JP2005006650A - Method for producing cadaverine-dicarboxylic acid salt - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for economically producing cadaverine as a diamine which is used as a raw material for producing a nylon, in such a form as to be easily applied to a polymerization reaction. <P>SOLUTION: This method comprises conducting enzymatic decarboxylation reaction of lysine, while adding a 4-10C dicarboxylic acid to a lysine solution so as to keep a pH value of the solution in a range suitable for the enzymatic decarboxylation reaction, for example, in a range of pH 4.0 to pH 8.0, so that the cadaverine-dicarboxylic acid salt is produced. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for producing cadaverine dicarboxylate. Cadaverine dicarboxylate can be used as a raw material for producing nylon.

  As a raw material for producing plastic, naphtha, a so-called fossil raw material, is used in many parts. When discarding plastic, whether it is recycled or not, disposal due to combustion or the like causes the release of carbon dioxide gas, which is becoming a problem in recent years. Therefore, in order to prevent global warming and form a recycling society, it is desired to replace the raw materials for plastic production with raw materials derived from biomass.

Polylactic acid is known as a plastic produced from biomass raw materials. The method for producing polylactic acid is to first extract starch or sugar from a plant, ferment them using them as a carbon source to produce lactic acid, and then chemically polymerize the obtained lactic acid. As uses of polylactic acid, various uses such as containers and packaging, clothing, and other industrial products are expected. However, since this polylactic acid has a melting point of about 190 ° C., it has a drawback that it is not suitable for use under high temperature conditions.

  On the other hand, nylon having high heat resistance includes polyamide, that is, polyamide. A commonly used nylon is nylon 66 produced by polymerizing hexamethylenediamine, which is a diamine having 6 carbon atoms, and adipic acid, which is a 6-carbon dicarboxylic acid, in a molar ratio of 1: 1. . Nylon 66 is a plastic material that can be used under high temperature conditions because its melting point is 250 ° C. or higher.

  By the way, the hexamethylenediamine is produced from benzene, propylene or butadiene obtained from naphtha, and a production method from biomass is not known. On the other hand, pentamethylenediamine having 5 carbon atoms is also called cadaverine and is known to be produced from lysine, which is one of amino acids, by lysine decarboxylase (LDC) (Non-patent Document 1). Therefore, by using nylon as a raw material instead of hexamethylenediamine having 6 carbon atoms as a raw material, a plastic material that can be used under high temperature conditions using a biomass-derived raw material Can be supplied.

  LDCs are bacteria such as Bacterium cadaveris (Non-patent Document 2) and Escherichia coli (E. coli) (Non-patent Document 3), and plants such as glass beans (Non-patent Document 4). Is known to exist. LDC can be extracted from these organisms and used for cadaverine production. In E. coli, the sequence of the LDC gene (cadA) has been clarified (Non-Patent Documents 5 and 6). Furthermore, a method for producing cadaverine by culturing a host in which the enzymatic activity of LDC or lysine-cadaverine antiporter is enhanced using such an LDC gene (Patent Document 1), and intracellular LDC A method of producing cadaverine by causing LDC derived from recombinant cells with increased activity to act on lysine has been proposed (Patent Document 2).

However, when LDC is allowed to act on lysine, carbon dioxide gas is liberated by decarboxylation by LDC, so that the pH during the reaction increases due to the production of cadaverine. Therefore, in order to prevent an increase in pH and maintain the optimum pH of the enzyme reaction, the reaction is carried out in a high concentration buffer, or acid is added to the reaction system sequentially to neutralize the alkali. Is required (Patent Documents 1 and 2). Usually, an inorganic acid such as hydrochloric acid, sulfuric acid or phosphoric acid, or an organic acid such as acetic acid is often used to neutralize the pH of the enzyme reaction. When neutralized by these, cadaverine obtained from the reaction solution becomes a salt such as cadaverine hydrochloride, cadaverine sulfate, cadaverine phosphate, or cadaverine acetate.

  As a method for producing nylon, a method of polycondensation of dicarboxylic acid dihalide and diamine in the presence of a base is conventionally known. As another method, a method is known in which a salt or a low-order condensate formed from a dicarboxylic acid and a diamine is polycondensed by heating under melting conditions (Non-patent Document 7). In any method, when cadaverine obtained by an enzymatic reaction is polymerized with a dicarboxylic acid, it is necessary to re-prepare free cadaverine from the salt of cadaverine, which is not economical because the process becomes complicated.

