JPH1075772A - New microorganism and production of l-aspartic acid, fumaric acid and/or l-malic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain fumaric acid and/or L-malic acid by treating maleic acid with the cells of a specific microorganism belonging to the genus Escherichia or Comamonas, the treated cells or their immobilized product. SOLUTION: Maleic acid is treated with the cells of Escherichia coli NSM-7 strain (FERM P-15713) or Comamonas testosteroni NSM-8 strain (FERM P-15714), the treated cells or their immobilized product. L-aspartic acid can be produced by treating maleic acid and ammonia with the above microorganisms (including the treated cells and their immobilized product).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing L-aspartic acid, fumaric acid and / or L-malic acid by using a microorganism belonging to the genus Escherichia or Comamonas.

[0002]

BACKGROUND OF THE INVENTION Fumaric acid, L
-Many microorganisms that produce malic acid or L-aspartic acid have been conventionally known. For example, microorganisms that produce L-malic acid include microorganisms belonging to the genera Alcaligenes, Pseudomonas, and Serratia (Japanese Unexamined Patent Publication No.
No. 60-102191, Agric. Biol. Chem., 50 (1) 84-94 (198
6)), Microorganisms that produce fumaric acid include those belonging to the genera Arthrobacter and Proteus (Asano et al.,
(April 1995 Agricultural Chemistry), as microorganisms producing L-aspartic acid, microorganisms belonging to the genera Alcaligenes, Pseudomonas, Achromobacter, Aerobacterium, Bacillus, and Previbacterium are known. (Japanese Patent Publication No. 38-2793, Japanese Patent Publication No. 42-11993,
Japanese Patent Publication No. 42-11994, Japanese Patent Publication No. 43-87104).

[0003]

As described above, many kinds of microorganisms produce fumaric acid, L-malic acid, or L-aspartic acid, and the microorganisms belonging to the genus Escherichia and Comamonas produce such substances. There was no known thing to do.

[0004] The present invention provides a means for producing fumaric acid and / or L-malic acid using maleic acid as a raw material, and producing L-aspartic acid using maleic acid and ammonia as raw materials, using microorganisms belonging to the genus Escherichia and Comamonas. It is intended to provide a means for doing so.

[0005]

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that NSM-7 strain belonging to the genus Escherichia and NSM-8 belonging to the genus Comamonas.
The strain was found to produce L-aspartic acid and fumaric acid and / or L-malic acid, and completed the present invention.

That is, a first aspect of the present invention is a novel microorganism Escherichia coli NSM-7 strain and Comamonas testosteroni NSM-8 strain having the ability to produce L-aspartic acid from maleic acid and ammonia.

A second aspect of the present invention is a microorganism, a treated microorganism, or an immobilized product of a microorganism belonging to the genus Escherichia or Comamonas and capable of producing L-aspartic acid from maleic acid and ammonia. A method for producing L-aspartic acid, comprising treating maleic acid and ammonia to produce L-aspartic acid.

A third aspect of the present invention is a microorganism, a treated bacterial cell, or a microorganism thereof belonging to the genus Escherichia or Comamonas and capable of producing fumaric acid and / or L-malic acid from maleic acid. A method for producing fumaric acid and / or L-malic acid, wherein maleic acid is treated with the immobilized product to produce fumaric acid and / or L-malic acid.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The above invention will be described below for each invention. (1) NSM-7 strain and NSM-8 strain The NSM-7 strain is a strain isolated from the soil in Tsukuba City and has the following bacteriological properties.

[0010]

The NSM-8 strain is a strain isolated from the soil in Tsukuba City and has the following mycological properties.

[0012]

The results of a search conducted on the mycological properties of the NSM-7 and NSM-8 strains above based on the Barjay's Manual of Systematic Bacteriology vol. 1 to 4 (1986) , NSM-7 strain, sugar fermentability, VP
-The test, hydrogen sulfide production, and citric acid assimilation proved to belong to Escherichia coli, and the NSM-8 strain contained urease, catalase, oxidase, OF test, sugar assimilation, From the assimilation of organic acids,
Comamonas testosteron
i). The NSM-7 strain was deposited with the National Institute of Advanced Industrial Science and Technology (AIST) under the deposit number FERM P-15713.
The NSM-8 strain has been deposited as deposit number FERM P-15714 (deposit date June 27, 1996) as of June 27, 1996.

