JPH09131196A - New microorganism and its utilization - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a compound useful as a raw material for various kinds of specialty chemicals, etc., by treating an aqueous maleic acid solution with the cells or enzyme of a new microorganism belonging to the genus Arthrobacter, the genus Proteus, etc., and capable of producing an maleic acid isomerase. SOLUTION: The cells of a microorganism belonging to the genus Arthrobacter (e.g. Arthrobacter globiformis IAM 12102), a microorganism belonging to the genus Proteus (e.g. Proteus vulgaris 10B-7), etc., and capable of producing a maleic acid-isomerizing enzyme, or the crude or pure enzyme fraction of the maleic acid-isomerizing enzyme collected from the culture solution of the microorganism are added to a maleic acid-containing aqueous solution to isomerize the maleic acid into fumaric acid. Thus, the fumaric acid useful as a raw material for various kinds of specialty chemicals such as aspartic acid, malic acid and alanine is efficiently obtained.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel microorganism and a method for producing fumaric acid using the same. For more information,
Utilizing a novel microorganism that produces maleic acid isomerase,
The present invention relates to a method for producing fumaric acid useful as a raw material for various fine chemicals such as aspartic acid, malic acid and alanine by cis-trans isomerization of a double bond of a conjugated dicarboxylic acid.

[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, regarding the production of fumaric acid by an enzymatic reaction, maleate isomerase (maleate cis-trans-is) is used.
is known to isomerize maleic acid to fumaric acid. Microorganisms that produce maleate isomerase include, for example, Pseudomonas sp.
monas sp.) [K. Otsuka, Agric.Biol.Chem., vol.25, N
o.9, p.726 (1961)], Alcaligenes faecalis (Al
caligenes faecalis) IB-14 [Takamura, Agric.Biol.Ch
em., Vol.33, p.718 (1969)], Pseudomonas fluorescens ATCC23728 [Sc
her, J. Biol. Chem., Vol.244, p.1878 (1969)], but details of the enzymological properties of the maleic acid isomerase produced by these are extremely unknown. The situation is poor.

[0004]

The maleic acid isomerases known so far are only known to be produced by the above-mentioned several aerobic Gram-negative bacteria. However, due to the fact that the specific activity is very limited and the specific activity is low, the industrial application has not been sufficiently examined.

It is an object of the present invention to provide a novel microorganism which produces a maleic acid isomerase and a method for producing fumaric acid using these microorganisms.

[0006]

[Means for Solving the Problems] In order to solve the above problems, the present inventors have conducted extensive studies to separate a novel microorganism having maleic acid isomerase activity, and as a result, maleic acid was used as a carbon source. The present invention has been completed by successfully separating a microorganism that has grown and has maleic acid isomerase activity.

[0007] That is, the present invention is an earth lobactor (Ar
throbacter) and Proteus (genus Proteus) belonging to the genus, which produces maleic acid isomerase, or a crude enzyme fraction or purification of maleic acid isomerase collected from the culture of this microorganism Provided is a method for producing fumaric acid, which comprises adding an enzyme to an aqueous solution containing maleic acid to isomerize maleic acid to produce fumaric acid.

In the above method, the microorganism belonging to the genus Arthrobacter or the genus Proteus is Arthrobacter globiformi.
s), Arthrobacter pasc
ens), Arthrobacte
r sulfureus), Arthrobacter ureafaciens, and Proteus vulgaris. More specific strains include Arthrobacter globiformis.
IAM12102, EarthLobacter Passage IAM12343, EarthLobacter Sulfureus IAM1488, EarthLobacter Urea Fasciens IAM1637, EarthLobacter
SP 3B-2, Proteus bulgaris 10B-7
Is mentioned.

The present invention also provides a novel microorganism that produces maleic acid isomerase, Arthrobacter sp.
robacter sp.) 3B-2, and Proteus vulgaris 10B-7.

