JPH08332092A - Production of fumaric acid - Google Patents

Abstract

PURPOSE: To efficiently produce fumaric acid useful for foods, medicines, industrial raw materials, etc., by suppressing the dissolved oxygen concentration to a specific value or below and carrying out the isomerizing reaction of a microorganism (a prepared substance) having maleic isomerase activities with maleic acid in an aqueous solution. CONSTITUTION: A microorganism having maleic isomerase activities (e.g. Alcaligenes faecalis IFO12669 strain) or a prepared substance thereof is mixed with maleic acid in an aqueous solution and the resultant reactional solution is then sealed with one or more gases such as N2 , Ar or He to maintain the dissolved oxygen in the reactional solution at <=4ppm. Enzymic isomerizing reaction is then carried out to isomerize the maleic acid into fumaric acid. Thereby, the objective fumaric acid useful as a raw material for producing L-aspartic acid, L-malic acid, L-alanine, etc., useful as foods, medicines or industrial raw materials is efficiently obtained in high yield.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for efficiently producing fumaric acid from maleic acid. Fumaric acid is
Mainly produced by isomerization of maleic acid, it is widely used as a raw material for medicines, foods, and industry. From fumaric acid, for example, L-aspartic acid, L-malic acid, and L-alanine useful as foods, pharmaceuticals, and industrial raw materials are produced by an enzymatic method or the like.

[0002]

BACKGROUND ART As a method for isomerizing maleic acid to produce fumaric acid, mainly chemical methods have been proposed (US Pat. Nos. 2,816,923, 2,955,136 and 2,33).
2,992), the conversion rate to fumaric acid is restricted by the reaction equilibrium, and because this method is a high temperature reaction, deterioration of maleic acid or fumaric acid occurs, and byproducts are formed, resulting in a decrease in yield. Has the problem of.

On the other hand, in the enzymatic method, it is known that maleate isomerase isomerizes maleic acid to produce fumaric acid (K. Otsuka Agric. Biol. Chem., 25 ,
(9), p726 (1961)) only examines the enzymatic properties, and little has been done from the viewpoint of industrial application.

[0004]

SUMMARY OF THE INVENTION The present invention has been made from the above viewpoints, and provides a practical method capable of efficiently producing fumaric acid from maleic acid by an enzymatic method in a high yield. The challenge is to provide.

[0005]

Means for Solving the Problems As a result of intensive studies to establish a method for efficiently producing fumaric acid, the present inventors have found that a microorganism having maleic acid isomerase activity or a microorganism having maleic acid isomerase activity is present. By mixing the preparation of (1) with maleic acid in an aqueous solution and producing fumaric acid from maleic acid by an enzymatic isomerization reaction, by maintaining the dissolved oxygen concentration in the reaction solution at 4 ppm or less, the reaction is carried out, It was found that fumaric acid can be produced in high yield, and the present invention has been completed.

That is, according to the present invention, a microorganism having maleate isomerase activity or a preparation of the microorganism having maleate isomerase activity and maleic acid are mixed in an aqueous solution, and maleic acid is converted to fumaric acid by an enzymatic isomerization reaction. Is a method for producing fumaric acid, which comprises reacting while maintaining the dissolved oxygen concentration in the reaction solution at 4 ppm or less.

To reduce the concentration of dissolved oxygen in the reaction solution, a method of sealing the reaction solution with one or more gases selected from N ₂ , Ar and He, and a method of adding sulfite to the reaction solution or its raw material , And a method of degassing the reaction liquid or the raw material thereof.

In the method of the present invention, the dissolved oxygen concentration in the reaction solution is preferably 0.5 ppm or less.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be specifically described below. The microorganism having maleate isomerase activity used in the present invention is not particularly limited as long as it is a microorganism having this activity, but is genus Alcaligenes, Pseudomonas.
Microorganisms belonging to the genus as), the genus Xanthomanas, and the genus Bacillus are preferably used. Especially Alcaligenes faecali
s) (for example, IFO12669, IFO13111, IAM1473),
Alcaligenes eutrophu
s), Pseudomonas fluorescens (Pseudomonas
fluolescens) (eg ATCC 23728), Xanthomonas
Xanthomonas maltophilia (eg A
TCC 13270), Bacillus stearothermophilus (Baci
llus stearothermophilus) [For example, MI-101 strain (FER
M P-14801)], Bacillus brevi (Bacillus brevi
s) [eg, MI-103 strain (FERM P-14803)] and the like are preferably used. Furthermore, there is no problem even if a mutant strain of the above microorganism or a modified strain obtained by gene recombination, a microorganism produced by introducing the maleate isomerase gene of the above microorganism into another microorganism, and the like are used.