The NSM-7 strain and the NSM-8 strain can be cultured and grown in the same manner as known microorganisms belonging to the genus Escherichia and the genus Comamonas, respectively. 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. Microorganisms cultured in a medium containing malonic acid, compared to microorganisms cultured in a medium without malonic acid,
Since the maleic acid isomerization activity is high, it is preferable to add malonic acid to the medium and grow the culture. The culture temperature is
The temperature is preferably 25 to 40 ° C., and the pH of the medium is preferably maintained at 5 to 9 with a mineral acid such as hydrochloric acid or an organic acid such as maleic acid. 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.

(2) Method for producing L-aspartic acid In the present invention, maleic acid and ammonia are treated with a microorganism belonging to the genus Escherichia or a microorganism belonging to the genus Comamonas to produce L-aspartic acid. The microorganism used belongs to the genus Escherichia or Comamonas,
Preferred are microorganisms having the ability to produce L-aspartic acid from maleic acid and ammonia.
A microorganism belonging to E. coli or Comamonas testosteroni and having the above properties is more preferable. Most preferred microorganisms include the NSM-7 and NSM-8 strains described above. In the present invention, "maleic acid" is intended to include, in addition to maleic acid itself, substances derived from maleic acid, for example, maleate ions, salts containing maleate ions, and the like. In addition to ammonia itself, it is intended to include substances derived from ammonia, for example, ammonium ions, salts containing ammonium ions, and the like. Therefore, the phrase "treating maleic acid and ammonia" includes treating ammonium maleate with microorganisms.

The microorganisms used in the present invention are cells,
It is used in the form of a processed bacterial cell or an immobilized product thereof. Here, the “processed bacterial cell” refers to a crushed bacterial cell, or an enzyme extracted from a culture (including bacterial cells and culture supernatant).

Examples of the method for producing L-aspartic acid using the cells include a method in which the cells of a microorganism are suspended in a substrate solution containing maleic acid and ammonia and reacted. Microbial cells can be obtained by culturing a microorganism belonging to the genus Escherichia or a microorganism belonging to the genus Comamonas by a method commonly used for the microorganism, followed by centrifugation or the like. The concentration of maleic acid in the substrate solution is preferably 5 to 30% by weight, and the concentration of ammonia is preferably about 1.5 to 10% by weight. The pH of the substrate solution during the reaction is preferably 7 to 10, and the reaction temperature is preferably 5 to 40 ° C. The reaction time is 0.
Preferably, it is 5 to 48 hours. As a method for collecting L-aspartic acid from the substrate solution (reaction solution) after completion of the reaction, a precipitation method can be exemplified. That is, the reaction solution is separated into cells and supernatant by centrifugation or the like, a large amount of a substance soluble in water is added to the supernatant, and L-aspartic acid is precipitated as a crystal, which is collected by suction filtration or the like. I do. In addition,
As a water-soluble substance, a mineral acid such as sulfuric acid can be used, but maleic acid is preferably used. This is because when maleic acid is used, after collecting L-aspartic acid, the supernatant can be used again as a substrate solution.

Examples of the method for producing L-aspartic acid using the treated cells include a method in which the treated cells of a microorganism are mixed with a substrate solution containing maleic acid and ammonia and reacted. The processed product of the microorganism can be obtained by culturing a microorganism belonging to the genus Escherichia or a microorganism belonging to the genus Comamonas by a method commonly used for the microorganism. The concentration of maleic acid in the substrate solution is preferably 5 to 30% by weight, and the concentration of ammonia is preferably about 1.5 to 10% by weight. The pH of the substrate solution during the reaction is preferably 7 to 10, and the reaction temperature is preferably 5 to 40 ° C. The reaction time is 0.5
It is preferable to set it to about 48 hours. Collection of L-aspartic acid can be performed in the same manner as in the case of using cells.

