JP3165040B2 - Novel microorganism and method for producing L-aspartic acid, fumaric acid and / or L-malic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention relates to a method for producing L-
The present invention relates to a method for efficiently producing aspartic acid, fumaric acid and / or L-malic acid.

[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, Aerobacter, Bacillus, Plevibacterium 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, but their production efficiency has not always been satisfactory. One of the causes is the low stability of maleate isomerase. That is, the microorganisms produce a target substance using a maleate isomerase, including those that produce L-malic acid and L-aspartic acid. Enzyme activity decreases. Therefore, there has been a demand for a microorganism having a more stable maleate isomerase. The present invention has been made to meet such demands,
An object of the present invention is to provide a microorganism having a highly stable maleate isomerase.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, the present inventors have found that an enzyme having remarkably high stability is obtained as compared with the maleic acid isomerase possessed by conventional microorganisms. A microorganism was found, and the present invention was completed. That is, the first of the present invention is a novel microorganism Pseudomonas alkaligenes XD-1 strain having the ability to produce fumaric acid and / or L-malic acid from maleic acid.

[0005] A second aspect of the present invention is to transform maleic acid and ammonia into cells of Pseudomonas alkaligenes strain XD-1, processed cells thereof, or immobilized products thereof and cells of microorganisms capable of producing aspartase. A method for producing L-aspartic acid, comprising treating L-aspartic acid by treating the treated product with a cell treated product or an immobilized product thereof.

Further, a third aspect of the present invention is to provide maleic acid,
Fumaric acid and / or L-malic acid characterized by producing fumaric acid and / or L-malic acid by treating with cells of Pseudomonas alkaligenes XD-1 strain, treated cells thereof, or immobilized products thereof. This is a method for producing malic acid.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The above invention will be described below for each invention. (1) Pseudomonas alkaligenes XD-1 strain The XD-1 strain is a strain isolated from soil in Tsukuba city and has the following mycological properties.

[0008]

As a result of conducting a search for the above mycological properties based on the Barjay's Manual of Systematic Bacteriology vol. 1 to 4 (1986),
The XD-1 strain was obtained from the OF test, catalase, oxidase, sugar assimilation and alkalinity, and organic acid assimilation, resulting in Pseudomonas alcalige (Pseudomonas alcalige).
nes). This strain was deposited at the National Institute of Bioscience and Biotechnology at the National Institute of Advanced Industrial Science and Technology (Deposit No. FERM P-157)
15 (deposited on June 27, 1996).

[0010] The XD-1 strain can be cultured and propagated in the same manner as a known microorganism belonging to the genus Pseudomonas. 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 45 ° C., and the pH of the medium is 5 to 5 with a mineral acid such as hydrochloric acid and 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.

(2) Method for producing L-aspartic acid In the present invention, maleic acid and ammonia are treated with an XD-1 strain and a microorganism capable of producing aspartase to produce L-aspartic acid. "Maleic acid" here means
In addition to maleic acid itself, it is meant to include substances derived from maleic acid, for example, maleate ions, salts containing maleate ions, etc. Similarly, "ammonia" means, in addition to ammonia itself, substances derived from ammonia, For example, it is intended to include ammonium ions, salts containing ammonium ions, and the like. Therefore, the phrase "treating maleic acid and ammonia" includes treating ammonium maleate with microorganisms. Note that XD-1
Not only the strain but also a microorganism capable of producing aspartase is used because the activity of aspartase produced by the XD-1 strain is very weak, and thus the production efficiency of L-aspartic acid by the XD-1 strain alone is low. Because it is bad. The microorganism that can produce aspartase is not particularly limited, and for example, Escherichia coli ATCC11303 strain can be used as used in Examples.

The XD-1 strain and the microorganism capable of producing aspartase are used in the form of cells, processed cells, or immobilized products 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).

As a method for producing L-aspartic acid using the cells, a method comprising suspending the cells of a microorganism capable of producing XD-1 strain and aspartase in a substrate solution containing maleic acid and ammonia and reacting the cells. Can be exemplified. The cells of the XD-1 strain can be obtained by culturing the cells by the method described in the section of "(1) Pseudomonas alkaligenes XD-1 strain" above, 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 45 ° C. The reaction time is preferably 0.5 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, and a large amount of a substance soluble in water is added to the supernatant.
Aspartic acid is precipitated as crystals, which are collected by suction filtration or the like. As a substance soluble in water, 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.

