KR100847984B1 - 26- Purification method of crude naphthalene dicarboxylic acid using recombinated microorganism and 26-naphthalene dicarboxylic acid in crystalline form obtained by using the same - Google Patents

Abstract

The present invention relates to the xanthine dehydrogenase gene pucABCDE or aldehyde dehydrogena derived from Bacillus subtilis HSB-21 having the ability to convert 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid. Recombinant microorganisms prepared using the genes aldX and aldY are reacted with crude naphthalene dicarboxylic acid to form 2-formyl-6-naphthoic acid, which is an impurity contained in the crude naphthalene dicarboxylic acid, as 2,6-naphthalene dicarboxylic acid. After conversion to acid and removal, acidic solution was added thereto under certain conditions, and then the crude naphthalene dicarboxylic acid was crystallized while stirring, followed by washing to remove other impurities, followed by drying the wash. A method for purifying crude naphthalene dicarboxylic acid characterized by obtaining 2,6-naphthalene dicarboxylic acid in a crystalline state. According to the present invention, there is an advantage that mass production of high purity crystalline 2,6-naphthalene dicarboxylic acid can be economically and environmentally friendly.

Recombinant microorganisms, crude Naphthalene dicarboxylic acid, 2-formyl-6-naphthoic acid, 2,6-naphthalene dicarboxylic acid dicarboxylic acid), crystallization

Description

Purification method of crude naphthalene dicarboxylic acid using recombinated microorganism and 2,6-naphthalene dicarboxylic acid in crystalline form obtained by using the same}

1 is a genetic map of the recombinant expression vector pBS- pucABCDE comprising the gene pucABCDE encoding xanthine dehydrogenase from Bacillus subtilis HSB-21 of the present invention,

2 is a genetic map of a recombinant expression vector pBS- alDX comprising a gene aldX encoding an aldehyde dehydrogenase derived from Bacillus subtilis HSB-21 of the present invention,

3 is a genetic map of the recombinant expression vector pBS- alDY containing the gene aldY encoding aldehyde dehydrogenase from Bacillus subtilis HSB-21 of the present invention,

4 is a micrograph of the crystallized cNDA obtained in the third step of Example 1 of the present invention,

Figure 5 illustrates an aspect of a purification apparatus system for carrying out the process for purifying crude naphthalene dicarboxylic acid according to the present invention.

* Description of the symbols for the main parts of the drawings *

A: microbial purification reactor B: microbial removal device

C: Crystallization Reactor D: Preheater

E: Solvent heating supply device F: Filtration and cleaning device

G: High pressure filtrate recovery device H: High pressure slurry recovery device

I: Powder Separator J: Drying Machine

The present invention relates to a method for purifying crude naphthalene dicarboxylic acid using a recombinant microorganism and 2,6-naphthalene dicarboxylic acid in a crystalline state obtained by the above method, and more specifically, 2-formyl-6- Bacillus subtilis having the ability to convert the naphthoic acid into 2,6-naphthalene dicarboxylic acid subtilis HSB-21 (Bacillus subtilis HSB-21 KCTC 11221) derived from the xanthine dehydrogenase gene pucABCDE or the aldehyde dehydrogenase genes aldX , aldY were reacted with crude naphthalene dicarboxylic acid to react the crude naphthalene dicarboxylic acid. 2-formyl-6-naphthoic acid, which is an impurity contained in the acid, is converted to 2,6-naphthalene dicarboxylic acid and removed, and then, an acidic solution is added thereto under constant conditions, and then the crude naphthalene dicarboxylic acid is stirred. Crystallization, washing it to remove other impurities, and drying the washing to obtain 2,6-naphthalene dicarboxylic acid in pure crystalline state, wherein the crude naphthalene dicarboxylic acid is purified. It is about.

Diesters of 2,6-naphthalene dicarboxylic acid and 2,6-naphthalene dicarboxylic acid are useful monomers for preparing various types of polymeric materials such as polyesters and polyamides. For example, poly (ethylene), which is a high performance polyester material, by condensation of a diester of 2,6-naphthalene dicarboxylic acid (hereinafter referred to as NDA) and 2,6-naphthalene dicarboxylic acid (hereinafter referred to as NDC) with ethylene glycol. 2,6-naphthalate) (hereinafter PEN) can be produced. Fibers and films made from PEN have higher strength and better thermal properties than poly (ethylene terephthalate) (hereinafter PET). This makes PEN a very good material for making commercial products such as thin films that can be used to make magnetic recording tapes and electronic components. Films made of PEN are also useful for making food containers, especially food containers for high temperature fillings, due to their good resistance to gas diffusion, in particular carbon dioxide, oxygen and water vapor. It can also be used to make reinforcing fibers useful for making tire cords.

Currently, NDC is produced by oxidizing 2,6-dimethylnaphthalene (hereinafter, 2,6-DMN) to produce crude naphthalene dicarboxylic acid (hereinafter, cNDA) and then esterifying it. Currently, NDC is used as a main raw material when synthesizing PEN, but there are some problems compared to using NDA as a raw material. First, water is produced during NDA condensation reaction, while methanol is a by-product in the case of NDC, and there is a risk of explosion. Second, NDA is esterified to obtain pure NDC during the NDC manufacturing process, and then NDC is produced through purification process. Therefore, one step process is needed compared to NDA, and thirdly, if you have existing PET production facilities, the use of NDC is not appropriate in terms of the use of existing facilities. Despite the disadvantages of NDC, NDC is used instead of NDA in PEN production because it is still difficult to produce purified NDA having the purity required for polymerization.

In the oxidation of 2,6-DMN, cNDA containing various impurities such as 2-formyl-6-naphthoic acid (hereinafter referred to as FNA), 2-naphthoic acid (hereinafter referred to as NA) and trimellitic acid (hereinafter referred to as TMLA) Is generated. In particular, the presence of FNA causes the polymerization reaction to stop in the middle, which leads to a decrease in the degree of polymerization, which adversely affects the physical properties of the polymer or causes the coloring of the polyester. Therefore, the FNA must be removed, but there is a difficult problem. .

Therefore, in order to remove FNA present in cNDA or to purify NDA, recrystallization method, oxidation process is performed once more, cNDA is prepared by NDC using methanol and then hydrated to produce NDA or purification by hydrogenation process. Various chemical methods have been studied, such as the preparation of NDA, and in addition, various purification methods such as solvent treatment, melted crystal, high pressure crystal, and supercritical extraction have been used.