In the present application, "maleate cis-trans-isomerase (EC5.2.1.1)" means
It means an enzyme that catalyzes the reaction of isomerizing maleic acid (cis type) to form fumaric acid (trans type).

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail along with its embodiments. The method for producing fumaric acid of the present invention is a bacterium belonging to a genus selected from the genus Arthrobacter and the genus Proteus, and produces a maleic acid isomerase from a bacterial cell or a culture of this microorganism. It is characterized in that the collected crude enzyme fraction of maleic acid isomerizing enzyme or purified enzyme is added to an aqueous solution containing maleic acid to isomerize maleic acid to produce fumaric acid. Conventionally, a microorganism belonging to the genus Arthrobacter or the genus Proteus that produces maleic acid isomerase has not been known, but the presence of the microorganism was shown for the first time by the present invention.

The microorganism used in the above method belongs to the genus Arthrobacter or the genus Proteus and expresses maleic acid isomerase constitutively, or inducibly by an inducer or culture temperature. No matter what, it is not particularly limited as long as the cells or culture solution obtained by culturing in the medium have the enzyme activity. Such microorganisms can be isolated / obtained from the natural world or selected / obtained from existing preserved strains, for example, by the following method. (1) Separation of maleic acid-assimilating bacterium A soil sample collected from the nature is used as a medium containing maleic acid as a carbon source, for example, medium A [composition: maleic acid 10 g,
Yeast extract 10g, K ₂ HPO ₄ 2g, NaCl 1
_{g, MgSO 4 · 7H 2 O} 0.2g, tap water 1L (p
H7)] 10 ml, and aerobically shake culture or anaerobically static culture at 30 ° C. for 2 days. A part of the culture after the culturing is subcultured in the same medium A and the enrichment culture is performed in the same manner. After performing this operation twice, it is smeared on a plate medium having the composition of the above-mentioned medium A containing 2% of agar, and the amount is 2 to 4 at 30 ° C.
Culture for one day. The colonies grown on the plate medium are isolated and subjected to the following maleate isomerase activity detection test to select microorganisms having maleate isomerase activity. With respect to existing stock strains, maleic acid assimilating bacteria can be selected in the same manner. (2) Selection of microorganism having maleic acid isomerase activity The microorganism isolated in (1) above is used in the above medium A and 3
After culturing with shaking at 0 ° C. for 1 day, the cells are recovered by centrifugation. Then, the obtained bacterial cells were suspended in 0.1M phosphate buffer (pH 7) containing 2-mercaptoethanol (5 mM) and dithiothreitol (1 mM), and the bacterial cells were disrupted by an ultrasonic disruptor, The crushed product is centrifuged and the supernatant is collected and used as an enzyme solution. This enzyme solution was used as reaction solution A [composition: sodium maleate 50 mM, dithiothreitol
1 mM, Tris-HCl buffer 100 mM (pH 8)], and reacted at 30 ° C. for 1 to 24 hours. By confirming the formation of fumaric acid in the reaction solution after the reaction, a microorganism having maleate isomerase activity can be selected.

The method for confirming fumaric acid is not particularly limited, but HPLC analysis in which an organic acid analysis column is used to detect with an ultraviolet detector, paper chromatography, or enzymes such as fumarase, malate dehydratase, and aspartase are used. It can be confirmed by an enzymatic method or the like.

