JP3450663B2 - Method for producing S, S-ethylenediamine-N, N'-disuccinic acid - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing S, S-ethylenediamine-N, N′-disuccinic acid from ethylenediamine and fumaric acid by the action of a microorganism.
S, S-ethylenediamine-N, N'-disuccinic acid has a unique property of capturing heavy metals, and is susceptible to biodegradation after being released to nature, so that it can be used as a chelating agent or a detergent builder. Expected. [0002] Stereoisomer mixtures of ethylenediamine-N, N'-disuccinic acid (S, S-, R, R-, meso)
Isomer from maleic acid and ethylenediamine by an organic synthetic method (see U.S. Pat. No. 3,158,635), and its optically active S, S-isomer is obtained from L-aspartic acid and dibromoethane [John A . Neal et al. Inor
ganic Chem. 7 , 2405 (1968)]. However, in the production method from L-aspartic acid and dibromoethane, the production raw materials are relatively expensive,
It is difficult to supply a cheap and versatile optically active substance. On the other hand, regarding production by microorganisms, S, S
-Ethylenediamine-N, N'-disuccinic acid was obtained from the culture of actinomycete MG417-CF17 phospholipase C
Has been isolated and identified as a specific inhibitor of T. Nishiki
ori et al., J. Antibiotics 37, 426 (1984)].
However, the productivity of this actinomycete is extremely low, making it difficult to achieve an industrial production method. On the other hand, the present inventors have previously made efficient use of the catalytic action of microorganisms to efficiently convert optically active diaminoalkylene-N, N from fumaric acid and various diamines. A new method for producing '-disuccinic acid and the like has been proposed (see Japanese Patent Application Laid-Open No. 9-140390). An object of the present invention is to improve the reaction yield of S, S-ethylenediamine-N, N'-disuccinic acid in the method. Means for Solving the Problems The inventors of the present invention have made intensive studies to solve this problem, and as a result, it has been found that S, S-ethylenediamine-N, N'-disuccinic acid formed in a reaction system is generated. Have been found to be able to dramatically improve the reaction yield of S, S-ethylenediamine-N, N'-disuccinic acid by the presence of a metal ion capable of coordinating and forming a complex, and reached the present invention. [0006] That is, the present invention provides a method for producing S, S-ethylenediamine from a mixture of ethylenediamine and fumaric acid by the action of a microorganism having lyase activity or the treated product.
In producing N, N'-disuccinic acid, the reaction system is composed of an alkaline earth metal (excluding magnesium) , iron,
A process for producing S, S-ethylenediamine-N, N'-disuccinic acid, characterized by the presence of ions of at least one metal selected from the group consisting of zinc, copper, nickel, aluminum, titanium and manganese. . [0007] The reaction mechanism of the present invention, which has the above-described point, is considered as follows. First of all,
S, S-ethylenediamine-N, N'-disuccinic acid is produced from fumaric acid and ethylenediamine by the action of a microorganism having lyase activity or the treated product, and then the produced S, S-ethylenediamine-N, N'-disuccinic acid is produced. The acid is more strongly coordinated to a metal ion present in the reaction system than fumaric acid or ethylenediamine as a substrate to form a stable complex, so that the chemical equilibrium point moves to the product side. That is, the addition of an equilibrium reaction to form a stable complex to an equilibrium reaction in which S, S-ethylenediamine-N, N'-disuccinic acid is generated from fumaric acid and ethylenediamine gives rise to free S, S-ethylenediamine-
It is presumed that the total yield of N, N'-disuccinic acid and its metal complex is improved as compared with the case where no metal ion is present. BEST MODE FOR CARRYING OUT THE INVENTION The metal ion in the present invention is
Alkaline earth metals (except magnesium) ,
Ions of iron, zinc, copper, nickel, aluminum, titanium and manganese, such as Ca (II) , Sr
(II), Ba (II), Fe (II), Fe (III), Zn (II)),
Cu (II), Ni (II), Al (III), Ti (IV) and Mn
(II) ions and various complex ions thereof. Examples of the ion sources of these metals include hydroxides and oxides of these metals, inorganic or organic acid salts such as sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, carbonic acid and acetic acid; Examples include compounds with fumaric acid and ethylenediamine, which are substrates of the invention. These compounds can be used as a mixture of two or more kinds. [0010] Some of these metal compounds have low or low solubility in water. S-ethylenediamine-N, N'-
It can be used because a considerable amount is solubilized by the coordination ability of disuccinic acid. That is, the compound as the “metal ion” source of the present invention is used as long as the metal ion coordinates with S, S-ethylenediamine-N, N′-disuccinic acid and the effects of the present invention can be obtained. it can. As described above, the present invention is based on the movement of the chemical equilibrium point from the substrate side to the product side by metal ions, but the chemical equilibrium point is generally not affected by the type of catalyst. Therefore, the value of the chemical equilibrium point in the present invention changes only depending on the type of metal ion, and shows a constant value for all catalysts unless side reactions or other reactions are involved. This is demonstrated by the examples and reference examples of the present invention. That is, the lyase which is the catalyst of the present invention is not particularly limited as long as it is derived from any microorganism as long as it has the ability to produce S, S-ethylenediamine-N, N'-disuccinic acid. Examples of microorganisms having lyase activity include the genus Burkholderia, the genus Acidovorax, and the genus Pseudomoas.