As a specific example, U.S. Patent No. 5,859,294 discloses that when cNDA is dissolved in an aqueous solution containing aliphatic amine or alicyclic amine, the impurity contained therein becomes 100 ppm or less. It is disclosed that a method for producing naphthalene dicarboxylic acid by removing the solution up to and then distilling the amine component by heating the aqueous solution containing the naphthalene dicarboxylic acid amine salt,

Another U. S. Patent No. 6,255, 525 discloses a mixture of aromatic carboxylic acid containing impurities and water at 277 to 316 DEG C, 77-121 kg / cm ² , and hydrogenation metal component in the presence of hydrogen gas. a high purity aromatic carboxylic acid by contacting with a carbon catalyst having no component) and then cooling the mixed solution to crystallize the aromatic carboxylic acid, and recovering the crystallized aromatic carboxylic acid from the cooled mixed solution. The method is disclosed.

Also in connection with the crystallization of NDA, U.S. Patent No. 6,087,531 discloses a poly (alkylene naphthalene dicarboxylate) [poly (alkylene naphthalene dicarboxylate)] at a basic solution (alkali metal basic solution, hydroxide) at 125 to 400 ° C. (hydroxide) or alkali metal carbonate) and neutralized with acid at 170-240 ° C. to recover NDA crystals, or the polyester material was dissolved in NaOH solution or KOH solution at 170-240 ° C. and then neutralized with acetic acid. To disclose how to recover an NDA decision,

U.S. Patent No. 6,426,431 describes KH-NDA by treating K ₂ -NDA aqueous solution with CO ₂ at 0-50 ° C. and 0-200 psi to form KH-NDA. Suspended and disclosed to increase the purification yield of 2,6-NDA to about 45% by treatment with CO ₂ again at 100 ° C. or higher (140-160 ° C.) and 100 psi or higher (175-250 psi) conditions. have.

However, in order to obtain high purity NDA crystals, the above-mentioned methods have a problem in that it is economically inadequate by requiring high temperature and high pressure conditions, a long time treatment, a large amount of expensive materials, etc., the purification yield and purity are very low, and the obtained NDA individual There is a problem that agglomeration phenomenon between crystals is difficult to apply to actual production.

The present invention is to solve the above-mentioned problems of the prior art, an object of the present invention by using a novel recombinant microorganism converting FNA to NDA to purify and crystallize cNDA at room temperature and atmospheric pressure conditions to determine the high yield and high purity NDA To provide a method for efficiently obtaining the.

Another object of the present invention is to provide a NDA crystal having a high purity uniform size obtained by the above method.

One aspect of the present invention for achieving the above object relates to a method for purifying crude naphthalene dicarboxylic acid using recombinant microorganisms.

More specifically, the present invention

(a) Bacillus subtilis HSB-21 represented by SEQ ID NO: 1 ( Bacillus a recombinant microorganism obtained by transformation with a xanthine dehydrogenase gene pucABCDE derived from subtilis HSB-21 KFCC 11221) or a recombinant expression vector comprising a gene having a homology of 90% or more with the gene; As a recombinant expression vector comprising an aldehyde dehydrogenase gene aldX derived from Bacillus subtilis HSB-21 KFCC 11221 represented by SEQ ID NO: 4 or a gene having a homology of 90% or more to the gene. Recombinant microorganisms obtained by transformation; Or a recombinant expression comprising an aldehyde dehydrogenase gene aldY derived from Bacillus subtilis HSB-21 KFCC 11221 represented by SEQ ID NO: 7 or a gene having at least 90% homology with the gene. Recombinant microorganisms obtained by transformation with a vector are reacted with crude naphthalene dimethylcarboxylic acid to convert 2-formyl-6-naphthoic acid in the crude naphthalene dicarboxylic acid to 2,6-naphthalene dicarboxylic acid. Switching to and removing;

(b) crystallizing the crude naphthalene dicarboxylic acid by adding an acidic solution to the reaction solution obtained in step (a) to adjust pH and then reacting with stirring;

(c) washing the crystallized crude naphthalene dicarboxylic acid to remove impurities contained therein, the step of dispersing the crystallized crude naphthalene dimethylcarboxylic acid in water, followed by a pressure of 1 to 60 kg / cm ² Agitating for 10 minutes to 1 hour at a temperature of 100 to 270 ° C., followed by filtration to remove the water several times; And

(d) a method of purifying crude naphthalene dicarboxylic acid using a recombinant microorganism comprising drying the wash to obtain 2,6-naphthalene dicarboxylic acid in a pure crystalline state. do.

Hereinafter, the method for purifying crude naphthalene dicarboxylic acid of the present invention will be described in detail step by step.

(Iii) First step: purification process using recombinant microorganisms

First, a recombination comprising a xanthine dehydrogenase gene pucABCDE derived from Bacillus subtilis HSB-21 KFCC 11221 represented by SEQ ID NO: 1 or a gene having a homology of 90% or more with the gene Recombinant microorganisms obtained by transformation with expression vectors; As a recombinant expression vector comprising an aldehyde dehydrogenase gene aldX derived from Bacillus subtilis HSB-21 KFCC 11221 represented by SEQ ID NO: 4 or a gene having a homology of 90% or more to the gene. Recombinant microorganisms obtained by transformation; Or Bacillus subtilis HSB-21 represented by SEQ ID NO: 7 ( Bacillus subtilis HSB-21 KFCC 11221) derived from the aldehyde dehydrogenase gene aldY or a recombinant microorganism obtained by transforming with a recombinant expression vector containing a gene having at least 90% homology with the gene, crude naphthalene dicarboxylic acid (crude) naphthalene dimethylcarboxylic acid) to remove 2-formyl-6-naphthoic acid in the crude naphthalene dicarboxylic acid by conversion to 2,6-naphthalene dicarboxylic acid.

At this time, in the present invention, "gene having a homology of 90% or more" means an enzyme gene used in the present invention, that is, a base sequence or amino acid sequence with the xanthine dehydrogenase gene pucABCDE or the aldehyde dehydrogenase gene aldX or aldY. A gene having a homology of 90% or more at the level means a gene having a nucleotide sequence or an amino acid sequence portion of the gene which is different within 10% but exhibits the same function as that of the enzymes. The different sequence portions may be modified in any case that can be modified by one of ordinary skill in the art without significantly affecting the inherent function of the enzymes, ie, xanthine dehydrogenase or aldehyde dehydrogenase. Include.

Specifically, the step 1) inoculates the recombinant microorganism into a liquid medium to induce the expression of xanthine dehydrogenase or aldehyde dehydrogenase, and then the cells recovered through centrifugation are suspended in physiological saline or distilled water. Preparing a reaction cell; 2) mixing the crude naphthalene dicarboxylic acid as a substrate with a buffer solution and then adding an alkaline solution to adjust the pH of the mixed solution to form a purification reaction solution; And 3) reacting the reaction cells prepared in step 1) with the reaction solution prepared in step 2) to convert 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid to 2,6-naphthalene. Increasing the purity of the dicarboxylic acid.