The mycological properties of the Arthrobacter sp. 3B-2 strain and the Proteus vulgaris 10B-7 strain thus isolated from nature by the present inventor are shown below. All of these are presumed to be novel microorganisms. A) 3B-2 strain 1. Morphological properties Morphology: Bacillus (coryneform) Spores: Not formed Gram stainability: Positive Motility: Yes 2. Physiological properties Attitude toward oxygen: Aerobic catalase: Positive oxidase: Negative dextrose (anaerobic): Negative dextrose (under aerobic): Positive urease: Negative β-galactosidase: Negative β-glucosidase: Positive phosphatase: Negative glucose fermentation: Positive Use of sugar: ・ Fructose: Positive ・ Maltose: Positive ・ Sucrose: Positive ・ Lactose: Negative ・ Xylose: Positive ・ Arabinose: Positive ・ Galactose: Negative ・ Mannose: Negative ・ Mannitol: Negative ・ Starch: Positive ・ Inulin: Positive ・Raffinose: Negative ・ Sorbitol: Negative ・ Trehalose: Negative ・ Salicin: Negative Use of amino acids ・ Phenylalanine: Negative ・ Arginine: Negative ・ Lysine: Negative ・ Ornithine: Negative ・ High-grade Minic acid: Positive Gelatin liquefaction: Positive Utilization of citric acid: Negative foss-proscar well test: Negative Indole production: Negative Growth at 30 ° C: Positive Growth at 37 ° C: Positive Growth at 41 ° C: Negative at 45 ° C Growth: Negative Analysis of cell wall components: -Mycolic acid: None-Diamino acid: Lysine-Fatty acid: Iso and anteisobranched maleic acid isomerase activity: Positive The above-mentioned mycological properties are described in Cowan and Steel's Manual for
the Identificationof Medical Bacteria, 3rd Eds. [C
ambridge Univ. Press, UK, (1993)] and Bergey's M
anual of Systematic Bacteriology Vol.2, [PHASne
ath, NSMair, MESharpe and JGHol (1986) William
& Wilkins]), it was found to be a novel strain belonging to Arthrobacter (genus Arthrobacter). The present inventors have named this bacterium as Arthrobacter sp. 3B-2 strain. The bacterium has been deposited at the Institute of Biotechnology, Institute of Biotechnology under the deposit number: FERM P-15241. B) 10B-7 strain 1. Morphological properties Morphology: Bacillus spores: Not formed Gram stainability: Negative Motility: Yes (swimming) 2. Physiological properties Attitude toward oxygen: Facultative anaerobic Growth under anaerobic: Growing catalase: Positive oxidase: Negative phenylalanine deaminase: Positive urease: Positive β-galactosidase: Negative arginine galactose: Negative regina carboxylase negative: : Negative triptophan aminase: Positive cytochrome oxydase: Negative glucose fermentation: Positive Utilization of citric acid: Negative fogz-proscarwell test: Negative hydrogen sulfide production: Positive indole production: Positive gelatin liquefaction: Positive acid production: Glucose and sucrose Produced from mannitol, inositol, sorbitol, rhamnose, melibiose, L-(+)-arabinose . A) Growth at 30 ° C. - Growth at positive 41 ° C. - Growth at positive 45 ° C.: Negative maleate isomerase activity: positive

The above-mentioned mycological properties are confirmed by Cowan and Steel '
s Manual for the Identificationof Medical Bacteri
a, 3rd Eds. [Cambridge Univ. Press, UK, (1993)] and Bergey's Manual of Systematic Bacteriology Vo.
l.1, (Noel R. Krieg and JGHolt (1984) Williams & W
ilkins]) and found to be a new strain belonging to Proteus vulgaris. The present inventors named this bacterium Proteus bulgaris 10B-7 strain. In addition, this bacterium has been deposited at the Institute of Biotechnology and Industrial Science with the deposit number: FERM P-15242.

[0016] Further, when the same separation operation as above was carried out from the preserved strains preserved in the strain preservation organization, Arthrobacter globiformis IAM12102, Arthrobacter pasense IAM12343, Arthrobacter sulphureus IAM1488, Arthrobacter Bactor Urea Faciens I
AM1637 was found to produce maleic acid isomerase.

An example of a method for producing a maleic acid isomerizing enzyme using the above microorganism and isomerizing maleic acid to produce fumaric acid will be described below.