By the way, as a method for producing lysine by fermentation, bacteria are cultured in a medium mainly composed of adipic acid, succinic acid, fumaric acid or salts thereof, and maintained at pH 7.5 to 8.2 with ammonium hydroxide. Is disclosed (Patent Document 3). In this method, the bacteria are subcultured to proliferate the bacteria, and after maintaining the dynamic equilibrium, the culture is performed by changing the medium conditions or a part of the culture conditions, and the fermentation is performed by shifting the equilibrium of the substance metabolism. The lysine is accumulated at a high concentration in the medium.
JP 2002-223770 A JP 2002-223771 A Japanese Patent Laid-Open No. 49-126871 Enzyme Handbook First Edition, 636 pages Asakura Shoten K. Soda et al., Biochem. Biophys. Res. Com., (1969) vol. 34, p. 34-39 DL Sabo et al., Biochemistry, (1974), vol.13, p.662-670 S. Ramakrishna et al., Phytochemistry, (1976) vol.15, p.83-86 N. Watson et al., Journal of bacteriology, (1992) vo.174, p.530-540 SY Meng et al. Journal of bacteriology (1992) vol.174, p2659-2669 Introduction to New Polymer Chemistry, page 22, Chemical Doujin (Ise Norio et al., 1995, ISBN 4-7598-0258-4)

  An object of the present invention is to provide a method for economically producing cadaverine as a diamine as a raw material for producing nylon in a form that can be easily used for a polymerization reaction.

  In order to solve the above-mentioned problems, the present inventors have conducted intensive studies. As a result, in enzymatic decarboxylation of lysine, free lysine was used as a raw material, and the pH of the lysine solution was adjusted to the optimum level by adding dicarboxylic acid. It is generated by decarboxylation of lysine by adjusting to pH, allowing LDC to act on it, and further decarboxylation while neutralizing the pH that rises during enzymatic decarboxylation by adding the dicarboxylic acid. It has been found that cadaverine can be obtained as a dicarboxylate, and the present invention has been completed.

That is, the present invention is as follows.
(1) Cadaverine dicarboxylate is obtained by carrying out enzymatic decarboxylation of lysine while adding dicarboxylic acid to the lysine solution so that the pH of the solution is maintained at a pH suitable for enzymatic decarboxylation. To produce cadaverine dicarboxylate.
(2) The method of (1), wherein the pH of the reaction solution is maintained at 4.0 to 8.0.
(3) The method of (1) or (2), wherein the dicarboxylic acid is a dicarboxylic acid having 4 to 10 carbon atoms.
(4) The method according to (3), wherein the dicarboxylic acid is adipic acid.
(5) The method according to any one of (1) to (4), wherein the enzymatic decarboxylation reaction is performed using lysine decarboxylase, a cell that produces lysine decarboxylase, or a treated product of the cell.
(6) The method according to (5), wherein the cell is a cell modified to increase lysine decarboxylase activity.
(7) When the cell increases the copy number of a gene encoding lysine decarboxylase, or the expression regulatory sequence of the gene is modified so that expression of the gene is enhanced, lysine decarboxylation The method according to (6), which is a recombinant cell having an increased enzyme activity.
(8) The method according to (7), wherein the cell is an Escherichia coli cell, and the gene encoding lysine decarboxylase is a cadA gene.
(9) Cadaverine dicarboxylate is obtained by performing enzymatic decarboxylation of lysine while adding dicarboxylic acid to the lysine solution so that the pH of the solution is maintained at a pH suitable for enzymatic decarboxylation. And a process for producing nylon by polycondensation of the cadaverine dicarboxylate obtained in the above process.

  According to the present invention, cadaverine dicarboxylate can be produced by a simple process. The cadaverine dicarboxylate obtained by the present invention can be used as it is for the polymerization reaction of nylon production.

Hereinafter, the present invention will be described in detail.
In the method of the present invention, the cadaverine-decarboxylation reaction is performed by adding a dicarboxylic acid to the lysine solution so that the pH of the solution is maintained at a pH suitable for the enzymatic decarboxylation reaction. A dicarboxylate is formed.

  The lysine used as a raw material is usually preferably a free base (lysine base), but may be a dicarboxylic acid salt of lysine. The lysine may be either L-lysine or D-lysine as long as it produces cadaverine by enzymatic decarboxylation reaction, but L-lysine is usually preferred. The lysine used in the method of the present invention may be a purified lysine, and if cadaverine produced by enzymatic decarboxylation can form a salt with a dicarboxylic acid, a fermentation broth containing lysine. Even so.