Cultivation of a microorganism having maleate isomerase activity can be carried out in an ordinary nutrient medium containing a carbon source, a nitrogen source, an inorganic salt, various vitamins and the like. Examples of the carbon source include maleic acid, glucose and sucrose. Sugars, fructose, maltose, and other sugars, ethanol, methanol, and other alcohols, citric acid, maleic acid, and other organic acids, spent molasses, and the like, preferably maleic acid or a mixture of maleic acid and other carbon sources, are used. As the nitrogen source, for example, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea and the like are used alone or in combination. Further, as the inorganic salt, for example, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and the like are used. In addition to this, nutrients such as various vitamins such as peptone, meat extract, yeast extract, corn stay liquor, casamino acid, and biotin can be added to the medium. Furthermore, maleic acid, malonic acid, tartronic acid, citraconic acid, mesaconic acid, etc. may be added to the medium as an inducer of maleic acid isomerase. The concentration of these inducers added is usually 10 to 200 mM, preferably 50 to 100 mM.

The culture conditions are not particularly limited as long as the temperature is a temperature at which a microorganism having maleate isomerase activity can grow under aerobic conditions such as aeration and agitation.
The pH during the culture is not particularly limited as long as the microorganism can grow. The pH adjustment during the culture can be performed by adding an acid or an alkali.

By collecting cells from the thus obtained culture by centrifugation or the like, cells containing maleate isomerase can be obtained. In the present invention, in the isomerization reaction of maleic acid to fumaric acid, not only the cells separated from the culture solution in which the above-mentioned cells are cultured, but also the culture solution, a preparation such as a crushed product or an extract of the cells. Can also be used. The cells may be used after being washed with, for example, a buffer solution (pH 7) such as a phosphate buffer solution.
A crude enzyme preparation obtained from microbial cells may be used, or a further purified enzyme preparation may be used. Further, the above-mentioned microbial cells, a crushed product or an extract thereof, or a purified enzyme immobilized on a carrier can also be used.
The term "preparation of a microorganism having maleate isomerase activity" as used herein means to include all such fractions derived from microbial cells and having maleate isomerase activity. When using the bacterial cells, the bacterial cells are frozen in advance, or Triton X-100, Tw is added to the buffer solution.
Use after increasing the permeability of the microbial cells by treating the microbial cells for 10 to 120 minutes at a temperature of 15 to 40 ° C in a liquid containing 0.01 to 0.2% of a surfactant such as een20. You can also do it.

In the present invention, "mixing a microorganism having maleate isomerase activity or a preparation of the microorganism having maleate isomerase activity with maleic acid in an aqueous solution" means microbial cells or a preparation thereof or Adding maleic acid or an aqueous solution containing maleic acid to an aqueous solution containing them, and further passing an aqueous solution containing maleic acid through a column packed with immobilized cells or immobilized enzyme .

As a method for extracting and purifying maleic acid isomerase from microbial cells, any known method for purifying an enzyme can be applied. For example, as the method for destroying the bacterial cells, ultrasonic disruption, mechanical disruption method using French press, homogenizer, etc., enzymatic disruption method using lysozyme and the like can be used. The soluble fraction of the disrupted cells or the fraction thereof thus obtained can be used as a crude enzyme fraction of maleate isomerase. In addition, a purified enzyme obtained by further purifying this crude enzyme fraction may be used. Maleic acid isomerase is usually purified from the crude enzyme fraction by (a) precipitation method, for example, ammonium sulfate precipitation method, (b) chromatography separation, for example, ion exchange chromatography, affinity adsorption chromatography, gel filtration chromatography. Etc. (C) Separation methods by electrophoresis, and any combination of these methods.

