WO2008004731A1

WO2008004731A1 - Purification method of crude naphthalene dicarboxylic acid using recombinated microorganism and 2,6-naphthalene dicarboxylic acid in crystalline form obtained by using the same

Info

Publication number: WO2008004731A1
Application number: PCT/KR2006/005729
Authority: WO
Inventors: Dong Sung Kim; Yong Bok Choi; Sung Kyoon Kim; Ik Hyun Kwon
Original assignee: Hyosung Corporation
Priority date: 2006-07-04
Filing date: 2006-12-27
Publication date: 2008-01-10
Also published as: KR20080004041A

Abstract

Disclosed is a method for purifying a crude naphthalene dicarboxylic acid using a recombinant microorganism. According to the purification method, a crude naphthalene dicarboxylic acid is purified by reacting a recombinant microorganism having the ability to convert 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid with a crude naphthalene dicarboxylic acid, adding an acidic solution to the reaction solution under particular conditions, allowing the mixed solution to react with stirring to crystallize the crude naphthalene dicarboxylic acid, washing the crystal of the crude naphthalene dicarboxylic acid, and drying the washed product to obtain 2,6-naphthalene dicarboxylic acid in a pure crystalline form. Advantageously, the purification method enables the production of high-purity crystalline 2,6-naphthalene dicarboxylic acid on an industrial scale in an economically feasible and environmentally friendly manner.

Description

Description PURIFICATION METHOD OF CRUDE NAPHTHALENE DI-

CARBOXYLIC ACID USING RECOMBINATED MICROORGANISM AND 2,6-NAPHTHALENE DICARBOXYLIC ACID IN CRYSTALLINE FORM OBTAINED BY USING THE

SAME Technical Field

[1] The present invention relates to a method for purifying a crude naphthalene di- carboxylic acid using a recombinant microorganism and 2,6-naphthalene dicarboxylic acid in a crystalline form produced by the method. More specifically, the present invention relates to a method for purifying a crude naphthalene dicarboxylic acid by reacting a recombinant microorganism having the ability to convert 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid with a crude naphthalene dicarboxylic acid to remove 2-formyl-6-naphthoic acid contained as an impurity in the crude naphthalene dicarboxylic acid, adding an acidic solution to the reaction solution under particular conditions, allowing the mixed solution to react with stirring to crystallize the crude naphthalene dicarboxylic acid, washing the crystal of the crude naphthalene dicarboxylic acid to remove other impurities contained therein, and drying the washed product to obtain 2,6-naphthalene dicarboxylic acid in a pure crystalline form. Background Art

[2] 2,6-Naphthalene dicarboxylic acid (NDA) and its diester (i.e. 2,6-naphthalene di- carboxylate (NDC)) are useful monomers for the preparation of a variety of polymeric materials, such as polyesters and polyamides. For example, NDA and NDC can be condensed with ethylene glycol to form poly (ethylene 2,6-naphthalate) (PEN), which is a high-performance polyester material. Fibers and films made from PEN exhibit high strength and superior thermal properties, compared to those made from poly(ethylene terephthalate) (PET). Based on these advantages, PEN is highly suitable for use in the production of commercial articles, such as thin films, which can be used for the manufacture of magnetic recording tapes and electronic components. In addition, since PEN is highly resistant to the diffusion of gases, particularly carbon dioxide, oxygen and water vapor, films made from PEN are useful for the manufacture of food containers, especially hot-fill food containers. PEN can also be used to produce reinforced fibers useful for the manufacture of tire cords.

[3] NDC is currently produced by oxidizing 2,6-dimethylnaphthalene (2,6-DMN) to obtain a crude naphthalene dicarboxylic acid (cNDA) and esterifying the cNDA. At present, NDC is used as a major raw material in the synthesis of PEN. However, some problems are presented when NDC is used as a raw material in the synthesis of PEN, compared to when 2,6-naphthalene dicarboxylic acid (NDA) is used. Firstly, water is formed as a by-product during the condensation of NDA, whereas methanol is formed as a by-product in the case of NDC, thus risking the danger of explosion. Secondly, since pure NDC is produced by esterifying NDA and purifying the esterification product, one additional processing step is involved, compared to the use of NDA. Thirdly, the use of NDC is not suitable in view of the use of existing PET production facilities. Despite the problems associated with the use of NDC, NDC is preferentially used to produce PEN because it is still difficult to produce purified NDA having a purity necessary for the synthesis of PEN.

[4] 2,6-Dimethylnaphthalene (2,6-DMN) is oxidized to form a cNDA containing various impurities, such as 2-formyl-6-naphthoic acid (FNA), 2-naphthoic acid (NA) and trimellitic acid. Particularly, the presence of FNA in a cNDA stops the polymerization for the production of PEN, resulting in a low degree of polymerization. Th is low degree of polymerization adversely affects the physical properties of the final polymer (i.e. PEN) and causes coloration of the polyester. It is thus essential to remove FNA present in a cNDA, but difficulties exist in removing FNA.

[5] Under these circumstances, research has been conducted on various chemical methods for the removal of FNA present in a cNDA or purification of NDA. For example, NDA is produced by i) recrystallizing a cNDA, ii) oxidizing a cNDA one more time, or iii) treating a cNDA with methanol to produce NDC and hydrating the NDC. Further, purified NDA is produced by hydrogenation of a cNDA. In addition to these chemical methods, many processes, e.g., solvent treatment, melting/crystallization, high-pressure crystallization and supercritical extraction, have been employed to purify NDA.

[6] For example, U.S. Patent No. 5,859,294 discloses a process for the production of a naphthalene dicarboxylic acid, which comprises dissolving a crude naphthalene dicarboxylic acid in an aqueous solution containing an aliphatic or alicyclic amine, removing heavy metal components contained as impurities until the content of the heavy metal components based on the crude naphthalene dicarboxylic acid is 100 ppm or less, and heating the aqueous solution containing a naphthalene dicarboxylic acid amine salt to distill off the amine.

[7] U.S. Patent No. 6,255,525 discloses a process for preparing an aromatic carboxylic acid having improved purity comprising the steps of contacting a mixture comprising an impure aromatic carboxylic acid and water at a pressure of 77 to 121 kg/cm and a temperature of 277 to 316⁰C in the presence of hydrogen with a carbon catalyst which is free of a hydrogenation metal component, cooling the mixture to form crystallized aromatic carboxylic acid, and recovering the crystallized aromatic carboxylic acid from the cooled mixture.

