JP2000300286A - Production of highly pure s,s-ethylenediamine-n,n'- disuccinic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing highly pure S,S-ethylenediamine-N, N'-disuccinic acid by an enzyme method while suppressing the formation of meso-ethylenediamine-N,N'-disuccinic acid inferior in the biodegradability. SOLUTION: The amount of ethylenediamene-N-monosuccinic acid formed at the time of preparation of an aqueous solution of fumaric acid or maleic acid, and ethylenediamine, which are substrates, is regulated so as to be <=4 mol% based on the theoretical amount of S,S-ethylenediamine-N,N'-disuccinic acid to be obtained to regulate the excessive rate of the S,S-ethylenediamine-N,N '-disuccinic acid in the product so as to be >=95% d.e.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing high-purity S, S-ethylenediamine-N, N'-disuccinic acid. S, S-ethylenediamine-N, N'-disuccinic acid [hereinafter abbreviated as "SS-EDDS"] has the most excellent biodegradation performance among the stereoisomers, and therefore, detergents,
It is a chelating agent expected to be used in fields such as photographic processing and pulp bleaching.

[0002]

2. Description of the Related Art As a method for producing SS-EDDS,
(1) Chemical synthesis from L-aspartic acid and dibromoethane in a basic aqueous medium of Neal and Rose et al. [Inorganic Chemistry, Vol. 7, No. 2405-2412 (1968)], (2) P
atel R. N. L- in the presence of calcium hydroxide
Chemical synthesis from aspartic acid and dibromoethane [W
O 95-12570], (3) T.I. Nishi
Kiori et al. [J. Antibiot. 37,426-
427 (1994)] and Z.A. Hans et al. [WO 9
No. 636725], a fermentation method using actinomycetes, (4)
Endo et al. Enzymatic method from fumaric acid and ethylenediamine [JP-A-9-140390 and JP-A-10-52292]
Publications] are known.

In the chemical synthesis methods (1) and (2), since a large amount of unreacted raw materials and by-products remain, high purity SS-
In order to obtain EDDS, purification such as crystallization must be performed. The fermentation method (3) has extremely low productivity and is unlikely to be an industrial production method. In the enzymatic method (4), since there are almost no by-products and the enzyme activity is high and the amount of unreacted raw materials is small,
This is a production method in which purification steps such as crystallization can be simplified or omitted, and commercialization is expected only by separating bacterial cells from the reaction solution.

[0004]

However, even in the above-mentioned enzymatic method, in the course of studying an industrial production method, the product has a biodegradability lower than that of SS-EDDS in the product.
A phenomenon was confirmed in which so-ethylenediamine-N, N'-disuccinic acid [hereinafter abbreviated as meso-EDDS] (R, S-ethylenediamine-N, N'-disuccinic acid) was mixed.

Accordingly, it is an object of the present invention to provide an efficient industrial method for producing SS-EDDS which suppresses the production of meso-EDDS and does not substantially require a purification step.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies on the cause of contamination of meso-EDDS in order to solve the above-mentioned problems. As a result, surprisingly, the racemate was prepared at the time of preparing the aqueous substrate solution before proceeding to the enzymatic reaction. Ethylenediamine-N-
Monosuccinic acid is produced, of which only R-ethylenediamine-N-monosuccinic acid undergoes an enzymatic reaction.
Converted to eso-EDDS (S-ethylenediamine-N-
Monosuccinic acid was converted to SS-EDDS), which was found to be contained in the product as it was, and the present invention was completed.

That is, the present invention relates to an ethylenediamine
In an enzymatic reaction by the action of N, N'-disuccinic acid ethylenediamine lyase, at least one alkali selected from fumaric acid and ethylenediamine, a hydroxide of an alkali metal, a hydroxide of an alkaline earth metal and ammonia as substrates. In the method for producing S, S-ethylenediamine-N, N'-disuccinic acid from the substrate in an aqueous solution containing, the amount of ethylenediamine-N-monosuccinic acid produced at the time of preparing the aqueous substrate solution is determined by obtaining S, S -Ethylenediamine-N, N'-disuccinic acid, the amount of S,
S-ethylenediamine-N, N'-disuccinic acid excess is 95% d. e. High purity S,
This is a method for producing S-ethylenediamine-N, N'-disuccinic acid.