Recombinant microorganisms used in the present invention, Bacillus subtilis HSB-21 represented by SEQ ID NO: 1 ( Bacillus subtilis HSB-21 KFCC 11221) xanthine dehydrogenase gene pucABCDE or gene having at least 90% homology with said gene; Bacillus subtilis HSB-21 represented by SEQ ID NO: 4 ( Bacillus subtilis HSB-21 KFCC 11221) aldehyde dehydrogenase gene aldX or a gene having at least 90% homology with the gene; Or a general method in which a aldehyde dehydrogenase gene aldY derived from Bacillus subtilis HSB-21 KFCC 11221, represented by SEQ ID NO: 7, or a gene having at least 90% homology with the gene is commonly known. After cloning and introducing into an expression vector to produce a recombinant expression vector, it can also be obtained by introducing into a microorganism and transforming it according to conventional methods known in the art.

Specifically, it can be obtained by the following method:

That is, in order to clone each of the enzyme genes, pucABCDE , aldX , or aldY , genomic DNA isolated from Bacillus subtilis HSB-21 is used as a template, and then, PCR is performed using the primers to obtain genes encoding each of the above enzymes. In this case, in some cases, according to a conventional method known in the art, it is also possible to obtain a gene in which some nucleotide sequences are modified.

Subsequently, the cloned pucABCDE , aldX , or aldY genes are operably linked to the promoter of the expression vector, respectively, in a conventional manner to prepare a recombinant expression vector.

At this time, the expression vector is not particularly limited, but it is preferable to use a vector that enables optimal expression of a gene in the host, and for example, it can be inserted into a plasmid, phage or other DNA. Examples of the plasmid include, but are not particularly limited to, E. coli , known pForexT vectors, pHCEIIB (Nde I), pLG338, pACYC184, pBR322, pUC119, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III ^138- B1, λgt11, or pBDCl, and the like in Streptomyces , pIJ101, pIJ364, pIJ702 or pIJ361, and in Bacillus . pUB110, pC194 or pBD214, and 2 μM in yeast, pAG-1. YEp6, YEp13, pEMBLYe23, and the like.

The promoter is cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacI ^q , T7, T5, T3, gal, trc, ara, SP6, λ- P _R or λ-P _L promoter and the like can be used, gram positive promoter amy and SPO2, or fungal or yeast promoter ADC1, MFα, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH promoter, etc. can be used. However, the present invention is not limited thereto.

It is also possible to further include regulatory sequences at the 3 'end and / or 5' end to increase optimal expression depending on the host organism or gene selected.

In this regard, Figure 1 shows a genetic map of the recombinant expression vector pBS- pucABCDE comprising the gene pucABCDE encoding xanthine dehydrogenase derived from Bacillus subtilis HSB-21 used in one embodiment of the present invention, Figure 2 shows a genetic map of the recombinant expression vector pBS- aldX comprising the gene aldX encoding aldehyde dehydrogenase derived from Bacillus subtilis HSB-21 used in another embodiment of the present invention, Figure 3 The gene map of the recombinant expression vector pBS- alDY containing the gene aldY encoding the aldehyde dehydrogenase derived from Bacillus subtilis HSB-21 used in another embodiment of the invention is shown.

Cloning of each enzyme gene into such a recombinant expression vector can be confirmed through restriction enzyme cleavage and sequence analysis.

Then, the recombinant microorganism is obtained by transforming the microorganism by introducing the prepared recombinant expression vector into the host microorganism according to a generally known method. At this time, all prokaryotic or eukaryotic organisms may be used as the host microorganism, and examples thereof include microorganisms such as bacteria, fungi or yeast, but are not necessarily limited thereto. For example, Gram-positive bacteria or Gram-negative bacteria, preferably Enterobacteriaceae or Norrcardiace bacteria can be used, and more preferably Escherichia, Pseudomonas, and Rhodokocos , Bacteria of the genus Bacillus can be used. Most preferably, the genus or species of E. Coli may be used, specifically, MC1061 ( E. coli ), JM109 ( E. coli ), XL1-Blue ( E. coli ), DH5α ( E. coli ) Etc. can be mentioned. As a transformation method, a heat treatment, an electric shock method, a micro injection method, a calcium chloride method, a rubidium chloride method, a spray method using a pressure, etc. may be used, but are not necessarily limited thereto.

Specifically, the recombinant microorganism is sufficiently preincubated at a normal culture temperature of 25 to 45 ° C., preferably 37 ° C., which is then 0.1 to 100 to 200 ml of liquid medium (eg, LB medium or LBA medium). Expression of each enzyme can be induced by inoculation to 1% (v / v) and shaken at 37 ° C. for a sufficient time. Cultivation of such recombinant microorganisms and expression of enzymes are not limited to the above methods, and any method known to those skilled in the art to which the present invention pertains may be used without limitation.

Since the recombinant microorganism of the present invention is capable of high expression of the enzyme as described above, it is easy to cultivate high concentrations and has an economically advantageous effect.

The recombinant microorganism with high expression of the enzyme recovers only the cells through centrifugation from the culture medium and suspends them in physiological saline or distilled water and uses them as reaction cells for purifying crude naphthalene carboxylic acid.

The buffer solution is not particularly limited, but water, sodium carbonate buffer solution, glycine buffer solution, potassium phosphate buffer solution, sodium phosphate buffer solution, succinic acid buffer solution, sodium acetate buffer solution, citric acid buffer solution, sodium pyrophosphate buffer solution, Boric acid buffer, sodium borate buffer, and the like can be used. Preferably, potassium phosphate buffer or boric acid buffer solution having a pH range of 6.0 to 9.0 is used. At this time, the concentration of the buffer solution is preferably used at a concentration of 0.01 to 100 mM.

Although not necessarily limited to the alkaline solution, it is preferable to use NaOH solution or KOH solution, and the pH of the mixed solution is preferably adjusted to 6 to 10, further 7 to 9.

Meanwhile, an organic solvent may be additionally added to the mixed solution in step 2) for the purpose of dissolving cNDA. Examples of preferred organic solvents include dimethylsulfoxide ("DMSO"), dimethylformamide (N, N-Dimethylformamide, "DMF"), dimethylacetamide (N, N-Dimethylacetamide, "DMA"), tetrahydrofuran (Tetrahydrofuran, "THF") and the like, among which dimethyl sulfoxide is most preferably used in terms of enzymatic activity. At this time, the concentration of the organic solvent is preferably 0.01 to 20% by weight, more preferably 0.1 to 10% by weight. Most preferably not added. On the other hand, the addition of more than 20% by weight of the organic solvent shows a result of inhibiting the reaction by dissolving the cell membrane of the microorganism.