In the present invention, a microorganism having maleic acid isomerase activity or a preparation thereof is added to an aqueous solution containing maleic acid to produce fumaric acid from maleic acid by an enzymatic isomerization reaction. Dissolved oxygen concentration is 4 ppm or less, particularly preferably 0.5 ppm
It is characterized in that the reaction is carried out while maintaining the following.

However, even if the dissolved oxygen level may momentarily exceed 4 ppm, it may be maintained at substantially 4 ppm or less for a long time. For example, even if the concentration temporarily exceeds 4 ppm immediately after starting the stirring or immediately after preparing the maleic acid-containing aqueous solution, it may be maintained at 4 ppm or less thereafter. In addition, the number of stirring is increased on the way, and it is 4p for a very short time.
Even if it exceeds pm, it may be maintained at 4 ppm or less for most of the entire reaction time.

As a method of maintaining the dissolved oxygen concentration in the reaction solution at 4 ppm or less, specifically, for example, N ₂ , Ar,
Examples include sealing the reaction liquid with one or more gases selected from He, adding sulfite to the reaction liquid or its raw material, and degassing the reaction liquid or its raw material. Also, these means may be combined.

The term "one or more gases selected from N ₂ , Ar and He" means a single gas or a combination of any two or more gases. Examples of the method for carrying out the maleic acid isomerase reaction in the atmosphere of these gases include, for example, an aqueous maleic acid solution or an enzymatic reaction solution (hereinafter, simply referred to as "reaction solution") during the enzymatic reaction and / or prior to the enzymatic reaction. A method such as continuously blowing a gas, blowing the gas into the reaction solution and then sealing the gas, introducing the gas into the reaction vessel after degassing the reaction solution, and the like are performed.
In the method of the present invention, the gas phase in contact with the reaction liquid is the above-mentioned gas atmosphere, that is, the reaction liquid is sealed with the above-mentioned gas, and it is not always necessary to blow the above-mentioned gas continuously into the reaction liquid or the reaction tank. Is not mandatory. Further, the gas phase in contact with the reaction solution is preferably completely replaced by the above gas, but even if a part of the gas phase is replaced, the effect of the replacement can be expected.

The sulfite used in the present invention may be a sulfite salt, for example, inorganic sulfites such as sodium sulfite, potassium sulfite, ammonium sulfite, ammonium hydrogen sulfite and calcium sulfite are preferable, but the maleic acid isomerase activity is preferred. Salts with mercury and copper, which are known to inhibit the above, are not used. The concentration of sulfite is 1 ppm to 1000 ppm, preferably 10 ppm
~ 500 ppm is used.

The method of lowering the dissolved oxygen concentration of the reaction solution or its raw material by degassing is not particularly limited, but, for example, a method of heating to about 55 ° C. under reduced pressure with an aspirator, ultrasonic vibration For example, a method of expelling air in the liquid under reduced pressure while applying it can be performed. The degassing may be carried out prior to the reaction by degassing the reaction solution raw material, for example, an aqueous solution of maleic acid or an aqueous solution containing cells or a preparation thereof, or further degassing the reaction solution during the reaction.

The enzymatic reaction has a pH of 5 to 10, preferably 6 to
9, the reaction is carried out at a temperature of 20 to 50 ° C., preferably 25 to 37 ° C. for usually 5 to 120 hours. If necessary, a divalent metal salt such as a calcium salt, a magnesium salt, a manganese salt can be added to the reaction solution. In addition, the reaction liquid may contain a buffering agent, a trace amount of an organic solvent, or the like.

The maleic acid concentration of the aqueous solution containing maleic acid used in the reaction is 1 to 40 W / V%, preferably 10 to
Although it is 30 W / V%, the maleic acid concentration may be maintained at about 0.001 to 1% in a continuous reaction or the like.

The amount of the bacterial cells or the preparation thereof used in the reaction is not particularly limited, but a bacterial cell weight (wet bacterial cells) of 1 to 30% is preferably used. The fumaric acid produced by the above method is separated from the cells and its preparation by ultrafiltration membrane separation, centrifugation, etc., then precipitated by a known method such as sulfuric acid isoelectric precipitation, washed with water and dried. As a result, it can be collected as a crystal.

The fumaric acid produced is an aspartase-containing bacterium such as Brevibacterium flavum.
L-aspartic acid can be produced by reacting the AB-41 strain (FERM BP-1498), Escherichia coli ATCC 11303 and the like,
L-aspartic acid can also be efficiently produced from maleic acid by adding an aspartase-containing bacterium to the reaction system and reacting it.