(3) Production of maleic acid isomerase and production of fumaric acid from maleic acid using maleic acid isomerase Microorganism having maleic acid isomerase activity separated as described in (2) above To isomerize maleic acid to form fumaric acid by adding a crude enzyme fraction or purified enzyme of maleic acid isomerizing enzyme collected from bacterial cells or a culture of this microorganism to an aqueous solution containing maleic acid. You can Here, the culture refers to the microbial cells and / or the culture medium after the culture (the culture supernatant when a liquid medium is used as the culture medium).

As the maleic acid isomerase used in the isomerization reaction of maleic acid to fumaric acid of the present invention, not only the cells separated from the culture solution in which the above-mentioned cultured cells are cultured but also the culture solution, A crushed body or an extract can also be used. Further, a crude enzyme preparation obtained from microbial cells may be used, or a further purified enzyme preparation may be used. Furthermore, those obtained by immobilizing the above-mentioned microbial cells, their crushed or extracted products, or purified enzymes on a carrier can also be used. In the present invention, "the addition of a crude enzyme fraction of maleic acid isomerase or a purified enzyme to a maleic acid-containing aqueous solution to a microbial cell" means an aqueous solution containing a microbial cell or a maleic acid isomerase. It is a concept including adding maleic acid to the solution and further passing an aqueous solution containing maleic acid through the column packed with the immobilized cells or immobilized enzyme as described above.

Cultivation of a microorganism having maleic acid 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 and glucose. , Sugars such as sucrose, fructose, maltose, alcohols such as ethanol and methanol, organic acids such as citric acid and maleic acid, molasses, etc., preferably maleic acid or a mixture of maleic acid and other carbon sources. To be Examples of the nitrogen source include organic nitrogen sources such as yeast extract, peptone, meat extract, casamino acid, corn steep liquor, and urea, or inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate, and preferably yeast extract. Used alone or as a mixture. Further, as the inorganic salt, for example, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and the like are used. In addition, nutrients of various vitamins such as biotin and thiamin can be added to the medium. Further, 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 culturing conditions are usually maleic isomerase activity under aerobic conditions such as aeration and stirring and shaking in the case of aerobic bacteria, and under anaerobic conditions by static culture in the case of anaerobic bacteria. There is no particular limitation as long as it is a temperature at which microorganisms can grow,
Also, 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.

The bacterial cells containing the maleic acid isomerase can be obtained by collecting the bacterial cells from the thus obtained culture by centrifugation or the like. As a method for extracting and purifying maleate isomerase from microbial cells, any of the known enzyme purification methods can be applied. For example, as a method for disrupting the cells, ultrasonic disruption, mechanical disruption using a French press, a homogenizer, or the like, or enzymatic disruption using lysozyme or the like can be used. The soluble fraction of the cell destruction thus obtained or the fraction thereof 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. Purification of maleic acid isomerase from the crude enzyme fraction is usually carried out by (a) a precipitation method, for example, ammonium sulfate precipitation method, (b) a chromatography method, for example, ion exchange chromatography,
It can be performed by affinity adsorption chromatography, gel filtration chromatography, etc., (C) a separation method by an electrophoretic method, and any combination of these methods. One example is described in detail in Test Example 1.

The maleic acid isomerase-containing fraction in the above-mentioned purification process can be detected by measuring the maleic acid isomerase activity according to the method of Otsuka et al. [Agric. Biol. Chem., Vol. 25, p. 72].
6 (1961)], it can be carried out by measuring the decrease in the substrate maleic acid as the decrease in absorbance at 240 nm in the presence of fumarase derived from pig heart. The maleic acid isomerase activity can also be measured by the W. Scher method. That is, the purified maleic acid isomerase was added to the reaction solution [composition: 200 mM potassium phosphate buffer (pH 7.2), 0.5 mM dithiothreitol, 10 mM].
mM disodium maleate], in addition, by measuring the rate of fumaric acid production at each temperature as the rate of increase in absorbance at 290 nm using a spectrophotometer for 10 minutes,
The specific activity of maleic acid isomerase can be determined.
Furthermore, maleic acid isomerase was added to the reaction solution [composition: sodium maleate 50 mM, dithiothreitol 1 m.
M, tris-hydrochloric acid buffer solution 100 mM (pH 8)] and then high performance liquid chromatography analysis (detection at 240 nm) using an organic acid analysis column can also be performed to measure the maleic acid isomerase activity. it can.