The reaction temperature and reaction time in the step 3) is preferably 25 to 80 ℃, 1 minute to 2 hours, more preferably 40 to 60 ℃, 10 minutes to 40 minutes to react. In particular, when the reaction temperature is less than 25 ° C or more than 80 ° C, the reactivity is significantly reduced and unsuitable.

On the other hand, there is a close relationship between the FNA content in the cNDA, the cNDA concentration of the reaction solution, and the amount of cells required for complete removal of the FNA. The higher the FNA content in the cNDA and the higher the concentration of cNDA in the reaction solution, the higher the amount of cells. Preference is given to using.

Specifically, in the present invention, the concentration of cNDA in the purification reaction solution is preferably 0.001 to 20% by weight, and the FNA content in cNDA is preferably used in an amount of 0.001 to 10% by weight, preferably 1 to 3% by weight.

In addition, in the present invention, the amount of cells required to completely remove 1 g of FNA is about 5 g. This is because the general microorganisms found to have the ability to convert FNA to NDA, that is, for example, the strains of the genus F-1, F-3, etc. (filed by the present inventors, domestic application No. 2002-0087819) In view of the fact that about 10g is required to completely remove 1g of FNA, it is supported that the purification of cNDA using the recombinant microorganism of the present invention is more economically effective.

(Ii) second step: crystallization process

In this step, an acidic solution is added to the reaction solution obtained in step (iii) to adjust the pH, and then reacted with stirring to crystallize the crude naphthalene dicarboxylic acid.

More specifically, in order to crystallize the cNDA in the amorphous state present in the cNDA purification reaction solution with FNA removed, an appropriate amount of an acidic solution is added to the reaction solution obtained in step (iii) in a reactor equipped with a stirrer. The pH is adjusted to a certain range, and then the reaction is maintained at an appropriate temperature with continued stirring, so that crystallization is performed at room temperature and atmospheric pressure.

According to the crystallization process of the present invention, uniform cNDA crystals having a size of 100 µm or more under normal temperature and atmospheric conditions can be obtained, and thus are economical in terms of manufacturing cost and process, and there is no agglomeration phenomenon between the obtained cNDA individual crystals. This has a high effect.

In this case, sulfuric acid, hydrochloric acid, glacial acetic acid, nitric acid, and the like may be used as the acid solution, and sulfuric acid or hydrochloric acid is more preferable in terms of the size and yield of cNDA crystals. That is, when sulfuric acid or hydrochloric acid is added, cNDA crystals having a uniform size of 100 µm or more can be obtained in high yield.

The pH range is preferably adjusted to 1 to 4 in terms of recovery rate, and the lower the pH, the higher the recovery rate to 99.9%.

In addition, the crystallization temperature is about 4 to 130 ℃, preferably 30 to 100 ℃, more preferably 50 to 90 ℃ suitable for carrying out the crystallization reaction, the reaction time is about 1 minute to 12 hours, preferably 2 Minutes to 5 hours, more preferably 10 minutes to 1 hour range is preferable in terms of the continuous process. Most preferred crystallization temperature and reaction time is 75 ° C., 20 to 30 minutes. If the crystallization time is too short, the degree of crystallinity is low, aggregation may occur between individual crystals during the polymerization reaction, and excessively long crystallization time may cause unnecessary energy loss.

As a suitable stirring speed which performs crystallization, it is preferable to carry out at 0 rpm and 1000 rpm or less, Preferably it is 0 rpm and 400 rpm or less, More preferably, it is performed at 30-200 rpm.

(Iii) step 3: cleaning process

In this step, the crude naphthalene dicarboxylic acid crystallized through step (ii) is washed to remove impurities contained therein.

After purification and crystallization using recombinant microorganisms, FNA is converted to NDA and removed, but other impurities such as 2-naphthoic acid (NA), methylnaphthoic acid (MNA), and trimellitic acid (TLMA) remain as they are without being removed. Done. Therefore, if the crystallized cNDA is dispersed in water and washed several times under certain conditions, it is possible to remove the other impurities by using the difference in solubility. At this time, not only the impurities but also the recombinant microorganisms used to purify cNDA are also removed.

In this step, it is preferable that the reaction by-products are dissolved in the solvent and separated as much as possible from NDA, and the temperature range is 100 to 270 ° C, preferably 150 to 240 ° C. When the solvent is separated at a temperature exceeding 270 ℃, the loss of purified NDA increases, there is a problem that the yield is significantly reduced, when the separation at less than 100 ℃ by-products such as NA, MNA, TLMA is well dissolved Not preferred.

More specifically, by dispersing the crystallized cNDA in water and then stirred for 10 minutes to 1 hour at a pressure of 1 to 60 kg / cm ² , temperature 100 to 270 ℃ and then filtered to remove the water several times Impurities can be removed, where the amount of solvent is preferably used 5 to 20 times the weight of NDA.

(Iii) 4th step: drying process

Finally, the NDA crystal from which impurities are removed through the washing process is dried at a constant temperature to obtain final pure NDA crystal. At this time, the drying treatment is preferably carried out at 40 to 200 ℃, preferably 50 to 150 ℃, the drying method can be used without limitation the conventional drying method known in the art to which the present invention belongs.

On the other hand, the crude naphthalene dicarboxylic acid purification method of the present invention may further comprise the step of removing the recombinant microorganism used in the step (iii) between the step (iii) and (ii).

As such, if the recombinant microorganism is removed after the purification of cNDA in advance to perform a crystallization reaction, the purity and recovery of NDA crystals may be further improved.

As a method for removing the recombinant microorganism, a conventionally known method may be used without limitation, and specifically, may be removed using a microfilter system, a continuous centrifuge or a decanter. In this case, in the case of the microfilter system, a filter having a pore size of 0.1 to 2.0 μm and made of a material such as ceramic, stainless steel, polypropylene, or PET may be used.

Another aspect of the present invention for achieving the above object relates to 2,6-naphthalene dicarboxylic acid in pure crystalline state obtained by the purification method of the present invention.

The 2,6-naphthalene dicarboxylic acid provided by the present invention is obtained according to the above-described purification method of the present invention and is obtained in high yield and high purity of 99.9% or more. Can be obtained in a crystalline state. In particular, in the case of the shape can have a lattice structure, but is not necessarily limited thereto.