nas), Paracoccus, Sphingomonas and Brevimonosa (Brev)
undimonas). Specifically, Burkholderia sp. Strain KK-5 [FERM BP-5412] and KK-9 strain [FER
MBP-5413], Acidovorax sp. TN-51 strain [FERM BP-5416], Pseudomonas sp. TN
-131 strain [FERM BP-5418], Paracoccus
sp. KK-6 strain [FERM BP-5415], Sphing
omonas sp. TN-28 strain [FERM BP-541
9], Brevundimonas sp. TN-30 strain [FERM B
P-5417] and the TN-3 strain [FERM BP-
5886]. [0014] These bacteria are newly isolated from nature by the present inventors and deposited under the above numbers with the Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry. The mycological properties of this bacterium are as follows. Mycological Properties KK-5 Strain KK-9 Strain Morphology Bacillus Bacillus Gram Staining − − Spore − − Motility ++ Flagella Poliophyta Atmospheric Oxygen Aerobic Aerobic Oxidase ++ Catalase ++ OF Test O O Quinone Q-8 Q-8 Cleavage of protocatechinic acid Ortho-type Ortho-type fluorescent dye--Nitrate reduction-+ Indole formation--Arginine dihydrolase--Urea decomposition--Esculin decomposition--Gelatin liquefaction--PNPG +-Acid production from xylose + + + Utilizing glucose + + L-arabinose + + D-mannose + + D-mannitol + + Maltose--Potassium gluconate + + n-capric acid + + Adipic acid--dl- Malic acid ++ citric acid ++ phenyl acetate ++ [0017] [0018] [0019] Strain TN-30 Strain TN-3 Strain Morphology Bacillus Bacillus Gram-staining--Spores--Motility ++ Flagella Polar hair Polar hair Oxygen attitude aerobic aerobic oxidase ++ catalase ++ OF test- Color tone Characteristic colorant Characteristic colorant does not generate and does not generate Fluorescent dye generation --- PHB accumulation ++ auxotrophy ++ quinone system Q-10 Q-10 Nitrate reduction ++ indole formation --- arginine dihydrolase --Urea decomposition--Esculin decomposition--Gelatin liquefaction--PNPG--Utilization glucose--L-arabinose--D-mannose--D-mannitol--N-acetyl---D-glucosamine maltose-- Potassium gluconate ++ n-capric acid--adipic acid ++ dl-malic acid-+ Enoic acid + + Phenyl acetate - - [0021] The above bacteriological properties, Bergey's Manual of S
ystematic Bacteriology Vol. 1 (1984) and Bergey's M
When classified according to the 9th edition of the anual of Determinative Bacteriology (1994), the KK-5 strain and the KK-9 strain belong to the genus Burkholderia, the TN-51 strain belongs to the genus Acidovorax, and the TN-131 strain. Belongs to the genus Pseudomonas, Bergey's Manual of S
According to ystematic Bacteriology Vol. 1 (1984), the KK-6 strain belongs to the genus Paracoccus, and Berge
y's Manual of Determinative Bacteriology 9th edition (199
When classified according to 4) and Microbiol. Immunol. 34, 99 (1990), the TN-28 strain is a Sphingomona strain.
s) In the genus, also in Bergey's Manual of Determinative Ba
cteriology 9th edition (1994) and Int. J. Syst. Bacteriol.