Further, the fumaric acid produced is a fumarase-containing bacterium such as Brevibacterium flavum A.
B-41 strain (FERM BP-1498), E. coli ATCC11303 and the like can be produced to produce L-malic acid, and by adding a fumarase-containing bacterium to the reaction system and reacting it, It is also possible to efficiently produce L-malic acid from maleic acid.

In the present invention, "manufacturing fumaric acid from maleic acid" does not necessarily mean accumulating fumaric acid, but L-asparagine from maleic acid via fumaric acid as described above is used. It means that at least the conversion of maleic acid to fumaric acid has occurred, such as the production of acid or L-malic acid.

[0027]

The present invention will be described in more detail with reference to the following examples. [Example 1] Fumaric acid production under N ₂ atmosphere (1) Cultivation of microorganism having maleate isomerase activity Meat extract: 10 g, peptone: 10 g, NaCl: 5
g, maleic acid 10 g, and distilled water: 1000 ml (adjusted to pH 7.0 with caustic soda), 100 ml of a medium was dispensed into a 500 ml Erlenmeyer flask, and the temperature was kept at 120 ° C. at 20 ° C.
What was sterilized for a minute, and Alcaligenes faecalis
The IFO 12669 strain was inoculated and shake-cultured at 30 ° C. for 24 hours.

1000 ml of the same medium as described above was placed in a 3 L jar fermenter and sterilized at 120 ° C. for 20 minutes, and 30 ml of the shaking culture solution was inoculated,
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) Production of fumaric acid from maleic acid A reaction liquid which was previously stirred for 30 minutes while blowing N ₂ gas and replaced with N ₂ [maleic acid 116 g, 5N caustic soda 400
ml (total volume made up to 1000 ml with water)] was transferred to a 3 L jar fermenter, and the recovered bacterial cells (IFO 12669
Strain) was added, and N ₂ was supplied to the gas phase at a rate of 0.02 vvm, stirred, and reacted at 30 ° C. for 36 hours. The dissolved oxygen concentration was maintained at 0.5 ppm or less during the reaction. After the reaction was completed, the supernatant was collected by centrifugation, and the obtained reaction supernatant was used as an organic acid analysis column (SCR-1 manufactured by Shimadzu Corporation).
01H column), high performance liquid chromatography analysis using a UV detector (210 nm) (Shimadzu Corporation, LC-5)
It was subjected to A). As a result, it was confirmed that 66 g / L of fumaric acid was produced. The amount of fumaric acid produced is
Performed using pimelic acid as an internal standard. Also,
Fumaric acid was precipitated by lowering the pH of the above reaction supernatant to 3 with sulfuric acid. The obtained fumaric acid crystal was 63 g.

[Example 2] Fumaric acid production under Ar atmosphere (1) Cultivation of microorganism having maleate isomerase activity Alcaligenes faecalis IFO 12669 strain was used in Example 1
It culture | cultivated similarly to. (2) Production of fumaric acid from maleic acid A reaction solution in which Ar gas had been preliminarily stirred for 30 minutes while being blown with Ar gas [maleic acid 116 g, 5N caustic soda 40
0 ml (total volume to 1000 ml with water)] was transferred to a 3 L jar fermenter, and the recovered bacterial cells (IFO 1266
9 strains) (20 g) was added, Ar was supplied to the gas phase at a rate of 0.02 vvm, and the mixture was stirred and reacted in a seal at 30 ° C. for 36 hours. The dissolved oxygen concentration was maintained at 0.5 ppm or less during the reaction. Fumaric acid was obtained at 65 g / L, and fumaric acid crystals were obtained in the same manner as in Example 1. The crystals obtained were 62.
It was 5 g.

