WO2008013347A1

WO2008013347A1 - Purification method of crude naphthalene dicarboxylic acid using benzaldehyde dehydrogenase from recombinated microorganism and 2,6-naphthalene dicarboxylic acid in crystalline form obtained by using the same

Info

Publication number: WO2008013347A1
Application number: PCT/KR2006/005730
Authority: WO
Inventors: Dong Sung Kim; Yong Bok Choi; Sung Kyoon Kim; Ik Hyun Kwon
Original assignee: Hyosung Corporation
Priority date: 2006-07-25
Filing date: 2006-12-27
Publication date: 2008-01-31
Also published as: KR100851742B1; KR20080009922A

Abstract

Disclosed is a method for purifying a crude naphthalene dicarboxylic acid using benzaldehyde dehydrogenase. According to the purification method, a crude naphthalene dicarboxylic acid is purified by reacting benzaldehyde dehydrogenase having the ability to convert 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid with a crude naphthalene di¬carboxylic acid, thereby removing the 2-formyl-6-naphthoic acid from 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, 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 environmentally friendly manner.

Description

Description PURIFICATION METHOD OF CRUDE NAPHTHALENE DI-

CARBOXYLIC ACID USING BENZALDEHYDE DEHYDROGENASE FROM 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 benzaldehyde dehydrogenase expressed in a recombinant microorganism and isolated and purified from the 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 benzaldehyde dehydrogenase having the ability to convert 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid with a crude naphthalene dicarboxylic acid to convert 2-formyl-6-naphthoic acid contained as an impurity in the crude naphthalene dicarboxylic acid to 2,6-naphthalene dicarboxylic acid, thereby removing the 2-formyl-6-naphthoic acid from 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 v arious 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. This 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 316 ⁰C 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 400 ⁰C, neutralizing the aqueous solution with an acid at 170 to 240 ⁰C, 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 400 ⁰C, 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 0-50 ⁰C 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 100 ⁰C (140-160 ⁰C) 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] 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 producing high-purity NDA in high yield by purifying and crystallizing a crude naphthalene dicarboxylic acid at ambient pressure and temperature conditions using benzaldehyde dehydrogenase 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 objects, there is provided a method for purifying a crude naphthalene dicarboxylic acid using benzaldehyde dehydrogenase expressed in a recombinant microorganism and isolated and purified from the recombinant microorganism.

[14] Specifically, the purification method of the present invention comprises the following steps:

[15] (a) reacting a crude naphthalene dicarboxylic acid with benzaldehyde dehydrogenase isolated and purified from a recombinant microorganism 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, or benzaldehyde dehydrogenase isolated and purified from a recombinant microorganism 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 convert 2-formyl-6-naphthoic acid present in the crude naphthalene dicarboxylic acid to 2,6-naphthalene dicarboxylic acid, thereby removing the 2-formyl-6-naphthoic acid from 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 allowing the mixed solution to react 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 benzaldehvde dehydrogenase derived from re- combinant microorganism

[22] First, a crude naphthalene dicarboxylic acid is reacted with benzaldehyde dehydrogenase isolated and purified from a recombinant microorganism 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, or benzaldehyde dehydrogenase isolated and purified from a recombinant microorganism obtained by transformation with a recombinant expression vector carrying the benzalde hyde 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 convert 2-formyl-6-naphthoic acid present in the crude naphthalene dicarboxylic acid to 2,6-naphthalene dicarboxylic acid, thereby removing the 2-formyl-6-naphthoic acid from 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 gene.

[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 the benzaldehyde dehydrogenase, centrifuging the culture broth to collect the cells in which the benzaldehyde dehydrogenase is expressed, suspending the cells in a buffer solution or physiological saline, disrupting the cells by sonication, centrifuging the resulting suspension to obtain a supernatant, and isolating and purifying the benzaldehyde dehydrogenase from the supernatant; 2) mixing a crude naphthalene dicarboxylic acid 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 benzaldehyde dehydrogenase obtained 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] The benzaldehyde dehydrogenase 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 ( xylQ 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 expression vector to construct a recombinant expression vector, introducing the recombinant expression vector into a microorganism, transforming the microorganism with the recombinant expression vector by a conventional known technique to obtain a recombinant microorganism, followed by isolation and purification from the recombinant microorganism by a conventional technique.