  As a solvent for preparing the lysine solution, water is preferably used. In the present invention, since the pH of the reaction solution is adjusted with dicarboxylic acid, it is not necessary to use other pH adjusting agents or buffering agents, but a buffer solution may be used as the solvent. Examples of such a buffer include sodium acetate buffer. However, from the viewpoint of forming a salt of cadaverine and dicarboxylic acid, it is preferable not to use a buffering agent or the like or to suppress it to a low concentration.

When free lysine is used as the lysine, dicarboxylic acid is added to the lysine solution to adjust the pH to be suitable for the enzymatic decarboxylation reaction. Specifically, the pH is usually about 4.0 to 8.0, preferably about 5.0 to 7.0, and more preferably about 5.5 to 6.5. When lysine dicarboxylate is used as lysine, it is not necessary to add dicarboxylic acid when preparing the reaction solution. Hereinafter, adjusting the pH of the reaction solution to a pH suitable for the enzymatic decarboxylation reaction as described above may be referred to as “neutralization”.

  The dicarboxylic acid is not particularly limited as long as it does not inhibit the enzymatic decarboxylation reaction of lysine and can form nylon by polycondensation reaction with cadaverine. For example, the dicarboxylic acid has 4 to 10 carbon atoms. Preferably, dicarboxylic acids having carboxyl groups at both ends of the linear molecule, specifically succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid and the like can be mentioned. The dicarboxylic acid is preferably a free acid.

The enzymatic decarboxylation reaction of lysine can be performed, for example, by adding LDC to the lysine solution neutralized as described above. The LDC is not particularly limited as long as it acts on lysine to produce cadaverine. As the LDC, a purified enzyme may be used, or a cell such as a microorganism, a plant cell, or an animal cell that produces LDC may be used. One type of LDC or a cell producing the same may be used, or a mixture of two or more types may be used. Further, the cells may be used as they are, or a cell treatment product containing LDC may be used. Examples of the cell treatment product include a cell disruption solution and a fraction thereof.
When performing enzymatic reactions using cells such as microorganisms, plant cells, or animal cells, using cells treated with organic solvents or surfactants may improve the permeability of the substrate and improve the reactivity. Is generally known. Also in the enzymatic decarboxylation reaction of lysine, the reactivity can be enhanced by treating cells producing LDC with an organic solvent, a surfactant or the like. Triton X-100, Tween 20, sodium cholate and CHAPS can be used as the surfactant to be treated, and acetone, xylene, toluene and the like can be used as the organic solvent. More specifically, when using Triton X-100, a concentration of 0.01% to 1.0% (w / v) is added, and treatment at 0 ° C. to 37 ° C. for 2 minutes to 1 hour is appropriate. .

  Examples of the microorganism include Escherichia bacteria such as E. coli, coryneform bacteria such as Brevibacterium lactofermentum, Bacillus bacteria such as Bacillus subtilis, Serratia marcescens. ) Such as Serratia bacteria, and eukaryotic cells such as Saccharomyces cerevisiae. Of these, bacteria, particularly E. coli, are preferred.

  The microorganism may be a wild strain or a mutant strain as long as it produces LDC. Further, a recombinant strain modified so as to increase LDC activity may be used. Plant cells or animal cells can also be recombinant cells that have been modified to increase LDC activity. The recombinant cell will be described later.

  After starting the reaction by adding LDC to the lysine solution, as the reaction proceeds, carbon dioxide released from the lysine is released from the reaction solution, and the pH rises. Therefore, dicarboxylic acid is added to the reaction solution so that the pH of the reaction solution is in the above range. Usually, this dicarboxylic acid is the same as the dicarboxylic acid used for neutralization of the raw material lysine. The dicarboxylic acid may be added continuously, or may be added in portions as long as the pH is maintained in the above range. The reaction conditions are not particularly limited as long as LDC acts on lysine to produce cadaverine, but the temperature is usually 20 ° C to 60 ° C, preferably 30 ° C to 40 ° C.

  The raw material lysine or lysine / dicarboxylic acid may be added in total to the reaction solution at the start of the reaction, or may be added in portions as the LDC reaction proceeds.

  When the enzymatic reaction is carried out batchwise, the dicarboxylic acid can be easily added. The reaction can also be carried out by moving bed column chromatography using a carrier on which LDC, LDC-producing cells or treatments thereof are immobilized. In that case, lysine and dicarboxylic acid may be injected into an appropriate portion of the column so that the reaction proceeds while the pH of the reaction system is maintained within a predetermined range.