In addition, the 2,6-naphthalene dicarboxylic acid crystal produced in the present invention has a large and uniform particle shape with an average particle diameter of 100 µm or more, and thus is very suitable for forming ethylene glycol and a low viscosity slurry during the PEN polymerization process. It is a crystalline form. If the average particle size is small, handling of the product is difficult and power consumption is problematic because the slurry formation ability is poor when the slurry is mixed with ethylene glycol in the PEN polymerization process. Preferably the 2,6-naphthalene dicarboxylic acid crystals of the present invention have an average particle diameter in the range of 100 to 200 μm, more preferably 110 to 170 μm.

On the other hand, Figure 5 shows an aspect of a purification apparatus system for carrying out the method for purifying crude naphthalene dicarboxylic acid according to the present invention. An embodiment of the method for purifying crude naphthalene dicarboxylic acid according to the present invention based on FIG. 5 will be described in more detail as follows.

First, a certain amount of buffer solution is injected into the microbial purification reactor (A), and cNDA is added thereto to add an alkali solution while stirring to adjust the pH of the reaction solution, and then a predetermined amount of water is added to prepare a purification reaction solution. do. While maintaining the reaction solution at a constant temperature, the prepared recombinant microbial cells for reaction are prepared and reacted. Through this process, FNA can be removed by converting FNA in cNDA into NDA in the microbial purification reactor (A).

The purified reaction solution is passed through the microbial removal device (B) to remove the recombinant microorganism used to remove the FNA. The recombinant microorganism removal process using this microorganism removal apparatus B can be omitted as needed. Even if the microorganism removal step is omitted, the recombinant microorganism can be removed by the rear filtration and washing apparatus (F).

The purified reaction solution from which the recombinant microorganisms have been removed is transferred to a crystallization reactor (C), and an acidic solution is added thereto and the crystallization reaction proceeds while stirring.

Then, the slurry containing the crystallized cNDA is heated to about 100 to 270 ℃ by the preheater (D) and introduced into the filtration and washing apparatus (F). The filtration and washing apparatus (F) is provided with heating means for maintaining the temperature of the slurry solution at 100 to 270 ℃. In addition, the pressure is maintained at 1 to 60 kg / cm ² , it is equipped with a filter of 10 to 100 ㎛ for filtration.

The filtration and cleaning device (F) is connected to the high pressure filtrate recovery device (G) to discharge the filtrate to the high pressure filtrate recovery device (G) and filter the solid phase into the filter.

Water preheated to 100 to 270 ° C. from the solvent heating supply device (E) was added to the filtration and washing device (D) again, stirred for a while, and then filtered through a high pressure filtrate recovery device (G). Release the liquid. If necessary, the above washing steps may be repeated one or two more times. The pure NDA remaining after washing forms a slurry with water pre-heated from 100 ° C. to 270 ° C. from the solvent heating supply device E, and is sent to the high pressure slurry recovery device H through the slurry discharge line. Pure NDA slurry sent to the high pressure slurry recovery device (H), the pressure is lowered to normal pressure, the solvent is removed through the powder separation device (I) and dried in the drying device (J), pure NDA can be recovered.

Hereinafter, the specific configuration and operation of the present invention will be described by Examples and Experimental Examples, which are intended to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[ Example  One]

Step 1: Preparation of Recombinant Microorganisms

(1) Cloning of the pucABCDE Gene

Bacillus subtilis HSB-21 ( Bacillus To clone the pucABCDE gene (SEQ ID NO: 1) encoding xanthine dehydrogenase from subtilis HSB-21 KCTC 11221), genome DNA comprising the pucABCDE gene sequence was first isolated (see Sambrook et al. al ., Molecular Cloning, A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Primer 1 (5'-ATGGGGAATTTTCACACGATGTTAGATGCG-3 'and SEQ ID NO: 2) prepared based on the DNA sequence of the pucABCDE (GenBank Sequence Database, BSUB0017) gene using the isolated genomic DNA as template DNA. Polymerase chain reaction was performed using (5'-TTAAAAGCCGGACTCCCGTCTACCCCCGTT-3 ': SEQ ID NO: 3).

In the polymerase chain reaction, the first denaturation step was performed once at 94 ° C. for 5 minutes, and then the second denaturation step was performed at 94 ° C. for 1 minute, and the annealing step at 60 ° C. for 1 minute and 30 seconds, The extension step was performed at 72 ° C. for 7 minutes and was repeated 35 times. The fragment obtained by the above method was isolated from a DNA fragment of about 5.2 kb represented by SEQ ID NO: 1 and cloned into a pHCEIIB (Nde I) vector (Takara) to prepare a recombinant expression vector pBS- pucABCDE as shown in FIG. .

(2) Cloned Gene Analysis

For sequencing of the cloned gene of the recombinant vector (pBS- pucABCDE ) prepared in (1), the recombinant vector was cleaved using various restriction enzymes based on a sequence map of two vectors M13mp18 and M13mp19, respectively. The fragments of were subcloned into M13mp18 and M13mp19, and they were sequenced using ABI PRISM BigDye primer cycle-sequencing kit (Perkin-Elmer, USA) using AmpliTaq DNA polymerase. At this time, in order to read both directions of both strands of DNA, a synthetic nucleotide was partially prepared, and the nucleotide sequence of the cloned DNA fragment gene was analyzed and compared with the nucleotide sequence registered in GenBank (GenBank Accession Number Z99120). It was confirmed that the pucABCDE gene was cloned.

(3) Preparation of transformant

Using the calcium chloride method (see Sambrook et. al., Molecular Cloning, A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press, were transformed Cold Spring Harbor, NY, 1989) E. coli XL1-Blue to pBS- pucABCDE vector. LB plate medium (yeast extract, 5 g / L; tryptone, 10 g / L; NaCl, 10 g / L) to which ampicillin (100 mg / L) and bacto-agar (15 g / L) were added. ) Was first selected for transforming strain XL1-Blue (pBS- pucABCDE ) resistant to empicillin .

The primary screened E. coli was inoculated into 5 ml LB liquid medium containing ampicillin (ampicillin, 100mg / L), and then shaken at 37 ° C. for a sufficient time, and 5% of xanthine and empicillin 100 μl of the culture solution was added to the included LB solid medium and cultured in a 37 ° C. incubator for 24 hours, and then the formation of hollow around the colonies was observed. As a result, it was confirmed that the hollow is formed around the colony of E. coli.

(4) Expression of the pucABCDE gene

In order to express the pucABCDE gene in the transformed microorganism, E. coli obtained in (3) was inoculated in 5 ml of LB medium containing empicillin (100 mg / L), preincubated for 12 hours at 37 ° C, and then again. Inoculate 1% (v / v) in 100 ml LB medium containing empicillin (100 mg / L) and incubate for 16 hours at 37 ° C. to induce the expression of the pucABCDE gene, and then incubate at 37 ° C. It was.