As a method for producing L-aspartic acid using the treated cells, the treated cells of the XD-1 strain and a microorganism capable of producing aspartase are mixed with a substrate solution containing maleic acid and ammonia. And a reaction method. The treated cells of the XD-1 strain are described in the above "(1) Pseudomonas alkaligenes XD-1 strain".
Can be obtained by culturing or the like by the method described in the section. 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 45 ° C. The reaction time is preferably about 0.5 to 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, an immobilized substance such as a cell of an XD-1 strain and a microorganism capable of producing aspartase is packed in a column, and ammonia and ammonia are added to the column. A method of flowing a substrate solution containing maleic acid can be exemplified. The cells of the XD-1 strain are described in the above "(1) Pseudomonas alkaligenes X".
After culturing by the method described in the section of "D-1 strain", it can be obtained by centrifugation or the like. The treated bacterial cell of the XD-1 strain can be obtained by culturing or the like by the method described in the section of "(1) Pseudomonas alkaligenes XD-1 strain" above. 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. In addition, 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, 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 flow rate of the solution varies depending on the size of the column and the amount of the immobilized substance.
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 In the present invention, maleic acid is treated with cells of the strain XD-1 or treated cells thereof, or immobilized products thereof to produce fumaric acid and / or L-malic acid. And / or produce L-malic acid. As a method for producing fumaric acid and / or L-malic acid using cells, XD-1
A method of suspending the bacterial cells of the strain in a substrate solution containing maleic acid and allowing the suspension to react can be exemplified. The cells of the XD-1 strain are
The above "(1) Pseudomonas alkaligenes XD-1"
The strain can be obtained by culturing by the method described in the section of "Strain", followed by centrifugation or the like. The concentration of maleic acid in the substrate solution is preferably about 5 to 30% by weight. The pH of the substrate solution during the reaction 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 6 hours. When L-malic acid is to be obtained, the reaction time is preferably 12 to 24 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 the XD-1 strain is mixed with a substrate solution containing maleic acid and reacted. The treated cells of the XD-1 strain are described in the above “(1) Pseudomonas alkaligenes XD”.
-1 strain "and the like. The maleic acid concentration of the substrate solution is preferably about 5 to 30% by weight. The pH of the substrate solution during the reaction is preferably 5 to 9 and the reaction temperature is preferably 5 to 45 ° C. As in the case of using cells, it is preferable to adjust the pH by using sodium hydroxide or the like. The reaction time is preferably 0.5 to 6 hours if mainly fumaric acid is to be obtained, and 12 to 24 hours if mainly L-malic acid is to be obtained. Recovery of fumaric acid and L-malic acid from the reaction solution can be performed in the same manner as in the case of using cells.

[0018] 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 cell of the XD-1 strain is packed in a column and a substrate solution containing maleic acid is allowed to flow. X
The cells of the D-1 strain can be obtained by culturing the cells by the method described in the section "(1) Pseudomonas alkaligenes XD-1" above, followed by centrifugation or the like. X
The treated bacterial cell of the D-1 strain can be obtained by culturing or the like by the method described in the section of "(1) Pseudomonas alkaligenes XD-1 strain" above. 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 depends on the type of gel, but the diameter is 1 to 10 mm
The degree is appropriate. In addition, 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. 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 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 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.

[0019]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. The technical scope of the present invention is not limited to the embodiments.

Example 1 Medium 3 having the composition shown in Table 1
L was charged into a jar fermenter having a total volume of 5 L, and Pseudomonas alkaligenes XD-1 strain was inoculated and cultured with aeration and stirring at 30 ° C. Twenty hours after the culturing, the culturing was completed, and the cells were collected by centrifugation.

Table 1 Composition of maleic acid-containing medium

A 3 L medium having the composition shown in Table 2 was used in a total volume of 5 L.
And inoculated with Escherichia coli ATCC11303, followed by aeration and stirring culture at 37 ° C. The culture was terminated 16 hours after the culture, and the cells were collected by centrifugation.