  As described above, the enzymatic reaction proceeds satisfactorily by neutralizing the pH, which increases with cadaverine production, sequentially with dicarboxylic acid by enzymatic decarboxylation of lysine. The cadaverine thus produced accumulates in the reaction solution as a dicarboxylate.

  The cadaverine dicarboxylate obtained by the LDC reaction can be isolated and produced from the reaction solution by combining known methods. For example, after the reaction solution is sterilized by an autoclave or the like, the supernatant is collected by centrifugation, and the supernatant is decolored using activated carbon or the like and concentrated as appropriate. The cadaverine dicarboxylate may be in a solution or a crystal depending on the use mode. The crystals of cadaverine dicarboxylate can be formed, for example, by precipitating cadaverine dicarboxylate by cooling the concentrated reaction solution. Since the crystals obtained as described above contain cadaverine and dicarboxylic acid in equimolar amounts, they are suitable as raw materials for producing nylon.

  The cadaverine dicarboxylate produced according to the present invention produces nylon by polycondensation by the same method as the usual method for producing nylon. That is, as one aspect of the present invention, an enzymatic decarboxylation reaction of lysine is performed while adding a dicarboxylic acid to a lysine solution so that the pH of the solution is maintained at a pH suitable for the enzymatic decarboxylation reaction. There is provided a method for producing nylon, comprising a step of producing cadaverine dicarboxylate and a step of producing nylon by polycondensation of the cadaverine dicarboxylate obtained in the above step.

  Next, a method for modifying microorganisms so that LDC activity is increased will be exemplified. For other cells, LDC activity can be similarly increased by appropriately modifying the following method so as to be suitable for it. it can.

  LDC activity is increased by, for example, enhancing expression of a gene encoding LDC (LDC gene). Enhanced expression of the LDC gene is achieved by increasing the copy number of the LDC gene. For example, an LDC gene fragment may be ligated with a vector that functions in a microorganism, preferably a multicopy vector, to produce a recombinant DNA, which is introduced into a suitable host and transformed.

  Increasing the LDC gene copy number can also be achieved by having multiple copies of the LDC gene on the chromosomal DNA of the microorganism. In order to introduce a gene in multiple copies on the chromosomal DNA of a microorganism, homologous recombination is performed using a sequence present on the chromosomal DNA as a target. As a sequence present in multiple copies on chromosomal DNA, repetitive DNA and inverted repeats present at the end of a transposable element can be used. Alternatively, as disclosed in JP-A-2-109985, a target gene can be mounted on a transposon, transferred, and introduced in multiple copies on chromosomal DNA.

In addition to the gene amplification described above, the increase in LDC activity can also be achieved by replacing expression control sequences such as LDC gene promoters on chromosomal DNA or plasmid with strong ones. For example, lac promoter, trp promoter, trc promoter and the like are known as strong promoters. Further, as disclosed in International Publication WO 00/18935, it is possible to introduce a base substitution of several bases into the promoter region of a gene and modify it to be more powerful. These promoter substitutions or modifications enhance LDC gene expression and increase LDC activity. Modification of these expression regulatory sequences may be combined with increasing the copy number of the gene.

  The replacement of the expression regulatory sequence can be performed, for example, in the same manner as the gene replacement using a temperature sensitive plasmid. Examples of a vector having a temperature-sensitive replication origin of E. coli include plasmid pMAN997 described in WO 99/03988 International Publication Pamphlet. The expression regulatory sequence can also be replaced by a method using red recombinase of λ phage (Datsenko, K.A., PNAS (2000) 97 (12), 6640-6645).

  The LDC gene is not particularly limited as long as the encoded LDC can be effectively used for lysine decarboxylation. For example, bacteria such as bacteria cadaveris and E. coli, plants such as glass beans, Includes the LDC gene of microorganisms described in JP-A-2002-223770.

When E. coli is used as the host microorganism, the LDC gene derived from E. coli is preferred. As the LDC gene of E. coli, the cadA gene and the ldc gene (US Pat. No. 5,827,698) are known, and among these, the cadA gene is preferable. The sequence of the cadA gene of E. coli is known (N. Watson et al., Journal of bacteriology, (1992) vo.174, p.530-540; SY Meng et al. Journal of bacteriology (1992) vol. .174, p2659-2669; GenBank accession M76411), and can be isolated from E. coli chromosomal DNA by PCR using primers prepared based on the sequence. Examples of such primers include primers having the base sequences shown in SEQ ID NOs: 1 and 2.
The nucleotide sequence of the cadA gene and the amino acid sequence encoded thereby are shown in SEQ ID NOs: 3 and 4.