Second step: using recombinant microorganisms cNDA Tablets

Centrifugation of the culture medium of the recombinant microorganism expressing the xanthine dehydrogenase obtained in the first step was recovered, washed with 0.85% saline solution and suspended again in 5 L of 0.85% saline solution for the reaction cells Was prepared.

Inject 80 L of 50 mM potassium phosphate buffer solution into the microbial purification reactor (A), add 10 kg of cNDA thereto, add KOH solution while stirring to adjust the pH of the reaction solution to 8.0, and then add water again. 95 L of a purification reaction solution (cNDA concentration: 10%, FNA content in cNDA: 1.57%) was prepared.

While maintaining the reaction solution at 50 ° C., 0.29 kg / 5 L of the reaction cell was added thereto, followed by reaction at 50 ° C. for 30 minutes, thereby converting FNA in the cNDA into NDA.

Step 3: Refined cNDA Crystallization

100 L of the cNDA purified solution obtained in the second step was transferred to a crystallization reactor (C) equipped with a stirring device, and adjusted to a pH of 3.0 by adding sulfuric acid solution thereto, and then the stirring speed was 50 rpm and the temperature was 75 The crystallization reaction proceeded for 25 minutes while maintaining at ℃.

After the crystallization was completed, the crystals were analyzed using a microscope and a particle size analyzer. As a result, the crystallization proceeded well, and it was confirmed that entanglement between the individual crystals did not occur. In addition, the average crystal size of crystallized cNDA was analyzed to be about 162.3 ㎛. A micrograph of the crystallized cNDA is shown in FIG. 4.

Step 4: Crystallize cNDA Washing and drying

The cNDA slurry solution crystallized in the third step was heated to 220 ° C. or higher through a preheater (D), and then charged into a filtration and washing apparatus (F) for 30 minutes at a pressure of 28 kg / cm ² and a temperature of 225 ° C. After stirring, the mixture was filtered to remove water with a high pressure filtrate recovery device (G). After adding 225 ° C. and 100 L of water from the solvent heating supply device (E) again, the mixture was stirred for 30 minutes under the same conditions and then filtered, and the process of removing water with the high-pressure filtrate recovery device (G) was repeated twice. It was. Then, 225 ° C. and 100 L of water were added from the solvent heating supply device (E) again, followed by stirring for 30 minutes or more to disperse evenly, and then transferred to a high pressure sludge recovery device (H) to depressurize to atmospheric pressure, and a powder separation device. Water was removed using a decanter (I), followed by drying at 130 ° C. in a drying apparatus (J) to obtain NDA in pure crystalline state.

[ Example  2]

Except that the first step was carried out in the same manner as in Example 1 to obtain a pure crystalline NDA.

(1) Cloning of the aldX Gene

Bacillus subtilis HSB-21 ( Bacillus To clone the aldX gene (SEQ ID NO: 4) encoding aldehyde dehydrogenase from subtilis HSB-21 KCTC 11221), genome DNA comprising the aldX gene sequence was first isolated (see Sambrook et al. al ., Molecular Cloning, A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). To the separation a genome DNA as the template DNA sex, aldX (reference: GenBank Sequence Database, BSUB0021) a first primer produced based on the DNA base sequence of the gene (5'-GAGGGGGATTTTCATATGGAACAGCAAGTA-3 ': SEQ ID NO: 5) and primer 2 Polymerase chain reaction was performed using (5'-GGAATTCCATATGCATCACCATCACCATCACATGGAACAGCAAGTAAAGGACGACA-3 ': SEQ ID NO: 6).

In the polymerase chain reaction, the first denaturation step was performed once at 94 ° C. for 5 minutes, and then the second denaturation step was performed at 94 ° C. for 1 minute, and the annealing step was performed at 56 ° C. for 1 minute, and extended ( extension) step was performed at 72 ° C. for 1.5 minutes, this was repeated 40 times, and then the last extension step was performed once at 72 ° C. for 10 minutes. In the fragment obtained in this manner, a DNA fragment of about 1.33 kb represented by SEQ ID NO: 4 was isolated and cloned into a pHCEIIB (Nde I) vector to prepare a recombinant expression vector pBS- aldX as shown in FIG. 2.

(2) Cloned Gene Analysis

For sequencing of the cloned gene of the recombinant vector (pBS- alDX ) prepared in (1), the recombinant vector was cleaved using various restriction enzymes based on a sequence map of two vectors M13mp18 and M13mp19, respectively. The fragments of were subcloned into M13mp18 and M13mp19, and they were sequenced using ABI PRISM BigDye primer cycle-sequencing kit (Perkin-Elmer, USA) using AmpliTaq DNA polymerase. At this time, in order to read both directions of both strands of DNA, a synthetic nucleotide was partially prepared, and the nucleotide sequence of the cloned DNA fragment gene was analyzed and compared with the nucleotide sequence registered in GenBank (GenBank Accession Number Z99124). Result, aldX It was confirmed that the gene was cloned.

(3) Preparation of transformant

Using the calcium chloride method (see Sambrook et. al., Molecular Cloning, A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press, were transformed Cold Spring Harbor, NY, 1989) E. coli XL1-Blue to pBS- aldX vector. LB plate medium (yeast extract, 5 g / L; tryptone, 10 g / L; NaCl, 10 g / L) to which ampicillin (100 mg / L) and bacto-agar (15 g / L) were added. ) Was first selected for transforming strain XL1-Blue (pBS- aldX ) resistant to empicillin .

The first selected E. coli was inoculated into 5 ml LB liquid medium containing ampicillin (ampicillin, 100 mg / L), pre-incubated at 37 ° C. for 12 hours, and the 0.1% culture was again 200 ml containing empicillin. The cells were inoculated in LB medium and shaken for 16 hours and centrifuged to obtain cells. The recovered cells were suspended in 50 mM potassium phosphate buffer (KH ₂ PO ₄ -KOH) at pH 7.0, destroyed by ultrasonication, and centrifuged again to obtain supernatant. The aldehyde dehydrogenase enzyme titer of the supernatant was measured and finally selected as an aldehyde dehydrogenase producing strain.

At this time, the enzyme titer was determined by adding 3 ml of NAD solution and 0.5 ml of 1% (v / v) benzaldehyde solution to 0.5 ml of enzyme solution for 10 minutes at 25 ° C., and measuring the absorbance at 340 nm. It was.

(4) expression of aldX gene

In order to express the aldX gene in the transformed microorganism, E. coli obtained in (3) was inoculated in 5 ml of LBA medium containing empicillin (100 mg / L), pre-incubated at 37 ° C. for 12 hours, and then again. 100 ml LBA medium containing empicillin (100 mg / L) was inoculated to 1% (v / v) was incubated for 16 hours at 37 ℃.

