Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Process involving micro-organisms of different genera in the...
Reexamination Certificate
2000-06-22
2001-07-17
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Process involving micro-organisms of different genera in the...
C435S128000, C435S170000, C435S232000, C435S252100, C435S252310, C435S252340, C435S252500, C435S252400, C435S253300, C435S252300
Reexamination Certificate
active
06261798
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field of the Invention
The present invention relates to a method of producing [S,S]-ethylenediamine-N,N′-disuccinate (hereinafter abbreviated as [S,S]-EDDS). More specifically, the present invention relates to a method of producing [S,S]-EDDS from maleic acid, maleic anhydride or maleic acid salt and ethylenediamine by action of microorganisms or enzymes.
2. Disclosure of the Related Art
[S,S]-EDDS has the high ability to capture heavy metals and is bio-degradable when released into the nature. Hence [S,S]-EDDS is expected to be used as a bleaching agent for photography, an electroless plating agent, or a builder for detergent.
The present inventors have already proposed methods of producing [S,S]-EDDS by microorganisms: (1) a method of producing [S,S]-EDDS from fumaric acid and ethylenediamine (JP-A-9-140390, U.S. Pat. No. 5,707,836, and European Patent No. 0731171), (2) a method of producing the same from maleic acid and ethylenediamine (JP-A-9-289895) and the method of (1) under the presence of metal ion (European Patent No. 0805211, JP-A-10-52292).
In terms of raw materials, the method of (2) has the advantage of providing a more economical method of manufacturing [S,S]-EDDS, since fumaric acid is industrially produced from maleic acid through an isomerization process by chemical methods (U.S. Pat. Nos. 2,816,923, 2,955,136, and 2,332,992).
The method disclosed in JP-A-9-289895, wherein maleic acid and ethylenediamine are used as substrates, is not a sufficient method because of its low yields.
Accordingly, an object of the present invention is to obtain [S,S]-EDDS in high yield using maleic acid as a raw material.
The present inventors have intensively investigated an application of malate isomerase (JP2664648, JP-A-8-51989, and European Patent No. 693557) to the production of [S,S]-EDDS. There are many biochemical studies about malate isomerase (W. Scher et al., J. Biol. chem., 244, 1878-1882 (1969), Y. Takamura et al., Agr. Biol. Chem., 33, 718-728 (1969), T. Kimura et al., Agr. Biol. Chem., 50, 89-94 (1986), and T. Nakajima-Kambe et al., J. Ferment. Bioeng., 84, 165-168 (1997)). Moreover, the malate isomerase has been proposed for the production of aspartic acid.
The present inventors have now found that the addition of both a microorganism having malate isomerase activity and a microorganism having ethylenediamine-N,N′-disuccinate ethylenediamine lyase (hereinafter referred to as EDDSase) activity to a solution containing maleic acid and ethylenediamine in the presence of a metal ion e.g., alkaline earth metal can largely increase the conversion rate of maleic acid to [S,S]-EDDS, although the addition of only these microorganisms to the solution results in a low conversion rate to [S,S]-EDDS.
SUMMARY OF THE INVENTION
present invention provides a method of producing [S,S]-ethylenediamine-N,N′-disuccinate, wherein a microorganism having malate isomerase activity or matter processed therefrom and a microorganism having ethylenediamine-N,N′-disuccinate ethylenediamine lyase activity or matter processed therefrom are allowed to act on a substrate solution containing maleic acid, maleic anhydride, or maleic acid salt, and ethylenediamine, in the presence of at least one metal ion selected from the group consisting of alkaline earth metals, iron, zinc, copper, nickel, aluminum, titanium and manganese ions, thereby obtaining [S,S]-ethylenediamine-N,N′-disuccinate.
High-yield production of [S,S]-EDDS is impossible when using only the presence of an enzyme converting maleic acid into fumaric acid and an enzyme converting fumaric acid into [S,S]-EDDS, because both enzymes catalyze a reversible equilibrium reaction independent of their source. However in the present invention, it is assumed that [S,S]-EDDS is changed into a metal complex in the presence of a metal ion such as an alkaline earth metal ion and shifted out of the enzymatic reaction system so that the reaction equilibrium is driven toward the production side, thereby accumulating a high concentration of [S,S]-EDDS in a high yield within the reaction system.
The conversion reactions from maleic acid into fumaric acid and from fumaric acid into [S,S]-EDDS are weak exothermic reactions. Thermodynamically, as the reaction temperature becomes lower, the equilibrium shifts to the side of producing fumaric acid. Thus in the reaction, in the absence of the metal ion as described above, manipulations such as lowering the reaction temperature in the latter period of the reaction are required to improve the yield. However, the presence of the metal ion can lead to high conversion yield without such manipulations.
The specification includes all or part of the contents as disclosed in the specification and/or drawings of Japanese Patent Application No. 9-364798 (filed on Dec., 22th, 1997), which is a priority claimed in the present application.
DETAILED DESCRIPTION OF THE INVENTION
The present invention will now be described in detail.
Metal ions used in the present invention include alkaline earth metals, iron, zinc, copper, nickel, aluminum, titanium and manganese ions. For example, Mg(II), 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 can be included.
Ion sources of these metals can include hydroxides and oxides of these metals, salts of inorganic or organic acids, e.g., sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, carbonic acid and acetic acid, minerals containing these metallic compounds, and compounds with maleic acid and ethylenediamine that are substrates of the present invention. Two or more of the compounds may be mixed for use in the present invention.
These metallic compounds vary in their solubility in water, some have low and some have slight water solubility. Both types of these compounds can be used in the present invention, because most of them are solubilized by a coordination ability of [S,S]-EDDS even when these compounds are present at more than saturation concentration, for example in a suspension state. In other words, any compound can be used as a source of “metallic ions” in the present invention so far as the metallic ion can coordinate to [S,S]-EDDS, by which the advantages of the present invention can be achieved.
Any microorganisms capable of isomerizing maleic acid into fumaric acid can be used as those having a malate isomerase activity in the present invention. These microorganisms may belong to, but not limited to, the genus Alcaligenes, the genus Pseudomonas, the genus Xanthomonas, and the genus Bacillus. Further genetically engineered recombinant microorganisms having malate isomerase genes from the above-mentioned microorganisms can be used as well.
Specific examples of strains for use in the present invention include, but not limited to,
Alcaligenes faecalis
IFO 12669, IFO 13111 and IAM 1473,
Alcaligenes eutrophus
IAM 12305,
Pseudomonas fluolescens
ATCC 23728,
Xanthomonas maltophilia
ATCC 13270, Bacillus sp. MI105 (FERM BP-5164),
Bacillus stearothermophirus
MI101 (FERM BP-5160) and MI102 (FERM BP-5161),
Bacillus brevis
MI103 (FERM BP-5162) and MI104 (FERM BP-5163).
These microorganisms are available from Institute for Fermentation (IFO), Osaka, Japan; IAM Culture Collection, Cell/Functional Polymer Center, Cell Biology, the University of Tokyo, Japan, and American Type Culture Collection (ATCC).
Any microorganisms capable of producing [S,S]-EDDS from fumaric acid and ethylenediamine can be used as those having EDDSase activity in the present invention. These microorganisms can include, but not limited to, the genus Brevundimonas, the genus Paracoccus, the genus Sphingomonas, the genus Acidovorax, the genus Pseudomonas, and the genus Burkholderia. Further, genetically engineered recombinant mic
Endo Takakazu
Kaneko Makoto
Kato Osamu
Lilling Herbert J.
Mitsubishi Rayon Co. Ltd.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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