  To prepare the recombinant DNA by ligating the obtained LDC gene and the vector, cut the vector with a restriction enzyme that matches the end of the LDC gene, and use the ligase such as T4 DNA ligase to combine the gene and the vector. What is necessary is just to connect. Examples of vectors for E. coli include pUC18, pUC19, pSTV29, pHSG299, pHSG399, pHSG398, RSF1010, pBR322, pACYC184, pMW219 and the like.

  The LDC gene may be a wild type or a mutant type. For example, the cadA gene encodes an LDC containing a substitution, deletion, insertion, or addition of one or several amino acids at one or more positions, as long as the activity of the encoded LDC is not impaired. May be. Here, “several” differs depending on the position and type of protein in the three-dimensional structure of amino acid residues, but specifically 2 to 50, preferably 2 to 30, more preferably 2 to 10 It is a piece. The aforementioned LDC mutation is a conservative mutation that maintains LDC activity. A substitution is a change in which at least one residue in the amino acid sequence is removed and another residue is inserted therein. Amino acids that replace the original amino acid of the LDC protein and are considered conservative substitutions include Ala to ser or thr, arg to gln, his or lys, asn to glu, gln, lys, his or asp substitution, asp to asn, glu or gln substitution, cys to ser or ala substitution, gln to asn, glu, lys, his, asp or arg substitution, glu to asn, gln, lys or asp substitution, gly to pro substitution, his to asn, lys, gln, arg or tyr substitution, ile to leu, met, val or phe, leu to ile, met, val or substitution from phe, substitution from lys to asn, glu, gln, his or arg, substitution from met to ile, leu, val or phe, substitution from phe to trp, tyr, met, ile or leu, from ser Substitution from thr or ala, substitution from thr to ser or ala, substitution from trp to phe or tyr, substitution from tyr to his, phe or trp, and substitution from val to met, ile or leu It is done.

  DNA encoding a protein substantially the same as LDC as described above may contain a substitution, deletion, insertion, addition, or inversion of an amino acid residue at a specific site, for example, by site-directed mutagenesis. , Obtained by modifying the base sequence of the cadA gene. The modified DNA as described above can also be obtained by a conventionally known mutation treatment. As the mutation treatment, a method of treating DNA before mutation treatment with hydroxylamine or the like in vitro, and a microorganism that retains the DNA before mutation treatment, such as an Escherichia bacterium, are irradiated with ultraviolet rays or N-methyl-N′-nitro-N -A method of treating with a mutagen that is usually used for mutagenesis such as nitrosoguanidine (NTG) or EMS.

  By expressing the DNA having the mutation as described above in an appropriate cell and examining the activity of the expression product, DNA encoding a protein substantially the same as LDC can be obtained. In addition, it hybridizes under stringent conditions with a DNA encoding a mutated LDC or a cell carrying the same with, for example, a sequence of the coding region of the cadA gene (GenBank accession M76411) or a probe having a part of the sequence. In addition, DNA encoding a protein having an activity equivalent to that of LDC can be obtained. The “stringent conditions” referred to herein are conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to clearly quantify this condition, for example, highly homologous DNAs, for example, 70% or more, preferably 80% or more, more preferably 90%, most preferably 95% or more. The conditions are such that DNAs having the same homology hybridize with each other and DNAs having lower homology with each other do not hybridize with each other, or normal Southern hybridization washing conditions at 60 ° C., 1 × SSC, 0.1% SDS Preferably, the conditions include hybridization at a salt concentration corresponding to 0.1 × SSC and 0.1% SDS.

  A partial sequence of the cadA gene can also be used as a probe. Such a probe can be prepared by PCR using an oligonucleotide prepared based on a known cadA gene base sequence as a primer and a DNA fragment containing the cadA gene as a template. When a DNA fragment having a length of about 300 bp is used as a probe, hybridization washing conditions include 50 ° C., 2 × SSC, and 0.1% SDS.

  Specifically, the DNA encoding a protein substantially identical to LDC is preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, most preferably the amino acid sequence encoded by a known cadA gene. Preferably, DNA encoding a protein having 95% or more homology and having LDC activity can be mentioned.