The purity and molecular weight of the purified maleic acid isomerase are determined by the Davis method [BJ Davis, Ann. NY Acad.
Sci., Vol.121, p.404 (1964)], or Laemmli method [UK Laemmli, Nature, Vol.227, p.680 (1970)], polyacrylamide gel electrophoresis is performed, and the gel is subjected to, for example, Coomassie Blue dye solution [Composition: 0.2% (v
/ V) Coomassie Brilliant Blue R250, 40%
(V / v) methanol, 10% (v / v) acetic acid] or a commercially available silver staining kit (for example, Wako Pure Chemical Industries, Ltd.) to stain and detect the protein.

When the cells of the microorganism producing a maleic acid isomerase obtained as described above are immobilized on a carrier, they may be recovered from the culture, or may be used in an appropriate buffer solution such as 0.02. ~ 0.2M phosphate buffer (p
H6-10) and the like can be used. Further, a crude enzyme fraction of maleic acid isomerase, which has been extracted and purified from microbial cells and immobilized on a carrier, can also be used in the method for producing fumaric acid of the present invention. .

Immobilization of these microbial cells, cell crushed products, extracts or purified products is carried out on a suitable carrier such as acrylamide monomer, alginic acid, or carrageenan according to a commonly used method known per se. It can be performed by a method.

The aqueous solution used for the isomerization reaction of maleic acid is an aqueous solution containing maleic acid or a suitable buffer solution,
For example, about 0.02-0.2M Tris-HCl buffer (p
H7 to 9). When the cells are used for the reaction as they are, toluene, xylene, a nonionic surfactant or the like is added to the reaction solution in an amount of 0.05 to 2% (w) in order to further increase the permeability of the cell membrane of the cells. / V) can also be added.

The concentration of maleic acid as a reaction raw material in the aqueous solution is preferably about 0.01 to 2M. As the form of maleic acid, not only the form of acid or salt but also its anhydride can be used. Maleic anhydride is easily converted to maleic acid by converting it into an aqueous solution. Examples of the maleic acid salt include a sodium salt, a potassium salt, an ammonium salt and the like.

The enzyme reaction temperature and pH in the above aqueous solution are not particularly limited, but are usually 20 to 40 ° C., preferably 30 ° C., and the pH of the reaction solution is 5 to 1
It can be 0, preferably around 6-9. Also,
The pH can be adjusted by adding an acid or an alkali.

The aqueous solution containing fumaric acid thus obtained can be used as it is as a raw material for producing various useful substances such as fine chemicals. For example, in the same reaction tank in which maleic acid is isomerized or in a separate reaction tank, various enzymes having fumaric acid as a substrate or cells containing these, for example, fumarase, aspartase, etc. or cells containing these enzymes It is also possible to produce malic acid, aspartic acid, alanine and the like by adding.

For example, after the maleic acid isomerase of the present invention is allowed to act on an aqueous solution containing maleic acid, aspartate (EC 4.3.1.1) is allowed to act in the presence of ammonia ions to generate aspartic acid. You can Furthermore, alanine can be produced by allowing aspartic acid decarboxylase to act on the aqueous solution containing aspartic acid.

Malic acid can be produced by reacting fumarase with an aqueous solution containing fumaric acid produced by the maleic acid isomerase of the present invention.
Aspartase, aspartate decarboxylase and fumarase used herein can be of any biological origin as long as they have these enzyme activities, and cell bodies containing these enzymes or As long as an enzymatic reaction can be carried out, such as a crude enzyme fraction of the enzyme or a purified enzyme, the enzyme can be actuated by any known method.