[0008] The present invention further provides a maleic acid and ethylenediamine as a substrate, an alkali metal hydroxide and an alkaline earth in an enzymatic reaction by the action of maleic acid isomerase and ethylenediamine-N, N'-disuccinic ethylenediamine lyase. In the method of producing S, S-ethylenediamine-N, N'-disuccinic acid from the substrate in an aqueous solution containing at least one alkali selected from metal hydroxides and ammonia, it is produced when the substrate aqueous solution is prepared. By making the amount of ethylenediamine-N-monosuccinic acid 4 mol% or less based on the theoretical amount of S, S-ethylenediamine-N, N'-disuccinic acid obtained, S, S-ethylenediamine in the product is obtained.
95% of N, N'-disuccinic acid excess d. e. High purity S, S-ethylenediamine-
This is a method for producing N, N'-disuccinic acid.

In a preferred embodiment of these production methods, the preparation of the aqueous substrate solution and the enzymatic reaction are continuously performed, and / or the temperature during the preparation of the aqueous substrate solution is set to 50 ° C. or lower. The S, S-ethylenediamine-N, N'-disuccinic acid excess ratio referred to here [hereinafter, "S
The “S-body excess rate” is a value defined by the following equation.

[0010] SS body excess rate (% de) = (SS body production amount-R
S-body production amount) / (SS-body production amount + RS-body production amount) x 100

[0011]

Next, a general embodiment of the present invention will be described.

Preparation of an aqueous substrate solution:
The reaction is carried out by dissolving a predetermined amount of fumaric acid or maleic acid, ethylenediamine, and at least one alkali selected from alkali metal hydroxides, alkaline earth metal hydroxides and substances, and ammonia in water. At this time, the temperature rises due to the generation of heat of neutralization, but the amount of ethylenediamine-N-monosuccinic acid generated is 4 mol% or less based on the theoretical amount of S, S-ethylenediamine-N, N'-disuccinic acid. The dissolution temperature is usually adjusted to 60 ° C. or lower, preferably 40 ° C. or lower. The lower limit of the temperature may be any as long as the dissolving operation is possible,
It is usually 0 ° C. or higher. In addition, it is desirable that the time from the preparation of the aqueous substrate solution to the start of the reaction after the preparation be reduced as much as possible for the above-mentioned purpose of suppressing the amount of ethylenediamine-N-monosuccinic acid to be produced. This time depends on the temperature, but is usually 20 hours or less, preferably 12 hours or less. The concentration of fumaric or maleic acid is usually 0.0
It is 1 to 3M, and even if a precipitate having a saturation solubility or higher exists in the solution, it dissolves with the progress of the reaction during the reaction. The concentration of ethylenediamine is usually 0.01 to 2
M. Further, the amount of alkali added is 0.8 to 1.2 times equivalent to fumaric acid or maleic acid.

In the preparation of the aqueous substrate solution, the addition of one of the substrates, particularly ethylenediamine, immediately before the reaction is more effective in suppressing the amount of ethylenediamine-N-monosuccinic acid formed. The preparation of the aqueous substrate solution may be batch or continuous, but a continuous method using a multi-tank continuous tank of two or more tanks, a line mixer such as a static mixer, or a combination thereof is particularly preferable. In the continuous method, it is possible to more efficiently perform divided addition, such as adding ethylenediamine immediately before the reaction.

Ethylenediamine-N, N'-disuccinate ethylenediamine lyase: The ethylenediamine-N, N'-disuccinate ethylenediamine lyase which can be used in the present invention is not particularly limited, and examples thereof include genus Burkholderia and Acidovorax. (Acidovorax), Pseudomonas (Pse
udomonas), Paracoccus
cus), Sphingomonas
as) genus and Bravendimonas
onas) bacteria, furthermore ethylenediamine-
Plasmids containing the gene DNA encoding N, N'-disuccinic acid ethylenediamine lyase were cloned into the genus Escherichia or Rhodococcus (Rh).
odococcus).