  In order to introduce the recombinant DNA into the microorganism, transformation methods that have been reported so far may be used. For example, as reported for Escherichia coli K-12, a method in which recipient cells are treated with calcium chloride to increase DNA permeability (Mandel, M. and Higa, A., J. Mol. Biol. , 53, 159 (1970)), and a method for preparing competent cells from proliferating cells and introducing DNA as reported for Bacillus subtilis (Duncan, CH, Wilson, GA and Young, FE, Gene, 1, 153 (1977)). Alternatively, DNA-receptive cells, such as those known for Bacillus subtilis, actinomycetes, and yeast, are introduced into the DNA-receptor cells in a protoplast or spheroplast state that readily incorporates recombinant DNA. (Chang, S. and Choen, SN, Molec. Gen. Genet., 168, 111 (1979); Bibb, MJ, Ward, JMand Hopwood, OA, Nature, 274, 398 (1978); Hinnen, A Hicks, JBand Fink, GR, Proc. Natl. Acad. Sci. USA, 75 1929 (1978)). Also, transformation of microorganisms can be performed by an electric pulse method (Japanese Patent Laid-Open No. 2-207791).

Culture to obtain microorganisms or cells that produce LDC depends on the microorganisms or cells used.
A method suitable for DC production may be used.
For example, the medium may be a normal medium containing a carbon source, a nitrogen source, inorganic ions, and other organic components as required. Carbon sources include glucose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, sugars such as ribose and starch hydrolysates, alcohols such as glycerol, mannitol and sorbitol, gluconic acid, fumaric acid, citric acid And organic acids such as succinic acid can be used. As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia and the like can be used. As organic micronutrients, it is desirable to contain appropriate amounts of required substances such as vitamins such as vitamin B1, nucleic acids such as adenine and RNA, or yeast extract. In addition to these, a small amount of calcium phosphate, magnesium sulfate, iron ion, manganese ion or the like is added as necessary.

  For example, in the case of Escherichia coli, the culture is preferably carried out under aerobic conditions for about 16 to 72 hours, and the culture temperature is controlled to 30 ° C. to 45 ° C., and the pH is controlled to 5 to 8 during the culture. In addition, an inorganic or organic acidic or alkaline substance, ammonia gas or the like can be used for pH adjustment.

In addition, when the expression of the LDC gene is regulated by an inducible promoter, an inducing agent is added to the medium.
After culturing, the cells can be recovered from the culture solution by collecting them with a centrifuge or a membrane. The cells may be used as they are, but when using a treated product containing LDC, the cells are disrupted by ultrasonication, French press, or enzymatic treatment to extract the enzyme, and a cell-free extract is obtained. In the case of purifying LDC from, it can be purified by using ammonium sulfate salting out and various chromatographies according to a conventional method.

  Hereinafter, the present invention will be described more specifically with reference to examples.

[Example 1] Construction of an LDC-amplified strain of Escherichia coli
E. Nucleotide sequence of LDC gene (cadA) of Escherichia coli (N. Watson et al., Journal of bacteriology, (1992) vo.174, p.530-540; SY Meng and GN Bennet, Journal of bacteriology,
(1992) vol.174, p.2659-2669), PCR primers having the nucleotide sequences shown in SEQ ID NOS: 1 and 2 were designed. A DNA fragment containing cadA was amplified by PCR using the chromosome of E. coli W3110 (ATCC39936) as a template.

  The amplified DNA fragment was cleaved with KpnI and SphI, and the obtained fragment (2468 bp) was inserted into the KpnI and SphI cleavage sites of pUC18 (Takara Shuzo Co., Ltd.) to prepare plasmid pcadA (FIG. 1). With this plasmid pcadA, Escherichia coli JM109 strain (Takara Shuzo Co., Ltd.) was transformed. Transformants were selected using ampicillin resistance as an index. It was named coli JM109 / pcadA.

[Example 2] Production of cadaverine adipate from lysine adipate using cadA amplified strain (1) Cultivation of cadA amplified strain
E. After pre-culturing Escherichia coli JM109 / pcadA in LB medium, 1 ml of jar fermenter containing 50 ml of culture solution containing 500 ml of double concentration LB medium (tryptone: 2%, yeast extract: 1%, NaCl: 1%) (Able Co., Ltd.) was inoculated and cultured with aeration and stirring at an aeration rate of 250 ml / min, 35 ° C. and 700 rpm. After culturing for 15 hours, the whole culture solution was inoculated into a 50 L jar fermenter containing 22 L of LB medium having a double concentration, and further cultured. The culture conditions in the 50 L jar were an aeration rate of 11 L / min, 35 ° C., an internal pressure of the jar of 50 kPa, and 250 rpm. At 4 hours of culture, 3 g of IPTG (isopropyl-β-D-thiogalactopyranoside) was dissolved in 50 ml of water and then added through a filter. Thereafter, the culture was continued for 22 hours.