Table 2 Composition of medium containing fumaric acid

The two kinds of cells were uniformly dispersed in 120 g of Polycap 172 (manufactured by Hercules) and 240 g of deionized water. 300 ml of ion exchange resin (Amberlite IRA-94S C1 type, manufactured by Organo) and 0.5 ml of a 6-L eggplant-shaped flask
Insert 200 inch Teflon spheres and place the dispersion
Insert 1/6, and rotate 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 to remove the Teflon spheres to obtain an immobilized biocatalyst.

The immobilized biocatalyst thus prepared is
After immersing overnight in a 20% ammonium maleate solution (pH 8.5) at 4 ° C., 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 reactor temperature at 30 ° C. . The substrate solution having the composition shown in Table 3 placed in the bottle with a lid was passed through a Teflon tube through a column at a rate of 20 ml / hour to carry out a continuous reaction. After the reaction starts
When the reaction solution was analyzed at 12 hours, L-aspartic acid equivalent to the consumed maleic acid was produced as a reaction product.

Table 3 Composition of ammonium maleate substrate solution

From the time when the conversion became 80% or less, the conversion was analyzed by HPLC, and the time change of the conversion was tracked. The half life of the enzyme activity for converting maleic acid to L-aspartic acid was calculated from the slope obtained by plotting the logarithmic value of the conversion against the reaction time, and was 43 hours.

Table 4 Calculation of conversion and half-life of column reaction

[Comparative Example 1] A microorganism to be used was prepared from Pseudomonas alkaligenes strain XD-1.
The active half-life was calculated to be 14 hours in the same manner as in Example 1 except that the strain was changed to Alcaligenes faecalis IFO13111 strain.

Comparative Example 2 The microorganism used was Pseudomonas alkaligenes strain XD-1 and Enterobacter agglomerans NSM-1.
The activity half-life was calculated in the same manner as in Example 1 except that the strain (FERMP-14447) was changed to 19 hours.

Example 2 An immobilized biocatalyst prepared in the same manner as in Example 1 was treated with a 5% sodium maleate solution (pH
After immersion in 8.3) 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 reactor temperature to 30 ° C.

The substrate solution having the composition shown in Table 5 in a bottle with a lid was passed through a column at a rate of 20 ml / h through a Teflon tube to perform a continuous reaction. When the reaction solution was analyzed 6 hours after the start of the reaction, maleic acid had disappeared, and L-malic acid was produced at a yield of 35 mol% and fumaric acid at a yield of 65 mol%.

Table 5 Composition of sodium maleate substrate solution

[0034]

The present invention provides a novel method for producing L-aspartic acid and fumaric acid and / or L-malic acid. The microorganism used in the present invention has a longer active half-life of the maleic isomerase than conventional microorganisms, so that the substance can be produced efficiently.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12P 7/46 C12R 1:38) (C12P 13/20 C12R 1:38) (72) Inventor Koichi Sakano Tsukuba, Ibaraki Pref. 1-25, Ichikannondai 12 Nippon Shokubai Co., Ltd. (56) References JP-A-8-51991 (JP, A) JP 42-11993 (JP, B1) JP 42-11994 (JP, B1) ) Agric. Biol. Chem. , 50 (1), 89-94 (1986) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 1/20 BIOSIS (DIALOG) WPI (DIALOG)

Claims (3)

(57) [Claims] 1. The method of claim 1 wherein maleic acid is converted to fumaric acid and / or L-
A novel microorganism Pseudomonas alkaligenes XD-1 strain capable of producing malic acid. 2. A method comprising the steps of: converting maleic acid and ammonia into cells of Pseudomonas alkaligenes strain XD-1;
Or production of L-aspartic acid, which comprises treating L-aspartic acid by treating the cells with microorganisms capable of producing immobilized products thereof and aspartase, treated microorganisms, or immobilized products thereof. Method. 3. A method of producing fumaric acid and / or L-malic acid by treating maleic acid with cells of Pseudomonas alkaligenes XD-1 strain, treated cells, or immobilized products thereof. A method for producing fumaric acid and / or L-malic acid.