Specifically, Burkholderia
sp. KK-5 strain [FERM BP-5412], K
K-9 strain [FERM BP-5413], Acidov
orax sp. TN-51 strain [FERM BP-54
16], Pseudomonas sp. TN-131
Strain [FERM BP-5418], Paracoccu
s sp. KK-6 strain [FERM BP-5415],
TNO-5 strain [FERM P-16435], Sph
ingomonas sp. TN-28 strain [FERM
BP-5419], Brevundimonas s
p. TN-30 strain [FERM BP-5417] and TN-3 strain [FERM BP-5886] (Japanese Unexamined Patent Publication No.
0-52292), E.C. coli JM10
9 / pEDS020 (FERM BP-6161) (described in JP-A-10-210984). These microorganisms are deposited under the above numbers with the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry.

The medium of the microorganism used in the present invention is not particularly limited, and there are no particular limitations on carbon sources, nitrogen sources, inorganic salts,
Furthermore, any of a synthetic medium and a natural medium may be used as long as they contain a small amount of organic nutrients and the like. In culturing, addition of amino acids such as ethylenediamine-N, N'-disuccinic acid, ethylenediamine-N-monosuccinic acid, aspartic acid, glutamic acid, histidine and the like, fumaric acid, etc., to the culture medium is a high activity of the desired cells. Is sometimes preferred. 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.
The culture may be performed aerobically for 1 to 10 days until the activity is maximized in the range of ° C.

Enzyme reaction: The reaction is carried out by adding the above-mentioned microbial cells or a processed product thereof (dried cells, crushed cells,
Crude / purified enzymes, immobilized cells / enzymes, etc.) are added, and the reaction is usually performed at 0 to 60 ° C, preferably 10 to 40 ° C. The pH ranges from 4 to 11, preferably from 6 to 10. 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. After completion of the reaction, an aqueous solution of the SS-EDDS salt can be obtained by removing the cells or the treated cells by filtration or centrifugation.

The aqueous solution of the SS-EDDS salt obtained by the present invention has a high yield and a high selectivity in addition to the reaction.
Since the so-EDDS content is low, it can be used as it is as a chelating agent having excellent biodegradability for various uses, but if necessary, it can be purified with an ion exchange resin or the like. Further, if necessary, the aqueous solution is concentrated, solidified,
Alternatively, SS-EDDS salt crystals can be obtained by spray drying, and SS-EDDS crystals can be obtained by adding a mineral acid.

[0019]

The present invention will be described below in detail with reference to examples.

(1) Preparation of cell catalyst Escherichia coli JM109 / pED
One loop of S020 was taken from the slant medium, and an LB medium containing 50 mg / L ampicillin (1% bactotryptone, 0.5% bactoeast extract, 0.5% NaC
1) and cultured with shaking at 37 ° C. for 8 hours. This was added to an LB medium (50 mg / L ampicillin, 1 mM i
Sopropyl-b-galactoside) was inoculated in an amount of 2.5%, followed by aerobically shaking culture at 37 ° C. for 30 hours. From 1 L of the culture solution, the cells are collected by centrifugation (7,000 rpm, 20 minutes).
The well was washed once with 500 mL of 50 mM borate buffer pH 7.75 containing M 1,4-diaminobutane. 500m
After resuspending the cells in L of the same buffer, 25% glutaraldehyde was gradually added to 25 mM in ice. Since the pH decreases, pN with 6N NaOH
After adjusting to H 7.75, it was left for 2 hours with stirring. Ethylenediamine is added to be 50 mM,
After adjusting the pH to 9.0 with 6N NaOH, the mixture was allowed to stand for 2 hours. Next, sodium borohydride was added to a concentration of 25 mM, and the mixture was left for 2 hours with stirring. further,
After adjusting the pH to 9.2 with 6N NaOH, heat treatment was performed at 45 ° C. for 4 hours in a water bath to remove fumarase activity.