(2) Separation of bacterial cells The bacterial cells were collected from the culture solution using a tubular separator under the conditions of 17,000 rpm and a feed rate of 550 ml / min. The collected microbial cells were scraped from the cylinder inner sheet and suspended in 1 L of physiological saline to obtain a microbial cell solution. The wet weight of the collected cells was 147 g.

(3) Manufacture of cadaverine adipate Addition of adipic acid to 50% (w / v) lysine base solution (Daiichi Fine Chemical) so that the pH is 6.0 Was prepared as a substrate solution. A substrate solution is added to water to a final concentration of 50 g / L in terms of lysine concentration, and pyridoxal phosphate is added to 0.1 mM to prepare a reaction solution. The reaction was started by adding a bacterial cell solution of Escherichia coli JM109 / pcadA (wet cell weight 147 g). The reaction was carried out by charging a 22 L reaction solution into a 50 L jar fermenter. The reaction conditions were 37 ° C., 1/10 vvm aeration, 250 rpm, and an internal pressure of 5 kPa. The pH of the reaction solution was controlled at 6.0 by adding adipic acid slurry (250 g / kg-H ₂ O).

  At the second and third hours of the reaction, a substrate solution corresponding to 1 kg of lysine was added and the reaction was further continued. In 6 hours of reaction, lysine was converted to almost 100% cadaverine. Lysine and cadaverine were measured by a post-column OPA method using HPLC (S. R. Vale and M. B. Gloria, Journal Of AOAC International (1997) vol. 80, p. 1006-1012). The measurement results are as follows.

Cadaverine concentration 69g / L (0.68M)
Adipic acid concentration 105g / L (0.72M)
Residual lysine concentration <1 g / l
Cadaverine yield 2.2 kg (21 mol)
Charge of lysine 3.1kg (21mol)
Conversion yield 100% (mol / mol)

  As described above, a cadaverine-adipate solution containing cadaverine and adipic acid in approximately equimolar amounts was obtained.

[Example 3] Acquisition of cadaverine / adipate crystals (1) Disinfection of cadaverine / adipate solution The cadaverine / adipate solution obtained in Example 2 was autoclaved at 120 ° C. for 10 minutes and then centrifuged. The supernatant was recovered.

(2) Decolorization and concentration To the supernatant, activated carbon was added at a weight of 20% by weight with respect to cadaverine, and decolorized while stirring at 20 ° C. for 1 hour. Activated carbon was removed with filter paper, and the obtained filtrate was concentrated 4-5 times under reduced pressure (55-60 ° C., 110-150 mmHg). The solid content of the concentrate was 70-77%.

(3) Crystallization and crystal separation of cadaverine adipate The concentrated solution was cooled from 60 ° C to 10 ° C at 4 ° C / hour to precipitate crystals. The crystallization rate was 40 to 45%. The precipitated crystals were separated and recovered with a centrifugal filter and then air-dried for several days in a desiccator. The obtained crystal was analyzed by X-ray crystal analysis (AFC-5S, manufactured by Rigaku Corporation, analysis program: TEXAN). As a result, it was cadaverine, adipate, dihydrate, and the purity was 99% or more. It was. This crystal is an equimolar crystal of cadaverine and adipic acid and can be used as it is for the polymerization reaction of nylon.

[Example 4] Production of cadaverine succinate from lysine succinate using cadA amplified strain
E. coli JM109 / pcadA was inoculated into a 500 ml Sakaguchi flask containing 50 ml of LB medium and 100 mg / L of ampicillin, and pre-cultured at 28 ° C. for 8 hours. 12 ml of this culture solution was inoculated into a 1 L jar fermenter (manufactured by Able) containing 500 ml of double LB medium, and aerated and stirred culture was performed at an aeration rate of 250 ml / min at 35 ° C. and 700 rpm. At 3.5 hours of culture, IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.67 mM. Thereafter, the culture was continued for 12.5 hours. Centrifugation was performed at 8,000 rpm for 10 minutes, the supernatant was removed, and the cells were collected from the culture solution. The collected cells were stored frozen at −80 ° C., thawed on ice at the time of use, and suspended in pure water to obtain an enzyme solution. The wet weight of the collected cells was 10.3 g / liter of culture solution.