The fumaric acid produced by the above method is separated from the cells and the treated product thereof by ultrafiltration membrane separation, centrifugation, etc. and then precipitated by a known method such as sulfuric acid isoelectric precipitation. Further, it can be collected as crystals by washing with water and drying.

[0034]

【Example】

[Example 1] Separation of microorganisms having maleic acid isomerase activity (1) Separation of maleic acid-assimilating microorganisms Soil samples collected from nature were used as medium A [composition: maleic acid 10 g, yeast extract 10 g, K ₂ HPO ₄ 2
g, NaCl 1 g, MgSO ₄ .7H ₂ O 0.2 g, tap water 1 L (pH 7)] 10 ml, and aerobically shake culture or anaerobically static culture at 30 ° C. for 2 days. . 1 ml of this culture was subcultured into 10 ml of the above-mentioned medium A, and further cultured for 2 days. The operation of this enrichment culture is 2
After repeating the operation, it was smeared on a plate medium having the composition of the above-mentioned medium A containing 2% of agar and cultured at 30 ° C. for 2 to 4 days. Colonies that grew on this plate medium were isolated.

Similarly, an existing preserved strain (obtained from Institute for Applied Microbiology (IAM) of the University of Tokyo) was cultured to select a maleic acid-assimilating microorganism. (2) Selection of microorganism having maleic acid isomerase activity The microorganism isolated in (1) above was shaken for 1 day at 30 ° C. or statically cultivated with 5 ml of the above-mentioned medium A, and centrifuged ( The cells were collected at 3,000 xg, 4 ° C, 20 minutes). Then, the obtained bacterial cells were suspended in 1 ml of a 0.1 M phosphate buffer (pH 7) containing 2-mercaptoethanol (5 mM) and dithiothreitol (1 mM), and an ultrasonic disrupter (Cosmo Bioruptor UCD-200T) was used. ), The cells were disrupted (4 ° C., 15 minutes), the disrupted product was centrifuged, and the supernatant was collected to obtain an enzyme solution.

To detect the maleic acid isomerase activity, 0.5 ml of the reaction solution A [composition: sodium maleate 50 mM, dithiothreitol 1 mM, Tris hydrochloric acid buffer 100 mM (pH 8)] was added to the enzyme solution obtained above. The resulting reaction solution was added to the organic acid analysis column [SequeTag ™ (0.5 × 25 cmMi
lliGen / Biosearch, USA], UV detector (240n
m) using high performance liquid chromatography analysis (device:
LC-5A manufactured by Shimadzu Corp., mobile phase: 35 mM methanol solution containing 5 mM KH ₂ PO ₄ and 5 mM H ₃ PO ₄ ). Among the microorganisms isolated in (1) above, the microorganism having maleic acid isomerase activity is
It was selected as a microorganism having a fumaric acid peak in addition to the raw maleic acid peak.

6 strains of microorganisms having maleic acid isomerase activity obtained by the above-mentioned separation operation, Arthrobacter
Arthrobacter globiformis IAM121
02, Arthrobacter pasce
ns) IAM12343, Arthrobacter Sulfleus (Arth
robacter sulfureus) IAM1488, Arthrobacter ureafaciens IAM163
7 and 2 unidentified strains isolated from nature were obtained.
Of these, two strains isolated from nature were further examined for their mycological properties as in the previous period, and as a result, they were found to be novel strains, and Arthrobacter sp. , Proteus vulgaris 10B-7 strain.