[Example 3] Fumaric acid production under He atmosphere (1) Cultivation of microorganism having maleate isomerase activity Alcaligenes faecalis IFO 12669 strain was used in Example 1
It culture | cultivated similarly to. (2) Production of fumaric acid from maleic acid A reaction liquid in which He gas was blown for 30 minutes while stirring and He substitution was performed [maleic acid 116 g, 5N caustic soda 40
0 ml (total volume to 1000 ml with water)] was transferred to a 3 L jar fermenter, and the recovered bacterial cells (IFO 1266
(9 strain) was added, and He was supplied to the gas phase at a rate of 0.02 vvm, stirred, and reacted in a seal at 30 ° C. for 36 hours. The dissolved oxygen concentration was maintained at 0.5 ppm or less during the reaction. Fumaric acid was obtained at 67 g / L, and fumaric acid crystals were obtained in the same manner as in Example 1. The crystals obtained were 63.
It was 5 g.

[Example 4] Fumaric acid production in reaction solution substituted with N ₂ (1) Cultivation of microorganism having maleate isomerase activity Alcaligenes faecalis IFO 12669 strain was used in Example 1
It culture | cultivated similarly to. (2) Production of fumaric acid from maleic acid A reaction liquid which had been N ₂ substituted by stirring while blowing N ₂ gas for 30 minutes in advance [maleic acid 116 g, 5N caustic soda 400
ml (total volume up to 1000 ml with water)] was transferred to a 3 L jar fermenter, 20 g of the bacterial cells (IFO 12669 strain) recovered in Example 1 was added and stirred, and the mixture was stirred at 30 ° C. for 3 days.
The reaction was carried out for 6 hours. During the reaction, the dissolved oxygen concentration is about 4p
It was maintained below pm. Fumaric acid was obtained in an amount of 52.4 g / L, and fumaric acid was precipitated by lowering the pH to 3 with sulfuric acid after centrifugation as usual. The obtained fumaric acid crystal was 49.2 g.

[Example 5] Production of fumaric acid in a reaction solution containing sodium sulfite (1) Cultivation of microorganism having maleate isomerase activity Alcaligenes faecalis IFO 12669 strain was used in Example 1
It culture | cultivated similarly to. (2) Production of fumaric acid from maleic acid Reaction solution [116 g of maleic acid, 400 m of 5N caustic soda
0.2 g / L for 1 (total volume is made up to 1000 ml with water)
Of sodium sulphite was added, and the mixture was transferred to a 3 L jar fermenter and recovered in Example 1 (IFO 12669 strain) 20.
g was added, the jar was sealed and stirred and reacted at 30 ° C. for 36 hours. During the reaction, the dissolved oxygen concentration is approximately 3 ppm
Maintained below. 54.5 g / L of fumaric acid was obtained,
After centrifugation as usual, fumaric acid was precipitated by lowering the pH to 3 with sulfuric acid. The obtained fumaric acid crystal was 51 g.

[Example 6] Fumaric acid production in degassed reaction solution (1) Cultivation of microorganism having maleate isomerase activity Alcaligenes faecalis IFO 12669 strain was used in Example 1
It culture | cultivated similarly to. (2) Production of fumaric acid from maleic acid Reaction solution [116 g of maleic acid, 400 m of 5N caustic soda
1 (total volume of 1000 ml with water)] was put in a pressure resistant bottle and degassed for 15 minutes while sucking with a water aspirator. After gently returning to normal pressure, the reaction solution was transferred to a 3 L jar fermenter, 20 g of the bacterial cells (Alcaligenes faecalis IFO 12669 strain) recovered in Example 1 was added, and the jar was sealed and stirred at 30 ° C. And reacted for 36 hours. The dissolved oxygen concentration was maintained below about 4 ppm during the reaction. Fumaric acid was obtained in an amount of 51.3 g / l, and fumaric acid was precipitated by lowering the pH to 3 with sulfuric acid after centrifugation in a conventional manner. The amount of fumaric acid crystals obtained was 48 g.

[Control Example] Fumaric acid production under normal air (1) Cultivation of microorganism having maleate isomerase activity Alcaligenes faecalis IFO 12669 strain was used in Example 1
It culture | cultivated similarly to. (2) Production of fumaric acid from maleic acid Reaction liquid in which N ₂ was not blown [maleic acid 116
400 ml of 5N caustic soda (total volume of water is 1000 m
1)]] to a 3L jar fermenter,
20 g of the recovered bacterial cells (IFO 12669 strain) was added, and the mixture was stirred without N ₂ sealing and reacted at 30 ° C. for 36 hours. Fumaric acid was obtained in an amount of 44 g / L, and fumaric acid crystals were obtained in the same manner as in Example 1. The crystals obtained were 41 g.