[8] In connection with the crystallization reaction of NDA, U.S. Patent No. 6,087,531 teaches a process for recovering a naphthalene dicarboxylic acid (NDA) crystal comprising the steps of dissolving poly(alkylene naphthalene dicarboxylate) in an aqueous basic solution (e.g., an aqueous solution of an alkali metal base, an aqueous hydroxide solution or an aqueous solution of an alkali metal carbonate) at a temperature of 125 to 400⁰C, neutralizing the aqueous solution with an acid at 170 to 24O⁰C, and recovering the NDA. For example, an NDA crystal is recovered by dissolving the polyester material in a NaOH or KOH solution at a temperature of 125 to 400⁰C, neutralizing the aqueous solution with acetic acid, and recovering the NDA.

[9] U.S. Patent No. 6,426,431 teaches a method of increasing the purification yield of

2,6-NDA by as high as 45%, the method comprising treating an aqueous solution of K -NDA at a CO pressure of 0-200 psi and a temperature of 0-50⁰C to form KH-NDA, suspending the KH-NDA in water in a weight ratio higher than 1:8 (KH-NDA: water), and further treating the suspension at a temperature above 100⁰C (140-160⁰C) and at a CO pressure above 100 psi (175-250 psi).

[10] However, since these processes require high-temperature and high-pressure conditions, time-consuming treatments and use of large amounts of expensive materials to produce a high-purity NDA crystal, they are economically disadvantageous. Other disadvantages of the processes are very low purification yield and purity of NDA and occurrence of aggregation between individual NDA crystal particles, which makes the processes difficult to practice industrially. Disclosure of Invention Technical Problem

[11] Therefore, the present invention has been made in view of the problems of the prior art, and it is one object of the present invention to provide a method for efficiently producing high-purity NDA in high yield by purifying and crystallizing a crude naphthalene dicarboxylic acid at ambient pressure and temperature conditions using a recombinant microorganism capable of converting FNA to NDA.

[12] It is another object of the present invention to provide a high-purity NDA crystal with a uniform size produced by the method. Technical Solution

[13] In accordance with one aspect of the present invention for achieving the above obj ects, there is provided a method for purifying a crude naphthalene dicarboxylic acid using a recombinant microorganism. [14] Specifically, the purification method of the present invention comprises the following steps:

[15] (a) reacting a crude naphthalene dicarboxylic acid with at least one recombinant microorganism selected from the group consisting of microorganisms obtained by transformation with a recombinant expression vector carrying the benzaldehyde dehydrogenase gene (xylC) of SEQ ID NO: 1 derived from Sphingomonas aromaticivorans (KCTC 2888) or a gene having a homology of at least 90% with the xylC gene and microorganisms obtained by transformation with a recombinant expression vector carrying the benzaldehyde dehydrogenase gene (xylC) of SEQ ID NO: 4 derived from Pseudomonas putida mt-2 (ATCC 33015) or a gene having a homology of at least 90% with the xylC gene, to remove 2-formyl-6-naphthoic acid present in the crude naphthalene dicarboxylic acid;

[16] (b) adding an acidic solution to the reaction solution prepared in step (a) to adjust the pH of the reaction solution and reacting the mixed solution with stirring to crystallize the crude naphthalene dicarboxylic acid;

[17] (c) washing the crystal of the crude naphthalene dicarboxylic acid to remove impurities contained therein; and

[18] (d) drying the washed product to obtain 2,6-naphthalene dicarboxylic acid in a pure crystalline form.

[19] A more detailed explanation of the respective steps of the purification method according to the present invention will be provided below.

[20]

[21] (a) First step: Purification using recombinant microorganism

[22] First, a crude naphthalene dicarboxylic acid is reacted with at least one recombinant microorganism selected from the group consisting of microorganisms obtained by transformation with a recombinant expression vector carrying the benzaldehyde dehydrogenase gene (xylC) of SEQ ID NO: 1 derived from Sphingomonas aromaticivorans (KCTC 2888) or a gene having a homology of at least 90% with the xylC gene and microorganisms obtained by transformation with a recombinant expression vector carrying the benzaldehyde dehydrogenase gene (xylC) of SEQ ID NO: 4 derived from Pseudomonas putida mt-2 (ATCC 33015) or a gene having a homology of at least 90% with the xylC gene, to remove 2-formyl-6-naphthoic acid present in the crude naphthalene dicarboxylic acid;

[23] The expression "gene having a homology of at least 90%" as used herein refers to a gene that has a homology of at least 90% with the benzaldehyde dehydrogenase gene used in the method of the present invention in the base or amino acid sequence level, and that shows the same activity as the benzaldehyde dehydrogenase despite the difference in base or amino acid sequence from the benzaldehyde dehydrogenase gene within 10%. The different sequence segments are intended to include all segments that may be modified by those skilled in the art without significantly affecting the inherent activity of the benzaldehyde dehydrogenase.

[24] Specifically, this step includes the sub-steps of 1) inoculating the recombinant microorganism into a liquid medium, culturing the cells to induce the expression of benzaldehyde dehydrogenase, centrifuging the culture broth to collect the cells in which benzaldehyde dehydrogenase is expressed, and suspending the cells in physiological saline or distilled water, 2) mixing a crude naphthalene dicarboxylic acid (cNDA) as a substrate with a buffer solution and adjusting the pH of the mixed solution by the addition of an alkaline solution to prepare a reaction solution for subsequent purification, and 3) reacting the cells prepared in 1) with the reaction solution prepared in 2) to convert 2-formyl-6-naphthoic acid contained in the crude naphthalene dicarboxylic acid to 2,6-naphthalene dicarboxylic acid, so that the purity of the 2,6-naphthalene dicarboxylic acid is increased.

[25] Recombinant microorganisms that can be used in the present invention may be obtained by cloning the benzaldehyde dehydrogenase gene (xylC) of SEQ ID NO: 1 derived from Sphingomonas aromaticivorans (KCTC 2888), a gene having a homology of at least 90% with the xylC gene of SEQ ID NO: 1, the benzaldehyde dehydrogenase gene (xylC) of SEQ ID NO: 4 derived from Pseudomonas putida mt-2 (ATCC 33015) or a gene having a homology of at least 90% with the xylC gene of SEQ ID NO: 4 in a manner known in the art, introducing the cloned gene into an e xpression vector to construct a recombinant expression vector, and introducing the recombinant expression vector into a microorganism, and transforming the microorganism with the recombinant expression vector by a conventional known technique.

[26] Specifically, the recombinant microorganisms can be obtained by the following procedure.

[27] In order to clone each benzaldehyde dehydrogenase gene (xylC), plasmid DNA or genomic DNA is isolated from each of the microorganisms and polymerase chain reaction (PCR) using the plasmid DNA or genomic DNA as a template is performed using a set of primers synthesized on the basis of the xylC gene to obtain a gene encoding benzaldehyde dehydrogenase. If needed, the base sequence of the gene encoding benzaldehyde dehydrogenase may be partially modified by a common technique known in the art.