(2) Measurement of ethylenediamine-N-monosuccinic acid concentration HPLC conditions Column: Inertsil ODS-3V 4.6 × 250 mm Detector: JASCO UV-970 (wavelength: 254 nm) Mobile phase: 2 mM Sodium 1-hexanesulfonate 50 mM H ₃ PO ₄ / Na ₂ HPO ₄ 2 mM CuSO ₄ Flow rate: 1 ml / min Column oven 40 ° C. (3) Measurement of ethylenediamine-N, N′-disuccinic acid concentration HPLC conditions Column: Develosil ODS-UG-5 4.6 × 250mm detector: JASCO UV-970 (wavelength 270 nm) mobile phase: 10mM Tetra-n-butylammonium Hy droxide 50mM H 3 PO 4 0.4mM CuSO 4 flow rate: 1 ml / min Karamuo (4) Sample preparation 0.42N NaOHaq. After 10-fold dilution with 1
Centrifugation at 2000 rpm for 5 minutes. Then, appropriately dilute with water to be within the quantitative range.

Example 1 (1) Preparation of Substrate Aqueous Solution The substrate aqueous solution was prepared such that water, fumaric acid (1.027 mol), ethylenediamine (0.513 mol), magnesium hydroxide and magnesium hydroxide were added so that a total of 1 kg at the time of adding the cells was obtained. 0.513 mol of NaOH and 0.513 mol of NaOH were prepared for 2 hours while forcibly cooling and adjusting the temperature to 15 ° C. The amount of ethylenediamine-N-monosuccinic acid in the aqueous substrate solution thus prepared was analyzed and found to be 0.65 micromol. This is about 0.0001 mol% based on the theoretical amount of SS-EDDS obtained.

(2) Reaction After the preparation of the aqueous substrate solution, the temperature was adjusted to 40 ° C., and then 100 g of cells (in terms of dry weight) were charged, and the reaction was carried out for 12 hours. The pH before the addition of the cells was about 8.5, but the pH dropped during the reaction.
The pH was maintained at 8.5. SS-ED in reaction completed solution
The DS concentration was 475 mM, and the meso-EDDS concentration was 0.1.
It was 95 mM. SS excess is 99.6% d. e.

Example 2 (1) Preparation of Substrate Aqueous Solution Example 1 was carried out without forcible cooling. As a result, the temperature after preparation rose to about 49 ° C (the temperature of the used water was about 25 ° C). The amount of ethylenediamine-N-monosuccinic acid in the aqueous substrate solution thus prepared was analyzed and found to be 1.56 mmol. This is about 0.3 mol% based on the theoretical amount of SS-EDDS obtained.

(2) Reaction Same as in Example 1. The SS-EDDS concentration in the reaction completed solution was 470 mM, and the meso-EDDS concentration was 1.68 mM. SS body excess ratio is 99.3% d. e. Example 3 (1) Preparation of Substrate Aqueous Solution In Example 1, the reaction was carried out while adjusting the temperature to 40 ° C. during the preparation, and further kept warm for 12 hours after the preparation until it was used for the reaction. The amount of ethylenediamine-N-monosuccinic acid in the aqueous substrate solution thus prepared was analyzed and found to be 6.4 mmol. This is the SS-
It is about 1.3 mol% based on the theoretical amount of EDDS.

(2) Reaction Same as in Example 1. The SS-EDDS concentration in the reaction completed solution was 472 mM, and the meso-EDDS concentration was 4.3 mM. SS body excess ratio is 98.2% d. e.

Example 4 (1) Preparation of Aqueous Substrate Solution The aqueous substrate solution was prepared such that water, fumaric acid 1.027 mol, magnesium hydroxide 0.513 mol, and NaOH .513 mol was prepared over 2 hours while forcibly cooling and adjusting the temperature to 40 ° C. Then, after keeping the temperature at 40 ° C. for about 24 hours, while adjusting the temperature to 40 ° C., ethylenediamine 0.5
13 mol was added and the reaction was carried out immediately.

(2) Reaction Same as in Example 1. The SS-EDDS concentration in the reaction completed solution was 474 mM, and the meso-EDDS concentration was 0.92 mM. SS excess is 99.6% d. e.