When classified according to 44, 499 (1994), strains TN-30 and TN-3 were identified as bacteria belonging to the genus Brevundimonas. Fumarase widely present in the living world hydrates fumaric acid, which is a substrate of the present reaction, to malic acid and causes a reduction in yield. Therefore, the fumarase activity of the strain used is weak or easily. It is desirable to remove or inhibit, except when inactivating. For example, from the cell lysate,
A method of removing by a technique such as chromatography, salting out, electrophoresis, a method of inhibiting with an inhibitor, a method of inactivating fumarase in the form of cells [I. Umehara et al., Appl Mi
crobiol Biotechnol 20, 291 (1984) and Yukawa et al., Agricultural Chemistry 59, 279 (1985)]. Next, a general embodiment of the present invention will be described. The culture medium of the microorganism used in the present invention is not particularly limited, and a synthetic medium as long as it appropriately contains assimilable carbon sources, nitrogen sources, inorganic salts, and a trace amount of organic nutrients. Any of natural media may be used. In the culture, addition of amino acids such as ethylenediamine-N, N'-disuccinic acid, ethylenediamine-N-monosuccinic acid, aspartic acid, glutamic acid, histidine and the like, and fumaric acid to the medium has a high desired activity. It is preferable because cells can be obtained. Culture conditions vary depending on the cells and medium, but the pH of the medium is in the range of 4 to 10, preferably 6 to 9, and the culture temperature is 20 to 45 ° C, preferably 25 to 35 ° C.
The reaction may be performed aerobically for 1 to 10 days, and cultured for 1 to 10 days until the activity is maximized. Generally, S, S-ethylenediamine-N,
The N'-disuccinic acid production reaction is carried out in an aqueous solution containing the above-mentioned metal compound and ethylenediamine and fumaric acid, by treating the cells or the treated cells (dried cells, crushed cells, crude / purified enzymes, Immobilized cells / enzymes, etc.), but can also be carried out by directly adding a metal compound, ethylenediamine and fumaric acid to the cell culture. The concentration of fumaric acid varies from 0.01 M to a saturated concentration, preferably
The range of 1.2M and the concentration of ethylenediamine is 0.1M.
The range is from 01M to 2M, preferably from 0.015 to 1M. The amount of the metal compound is usually 0.01 to 2 times as much as the metal in relation to the theoretical amount of S, S-ethylenediamine-N, N'-disuccinic acid to be produced. The metal compound may be added all at once at the start of the reaction, or may be added during the reaction. The amount of the microorganism or the like to be used is usually 0.01 to 5% by weight in terms of dry cells based on the substrate. P of reaction solution
H ranges from 4 to 11, preferably from 6 to 10. The reaction is usually carried out at 5 to 60 ° C., preferably at 10
The reaction is carried out at a temperature in the range of -55 ° C, but a high temperature is advantageous in terms of the reaction rate. However, since the reaction for producing S, S-ethylenediamine-N, N'-disuccinic acid from fumaric acid and ethylenediamine and the reaction for forming a metal complex are exothermic reactions, the reaction yield is low in terms of reaction yield. Temperature is advantageous. Thus, the reaction can be carried out at an elevated temperature initially and at a lower temperature later. The reaction can be carried out either batchwise or continuously. Also, regardless of which raw material is used, even if a reaction system capable of synthesizing ethylenediamine and fumaric acid from another compound is allowed to coexist in the present reaction system, there is no problem as long as the effects of the present invention are obtained. When a metal complex of S, S-ethylenediamine-N, N'-disuccinic acid is required, the reaction is carried out in the presence of a predetermined metal ion, followed by pH adjustment, concentration,
The target compound can be directly obtained by other operations. On the other hand, S, S-ethylenediamine-
In order to collect N, N′-disuccinic acid, acid precipitation with a mineral acid is usually performed. However, in a system in which a stable complex is formed at the pH of acid precipitation, such as when a reaction is performed in the presence of ions of a heavy metal such as iron, the operation of removing the metal ions before acid precipitation is performed. is necessary. Therefore, when S, S-ethylenediamine-N, N'-disuccinic acid is required, an alkaline earth metal ( such as calcium), such as calcium, which can omit the above-described removal operation during acid precipitation.
However, it is efficient to carry out the reaction in the presence of ions ( excluding magnesium) . Next, the present invention will be described in detail with reference to examples. Example 1 (1) Culture Brevundimonas sp. TN-3 strain, Sphingomonas sp. TN
-28 strains and one strain of Pseudomonas sp. TN-131 were picked from a slant medium, inoculated into the following medium, and incubated at 30C
The cells were cultured aerobically with shaking for 4 days. Medium composition (pH 7.5, 100 ml) Ethylenediamine-N, N'-disuccinic acid 0.2 g glucose 0.2 g yeast extract 0.1 g polypeptone 0.05 g magnesium sulfate 7H ₂ O 0.1 g sodium sulfate 0. 28 g Phosphate buffer 25 mM metal salt mixture solution ^* 0.5 ml * Metal salt mixture solution (100 ml); magnesium chloride 6H ₂ O 8 g, calcium chloride 0.