[28] Subsequently, the cloned xylC gene is functionally linked to a promoter of an expression vector by a conventional technique to construct a recombinant expression vector.

[29] Any expression vector that can allow the optimum expression of the gene in a host may be available without any particular limitation. For example, the gene can be inserted into a plasmid, phage or another DNA as the expression vector. Examples of suitable plasmids include, but are not specially limited to: known pForexT, pLG338, pACYC184, pBR322, pUC119, pUC18, pUC19, pKC30, _PRep4, pHSl, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III¹³⁸-Bl, λgtl l and pBDCl vectors in E. colϊ, pIJlOl, pIJ364, pIJ702 and pIJ361 vectors in Streptomyces; pUBl lO, pC194 and pBD214 vectors in Bacillus; and 2D, pAG-1. YEp6, YEpl3 and pEMBLYe23 vectors in yeast.

[30] Examples of promoters that can be used in the present invention include, but are not limited to: promoters suitable for use in gram-negative bacteria, such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacf, T7, T5, T3, gal, trc, ara, SP6, λ-PR and λ-PL promoters; gram-positive promoters, such as amy and SPO2 promoters; and fungi or yeast promoters, such as ADCl, MFa, AC, P-60, CYCl, GAPDH, TEF, rp28 and ADH promoter.

[31] To ensure the optimum expression of the gene depending on the type of the selected host organism, a regulatory sequence may be further included in the 3' end and/or 5' end of the gene.

[32] In this connection, FIG. 1 shows a genetic map of a recombinant expression vector

(pET20-xy/C) carrying an xylC gene encoding benzaldehyde dehydrogenase derived from Sphingomonas aromaticivorans used in Example 1 of the present invention, and FIG. 2 shows a genetic map of a recombinant expression vector (pUC18-xy/C) carrying an xylC gene encoding benzaldehyde dehydrogenase derived from Pseudomonas putida used in Example 3 of the present invention.

[33] The cloning of the xylC gene into the corresponding recombinant expression vector may be confirmed by various techniques, such as restriction enzyme cleavage and base sequencing.

[34] Then, the recombinant expression vector thus constructed is introduced into a host microorganism by any conventional technique known in the art to transform the microorganism. As a result, a recombinant microorganism is prepared. As the host microorganism, any prokaryotic or eukaryotic organism may be used. Examples of preferred host microorganisms include, but are not necessarily limited to, bacteria, fungi and yeasts. The host microorganism may be typically a gram-positive or gram- negative bacterial species, preferably a species of Enterobacteriaceae or Nocardiaceae family, more preferably a bacterial species of the genus Escherichia, Pseudomonas, Rhodococcus or Bacillus, and most preferably a bacterial species of the genus E. coli. Specific examples of the host microorganism include MC 1061 (E. coli), JM 109 (E. coli), XLl -Blue (E. coli) and DH5 (E. coli). The transformation may be performed by any suitable technique, such as heat treatment, electric shock, microinjection, calcium chloride method, rubidium chloride method or pressure spraying, but the present invention is not necessarily limited to these techniques.

[35] According to the method of the present invention, 2-formyl-6-naphthoic acid is converted to 2,6-naphthalene dicarboxylic acid with the help of the recombinant microorganism transformed with the expression vector of benzaldehyde dehydrogenase. To this end, it is preferred that benzaldehyde dehydrogenase be expressed at a high level within the recombinant microorganism. For example, since the pUC 18 vector used in the present invention is used to express the gene under the control of a lac promoter and the vector pET-20b(+) is used to express the gene under the control of a T7 promoter, isopropyl-β-D-thiogalactopyranoside (IPTG) can be used to induce the expression of benzaldehyde dehydrogenase.

[36] Specifically, the recombinant microorganism is sufficiently cultured in a typical culture temperature range (25-45⁰C), preferably at 37⁰C, and is then inoculated into a 100 ml of a liquid medium (e.g., an LB medium) in such an amount that the concentration is 1% (v/v). When the OD value reaches 0.4 to 0.5, IPTG is added until

600 the concentration is 0.1-2.0 mM and preferably 0.5 mM to induce the expression of the benzaldehyde dehydrogenase, followed by culture at 37⁰C. It is to be appreciated that the culture and the protein expression of the recombinant organism can be achieved, without any limitation to the foregoing techniques, by any technique known to those skilled in the art.

[37] The recombinant microorganism used in the method of the present invention enables the expression of benzaldehyde dehydrogenase. Therefore, the high- concentration culture of the recombinant microorganism is facilitated, which provides an economic advantage.

[38] The culture solution of the recombinant microorganism in which the protein is expressed at a high level is centrifuged to collect the cells, and then the cells are suspended in physiological saline or distilled water. The suspension is used as a reaction solution for the purification of a crude naphthalene dicarboxylic acid in the subsequent step.

[39] The kind of the buffer solution is not especially restricted. As the buffer solution, there can be used, for example, water, a sodium carbonate buffer (Na CO /NaHCO ), a glycine buffer (glycine/NaOH), a potassium phosphate buffer (KH PO /KOH), a sodium phosphate buffer (Na HPO /NaH PO ), a succinic acid buffer (succinic acid/ NaOH), a sodium acetate buffer (sodium acetate/acetic acid), a citric acid buffer (citric acid/sodium citrate), a sodium pyrophosphate buffer (Na P O /HCl), a boric acid buffer (boric acid/NaOH), or a sodium borate buffer (sodium borate/HCl). Preferred is a potassium phosphate buffer (KH PO -KOH) or a boric acid buffer (boric acid/NaOH), which has a pH range of 6.0-9.0. It is preferred that the concentration of the buffer solution be in the range of 0.01 to 100 mM.

[40] The alkaline solution is preferably a NaOH or KOH solution, but is not limited thereto. The pH of the mixed solution is preferably adjusted to the range of 6 to 10, preferably 8 to 10.

[41] In the second sub-step, an organic solvent may be additionally added to the mixed solution for the purpose of dissolving the cNDA. Examples of preferred organic solvents include dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), and tetrahydrofuran (THF). Of these, dimethylsulfoxide is most preferred in terms of enzymatic activity. The organic solvent is preferably added at a concentration of 0.01 to 20% and more preferably 0.1 to 10%. It is most preferred that no organic solvent be added. The addition of the organic solvent at a concentration exceeding 20% causes lysis of the cell membranes of the microorganism, resulting in inhibition of the reaction.