Reference Example 1 Production of Aspartic Acid (1) Cultivation of Microorganism Having Maleate Isomerase Activity Alcaligenes faecalis IFO 12669 strain was used in Example 1
It culture | cultivated similarly to. (2) Cultivation of a microorganism capable of producing aspartic acid from fumaric acid Urea: 4 g, (NH ₄ ) ₂ SO ₄ : 14 g, KH ₂ PO ₄ :
_{0.5g, K 2 HPO 4: 0.5g} , MgSO 4 · 7H 2
_{O: 0.5g, FeSO 4 · 7H} 2 O: 20mg, MnS
O ₄ · nH ₂ O: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 100 μg, yeast extract 1 g, casamino acid 1 g and distilled water: 100 ml medium consisting of 1000 ml (pH 6.6) was dispensed into a 500 ml Erlenmeyer flask. Then, sterilized at 120 ° C for 15 minutes, 4 ml of sterilized 50% aqueous glucose solution was added to inoculate Brevibacterium flavum AB-41 strain (FERM BP-1498), and shaken at 33 ° C for 24 hours. Cultured.

1000 ml of the same medium as described above was placed in a 2 L jar fermenter and sterilized at 120 ° C. for 20 minutes, and then 20 ml of the above shaking culture solution and 200 ml of sterilized 50% glucose aqueous solution were added.
The cells were cultured at 0 ° C for 24 hours. The obtained culture solution was centrifuged (8000 rpm, 15 minutes, 4 ° C.) to collect the cells. Contaminant malic acid by-productivity was removed from the obtained bacterial cells by the following method. That is, aspartic acid: 100 g, ammonia: 180 ml, calcium chloride: 2.2 g, Tw
The collected cells were suspended in a composition solution consisting of een20: 0.8 g (total volume of 1 L with water), shaken at 45 ° C for 3 hours, and centrifuged (8000 rpm, 15 minutes, 4 ° C) to remove the cells. Recovered.

(3) Aspartic acid production from maleic acid and ammonia: N ₂ gas was previously stirred for 30 minutes while stirring, and the reaction solution was N ₂ substituted [116 g of maleic acid and 153 ml of ammonia.
(To bring the total volume to 1000 ml with water)] was transferred to a 3 L jar fermenter, and both recovered bacterial cells (IFO 12669 strain 20 g, AB-41 strain 120 g) were added, and N ₂ was 0.02.
Feed at a speed of vvm, stir and stir in a seal at 30 ° C for 3
The reaction was carried out for 6 hours. After completion of the reaction, the supernatant was recovered by centrifugation, and the obtained reaction supernatant was subjected to thin-layer chromatography [developing solvent n-butanol: acetic acid: water = 4: 1: 1].
(Volume ratio), color former: ninhydrin reagent]. As a result, it was confirmed that 128 g / L of L-aspartic acid was produced. To this ammonium aspartate solution, sulfuric acid was added to adjust the pH to 3, aspartic acid was precipitated, washed with water and dried to obtain aspartic acid crystals. The crystals obtained were 125 g.

[Reference Example 2] Production of L-malic acid (1) Cultivation of microorganism having maleate isomerase activity Alcaligenes faecalis IFO 12669 strain was used in Example 1
It culture | cultivated similarly to. (2) Cultivation of microorganism having L-malic acid-forming ability from fumaric acid Urea: 4 g, (NH ₄ ) ₂ SO ₄ : 14 g, KH ₂ PO ₄ :
_{0.5g, K 2 HPO 4: 0.5g} , MgSO 4 · 7H 2
_{O: 0.5g, FeSO4 · 7H 2} O: 20mg, MnS
O ₄ · nH ₂ O: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 100 μg, yeast extract: 1 g, casamino acid: 1 g, and distilled water: 1000 ml (pH 6.6) 100 ml of a medium was divided into a 500 ml Erlenmeyer flask. Note,
Sterilized for 15 minutes at 120 ° C, 50% sterilized
Add 4 ml of glucose solution and add Brevibacterium
Inoculated with flavum AB-41 strain (FERM BP-1498),
The culture was performed at 33 ° C. for 24 hours with shaking.