Example 2 Production of Fumaric Acid by Arthrobacter sp. 3B-2 Strain 3B-2 strain isolated in Example 1 was inoculated into the above-mentioned medium A, and 30 The cells were shaken at 1 ° C for 1 day. After culturing, cells were collected from the culture solution by centrifugation (3,000 xg, 4 ° C, 20 minutes), and the obtained cells were treated with 2-mercaptoethanol (5 mM) and dithiothreitol (1 mM). After suspending in 1 ml of 0.1 M phosphate buffer (pH 7),
An enzyme solution was prepared in the same manner as in Example 1 using an ultrasonic crusher. The obtained enzyme solution was added to 0.5 ml of the above reaction solution A and reacted at 30 ° C. for 3 hours. After completion of the reaction, the reaction solution was subjected to high performance liquid chromatography analysis as in the previous case. As a result, the presence of fumaric acid was confirmed, and the maleic acid isomerase activity was 204 U / L culture solution. The enzyme activity of 1 U as used herein means the amount of enzyme that produces 1 μmol of fumaric acid in 1 minute.

[Example 3] Production of fumaric acid by Arthrobacter globiformis strain IAM12102 The above-mentioned medium A was inoculated with the strain IAM12102 isolated in Example 1 at 30 ° C for 1 day. Shake culture was performed. After culturing, cells were collected from the culture solution by centrifugation (3,000 xg, 4 ° C, 20 minutes), and the obtained cells were treated with 2-mercaptoethanol (5 mM) and dithiothreitol (1 mM). After suspending in 1 ml of 0.1 M phosphate buffer (pH 7),
An enzyme solution was prepared in the same manner as in Example 1 using an ultrasonic crusher. The obtained enzyme solution was added to 0.5 ml of the above reaction solution A and reacted at 30 ° C. for 3 hours. After completion of the reaction, the reaction solution was subjected to high performance liquid chromatography analysis as in the previous case. As a result, the presence of fumaric acid was confirmed, and the maleic acid isomerase activity was 142 U / L culture solution. The enzyme activity of 1 U as used herein means the amount of enzyme that produces 1 μmol of fumaric acid in 1 minute.

[Example 4] Production of fumaric acid by Arthrobacter pascens IAM12343 strain The above-mentioned medium A was inoculated with the IAM12343 strain isolated in Example 1 at 30 ° C for 1 day. Shake culture was performed. After culturing, cells were collected from the culture solution by centrifugation (3,000 xg, 4 ° C, 20 minutes), and the obtained cells were treated with 2-mercaptoethanol (5 mM) and dithiothreitol (1 mM). After suspending in 1 ml of 0.1 M phosphate buffer (pH 7),
An enzyme solution was prepared in the same manner as in Example 1 using an ultrasonic crusher. The obtained enzyme solution was added to 0.5 ml of the above reaction solution A and reacted at 30 ° C. for 3 hours. After completion of the reaction, the reaction solution was subjected to high performance liquid chromatography analysis as in the previous case. As a result, the presence of fumaric acid was confirmed, and the maleic acid isomerase activity was 104 U / L culture solution. The enzyme activity of 1 U as used herein means the amount of enzyme that produces 1 μmol of fumaric acid in 1 minute.

[Example 5] Production of fumaric acid by Arthrobacter sulfureus IAM1488 strain [0041] The above-mentioned medium A was inoculated with the IAM1488 strain isolated in Example 1, and the mixture was incubated at 30 ° C for 1 day. Shake culture was performed. After culturing, cells were collected from the culture solution by centrifugation (3,000 xg, 4 ° C, 20 minutes), and the obtained cells were treated with 2-mercaptoethanol (5 mM) and dithiothreitol (1 mM). After suspending in 1 ml of 0.1 M phosphate buffer (pH 7),
An enzyme solution was prepared in the same manner as in Example 1 using an ultrasonic crusher. The obtained enzyme solution was added to 0.5 ml of the above reaction solution A and reacted at 30 ° C. for 3 hours. After completion of the reaction, the reaction solution was subjected to high performance liquid chromatography analysis as in the previous case. As a result, the presence of fumaric acid was confirmed, and the maleic acid isomerase activity was 125 U / L culture solution. The enzyme activity of 1 U as used herein means the amount of enzyme that produces 1 μmol of fumaric acid in 1 minute.