[42] In the third sub-step, the reaction is preferably conducted at 25-7O⁰C for 1 min.-l hour and more preferably at 40-60⁰C for 10-40 minutes. When the reaction temperature is lower than 25⁰C or higher than 7O⁰C, a marked decrease in the reactivity is undesirably caused.

[43] On the other hand, there is an intimate relationship among the FNA content in the cNDA, the concentration of the cNDA in the reaction solution and the amount of the microorganism necessary to completely remove the FNA. That is, as the FNA content in the cNDA and the concentration of the cNDA in the reaction solution increase, a larger amount of the microorganism is required to remove the FNA.

[44] Specifically, the concentration of the cNDA in the reaction solution is preferably between 0.001% and 20%, and the FNA content in the cNDA is between 0.001% and 10% and preferably 9%.

[45] The amount of the microorganism necessary to completely remove Ig of the FNA is approximately 5g. In view of the fact that a microorganism (e.g., Bacillus sp. F-I or F- 3 described in Korean Patent Application No. 2002-0087819, which was filed by the present inventors) having the ability to convert FNA to NDA is generally used in an amount of about 1Og in order to completely remove Ig of FNA, it turns out that the purification of cNDA using the recombinant microorganism in accordance with the present invention is more efficient from an economic viewpoint.

[46]

[47] (b) Second step: Crystallization

[48] In this step, an acidic solution is added to the reaction solution prepared in step (a) to adjust the pH of the reaction solution, and is then reacted with the reaction solution with stirring to crystallize the crude naphthalene dicarboxylic acid.

[49] More specifically, an appropriate amount of an acidic solution is added to the reaction solution prepared in step (a) in a reactor equipped with an stirrer to adjust the pH of the reaction solution to a desired range, and then the mixed solution is reacted with continuous stirring while maintaining the temperature of the mixed solution constant, so that the cNDA in an amorphous form present in the FNA-free reaction solution is crystallized at ambient pressure and temperature conditions.

[50] Since this crystallization enables production of a cNDA crystal having a uniform size of 100 D or more at ambient pressure and temperature conditions, the purification method of the present invention is economically advantageous in terms of production cost and processing. In addition, since no aggregation between the individual cNDA crystal particles occurs, the purification method of the present invention is suitable for actual use and is advantageous in terms of a high recovery rate of the final product.

[51] As the acidic solution, there may be used, for example, sulfuric acid, hydrochloric acid, glacial acetic acid or nitric acid. The use of sulfuric acid or hydrochloric acid is preferred because it leads to large size and high yield of the cNDA crystal. That is, the addition of sulfuric acid or hydrochloric acid enables the production of a cNDA crystal having a uniform size of 100 D or more in high yield.

[52] The pH of the reaction solution is preferably adjusted to 1-4 to increase the recovery rate of the final product. As the pH of the reaction solution decreases, the recovery rate of the final product tends to increase from about 94% to about 99.9%.

[53] The crystallization is performed at about 4⁰C to about 15O⁰C, preferably 3O⁰C to

13O⁰C and more preferably 5O⁰C to 12O⁰C. The crystallization is performed for about 1 minute to about 10 hours, preferably 2 minutes to 5 hours and more preferably 10 minutes to one hour in terms of continuous processing. The most preferable conditions for the crystallization are 8O⁰C and 20-30 minutes. Too short crystallization time may cause a low degree of crystallization of the cNDA, resulting in aggregation between the individual cNDA crystal particles upon polymerization. Meanwhile, too long crystallization time may cause an unnecessary energy loss.

[54] The stirring required to perform the crystallization of the cNDA is typically performed at a rate of 0-1,000 rpm, preferably 0-400 rpm and more preferably 50-150 rpm.

[55]

[56] (c) Third step: Washing

[57] In this step, the crystal of the crude naphthalene dicarboxylic acid obtained in step

(b) is washed to remove impurities contained therein.

[58] Although the FNA is converted to NDA and is completely removed through the purification reaction using the recombinant microorganism and the crystallization reaction, other impurities, including 2-naphthoic acid (NA), methylnaphthoic acid (MNA) and trimellitic acid (TLMA), remain unremoved. When the cNDA crystal is dispersed in water and washed several times under given conditions to completely remove the remaining impurities. The complete removal of the impurities is based on a difference in solubility of the cNDA and the impurities in water. At this time, the recombinant microorganism used to purify the cNDA is also removed by the washing.

[59] In this step, it is desirable to practice the separation in a state in which the reaction by-products are dissolved in the solvent and NDA is precipitated as much as possible. The temperature range for the separation is between 100⁰C and 27O⁰C and preferably between 15O⁰C and 24O⁰C. The separation of the solvent at a temperature higher than 27O⁰C causes increased loss of the purified NDA, leading to a considerable drop in yield. Meanwhile, the separation of the solvent at a temperature lower than 100⁰C unfavorably causes low dissolution of the impurities, including NA, MNA and TLMA.

[60] More specifically, the cNDA crystal is dispersed in water, stirred at a pressure of 1 to 60 kg/cm and a temperature of 100 to 27O⁰C for 10 minutes to 1 hour, and filtered to remove the water. This procedure may be repeated several times to remove the impurities. At this time, the solvent is preferably used in an amount of 5-20 times of the weight of the cNDA crystal.

[61]

[62] (d) Fourth step: Drying

[63] In this step, the NDA crystal, from which the impurities are removed by the washing, is dried at a specified temperature to obtain NDA in a pure crystalline form. At this time, the drying is preferably performed at 30 to 200⁰C. The drying is performed, without limitation, by a conventional technique known in the art.

[64] The purification method of the present invention may further comprise the step of removing the recombinant microorganism used in step (a) after step (a) and prior to step (b).

[65] The previous removal of the recombinant microorganism after the purification of the cNDA and prior to the crystallization of the cNDA can contribute to improvements in purity and recovery rate of the NDA crystal.

[66] The removal of the recombinant microorganism is achieved, without limitation, by a conventional technique known in the art. For example, a microfilter system, a continuous type centrifugal separator or a decanter may be used to remove the recombinant microorganism. The microfilter system uses a filter having a pore size of 0.1-0.5 D and made of a material selected from ceramic, stainless steel, polypropylene and polyethylene terephthalate (PET).

[67] In another aspect, the present invention provides 2,6-naphthalene dicarboxylic acid in a pure crystalline form produced by the purification method.

[68] The purification method of the present invention enables the production of high- purity 2,6-naphthalene dicarboxylic acid in a high yield of 99.9% or more. Depending on the intended applications and needs, the treatment conditions may be varied to produce 2,6-naphthalene dicarboxylic acid in a regular or random crystalline form. The 2,6-naphthalene dicarboxylic acid in a crystalline form may have a lattice structure.