[ Example  3]

Except that the first step was carried out in the same manner as in Example 1 to obtain a pure crystalline NDA.

(1) Cloning of the aldY Gene

Bacillus subtilis HSB-21 ( Bacillus To clone the aldY gene (SEQ ID NO: 7) encoding aldehyde dehydrogenase from subtilis HSB-21 KCTC 11221), genome DNA comprising the aldY gene sequence was first isolated (see Sambrook et al. al ., Molecular Cloning, A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Primer 1 (5'-AAGGAGGGTCATATGAGTTTCGAAACG-3 ': SEQ ID NO: 8) and Primer 2 (5) prepared based on the DNA base sequence of the GenBank Sequence Database (BSUB0020) gene. The polymerase chain reaction was carried out using '-CCCAAGCTTTTAATGGTGATGGTGATGGTGGAACGGATATTTGCGGTATTGCTTC-3': SEQ ID NO: 9). At this time, the primer 2 was prepared by attaching a histidine tag to the aldY gene.

In the polymerase chain reaction, the first denaturation step was performed once at 94 ° C. for 5 minutes, and the second denaturation step was performed at 94 ° C. for 1 minute, and the annealing step was performed at 60 ° C. for 1 minute and 30 seconds, The extension step was carried out at 72 ° C. for 2 minutes and was repeated 35 times. DNA fragments of about 1.5 kb represented by SEQ ID NO: 1 were isolated from the fragments obtained by the above method, and cloned into pHCEIIB (Nde I) to prepare a recombinant expression vector pBS- aldY as shown in FIG. 3.

(2) Cloned Gene Analysis

For sequencing of the cloned gene of the recombinant vector (pBS- alDY ) prepared in (1), the recombinant vector was cleaved using various restriction enzymes based on a sequence map of two vectors, M13mp18 and M13mp19, respectively. The fragments of were subcloned into M13mp18 and M13mp19, and they were sequenced using ABI PRISM BigDye primer cycle-sequencing kit (Perkin-Elmer, USA) using AmpliTaq DNA polymerase. At this time, in order to read both directions of both strands of DNA, a synthetic nucleotide was partially prepared, and the nucleotide sequence of the cloned DNA fragment gene was analyzed and compared with the nucleotide sequence registered in GenBank (GenBank Accession Number Z99123). It was confirmed that the aldY gene was cloned.

(3) Preparation of transformant

Using the calcium chloride method (see Sambrook et. al., Molecular Cloning, A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press, were transformed Cold Spring Harbor, NY, 1989) E. coli XL1-Blue to pBS- aldY vector. LB plate medium (yeast extract, 5 g / L; tryptone, 10 g / L; NaCl, 10 g / L) to which ampicillin (100 mg / L) and bacto-agar (15 g / L) were added. ), And transformed strain XL1-Blue (pBS- aldyY ) resistant to empicillin was selected.

(4) expression of aldY gene

In order to express the aldY gene in the transformed microorganism, E. coli obtained in (3) was inoculated in 5 ml of LB medium containing empicillin (100 mg / L), pre-incubated at 37 ° C. for 12 hours, and then again. 200 ml of LB medium containing empicillin (100 mg / L) was inoculated to 0.1% (v / v) was incubated for 16 hours at 37 ℃.

[ Example  4]

Between the second and third steps of Example 1, except that the cNDA purification solution obtained in the second step was used for the crystallization reaction after removing the recombinant microorganism using a 0.5 탆 polypropylene filter, The same procedure as in Example 1 was carried out to obtain NDA in pure crystalline state.

Experimental Example  1: Component Analysis

The components were analyzed for the first cNDA used in Examples 1 to 3, the cNDA purification solution obtained after the second purification step, and the pure crystalline NDA obtained after the fourth step washing and drying step. After the results are shown in Tables 1 to 3, respectively.

When the cNDA purification method using the recombinant microorganism of the present invention is used from the results of Tables 1 to 3, impurities in the cNDA can be almost completely removed, and in particular, FNA is 100% converted to NDA and thus 2,6- high purity of 99.99% or more. It was confirmed that naphthalene dicarboxylic acid crystals could be obtained.

Meanwhile, in order to find the optimum reaction conditions of the crystallization step of the cNDA purification solution, especially among the purification steps of the cNDA using the recombinant microorganism of the present invention, experiments were carried out by varying the reaction conditions as follows, and the comparative analysis and crystallization reaction and recovery rate accordingly It was.

Experimental Example  2: acid type and At the stirring speed  Crystallization reaction

100 ml of each cNDA purification solution obtained in the second step of Examples 1 to 3 were put in a reactor equipped with a stirring device, and sulfuric acid, hydrochloric acid, glacial acetic acid, and nitric acid solution were added thereto so that the pH was 3.0. After the adjustment, the stirring speed was maintained at 0, 50, 100, 200, 400, 800, 1000 rpm. The temperature was 75 ° C., and the crystallization reaction was performed for 25 minutes under the above conditions.

After the crystallization was completed, the crystals were analyzed using a microscope and a particle size analyzer. As a result, all crystallizations were confirmed, and it was confirmed that entanglement between individual crystals did not occur. In addition, the average size of cNDA crystals obtained under each of the above conditions is shown in Tables 4 to 6 below. As can be seen from the results of Tables 4 to 6, sulfuric acid and hydrochloric acid showed the best results in the crystallization reaction among the four acid solutions, and the preferred stirring speed was more than 0 rpm and less than 400 rpm, and more preferable stirring speed. Was determined to be about 30 to 200 rpm.

Experimental Example  3: crystallization reaction according to acid type and temperature condition

The stirring speed was 50 rpm, and the reaction temperature was 4, 30, 50, 75, 100, 130 ℃ except that the experiment was carried out in the same manner as in Experiment 2.

After crystallization was completed, the crystals were analyzed using a microscope and a particle size analyzer. As a result, all crystallizations were confirmed, and it was confirmed that entanglement between individual crystals did not occur. In addition, the average size of cNDA crystals obtained under each of the above conditions is shown in Tables 7 to 9 below. As can be seen from the results of Tables 7 to 9, sulfuric acid and hydrochloric acid showed the best results for the crystallization reaction among the four acid solutions, and the preferred reaction temperature was 50 to 90 ° C, and more preferably the reaction temperature was It was judged to be 75 ° C.

Experimental Example  4: mountain type and pH  Recovery rate according to condition

Experiment was carried out in the same manner as in Experiment 2 except that the stirring speed was 50 rpm, the reaction temperature was 75 ℃, pH range was adjusted to 1, 2, 3, 4, 5, 6. That is, after performing the crystallization reaction for 25 minutes, a certain amount was taken to compare the recovery. The results are shown in Tables 10 to 12 below. As can be seen from the results of Tables 10 to 12, sulfuric acid and hydrochloric acid showed the best results in the recovery rate, and a recovery rate of 99% or more was obtained under the condition of pH 3 or less. At this time, the lower the pH was, the higher the recovery was.