8 g, manganese sulfate · 4H ₂ O 0.6 g, ferric · 6H ₂ O 0.12 g chloride, zinc 0.06g [0033] sulfuric acid (2) removing fumarase like active fraction acquired bacterial cultures 20ml Into a centrifuge tube, 10,000 rpm
After centrifugation at 5 ° C for 15 minutes to collect the cells, 50 m
Washed twice with M phosphate buffer. 5 against the obtained cells
0 W, sonication for 5 minutes, 10,000 rpm
A crude enzyme solution was obtained by centrifugation at m for 20 minutes. Further, after 60% saturated ammonium sulfate precipitation and desalting by dialysis, 50 m
Adsorbed on DEAE-Sephacel [Pharmacia] equilibrated with M phosphate buffer.
Elution was carried out by a linear gradient method up to the same buffer containing sodium chloride. Furthermore, if necessary, under the same conditions, HPLC [TSK-gel DEAE-5PW (Tosoh)] was used instead of DEAE-Sephacel to obtain a fraction from which fumaric acid-reducing activities such as fumarase were removed as much as possible. (3) Reaction The reaction solution was 68.4 mM fumaric acid and 34.2 mM ethylenediamine, 200 mM borate buffer, 17.1 mM
The reaction was carried out at 30 ° C. for 10 days using a metal compound shown in Table 1 (concentration as a metal) and the above active fraction, adjusted to pH 8 with 6N sodium hydroxide. The pH of the reaction solution was maintained at 8 using 6N sulfuric acid and 6N sodium hydroxide. For comparison, a system containing no metal compound was similarly reacted. The amount of the product S, S-ethylenediamine-N, N'-disuccinic acid (S, S-EDDS) in the reaction-terminated liquid was 15,000 rpm.
The supernatant after centrifugation at 5 ° C. for 5 minutes was subjected to WAKOSIL 5C8 (Wako Pure Chemical Industries) [eluent: 50 mM containing 10 mM tetra-n-butylammonium hydroxide and 0.4 mM CuSO ₄
M phosphoric acid pH2] and MCI GEL CRS 10W (Mitsubishi Chemical)
[Eluent: 10 mM CuSO ₄ ] was used for liquid chromatography. Separation and purification of S, S-EDDS is described in T, Nishikiori et al., J. Antibiotics 37, 426.
(1984), and after obtaining crystals, the chemical structure was confirmed by NMR and mass spectrometry analysis. (4) Result Example 2 (1) Culture Culture was performed in the same manner as in Example 1. (2) Acquisition of active fraction from which fumarase and the like were removed The procedure was the same as in Example 1. (3) The procedure was performed in the same manner as in Example 1 except that calcium chloride and manganese sulfate were used as the reaction metal compounds. (4) Result Reference Example 1 Next, it was confirmed that the S, S-EDDS production amount (mM) obtained in Examples 1 and 2 was the production amount at the equilibrium point. (1) Culture Brevundimonas sp. TN-3 strain was cultured under the same conditions as in Example 1. (2) Acquisition of active fraction from which fumarase and the like were removed The procedure was the same as in Example 1. (3) Reaction The reaction solution was 34.2 mM, S-EDDS, 200 mM.
A borate buffer, containing 17.1 mM (concentration as a metal) of the metal compound shown in Table 3 and the above active fraction, adjusted to pH 8 with 6N sodium hydroxide, was used.
The reaction was carried out at 10 ° C. for 10 days until the decomposition of S, S-EDDS was almost not observed. The pH of the reaction solution is 6N sulfuric acid, 6N
It was kept at 8 with sodium hydroxide. For comparison, the same reaction was carried out for those containing no metal compound. S, S-EDDS in the reaction solution was quantified by the same method as in Example 1. [0040] Example 3 (1) Culture Brevundimonas sp. TN-3 strain was cultured under the same conditions as in Example 1. (2) Acquisition of active fraction from which fumarase and the like were removed The procedure was the same as in Example 1. (3) Reaction The reaction solution contains 68.4 mM fumaric acid and 34.2 mM ethylenediamine, 200 mM borate buffer, ferric sulfate at the concentration shown in Table 4, and the above-mentioned active fraction. Use a solution adjusted to pH 8 at 30 ° C.