[69] The 2,6-naphthalene dicarboxylic acid crystal of the present invention has an average particle diameter not smaller than 100 D and a uniform particle shape. Accordingly, the 2,6-naphthalene dicarboxylic acid crystal is very suitable for the formation of a low- viscosity slurry with ethylene glycol when it is polymerized with the ethylene glycol to produce PEN. A 2,6-naphthalene dicarboxylic acid crystal having an average particle diameter smaller than 100 D is difficult to treat and exhibits a poor ability to form a slurry with ethylene glycol when it is mixed with the ethylene glycol to prepare PEN, causing an increase in power consumption. The 2,6-naphthalene dicarboxylic acid crystal of the present invention preferably has an average particle diameter of 100-200 D and more preferably 110-170 D.

[70] FIG. 5 shows a purification system for implementing a method for purifying a crude naphthalene dicarboxylic acid according to an embodiment of the present invention. With reference to FIG. 5, the purification method according to the embodiment of the present invention will be explained in more detail below.

[71] First, a specified amount of a buffer solution is introduced into a reactor A where a crude naphthalene dicarboxylic acid is purified using a microorganism. A cNDA is added to the reactor, and subsequently, an alkaline solution is added to the reactor with stirring to adjust the pH of the reaction solution. A specified amount of water is added to the reactor to prepare a reaction solution for subsequent purification. The cells are added to react with the reaction solution while maintaining the temperature of the reaction solution at a constant level. By this procedure, FNA contained in the cNDA is converted to NDA in the reactor A, and can be finally removed.

[72] After completion of the reaction, the reaction solution is passed through a unit B where the recombinant microorganism used to remove the FNA is removed. The removal of the recombinant microorganism using the unit B may be omitted, if needed, because the removal of the recombinant microorganism can be achieved in a downstream filtering/cleaning unit F.

[73] The reaction solution, from which the microorganism is removed, is transferred to a crystallization reactor C. An acidic solution is added to the crystallization reactor C with stirring to conduct a crystallization reaction.

[74] A slurry containing the cNDA crystal is obtained after the crystallization reaction.

The slurry is heated to about 100 to about 27O⁰C using a preheater D, and is then fed into a filtering/cleaning unit F where a heater is provided to maintain the temperature of the slurry at 100-270⁰C. The pressure of the filtering/cleaning unit F is maintained at 1-60 kg/cm . The filtering/cleaning unit F is equipped with a filter (pore size: 10-100 D).

[75] The filtering/cleaning unit F is connected to a high-pressure filtrate collector G. A filtrate is discharged from the filtering/cleaning unit F into the high-pressure filtrate collector G and solid components are filtered by the filter included in the filtering/ cleaning unit F.

[76] Water (100-270⁰C) is preheated in a solvent heating/supply unit E and supplied to the filtering/cleaning unit F. After stirring is continued in the filtering/cleaning unit F for a given time, a secondary filtrate is discharged into the high-pressure filtrate collector G. If necessary, the cleaning step is repeated once or twice. Pure NDA remaining after the cleaning is mixed with preheated water (100-270⁰C) to form a slurry. The slurry is sent to a high-pressure slurry collector H via a slurry discharge line. After the pressure of the high-pressure slurry collector H drops to ambient pressure, the slurry containing the pure NDA is transferred to a powder separator I where the solvent is removed from the slurry, followed by drying in a dryer J to collect the pure NDA only.

Advantageous Effects

[77] According to the purification method of the present invention, a crude naphthalene dicarboxylic acid is purified and crystallized under respective suitable conditions using a recombinant microorganism capable of converting FNA to NDA. Therefore, the purification method of the present invention enables the production of high-purity crystalline 2,6-naphthalene dicarboxylic acid on an industrial scale in an economically feasible and environmentally friendly manner. Brief Description of the Drawings

[78] FIG. 1 is a genetic map of a recombinant expression vector (pET20-xy/C) carrying an xylC gene encoding benzaldehyde dehydrogenase derived from Sphingomonas aro- maticivorans;

[79] FIG. 2 is a genetic map of a recombinant expression vector (pUC18-xy/C) carrying an xylC gene encoding benzaldehyde dehydrogenase derived from Pseudomonas putida;

[80] FIG. 3 is a micrograph of a cNDA crystal obtained in the third step of Example 1 of the present invention;

[81] FIG. 4 is a micrograph of a cNDA crystal obtained by adding a sulfuric acid solution to a reaction solution of a purified cNDA prepared in Example 1 and allowing the mixed solution to react at 8O⁰C in Experimental Example 3 of the present invention; and

[82] FIG. 5 shows a purification system for implementing a method for purifying a crude naphthalene dicarboxylic acid according to an embodiment of the present invention. [83]

[84] * Brief explanation of essential parts of the drawings *

[85] A: Purification reactor using microorganism

[86] B: Microorganism removing unit C: Crystallization reactor

[87] D: Preheater E: Solvent heating/supply unit

[88] F: Filtering/cleaning unit G: High-pressure filtrate collector

[89] I: Powder separator J: Dryer

Mode for the Invention

[90] Hereinafter, the constitutions and effects of the present invention will be concretely explained in detail with reference to the following examples. However, these examples are given for the purpose of illustration and are not to be construed as limiting the scope of the invention.

[91]

[92] EXAMPLES

[93] Example 1

[94] First Step: Preparation of Recombinant Microorganism

[95] (1) Cloning of xylC Gene

[96] First, in order to clone an xylC gene encoding benzaldehyde dehydrogenase derived from Sphingomonas aromaticivorans (KCTC 2888), a plasmid DNA (pNLl) carrying a sequence of the xylC gene was isolated (see, Sambrook et al., Molecular Cloning, A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Polymerase chain reaction (PCR) using the isolated plasmid DNA (pNLl) as a template was performed using primer 1,

5'-GGAGAATTCATATGGCTACGCAGT-S' (SEQ ID NO: 2) and primer 2, 5'-GTCTTGCAGTGAGCTCGTTTCTCC-S' (SEQ ID NO: 3), which were synthesized on the basis of the DNA base sequence of the xylC gene (available from GenBank Sequence Database, NC002033). In the PCR, denaturation was twice carried out at 94⁰C for 5 minutes (first denaturation) and at 94⁰C for one minute (second denaturation), annealing was carried out at 56⁰C for one minute, and extension was carried out at 72⁰C for 1.5 minutes. This procedure was repeated forty times. Finally, extension was once more carried out at 72⁰C for 10 minutes to obtain a fragment. From the fragment, the DNA fragment (about 1.5 kbp long) set forth in SEQ ID NO: 1 was isolated, cleaved with restriction enzymes Ndel and Sail, and cloned into the plasmid vector pET-20b(+), which was previously cleaved with the same restriction enzymes, to construct the recombinant expression vector pET20-xy/C shown in FIG. 1.