As a method for producing L-aspartic acid using an immobilized substance such as a cell, a method in which an immobilized substance such as a cell of a microorganism is packed in a column and a substrate solution containing ammonia and maleic acid is passed through the column. Examples can be given. Microbial cells can be obtained by culturing a microorganism belonging to the genus Escherichia or a microorganism belonging to the genus Comamonas by a method commonly used for the microorganism, followed by centrifugation or the like. The processed product of the microorganism can be obtained by culturing a microorganism belonging to the genus Escherichia or a microorganism belonging to the genus Comamonas by a method commonly used for the microorganism. Examples of the means for immobilizing bacterial cells and the like include means for inclusive immobilization with a gel, means for immobilization by being carried on an ion exchanger, and the like. Examples of the gel used include carrageenan gel, agar gel, mannan gel, PVA gel, and polyacrylamide gel. The size of the gel sphere varies depending on the type of gel, but a diameter of about 1 to 10 mm is appropriate. Also,
Examples of the ion exchanger include a cellulose-based, styrene-divinylbenzene-based, and phenol-formalin-based ion exchanger. The concentration of maleic acid in the substrate solution is preferably 5 to 30% by weight, and the concentration of ammonia is preferably 1.5 to 10% by weight. In addition, the solution contains S such as mercaptoethanol, cysteine, and glutathione.
An H compound, a reducing agent such as a sulfite, an enzyme activator such as a magnesium ion or a manganese ion, or the like can also be added. The flow rate of the solution varies depending on the size of the column and the amount of the immobilized substance, but SV = 0.05 to 10 is appropriate. The solution passed through the column contains a large amount of L-aspartic acid due to the action of microorganisms. Collection of L-aspartic acid from the solution can be performed in the same manner as in the case of using cells.

(3) Method for producing fumaric acid and / or L-malic acid According to the present invention, maleic acid is treated with microorganisms belonging to the genus Escherichia or the genus Comamonas, treated microorganisms, or immobilized products thereof. To produce fumaric acid and / or L-malic acid. The microorganism to be used is preferably a microorganism belonging to the genus Escherichia or Comamonas and having an ability to produce fumaric acid and / or L-malic acid from maleic acid, and a microorganism belonging to Escherichia coli or Comamonas testosteroni and having the above properties. Is more preferred. The most preferred microorganisms include the NSM-7 strain described above and
NSM-8 strain can be mentioned.

Examples of the method for producing fumaric acid and / or L-malic acid using the cells include a method in which the cells of a microorganism are suspended in a substrate solution containing maleic acid and reacted. Microbial cells can be obtained by culturing a microorganism belonging to the genus Escherichia or a microorganism belonging to the genus Comamonas by a method commonly used for the microorganism, followed by centrifugation or the like. The concentration of maleic acid in the substrate solution is preferably about 5 to 30% by weight. P of substrate solution during reaction
H is preferably 5 to 9 and the reaction temperature is preferably 5 to 40 ° C.
As the alkaline substance added for adjusting the pH, it is preferable to use sodium hydroxide or the like instead of ammonia. This is because if ammonia is present in the substrate solution, L-aspartic acid is generated and the yield of L-malic acid is reduced. After reacting the cells with the substrate solution under the above conditions, fumaric acid and / or L-malic acid are recovered from the reaction solution. Since maleic acid changes to L-malic acid via fumaric acid, the ratio of L-malic acid increases if the reaction time is long, and the ratio of fumaric acid increases if the reaction time is short. Therefore, if fumaric acid is mainly intended to be obtained, the reaction time should be shortened and L-
If malic acid is to be obtained, increase the reaction time. Specifically, when fumaric acid is to be obtained, the reaction time is preferably 0.5 to 12 hours. When L-malic acid is to be obtained, the reaction time is preferably 6 to 48 hours. preferable. The method for recovering fumaric acid or L-malic acid from the reaction solution is not particularly limited. Fumaric acid can be recovered, for example, by the following method. The reaction solution is separated into cells and supernatant by centrifugation or the like, a large amount of a substance soluble in water is added to the supernatant, and fumaric acid is precipitated as crystals.
This is collected by suction filtration or the like. As a water-soluble substance, a mineral acid such as sulfuric acid can be used, but maleic acid is preferably used. This is because, when maleic acid is used, the supernatant can be used again as a substrate solution after fumaric acid is recovered. On the other hand, L-malic acid can be recovered by the following method. The reaction solution is separated into cells and supernatant by centrifugation or the like, and fumaric acid is precipitated by adding a mineral acid to the supernatant to make it acidic. To the filtrate from which fumaric acid has been removed by suction filtration, malic acid can be extracted by adding a solvent that dissolves malic acid well, and the solvent phase can be concentrated to obtain malic acid. It is preferable to use inexpensive sulfuric acid as the mineral acid. Ether or the like can be used as a solvent for the extraction. Further, a purification method using an ion exchange resin instead of the extraction can also be adopted.