The reaction was continued for 10 to 10 days until almost no S, S-EDDS generation was observed. The pH of the reaction solution was maintained at 8 using 6N sulfuric acid. For comparison, the same reaction was carried out for those containing no ferric sulfate. S, S- in the reaction solution
EDDS was quantified in the same manner as in Example 1. [0043] Example 4 (1) Culture The strain Brevundimonas sp. TN-3 was cultured under the same conditions as in Example 1. (2) Acquisition of active fraction from which fumarase and the like were removed The procedure was the same as in Example 1. (3) Reaction The reaction solution contains 68.4 mM fumaric acid and 34.2 mM ethylenediamine, 200 mM borate buffer, 17.1 mM ferric sulfate (34.2 mM as iron), and the above-mentioned active fraction. S, S was adjusted to pH 8 with sodium hydroxide at 20, 30, 40 ° C for 4 to 10 days.
-The reaction was allowed to proceed until almost no EDDS was formed. The pH of the reaction solution was maintained at 8 using 6N sulfuric acid. still,
For comparison, a reaction containing no ferric sulfate was similarly reacted. S, S-EDDS in the reaction solution was quantified in the same manner as in Example 1. [0046] Example 5 (1) Culture The strain Brevundimonas sp. TN-3 was cultured under the same conditions as in Example 1. (2) Acquisition of active fraction from which fumarase and the like have been removed Same as in Example 1. (3) Reaction The reaction solution contains 68.4 mM fumaric acid and 34.2 mM ethylenediamine, 200 mM borate buffer, 17.1 mM ferric sulfate (34.2 mM as iron), and the above active fraction, and contains 6N PH 6, 7, 89 by sodium hydroxide
S, S at 30 ° C for 4 to 10 days
-The reaction was allowed to proceed until almost no EDDS was formed. The pH of the reaction solution was kept constant using 6N sulfuric acid. For comparison, the same reaction was carried out for those containing no metal compound. S, S-EDD in reaction solution
S was quantified in the same manner as in Example 1. [0049] Example 6 (1) Culture The strain Brevundimonas sp. TN-3 was cultured under the same conditions as in Example 1. (2) Acquisition of active fraction from which fumarase and the like were removed The procedure was the same as in Example 1. (3) Reaction The reaction solution contains 342 mM fumaric acid, 171 mM ethylenediamine, 171 mM calcium hydroxide or ferric hydroxide as a metal, and the above active fraction, and is adjusted to the pH shown in Table 7 with 6N sodium hydroxide. Using the adjusted one,
The reaction was carried out at 30 ° C. for 10 to 20 days until the generation of S, S-EDDS was almost not observed. The pH of the reaction solution is 6N
Each was kept constant using sodium hydroxide or 6N sulfuric acid. For comparison, the same reaction was carried out for those containing neither calcium hydroxide nor ferric hydroxide. S, S-EDDS in the reaction solution was quantified in the same manner as in Example 1. [0052] Example 7 (1) Cultured Burkholderia sp. Strains KK-5 and KK-9, Acidovor
ax sp. TN-51 strain, Pseudomonas sp. TN-131
Strain, Paracoccus sp. KK-6 strain, Sphingomonas sp. TN
-28 strain, Brevundimonas sp. TN-30 strain and T strain
One platinum loop of the N-3 strain was removed from the slant medium, inoculated into the medium described in Example 1, and cultured at 30 ° C. for 3 days under aerobic shaking. (2) Acquisition of bacterial cells Transfer 20 ml of the bacterial cell culture into a centrifuge tube, and rotate at 10,000 rpm.
After centrifugation at 5 ° C. for 15 minutes to collect the cells, the cells were washed twice with 50 mM borate buffer, pH 8.0. (3) Reaction The reaction solution contains 200 mM fumaric acid and 200 mM ethylenediamine, 100 mM ferrous hydroxide or ferric hydroxide, and the above-mentioned cells, and is added with 6N sodium hydroxide.
The mixture was adjusted to H8 and reacted at 30 ° C. with shaking for 24 hours. For comparison, a reaction containing no ferrous hydroxide and ferric hydroxide was similarly reacted. S, S-EDDS in the reaction solution was quantified by the same method as in Example 1. [0056] The present invention improves the reaction yield by allowing the presence of a specific metal ion, and efficiently converts S, S-ethylenediamine-N, N'-disuccinic acid or its metal complex. Can be manufactured.

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Claims (1)

(57) [Claim 1] S, S-ethylenediamine-N, N'-disuccinic acid is produced from a mixture of ethylenediamine and fumaric acid by the action of a microorganism having lyase activity or the treated product. At this time, an alkaline earth metal (
However, except for magnesium), iron, S to zinc, copper, nickel, aluminum, characterized in that the presence of at least one metal ion selected from the group consisting of titanium and manganese, S- ethylenediamine -N,
A method for producing N'-disuccinic acid.