[97]

[98] (2) Analysis of Cloned Gene [99] For base sequencing of the cloned gene of the recombinant vector pET20-xy/C in

Example 1, the recombinant vector was cleaved using various restriction enzymes on the basis of the genetic maps of two vectors M13mpl8 and M13mpl9 to obtain respective fragments. The fragments were subcloned into M13mpl8 and M13mpl9. The resulting subclones were subjected to base sequencing using an ABI PRISM BigDye primer cycle- sequencing kit (Perkin-Elmer, U.S.) with AmpliTaq DNA polymerase. In order to read the double-stranded DNA in both directions, nucleotide fragments were partially synthesized. The base sequence of the fragments of the cloned DNA was analyzed through the nucleotide fragments and compared with the base sequence available from GenBank (GenBank Accession Number: AF073917 or NC002030). As a result, the cloned DNA was confirmed to be xylC gene.

[100]

[101] (3) Preparation of Transformant

[102] E. coli XLl -Blue was transformed with the pET20-xy/C vector by a calcium chloride method (see, Sambrook et al., Molecular Cloning, A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and grown by culture in an LB plate medium (yeast extract 5 g/L, trypton 10 g/L and NaCl 1O g/ L) supplemented with ampicillin (100 mg/L), X-gal, IPTG and bacto-agar (15 g/L) to select transformed E. coli XLl-Blue (pET20-xy/C).

[103]

[ 104] (4) Expression of xylC Gene

[105] To express the xylC gene in the transformed microorganism, the E. coli obtained in

(3) above was inoculated into an LB test tube, allowed to sufficiently grow at 37⁰C, and inoculated into another 100 ml LB medium in such an amount that the concentration is 1% (v/v). When the OD value reached 0.4-0.5, IPTG was added until

600 the concentration was 0.5 mM to induce the expression of xylC, followed by culture at 37⁰C.

[106]

[107] Second step: Purification of cNDA Using Recombinant Microorganism

[108] The culture of the recombinant microorganism in which benzaldehyde dehydrogenase was expressed, which was obtained in the first step, was centrifuged to collect the cells. The collected cells were washed with a 0.85% physiological saline solution and suspended in 5 L of a 0.85% physiological saline solution.

[109] 80 L of a 50 mM potassium phosphate buffer was injected into a reactor A where a cNDA is purified by the recombinant microorganism. 10 kg of a cNDA was added to the reactor A and then KOH or NaOH was added thereto with stirring to adjust the pH of the reaction solution to 8.0. Water was added to the mixed solution to prepare a reaction solution for subsequent purification (cNDA concentration: 10%, FNA content in the cNDA: 0.58%).

[110] 0.29 kg/5L of the cells was fed to the reaction solution to react with the reaction solution at 5O⁰C for 30 minutes while maintaining the temperature of the reaction solution at 5O⁰C. As a result of the reaction, FNA contained in the cNDA was converted to NDA in the reactor A.

[I l l]

[112] Third step: Crystallization of Purified cNDA

[113] 100 L of the solution of the purified cNDA prepared in the second step was transferred to a crystallization reactor C equipped with an agitator. After a sulfuric acid solution was added to the crystallization reactor to adjust the pH of the solution of the purified cNDA to 3.0, a crystallization reaction was conducted with stirring at a rate of 50 rpm for 25 minutes while maintaining the temperature at 8O⁰C.

[114] After completion of the crystallization, analysis of the cNDA crystal was conducted using a microscope and a particle size analyzer. The analytical results reveal that the cNDA was satisfactorily crystallized without any aggregation between the individual crystal particles. The cNDA crystal was measured to have an average particle size of about 162.5 D. FIG. 3 shows a micrograph of the cNDA crystal.

[115]

[116] Fourth step: Washing and Drying of cNDA Crystal

[117] The slurry of the cNDA crystal prepared in the third step was heated to above 22O⁰C using a preheater D, fed into a filtering/cleaning unit F, stirred at a pressure of 28 kg/ cm and a temperature of 225⁰C for 30 minutes, and filtered. The filtrate (i.e. water) was discharged into a high-pressure filtrate collector G. 100 L of water at 225⁰C from a solvent heating/supply unit E was added to the filtering/cleaning unit F, stirred under the same conditions as above for 30 minutes, and filtered. The filtrate (i.e. water) was discharged into the high-pressure filtrate collector G. This procedure was repeated a total of two times. 100 L of water at 225⁰C from the solvent heating/supply unit E was added to the filtering/cleaning unit F and stirred for at least 30 minutes to homogeneously disperse pure NDA. The slurry containing the pure NDA was transferred to a high-pressure slurry collector H. After the pressure of the high-pressure slurry collector H dropped to ambient pressure, the slurry was transferred to a powder separator I (i.e. a decanter) where the water was removed from the slurry, followed by drying in a dryer J at 12O⁰C to collect the NDA in a pure crystalline form.

[118]

[119] Example 2

[120] NDA in a pure crystalline form was collected in the same manner as in Example 1, except that the recombinant microorganism was previously removed from the solution of the purified cNDA, which was prepared in the second step, using a polypropylene filter (pore size: 0.2 D) after the second step and prior to the third step.

[121] [122] Example 3 [123] NDA in a pure crystalline form was collected in the same manner as in Example 1, except that a microorganism JM109 (pUC18-xy/C) obtained by transformation with the recombinant expression vector (pUC18-xy/C) shown in FIG. 2 carrying the ben- zaldehyde dehydrogenase gene (see, GenBank Sequence Database, D63341) of SEQ ID NO: 4 derived from Pseudomonas putida mt-2 (ATCC 33015) was used as the recombinant microorganism. Details of a method for preparing the recombinant microorganism JM109 (pUC18-xy/C) are found in Korean Patent Unexamined Publication No. 2005-71188.

[124] [125] Example 4 [126] NDA in a pure crystalline form was collected in the same manner as in Example 2, except that JM 109 (pUC18-xy/C) was used as the recombinant microorganism.

[127] [128] Experimental Example 1 : Component Analysis [129] The unpurified cNDA, the solution of the purified cNDA obtained after the second step (purification step), and the NDA in a pure crystalline form obtained after the fourth step (washing/drying step) of each of Examples 1 and 3 were analyzed for the contents of the components therein. The respective analytical results are shown in Tables 1 and 2.