Fumaric acid and / or L
Examples of the method for producing malic acid include a method in which a treated product of a microorganism is mixed with a substrate solution containing maleic acid and reacted. The treated product of the microorganism can be obtained by culturing a microorganism belonging to the genus Escherichia or a microorganism belonging to the genus Comamonas by a method commonly used for the microorganism. The concentration of maleic acid in the substrate solution is preferably about 5 to 30% by weight. P of substrate solution during reaction
H is preferably 5 to 9 and the reaction temperature is preferably 5 to 40 ° C.
As in the case of using cells, it is preferable to adjust the pH using sodium hydroxide or the like. The reaction time is preferably short if it is intended mainly to obtain fumaric acid, and long if it is intended mainly to obtain L-malic acid.
Recovering fumaric acid and L-malic acid from the reaction solution
It can be carried out in the same manner as when using cells.

[0023] Fumaric acid and / or
Alternatively, examples of a method for producing L-malic acid include a method in which an immobilized substance such as a microbial cell is packed in a column and a substrate solution containing maleic acid is allowed to flow. Microbial cells can be obtained by culturing a microorganism belonging to the genus Escherichia or a microorganism belonging to the genus Comamonas by a method commonly used for the microorganism, followed by centrifugation or the like. The treated product of the microorganism can be obtained by culturing a microorganism belonging to the genus Escherichia or a microorganism belonging to the genus Comamonas by a method commonly used for the microorganism. Examples of the means for immobilizing bacterial cells and the like include means for inclusive immobilization with a gel, means for immobilization by being carried on an ion exchanger, and the like. Examples of the gel used include carrageenan gel, agar gel, mannan gel, PVA gel, and polyacrylamide gel. The size of the gel sphere varies depending on the type of gel, but a diameter of about 1 to 10 mm is appropriate. Also,
Examples of the ion exchanger include a cellulose-based, styrene-divinylbenzene-based, and phenol-formalin-based ion exchanger. The concentration of maleic acid in the substrate solution is preferably 5 to 30% by weight. Further, in the solution, SH compounds such as mercaptoethanol, cysteine and glutathione, reducing agents such as sulfites,
An enzyme activator such as a magnesium ion or a manganese ion may be added. The flow rate of the solution varies depending on the size of the column and the amount of the immobilized substance.
= 0.05 to 10 is appropriate. The solution that has passed through the column contains a large amount of fumaric acid and / or L-malic acid due to the action of microorganisms. The collection of fumaric acid and / or L-malic acid from the solution can be performed in the same manner as in the case of using cells.

[0024]

【Example】

[Example 1] Escherichia coli was added to 10 ml of the medium shown in Table 1.
After inoculating the NSM-7 strain and culturing it for 24 hours, the cells were collected by centrifugation, suspended in 5 ml of an ammonium maleate substrate solution shown in Table 2, and then gently shaken at 30 ° C. Allowed to react for hours. Analysis of the reaction mixture revealed that maleic acid had disappeared and L-aspartic acid had been produced at a concentration of 20.45% by weight. As a by-product, fumaric acid present in equilibrium with L-aspartic acid was produced at a concentration of 0.24% by weight.

Table 1 Medium for culturing bacterial cells

Table 2 Composition of ammonium maleate substrate solution

Example 2 Comamonas testosteroni NS was added to 10 ml of the medium shown in Table 1.
After inoculating the M-8 strain (FERM P-15714) and culturing for 24 hours, the cells were collected by centrifugation, suspended in 5 ml of an ammonium maleate substrate solution shown in Table 2, and then slowly cooled at 30 ° C. And reacted for 24 hours.

Analysis of the reaction mixture revealed that maleic acid had disappeared and L-aspartic acid had been produced at a concentration of 20.5% by weight. As a by-product, fumaric acid present in equilibrium with L-aspartic acid was produced at a concentration of 0.13% by weight.