[Example 6] Production of fumaric acid by Arthrobacter ureafaciens strain IAM1637 The above medium A was inoculated with the strain IAM1637 isolated in Example 1, and the mixture was incubated at 30 ° C. Culture was carried out with shaking for 1 day. After culturing, cells were collected from the culture solution by centrifugation (3,000 xg, 4 ° C, 20 minutes), and the obtained cells were treated with 2-mercaptoethanol (5 mM) and dithiothreitol (1 mM). After suspending in 1 ml of 0.1 M phosphate buffer (pH 7),
An enzyme solution was prepared in the same manner as in Example 1 using an ultrasonic crusher. The obtained enzyme solution was added to 0.5 ml of the above reaction solution A and reacted at 30 ° C. for 3 hours. After completion of the reaction, the reaction solution was subjected to high performance liquid chromatography analysis as in the previous case. As a result, the presence of fumaric acid was confirmed, and the maleic acid isomerase activity was 87 U / L culture solution. The enzyme activity of 1 U as used herein means the amount of enzyme that produces 1 μmol of fumaric acid in 1 minute.

[Embodiment 7] Proteus bulgaris (Pr
Production of fumaric acid by strain oteus vulgaris) 10B-7 The medium 10 was inoculated with the strain 10B-7 isolated in Example 1 and statically cultured at 30 ° C for 1 day. After culturing, cells were collected from the culture solution by centrifugation (3,000 xg, 4 ° C, 20 minutes), and the obtained cells were treated with 2-mercaptoethanol (5 mM) and dithiothreitol (1 mM). After suspending in 1 ml of a 0.1 M phosphate buffer solution (pH 7) contained therein, an enzyme solution was prepared in the same manner as in Example 1 using the ultrasonic crusher. The obtained enzyme solution is used as the reaction solution A 0.5 m
It was added to 1 and reacted at 30 ° C. for 3 hours. After completion of the reaction, the reaction solution was subjected to high performance liquid chromatography analysis as in the previous case. As a result, the presence of fumaric acid was confirmed, and the maleic acid isomerase activity was 8 U / L culture solution. still,
The enzyme activity of 1 U as used herein means the amount of enzyme that produces 1 μmol of fumaric acid in 1 minute.

[0044]

By the method of the present invention, fumaric acid can be efficiently produced from maleic acid. Further, the novel microorganism of the present invention produces a maleic acid isomerase and can be suitably used in the above method.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12R 1:37) (C12N 1/20 C12R 1:06) (C12N 1/20 C12R 1:37)

Claims (5)

[Claims] 1. A genus selected from the genus Arthrobacter and the genus Proteus,
A microbial cell producing a maleic acid isomerase, or a crude enzyme fraction or a purified enzyme of a maleic isomerase collected from a culture of this microorganism is added to an aqueous solution containing maleic acid, and maleic acid is added. A method for producing fumaric acid, which comprises isomerizing to produce fumaric acid. 2. The microorganism is Arthrobacter globiformis, Arthrobacter pascens, Arthrobacter sulfureus.
s), Arthrob, Urea Fasciens
acter ureafaciens), Proteus bulgaris (Prote
us vulgaris). 1.
The described method. 3. The microorganisms are Arthrobacter globiformis IAM12102 and Arthrobacter pascens IAM1.
The method according to claim 1, which is selected from 2343, Arthrobacter sulphreus IAM1488, Arthrobacter ureafaciens IAM1637, Arthrobacter sp. 3B-2, and Proteus vulgaris 10B-7. 4. Arthrobacter sp. 3B-2 which produces maleic acid isomerase. 5. Proteus vulgaris 10B-7 which produces a maleic acid isomerase.