[130] TABLE 1: Component Analysis Data in Example 1 [131]

[132] TABLE 2: Component Analysis Data in Example 3 [133]

[134] From the results of Tables 1 and 2, it could be confirmed that the purification method using the recombinant microorganism enabled substantially complete removal of impurities contained in cNDA. Particularly, FNA was converted to NDA in a yield of 100%. As a result, 2,6-naphthalene dicarboxylic acid was produced with a purity of 99.99% or higher.

[135] To determine optimal reaction conditions for the purification of the cNDA using the recombinant microorganism, particularly those for the crystallization of the solution of the purified cNDA, a series of experiments was conducted by varying reaction conditions as follows, and the average particle sizes and recovery rates of cNDA crystals obtained after respective crystallization reactions according to the different reaction conditions were compared and analyzed.

[136] [137] Experimental Example 2: Crystallization Reactions at Different Kinds of Acids and Stirring Rates

[138] 100 ml of the solution of the purified cNDA prepared in the second step of each of Examples 1 and 3 was added to reactors, each of which was equipped with an agitator, and then sulfuric acid, hydrochloric acid, glacial acetic acid and nitric acid solutions were added to the reactors to adjust the pH of the solutions of the purified cNDA to 3.0. The mixed solutions were stirred at different rates of 0, 50, 100, 200, 400, 800 and 1,000 rpm. At this time, the temperature of the mixed solutions was maintained at 8O⁰C. Under these reaction conditions, crystallization reactions were conducted for 25 minutes to obtain cNDA crystals.

[139] After completion of the crystallization, analysis of the cNDA crystals was conducted using a microscope and a particle size analyzer. The analytical results reveal that the cNDA was satisfactorily crystallized without any aggregation between the individual crystal particles. The average particle sizes of the cNDA crystals obtained under the respective conditions were measured, and the results are shown in Tables 3 and 4. As can be seen from the data shown in Tables 3 and 4, the cNDA crystals obtained after the addition of the sulfuric acid or hydrochloric acid solution and stirring at a rate of 0 to 400 rpm, particularly 50 to 150 rpm, showed better results.

[140] TABLE 3: Crystal sizes at different kinds of acids and stirring rates in Example 1 [141]

Acid Sulfuric acid

Stirring rate (rpm) 0 50 100 200 400 800 1000

Average particle size (/m) 116 .6 135. 8 125.8 118.7 110.6 42.7 25.1

Acid Hydrochloric acid

St irring rate (rpm) 0 50 100 200 400 800 1000

Average particle size (/an) 117 .2 135. 4 126.2 119.4 99.8 40.3 20.0

Acid Glacial acetic acid

Stirring rate (rpm) 0 50 100 200 100 800 1000

Average particle size (jean) ( O . 4 91. 2 81.5 74.3 56.6 15.1 4.9

Acid Nitric acid

Stirring rate (rpm) 0 50 100 200 400 800 1000

Average particle size (AMI) 92. 1 110. 8 99.1 94.7 80.6 23.1 17.3

[142] TABLE 4: Crystal sizes at different kinds of acids and stirring rates in Example 3 [143]

Acid Sulfuric tacid

Stirring rate (rpm) 0 50 100 200 100 800 1000

Average part icle size (jaw) 118 .5 136.8 127. I 120.2 118.8 45.7 28.1

Acid Hydrochloric acid

St irring rate (rpm) 0 50 100 200 400 800 1000

Average particle size (jum) 121 .1 137.2 129. 5 120.3 101.7 48.5 30.0

Acid Gl acial acet c acid

St irring rate (rpm) 0 50 100 200 400 800 1000

Average particle size (/ail) 76. 4 99.7 86..^r 79.3 61.7 32.1 10.9

Acid Nitric acid

Stirring rate (rpm) 0 50 100 200 400 800 1000

Average particle size (μm) 92. 1 119.7 113. ') 95.7 81.1 25.1 18.6

[144] Experimental Example 3: Crystallization Reactions at Different Kinds of Acids and Temperatures [145] The procedure of Experimental Example 2 was repeated, except that the mixed solutions were stirred at a fixed rate of 50 rpm at different reaction temperatures of 4, 15, 50, 80, 120 and 15O⁰C.

[146] After completion of the crystallization, analysis of the cNDA crystals was conducted using a microscope and a particle size analyzer. The analytical results reveal that the cNDA was satisfactorily crystallized without any aggregation between the individual crystal particles. FIG. 4 is a micrograph of the cNDA crystal obtained by adding the sulfuric acid solution to the reaction solution of the purified cNDA prepared in Example 1 and allowing the mixed solution to react at 8O⁰C. The average particle sizes of the cNDA crystals obtained under the respective conditions were measured, and the results are shown in Tables 5 and 6. As can be seen from the data shown in Tables 5 and 6, the cNDA crystals obtained after the addition of the sulfuric acid or hydrochloric acid solution and reaction at a temperature of 50-120⁰C, particularly 8O⁰C, showed better results.

[147] TABLE 5: Crystal sizes at different kinds of acids and stirring rates in Example 1 [148]

[149] TABLE 6: Crystal sizes at different kinds of acids and stirring rates in Example 3 [150]

Acid Sulfuric acid

Temp. (⁰C) 4 15 50 80 120 150

'\vcragc particle size (μm) 112 .4 121.9 149.8 166.1 150.8 135.1

Acid Hydrochloric acid lemp. CC) 4 15 50 80 120 150

Average particle size OiITl) 115. O 12.1.4 118.5 164.5 151.3 136.1

Acid Glacial icetic acid

Temp. (⁰C) 4 15 50 80 120 150

Average particle size (AMI) 49. 1 65.5 80.6 92.7 72.3 52.3

Acid Nitric acid

Temp. CC) 4 15 50 80 120 150

Average particle size OiITl) 67. 7 93.8 125..3 145.9 121.9 89.2

[151] Experimental Example 4: Recovery Rates at Different Kinds of Acids and pH Values [152] The procedure of Experimental Example 2 was repeated, except that the mixed solutions were stirred at a fixed rate of 50 rpm, a fixed reaction temperature of 8O⁰C and varying pH values of 1, 2, 3, 4, 5 and 6. After the crystallization reactions were conducted for 25 minutes, equal amounts of the reaction solutions were taken out of the respective reactors. The recovery rates of the cNDA crystals were measured, and the results are shown in Tables 7 and 8. The results of Tables 7 and 8 show that the recovery rates of the cNDA crystals were not lower than 99% at a pH not higher than 3. The recovery rates of the cNDA crystals showed a tendency to increase with decreasing pH.