Example 3 Escherichia coli NSM-7 strain (FERM) was added to 10 ml of the medium shown in Table 1.
24 hours later, the cells were collected by centrifugation, suspended in 10 ml of the maleic acid substrate solution shown in Table 3, and then gently shaken at 30 ° C. The reaction was performed for 4 hours. When the solution was analyzed by high performance liquid chromatography after the reaction, maleic acid had disappeared and fumaric acid was found to be 7.0.
% And malic acid at a concentration of 3.5%. After centrifuging the reaction solution to remove cells, sulfuric acid is added to adjust the pH.
When the mixture was adjusted to 1.8 and stirred, a white precipitate of fumaric acid was formed. The precipitate was filtered, washed with a small amount of distilled water, and dried to obtain 0.62 g of fumaric acid powder. The purity of this fumaric acid was 99.4% by weight.

Table 3 Composition of maleic acid substrate solution

Example 4 Comamonas testosteroni NS was added to 10 ml of the medium shown in Table 1.
The M-8 strain (FERM P-15714) was inoculated, and after 24 hours, the cells were collected by centrifugation, and 10 ml of a maleic acid substrate solution shown in Table 3 was collected.
After suspending the cells, the cells were gently shaken at 30 ° C. and reacted for 4 hours. After the reaction, the solution was analyzed by high performance liquid chromatography. As a result, maleic acid had disappeared, and fumaric acid was produced at a concentration of 7.1% and malic acid at a concentration of 3.4%.
The reaction solution was centrifuged to remove the cells, sulfuric acid was added to adjust the pH to 1.8, and the mixture was stirred to produce a white precipitate of fumaric acid. The precipitate was filtered, washed with a small amount of distilled water, and dried to obtain 0.63 g of fumaric acid powder. The purity of this fumaric acid was 99.4% by weight.

Example 5 Escherichia coli NSM-7 strain (FERM) was added to 10 ml of the medium shown in Table 4.
24 hours later, the cells were collected by centrifugation, suspended in 10 ml of the maleic acid substrate solution shown in Table 3, and then gently shaken at 30 ° C. The reaction was performed for 24 hours. After the reaction, the solution was analyzed by high performance liquid chromatography.
% And fumaric acid at a concentration of 2.5%.

The obtained malic acid was subjected to a liquid chromatography column CROWNPAK CR (+) (4.0) for optical resolution manufactured by Daicel Chemical Industries, Ltd.
(150 mm), the D-form was not formed and only the L-form was formed.

Table 4 Composition of culture medium for cell culture

Example 6 Comamonas testosteroni NS was added to 10 ml of the medium shown in Table-4.
The M-8 strain (FERM P-15714) was inoculated, and after 24 hours, the cells were collected by centrifugation, and 10 ml of a maleic acid substrate solution shown in Table 3 was collected.
After suspending the cells, the cells were gently shaken at 30 ° C. and reacted for 24 hours. Analysis of the solution after the reaction revealed that maleic acid had disappeared, L-malic acid was 8.3%, and fumaric acid was
It was formed at a concentration of 2.5%.

The obtained malic acid was converted to a liquid chromatography column for optical resolution CROWNPAK CR (+) (4.0) manufactured by Daicel Chemical Industries, Ltd.
(150 mm), the D-form was not formed and only the L-form was formed.

[0037]

According to the present invention, there is provided a method for producing the above substance using a microorganism which is not known to produce L-aspartic acid and fumaric acid and / or L-malic acid.

Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical indication (C12N 1/20 C12R 1:01) (C12P 7/46 C12R 1:19) (C12P 7/46 C12R 1: 01) (C12P 13/20 C12R 1:19) (C12P 13/20 C12R 1:01)

Claims (4)

[Claims] 1. A novel microorganism Escherichia coli NSM-7 having the ability to produce L-aspartic acid from maleic acid and ammonia. 2. A novel microorganism, Comamonas testosteroni NSM-8, having the ability to produce L-aspartic acid from maleic acid and ammonia. 3. A method for producing maleic acid and ammonia using a microorganism, a treated microorganism, or an immobilized product of a microorganism belonging to the genus Escherichia or Comamonas and capable of producing L-aspartic acid from maleic acid and ammonia. L-aspartic acid to produce L-aspartic acid
-A process for producing aspartic acid. 4. A microorganism, a treated microorganism, or an immobilized product of a microorganism belonging to the genus Escherichia or Comamonas and capable of producing fumaric acid and / or L-malic acid from maleic acid. A method for producing fumaric acid and / or L-malic acid, comprising treating an acid to produce fumaric acid and / or L-malic acid.