Example 5 (1) Preparation of Aqueous Substrate Solution The aqueous substrate solution was prepared such that water, fumaric acid 2.054 mol, magnesium hydroxide 1.026 mol, and NaOH 0.026 mol was prepared over 2 hours while forcibly cooling and adjusting the temperature to 40 ° C. Then, after keeping the temperature at 40 ° C. for about 24 hours, while adjusting the temperature to 40 ° C., ethylenediamine 0.5
13 mol was added and the reaction was carried out immediately.

(2) Reaction Same as in Example 1. The SS-EDDS concentration in the reaction completed solution was 912 mM, and the meso-EDDS concentration was 2.13 mM. SS body excess ratio is 99.5% d. e.

Comparative Example-1 (1) Preparation of Substrate Aqueous Solution The aqueous substrate solution was prepared such that water, 2.054 mol of fumaric acid, 1.026 mol of ethylenediamine, and 1.06 mol of hydroxylamine were added so that the total at the time of adding the cells was 1 kg. 1.026 mol of magnesium and 1.026 mol of NaOH were mixed without temperature control over 2 hours. At this time, the temperature of the prepared liquid is about 76 ° C.
(The temperature of the water before blending was about 25 ° C.). When the amount of ethylenediamine-N-monosuccinic acid in the aqueous substrate solution thus prepared was analyzed, it was 56.8 mmol. This is about 5.5 mol% based on the theoretical amount of SS-EDDS obtained.

(2) Reaction Same as in Example 1. The SS-EDDS concentration in the reaction completed solution was 909 mM, and the meso-EDDS concentration was 30.9 mM. The SS body excess was 93.4% d. e.

[0033]

According to the present invention, since the production of meso-EDDS can be suppressed, a high-purity SS-EDDS salt can be provided by a simplified process that does not substantially require a purification process.

Continued on the front page F-term (reference) 4B064 AE01 CA21 CC06 CD01 CD07 CD12 DA19 4H006 AA02 BB31 BE10 BE11 BE14 BS10 BU32 NB10

Claims (4)

[Claims] 1. An enzyme reaction by the action of ethylenediamine-N, N'-disuccinate ethylenediamine lyase, wherein fumaric acid and ethylenediamine as substrates, alkali metal hydroxide, alkaline earth metal hydroxide and ammonia are used as substrates. In a method for producing S, S-ethylenediamine-N, N'-disuccinic acid from the substrate in an aqueous solution containing at least one selected alkali, ethylenediamine formed at the time of preparing the aqueous substrate solution is prepared.
The amount of N-monosuccinic acid is 4 mol% with respect to the theoretical amount of S, S-ethylenediamine-N, N'-disuccinic acid obtained.
The S, S-ethylenediamine-N, N'-disuccinic acid excess in the product is 95% d. e. A process for producing high-purity S, S-ethylenediamine-N, N'-disuccinic acid, characterized in that: 2. An enzyme reaction by the action of maleate isomerase and ethylenediamine-N, N'-disuccinate ethylenediamine lyase, maleic acid and ethylenediamine as substrates, and hydroxides of alkali metals and hydroxylation of alkaline earth metals. A process for producing S, S-ethylenediamine-N, N'-disuccinic acid from the substrate in an aqueous solution containing at least one alkali selected from a product and ammonia, wherein ethylenediamine-N- By making the amount of monosuccinic acid 4 mol% or less based on the theoretical amount of S, S-ethylenediamine-N, N'-disuccinic acid to be obtained, S, S-ethylenediamine-N, N'-disuccinic acid in the product is obtained. Excess 95% d. e. A process for producing high-purity S, S-ethylenediamine-N, N'-disuccinic acid, characterized in that: 3. The method for producing high-purity S, S-ethylenediamine-N, N′-disuccinic acid according to claim 1, wherein the preparation of the aqueous substrate solution and the enzymatic reaction are carried out continuously. 4. The method for producing high-purity S, S-ethylenediamine-N, N′-disuccinic acid according to claim 1, wherein the temperature during preparation of the aqueous substrate solution is 50 ° C. or lower.