[153] TABLE 7:Recovery rates at different kinds of acids and pH values in Example 1 [154]

[155] TABLE 8:Recovery rates at different kinds of acids and pH values in Example 3 [156]

Industrial Applicability

[157] According to the purification method of the present invention, a crude naphthalene dicarboxylic acid is purified and crystallized under respective suitable conditions using a recombinant microorganism capable of converting FNA to NDA. Therefore, the purification method of the present invention enables the production of high-purity crystalline 2,6-naphthalene dicarboxylic acid on an industrial scale in an economically feasible and environmentally friendly manner. Sequence Listing

[158] SEQ ID NO: 1 is a base sequence of a benzaldehyde dehydrogenase gene (xylC) derived from Sphingomonas aromaticivorans (KCTC 2888).

[159] SEQ ID NO: 2 shows primer 1 synthesized on the basis of a DNA base sequence of a benzaldehyde dehydrogenase gene (xylC) derived from Sphingomonas aromaticivorans (KCTC 2888) to clone the xylC gene.

[160] SEQ ID NO: 3 shows primer 2 synthesized on the basis of a DNA base sequence of a benzaldehyde dehydrogenase gene (xylC) derived from Sphingomonas aromaticivorans (KCTC 2888) to clone the xylC gene.

[161] SEQ ID NO: 4 is a base sequence of a benzaldehyde dehydrogenase gene (xylC) derived from Pseudomonas putida mt-2 (ATCC 33015).

Claims

[1] A method for purifying a crude naphthalene dicarboxylic acid using a recombinant microorganism, the method comprising the steps of:

(a) reacting a crude naphthalene dicarboxylic acid with at least one recombinant microorganism selected from the group consisting of microorganisms obtained by transformation with a recombinant expression vector carrying the ben- zaldehyde dehydrogenase gene (xylC) of SEQ ID NO: 1 derived from Sph- ingomonas aromaticivorans (KCTC 2888) or a gene having a homology of at least 90% with the xylC gene and microorganisms obtained by transformation with a recombinant expression vector carrying the benzaldehyde dehydrogenase gene (xylC) of SEQ ID NO: 4 derived from Pseudomonas putida mt-2 (ATCC 33015) or a gene having a homology of at least 90% with the xylC gene, to remove 2-formyl-6-naphthoic acid present in the crude naphthalene dicarboxylic acid;

(b) adding an acidic solution to the reaction solution prepared in step (a) to adjust the pH of the reaction solution and reacting the mixed solution with stirring to crystallize the crude naphthalene dicarboxylic acid;

(c) washing the crystal of the crude naphthalene dicarboxylic acid to remove impurities contained therein; and

(d) drying the washed product to obtain 2,6-naphthalene dicarboxylic acid in a pure crystalline form.

[2] The method according to claim 1, wherein step (a) includes the sub-steps of:

1) inoculating the recombinant microorganism into a liquid medium, culturing the cells to induce the expression of benzaldehyde dehydrogenase, centrifuging the culture broth to collect the cells in which benzaldehyde dehydrogenase is expressed, and suspending the cells in physiological saline or distilled water;

2) mixing a crude naphthalene dicarboxylic acid (cNDA) as a substrate with a buffer solution and adjusting the pH of the mixed solution by the addition of an alkaline solution to prepare a reaction solution for subsequent purification; and

3) reacting the cells prepared in 1) with the reaction solution prepared in 2) to convert 2-formyl-6-naphthoic acid contained in the crude naphthalene dicarboxylic acid to 2,6-naphthalene dicarboxylic acid, so that the purity of the 2,6-naphthalene dicarboxylic acid is increased.

[3] The method according to claim 2, wherein the microorganism is a bacterium belonging to the genus Escherichia, Pseudomonas, Rhodococcus or Bacillus.

[4] The method according to claim 2, wherein the microorganism is E. coli.

[5] The method according to claim 2, wherein the buffer solution is selected from the group consisting of water, sodium carbonate buffers (Na CO /NaHCO ), glycine buffers (glycine/NaOH), potassium phosphate buffers (KH PO /KOH),

2 4 sodium phosphate buffers (Na HPO /NaH PO ), succinic acid buffers (succinic

2 4 2 4 acid/NaOH), sodium acetate buffers (sodium acetate/acetic acid), citric acid buffers (citric acid/sodium citrate), sodium pyrophosphate buffers (Na P O / HCl), boric acid buffers (boric acid/NaOH), sodium borate buffers (sodium borate/HCl), and mixtures thereof; and has a concentration of 0.01 to 100 mM.

[6] The method according to claim 2, wherein the alkaline solution is a NaOH or

KOH solution.

[7] The method according to claim 2, wherein the mixed solution further contains an organic solvent.

[8] The method according to claim 7, wherein the organic solvent is selected from the group consisting of dimethylsulf oxide, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, and mixtures thereof; and is added at a concentration of 0.01 to 20%.

[9] The method according to claim 2, wherein the reaction is conducted at 25 to

7O⁰C for 1 minute to 1 hour.

[10] The method according to claim 2, wherein the crude naphthalene dicarboxylic acid is present at a concentration of between 0.001% and 20% in the reaction solution.

[11] The method according to claim 1, wherein the acidic solution is selected from the group consisting of sulfuric acid, hydrochloric acid, glacial acetic acid, nitric acid, and mixtures thereof.

[12] The method according to claim 1, wherein the pH of the reaction solution is adjusted to the range of 1 to 4.

[13] The method according to claim 1, wherein the reaction is carried out at 4⁰C to

15O⁰C for 1 minute to 10 hours.

[14] The method according to claim 1, wherein the stirring is performed at a rate of 0 to 1,000 rpm.

[15] The method according to claim 1, wherein step (c) is carried out by dispersing the cNDA crystal in water, stirring the dispersion at a pressure of 1 to 60 kg/cm and a temperature of 100 to 27O⁰C for 10 minutes to 1 hour, filtering the cNDA crystal to remove the water, and repeating the above procedure.

[16] The method according to claim 1, wherein the drying is performed at 30 to

200⁰C.

[17] The method according to claim 1, further comprising the step of removing the recombinant microorganism used in step (a) after step (a) and prior to step (b).

[18] The method according to claim 17, wherein the removal of the microorganism is achieved using a microfilter system, a continuous type centrifugal separator or a decanter. [19] The method according to claim 18, wherein the microfilter system uses a filter having a pore size of 0.1 to 0.5 D and made of ceramic, stainless steel, polypropylene or polyethylene terephthalate (PET). [20] 2,6-Naphthalene dicarboxylic acid in a pure crystalline form produced by the method according to any one of claims 1 to 19. [21] The 2,6-naphthalene dicarboxylic acid according to claim 20, wherein the

2,6-naphthalene dicarboxylic acid is in a regular crystalline form.