Further, 1000 ml of the same medium as described above was added to 2 ml.
The mixture was placed in an L volume jar fermenter and sterilized at 120 ° C. for 20 minutes, 20 ml of the shaking culture solution and 200 ml of a sterilized 50% glucose aqueous solution were added, 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.
Contaminant aspartase and succinic acid by-productivity of this bacterium were removed by the following method. That is, disodium fumarate: 32 g, Tween 20: 0.8 g (total amount 1 with water)
L)) and the collected cells are suspended in a composition liquid consisting of
Shake for 2 hours, centrifuge (8000 rpm, 15 minutes,
The cells were recovered at 4 ° C).

(3) Production of L-malic acid from maleic acid A mixture of N ₂ -substituted reaction liquid was previously stirred with N ₂ gas blown therein for 30 minutes [116 g of maleic acid, 200 g of 5N sodium hydroxide (total amount to 1000 ml with water). )] Was transferred to a 3 L jar fermenter, and both cells recovered in Example 1 and (1) above (IFO 12669 strain 20 g, AB-
41 strains (50 g) were added, N ₂ was supplied at a rate of 0.02 vvm, and the mixture was stirred and reacted at 30 ° C. for 36 hours in a seal. L-malic acid was obtained in an amount of 104 g / L, and calcium chloride was added to L-sodium malate solution to precipitate calcium malate. After washing with water, L-malic acid was separated with an ion exchange resin and dried. , L-malic acid crystals were obtained. The crystals obtained were 90 g.

[0042]

According to the method of the present invention, the enzymatic method is used.
Fumaric acid can be efficiently produced in high yield from maleic acid.

[Procedure amendment]

[Submission date] May 9, 1996

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0009

[Correction method] Change

[Correction content]

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be specifically described below. The microorganism having maleate isomerase activity used in the present invention is not particularly limited as long as it is a microorganism having this activity, but is genus Alcaligenes, genus Pseudomonas, xanthomonas (Xa).
genus nthomanas, Bacillus
s) Microorganisms belonging to the genus are preferably used. Especially Alcaligenes fa
ecalis) (for example, IFO12669, IFO)
13111, IAM 1473), Alcaligenes eutrohus (ΛI caligenes eutro
Phus), Pseudomonas fluorescens (Ps
eudomonas fluolescens (eg ATCC 23728), Xanthomonas maltophile
ia) (eg ATCC 13270), Bacillus stearothermophilus (Bacillus stearus)
other mofilus) [for example, the same MI-10
1 strain (FERM BP-5160 )], Bacillus brevis (Bacillus brevis) [for example, the same MI-103 strain (FERM BP-5162 )] and the like are preferably used. Furthermore, there is no problem even if a mutant strain of the above microorganism or a modified strain obtained by gene recombination, a microorganism produced by introducing the maleate isomerase gene of the above microorganism into another microorganism, and the like are used.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12R 1:64) (C12P 7/46 C12R 1:05) (C12P 7/46 C12R 1:07) (C12P 7/46 C12R 1:08) (72) Inventor Hideaki Yukawa 8-3-1 Chuo, Ami-machi, Inashiki-gun, Ibaraki Mitsubishi Kagaku Co., Ltd. Tsukuba Research Center

Claims (5)

[Claims] 1. A fumaric acid is produced from maleic acid by an enzymatic isomerization reaction by mixing maleic acid with a microorganism having maleate isomerase activity or a preparation of the microorganism having maleate isomerase activity in an aqueous solution. In this case, a method for producing fumaric acid, characterized in that the dissolved oxygen concentration in the reaction solution is maintained at 4 ppm or less for the reaction. 2. The fumaric acid according to claim 1, wherein the dissolved oxygen concentration in the reaction solution is lowered by sealing the reaction solution with one or more gases selected from N ₂ , Ar and He. Manufacturing method. 3. The method for producing fumaric acid according to claim 1, wherein the dissolved oxygen concentration in the reaction solution is reduced by adding sulfite to the reaction solution or its raw material. 4. The method for producing fumaric acid according to claim 1, wherein the dissolved oxygen concentration in the aqueous solution containing maleic acid is reduced by degassing the reaction solution or its raw material. 5. The dissolved oxygen concentration in the reaction solution is 0.5 ppm.
The method for producing fumaric acid according to any one of claims 1 to 4, wherein: