter, and Pseudomonas fluorescens NSM-4 strain (FERM) was used.
P-15560) and inoculated at 30 ° C. with aeration and stirring. culture
After 20 hours, the culture was terminated, and the cells were collected by centrifugation. The cells are polycap 172 (Hercules)
It was dispersed uniformly in 60 g and 120 g of deionized water. Ion exchange resin (Amberlite IRA-94S)
CI Type Organo) 150ml and 0.5 inch Teflon bulb 10
0 cells were put therein, 1/6 of the above-mentioned dispersion liquid was added thereto, and the mixture was dried under reduced pressure with an evaporator for 1 hour while rotating at 30 ° C., and the cells were coated with an ion exchange resin. Do this six times,
Teflon spheres were removed to obtain an immobilized product.

The immobilized product thus prepared was immersed in a 20% ammonium maleate solution (pH 8.5) at 4 ° C. overnight, and 100 ml thereof was packed in a jacketed column.
Circulate warm water of 30 ° C through the jacket to reduce the reactor temperature to 30 ° C.
Set to ° C. The substrate solution having the composition shown in Table 2 placed in the bottle with a lid was passed through the column at a rate of 20 ml / h through a Teflon tube to perform a continuous reaction. When the reaction solution was analyzed 12 hours after the start of the reaction, L-aspartic acid was produced as a reaction product in an equimolar amount with the consumed maleic acid.
When the conversion rate became 90% or less, the conversion rate was analyzed by HPLC, and the time change of the conversion rate was tracked. The half life of the enzyme activity for converting maleic acid to L-aspartic acid was measured from the slope of the logarithmic value of the conversion rate plotted against the reaction time, and was 30 hours (Table 3).

[0023]

[Table 1]

[0024]

[Table 2]

[0025]

[Table 3]

Comparative Example 1 The microorganism used was Alcaligenes faecalis IFO13111.
The activity half-life was calculated in the same manner as in Example 1 except that the strain was changed to 14 hours.

[Comparative Example 2] The microorganism used was Enterobacter agglomerans (Enterobacter).
agglomerans) NSM-1 strain (FERM P-144)
The active half-life was calculated in the same manner as in Example 1 except for changing to (47), and it was 19 hours.

Example 2 An immobilized product prepared in the same manner as in Example 1 was placed in a 5% sodium maleate solution (pH 8.3).
After immersion at 4 ° C. overnight, 100 ml of the solution was packed in a jacketed column, and warm water of 30 ° C. was circulated through the jacket to set the temperature of the reactor at 30 ° C. A substrate solution having the composition shown in Table 4 in a bottle with a lid was passed through a Teflon tube at a rate of 20 hours / hour.
The mixture was passed through the column at a rate of ml to perform a continuous reaction. When the reaction solution was analyzed 12 hours after the start of the reaction, maleic acid had disappeared, L-malic acid was 65 mol%, fumaric acid 35
It was formed in a yield of mol%.

[0029]

[Table 4]

Example 3 Medium 3 having the composition shown in Table 1
L into a 5L jar fermenter, Citrobacter amalanatic
us) NSM-10 strain was inoculated and aerated and stirred at 30 ° C. Twenty hours after the culturing, the culturing was terminated, and the cells were collected by centrifugation. The cells were uniformly dispersed in 60 g of Polycap 172 (manufactured by Hercules) and 120 g of deionized water. 150 ml of ion exchange resin (Amberlite IRA-94S) was added to a 3 L eggplant type flask.
Insert 100 5 inch Teflon balls and put 1 /
6 and rotate it at 30 ° C.
After drying under reduced pressure for a time, the cells were coated with an ion exchange resin.
This operation was performed six times, and Teflon spheres were removed to obtain an immobilized product.

[0031] The immobilized material thus produced is reduced to 20%.
After immersion in an ammonium maleate solution (pH 8.5) at 4 ° C. overnight, 100 ml of the solution was packed in a jacketed column, and warm water at 30 ° C. was circulated through the jacket to set the temperature of the reactor at 30 ° C. The substrate solution having the composition shown in Table 2 placed in a bottle with a lid was passed through a Teflon tube at an hourly rate of 2.
The mixture was passed through the column at a rate of 0 ml to perform a continuous reaction. When the reaction solution was analyzed 12 hours after the start of the reaction, L-aspartic acid in an amount equal to that of the consumed maleic acid was produced as a reaction product. When the conversion rate became 90% or less, the conversion rate was analyzed by HPLC, and the time change of the conversion rate was tracked. The half-life of the enzyme activity for converting maleic acid to L-aspartic acid was measured from the slope of the logarithmic value of the conversion rate plotted against the reaction time.
Approximately 42 hours.

[0032]

Table 5 Calculation of conversion and half-life of column reaction Reaction time (hour) 12 17 40 66 Maleic acid conversion (%) 100 81.9 63.5 36.9 Logarithm of conversion 1.91 1.80 1.57 Slope of logarithm of conversion rate plotted against reaction time -0.0071 Correlation coefficient R ² 0.9712 Active half-life = -0.693 / (slope x 2.303) = 42.6 hours

Comparative Example 3 The microorganism used was Citrobacter freundii NSM-2 strain F
The results are shown in Table 6, as in Example 3, except that the strain was changed to ERM P-14448 strain.

[0034]

Table 6 Calculated conversion time and half-life of column reaction Reaction time (hours) 12 37 43 69 Maleic acid conversion rate (%) 99.9 40 33.1 5.7 Logarithm of conversion rate 1.6020 1.5198 0.7558 Plot logarithm of conversion rate versus reaction time Slope -0.0273 Correlation coefficient R ² 0.9921 Active half-life = -0.693 / (slope x 2.303) = 11 hours

Comparative Example 4 The microorganism used was Klebsiella planticola NSM-3 strain (F
Table 7 shows the results obtained by performing the same operation as in Example 3 except for changing to ERM P-15144).

[0036]

Table 7 Calculated conversion time and half-life of column reaction Reaction time (hours) 12 87 95 118 Maleic acid conversion rate (%) 99.9 66.4 37.9 22.0 Logarithm of conversion rate 1.8222 1.5786 1.3424 Logarithm of conversion rate versus reaction time Slope -0.0143 Correlation coefficient R ² 0.9229 Active half-life = -0.693 / (slope x 2.303) = 21 hours

[Comparative Example 5] The microorganism used was Comamonas testosteroni NSM-8 (FE
RM P-15714), except that the results were as shown in Table 8.

[0038]

Table 8 Calculation of conversion rate and half-life of column reaction Reaction time (hour) 12 17 41 69 Conversion rate of maleic acid (%) 99.9 86.2 30.7 8.2 Logarithm of conversion rate 1.9356 1.4871 0.9138 Plot logarithm of conversion rate against reaction time -0.0197 Correlation coefficient R ² 0.9993 Active half-life = -0.693 / (slope x 2.303) = 15 hours

[Example 4] A 5% sodium maleate solution (pH 8.
After immersing in 3) at 4 ° C. overnight, 100 ml thereof was packed in a jacketed column, and warm water of 30 ° C. was circulated through the jacket to set the temperature of the reactor at 30 ° C. The substrate solution having the composition shown in Table 4 placed in the bottle with a lid was passed through the column at a rate of 20 ml / h through a Teflon tube to perform a continuous reaction. When the reaction solution was analyzed 12 hours after the start of the reaction, maleic acid had disappeared and L-malic acid was 64 mol%. Fumaric acid was produced in a yield of 36 mol%.

[0040]

According to the present invention, L-aspartic acid and / or fumaric acid and / or L-
-To provide a novel method for producing malic acid.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12N 1/20 C12R 1:39) (C12P 7/46 C12R 1:39) (C12P 7/46 C12R 1:01) (C12P 13/20 C12R 1:01) (C12P 13/20 C12R 1:39) (72) Inventor Takaya Hayashi 1-2-2 Uchisaiwaicho, Chiyoda-ku, Tokyo Nippon Shokubai Co., Ltd. (56) References JP-A-8-08 84594 (JP, A) JP-A-8-33491 (JP, A) JP-A-8-149983 (JP, A) JP-A-8-51989 (JP, A) JP-A-8-51991 (JP, A) (58) Field surveyed (Int. Cl. ⁷ , DB name) C12P 13/20 C12N 1/20 C12P 7/46 BIOSIS (DIALOG) WPI (DIALOG)

Claims (4)

(57) [Claims] 1. The method of claim 1, wherein maleic acid and ammonia are combined with Pseudomonas fluorescens NSM.
-4 strain (FERM P-15560) or Citrobacter amalanaticus NSM-10 strain (FERM P -15P)
-16142) , a method for producing L-aspartic acid, comprising producing L-aspartic acid by treating the microorganism with a cell, a treated cell thereof, or an immobilized product thereof. 2. The method according to claim 2, wherein the maleic acid is used as a Pseudomonas fluorescens NSM-4 strain (FERM P.
-15560) or Citrobacter amaronaticas (Citr
a fumaric acid and / or L-malic acid produced by treating the bacterium of the bacterium of Bacterium amalanaticus) NSM-10 strain (FERM P-16142), a treated product thereof, or an immobilized product thereof to produce fumaric acid and / or L-malic acid. A method for producing an acid and / or L-malic acid. 3. L-aspartic acid from maleic acid and ammonia, or fumaric acid and / or L-aspartic acid from maleic acid.
Pseudomonas fluorescens NSM-4, a microorganism capable of producing malic acid
(FERM P-15560) . 4. L-aspartic acid from maleic acid and ammonia, or fumaric acid and / or L-aspartic acid from maleic acid.
A microorganism capable of producing malic acid, Citrobacter amalanaticus NSM-
10 strains (FERM P-16142) .