  A lysine / succinate solution was prepared by adding succinic acid to a 50% (w / v) lysine base solution (manufactured by Daiichi Fine Chemical Co., Ltd.) so that the pH was 6.0. Water is added to the substrate solution to a final concentration of 100 g / L in terms of lysine concentration, and pyridoxal phosphate is further added to a concentration of 0.1 mM to prepare a reaction solution, to which E. coli JM109 / pcadA is added. Body fluid (wet cell weight 0.309 g) was added to initiate the reaction. The reaction was performed by charging 0.3 L of the reaction liquid into a 1 L jar fermenter. The reaction conditions were 37 ° C., 1/10 vvm aeration, and 200 rpm. The pH of the reaction solution was controlled to 6.0 by adding succinic acid crystals to an equimolar amount (24.23 g) with respect to the amount of lysine charged, and then reacted for 3 hours without pH control. The pH at the end of the reaction was 7.2.

  The results of measuring lysine and cadaverine by the post-column OPA method using HPLC are shown below. The decarboxylation reaction proceeded well, and a cadaverine / succinate solution containing cadaverine and succinic acid in approximately equimolar amounts was obtained.

Cadaverine concentration 69.5g / L (0.685M)
Residual lysine concentration 0.17 g / L
Succinic acid added concentration 78.3 g / L (0.663M)
Cadaverine yield 21.6 g (213 mmol)
Lysine charge 30g (204mmol)
Conversion yield 103%

[Example 5] Production of cadaverine sebacate from lysine sebacate using cadA amplified strain In the same manner as in Example 4, 1 L jar fermenter (manufactured by Able) of E. coli JM109 / pcadA was used. And the cells were collected. Sebacic acid was added to a 50% (w / v) lysine base solution (manufactured by Daiichi Fine Chemical) so that the pH was 6.6 to prepare a lysine / sebacate salt solution, which was used as a substrate solution. Water is added to the substrate solution to a final concentration of 100 g / L in terms of lysine concentration, and pyridoxal phosphate is further added to a concentration of 0.1 mM to prepare a reaction solution, to which E. coli JM109 / pcadA is added. Body fluid (wet cell weight 0.309 g) was added to initiate the reaction. The reaction was performed by charging 0.3 L of the reaction liquid into a 1 L jar fermenter. The reaction conditions were 37 ° C., 1/10 vvm aeration, and 200 rpm. The pH of the reaction solution was controlled to 6.6 by adding sebacic acid crystals to an equimolar amount (41.51 g) with respect to the amount of charged lysine, and then reacted for 3 hours without pH control. The pH at the end of the reaction was 7.1.

  The results of measuring lysine and cadaverine by the post-column OPA method using HPLC are shown below. The decarboxylation reaction proceeded well, and a cadaverine / sebacate solution containing cadaverine and sebacic acid in approximately equimolar amounts was obtained.

Cadaverine concentration 63.4g / L (0.622M)
Residual lysine concentration 0.17 g / L
Sebacic acid concentration 128.9 g / L (0.637M)
Cadaverine yield 20.2g (198mmol)
Lysine charge 30g (204mmol)
Conversion yield 97%

The figure which shows the structure of plasmid pcadA which has the cadA gene of E. coli.

Claims (9)

Cadaverine dicarboxylate is formed by performing enzymatic decarboxylation of lysine while adding dicarboxylic acid to the lysine solution so that the pH of the solution is maintained at a pH suitable for enzymatic decarboxylation. , Method for producing cadaverine dicarboxylate. The method according to claim 1, wherein the pH is 4.0 to 8.0. The method according to claim 1 or 2, wherein the dicarboxylic acid is a dicarboxylic acid having 4 to 10 carbon atoms. The method of claim 3, wherein the dicarboxylic acid is adipic acid. The method according to any one of claims 1 to 4, wherein the enzymatic decarboxylation reaction is performed using lysine decarboxylase, a cell that produces lysine decarboxylase, or a treated product of the cell. The method according to claim 5, wherein the cell is a cell modified to increase lysine decarboxylase activity. The cell has increased the copy number of a gene encoding lysine decarboxylase, or the expression regulatory sequence of the gene has been modified so that expression of the gene is enhanced, whereby lysine decarboxylase activity is increased. 7. The method of claim 6, wherein the method is an elevated recombinant cell. The method according to claim 7, wherein the cell is an Escherichia coli cell, and the gene encoding lysine decarboxylase is a cadA gene. Cadaverine dicarboxylate is formed by performing enzymatic decarboxylation of lysine while adding dicarboxylic acid to the lysine solution so that the pH of the solution is maintained at a pH suitable for enzymatic decarboxylation. The manufacturing method of nylon including the process and the process of producing | generating nylon by polycondensing the cadaverine dicarboxylate salt obtained at the said process.

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