As described in detail through the above examples, the present invention provides xanthine di derived from Bacillus subtilis HSB-21 having the ability to convert 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid. Purity and crystallization of crude naphthalene dicarboxylic acid under suitable conditions using recombinant microorganisms prepared using the hydrogenase gene pucABCDE or the aldehyde dehydrogenase genes aldX and aldY to obtain high purity crystalline 2,6-naphthalene dica There is an advantage that mass production of leric acid is economical and environmentally friendly.

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Claims (20)

(a) a recombinant microorganism obtained by transformation with a recombinant expression vector comprising a xanthine dehydrogenase gene pucABCDE from Bacillus subtilis HSB-21 KFCC 11221 represented by SEQ ID NO: 1; A recombinant microorganism obtained by transformation with a recombinant expression vector comprising an aldehyde dehydrogenase gene aldX from Bacillus subtilis HSB-21 KFCC 11221 represented by SEQ ID NO: 4; Alternatively, the recombinant microorganism obtained by transforming the recombinant microorganism obtained by transforming with a recombinant expression vector containing an aldehyde dehydrogenase gene aldY derived from Bacillus subtilis HSB-21 KFCC 11221 represented by SEQ ID NO: 7 is crude naphthalene dicar Reacting with crude naphthalene dimethylcarboxylic acid to convert 2-formyl-6-naphthoic acid in the crude naphthalene dicarboxylic acid to 2,6-naphthalene dicarboxylic acid to remove it; (b) crystallizing the crude naphthalene dicarboxylic acid by adding an acidic solution to the reaction solution obtained in step (a) to adjust pH and then reacting with stirring; (c) washing the crystallized crude naphthalene dicarboxylic acid to remove impurities contained therein, the step of dispersing the crystallized crude naphthalene dimethylcarboxylic acid in water, followed by a pressure of 1 to 60 kg / cm ² Agitating for 10 minutes to 1 hour at a temperature of 100 to 270 ° C., followed by filtration to remove the water several times; And (d) Purifying crude naphthalene dicarboxylic acid using a recombinant microorganism comprising the step of drying the wash to obtain 2,6-naphthalene dicarboxylic acid in a pure crystalline state. The method of claim 1, wherein step (a) 1) inoculating the recombinant microorganism in a liquid medium to induce the expression of xanthine dehydrogenase or aldehyde dehydrogenase, and then, the cells recovered through centrifugation are suspended in physiological saline or distilled water to prepare cells for reaction. step; 2) mixing the crude naphthalene dicarboxylic acid as a substrate with a buffer solution and then adding an alkaline solution to adjust the pH of the mixed solution to form a purification reaction solution; And 3) reacting the reaction cells prepared in step 1) and the reaction solution prepared in step 2) to convert 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid to convert 2,6-naphthalene dica Purification method of crude naphthalene dicarboxylic acid using a recombinant microorganism, comprising the step of increasing the purity of the carboxylic acid (2,6-Naphthalene dicarboxylic acid). The method for purifying crude naphthalene dicarboxylic acid using a recombinant microorganism according to claim 2, wherein the microorganism is a bacterium of Escherichia, Pseudomonas or Bacillus. The method of claim 2, wherein the buffer solution is water, sodium carbonate buffer solution, glycine buffer solution, potassium phosphate buffer solution, sodium phosphate buffer solution, succinic acid buffer solution, sodium acetate buffer solution, citric acid buffer solution, sodium pyrophosphate buffer solution, boric acid Purification method of crude naphthalene dicarboxylic acid using a recombinant microorganism, characterized in that at least one selected from the group consisting of a buffer solution and sodium borate buffer solution, the concentration is 0.01 to 100 mM. The method for purifying crude naphthalene dicarboxylic acid using a recombinant microorganism according to claim 2, wherein the alkali solution is a NaOH solution or a KOH solution. The method of claim 2, wherein the mixed solution of step 2) further comprises an organic solvent. The method of claim 6, wherein the organic solvent is selected from the group consisting of dimethylsulfoxide, dimethylformamide (N, N-Dimethylformamide), dimethylacetamide (N, N-Dimethylacetamide) and tetrahydrofuran (Tetrahydrofuran). Purified crude naphthalene dicarboxylic acid using a recombinant microorganism, characterized in that at least one, and the addition concentration is 0.01 to 20% by weight. The method of claim 2, wherein the reaction temperature in step 3) is 25 to 60 ℃, the reaction time is 1 minute to 2 hours, characterized in that the purification method of crude naphthalene dicarboxylic acid using a recombinant microorganism. The method for purifying crude naphthalene dicarboxylic acid according to claim 2, wherein the concentration of crude naphthalene dicarboxylic acid in the reaction solution is 0.001 to 20% by weight. The method of claim 1, wherein the acidic solution in step (b) is at least one selected from the group consisting of sulfuric acid, hydrochloric acid, glacial acetic acid and nitric acid, the method of purifying crude naphthalene dicarboxylic acid using a recombinant microorganism. The method of claim 1, wherein in step (b), the pH is adjusted to a range of 1 to 4. The method for purifying crude naphthalene carboxylic acid using recombinant microorganisms according to claim 1. The method of claim 1, wherein the reaction temperature in the step (b) is in the range of 4 to 130 ℃, the reaction time is 1 minute to 12 hours the purification method of crude naphthalene dicarboxylic acid using a recombinant microorganism. The method of claim 1, wherein the stirring speed in the step (b) is in the range of more than 0 rpm 1000 rpm or less using crude naphthalene dicarboxylic acid using a recombinant microorganism. delete The method of claim 1, wherein the drying temperature of step (d) is in the range of 40 to 200 ℃ the purification method of crude naphthalene dicarboxylic acid using a recombinant microorganism. The crude naphthalene dicarboxylic acid using a recombinant microorganism according to claim 1, further comprising the step of removing the recombinant microorganism used in the step (a) between the steps (a) and (b). Purification method. The method for purifying crude naphthalene dicarboxylic acid using a recombinant microorganism according to claim 16, wherein the removal of the recombinant microorganism is performed using a microfilter system, a continuous centrifuge or a decanter. 18. The bath according to claim 17, wherein the microfilter system has a pore size of 0.1 to 2.0 [mu] m and uses a filter made of ceramic, stainless, polypropylene or PET. Method for Purifying Naphthalene Dicarboxylic Acid. delete delete

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