[26] Specifically, the benzaldehyde dehydrogenase can be obtained in accordance with 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 2μM, 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-P and λ-P

R L 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 2 of the present invention.

[33] The cloning of the enzymegene 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] Then, the recombinant microorganism is sufficiently cultured, and then each ben- zaldehyde dehydrogenase is separated and purified therefrom. The separation and purification can be accomplished by any known technique. According to the method of the present invention, 2-formyl-6-naphthoic acid is converted to 2,6-naphthalene di- carboxylic acid with the help of the benzaldehyde dehydrogenase obtained from the recombinant microorganism. To this end, it is preferred that each benzaldehyde dehydrogenase be expressed at a high level within the corresponding recombinant microorganism. Therefore, it is desirable to induce the expression of each enzyme by shaking culture or IPTG addition and isolate and purify the expressed enzyme from the recombinant microorganism.

[36] Specifically, the recombinant microorganism is sufficiently cultured in a typical culture temperature range (25-45 ⁰C), preferably at 37 ⁰C, 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. The recombinant microorganism, in which the benzaldehyde dehydrogenase is expressed, is further cultured at 37 ⁰C, centrifuged to collect the cells, suspended in a buffer solution or physiological saline, disrupted by sonication, and re-centrifuged to obtain a supernatant. The supernatant is purified by chromatography on an ion-exchange resin to obtain the benzaldehyde dehydrogenase. The centrifugation and suspension of the cells may be repeated once or twice, if needed.

[37] Any technique known to those skilled in the art may be employed, without limitation, to culture the recombinant microorganism and express and purify the protein. [38] According to the purification method of the present invention, since the ben- zaldehyde dehydrogenase is isolated and purified from the corresponding recombinant microorganism after being expressed at a high level within the recombinant microorganism, it can be cultured at a high concentration. As a result, an advantage of the method according to the present invention is that the benzaldehyde dehydrogenase can be obtained in a large quantity at a high recovery rate.

[39] Further, any suitable technique, such as histidine tagging, can be utilized to obtain the benzaldehyde dehydrogenase. For example, according to the histidine tagging technique, the benzaldehyde dehydrogenase is obtained by attaching a histidine tag to each enzyme gene to construct a reverse primer, using the reverse primer to obtain a gene, using the gene to construct a recombinant expression vector, using the recombinant expression vector to obtain a transformed microorganism, and isolating and purifying histidine-tagged benzaldehyde dehydrogenase from the transformed microorganism. The use of histidine tagging makes the isolation and purification of the enzyme after subsequent expression considerably simpler.

[40] The kind of the buffer solution used in the present invention is not especially restricted. As the buffer solution, there can be used, for example, water, a sodium carbonate buffer (Na O /NaHCO ), a glycine buffer (glycine/NaOH), a potassium phosphate buffer (KU POJKOU), a sodium phosphate buffer (Na₂HPO₄ZNaH₂PO₄), 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

2 4 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.

[41] 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.

[42] 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% degrades the activity of the enzyme, resulting in inhibition of the reaction.

[43] In the third sub-step, the reaction is preferably conducted at 25-50 ⁰C for 1 min. - 1 hour and more preferably at 35-45 ⁰C for 10-40 minutes. When the reaction temperature is lower than 25 ⁰C or higher than 50 ⁰C, a marked decrease in the reactivity is undesirably caused.

[44] 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 enzyme 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, it is preferred to use a larger amount of the enzyme to remove the FNA.

[45] 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 1%.

[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 μm 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 μm or more in high yield.

[52] The pH of the reaction solution is preferably adjusted to 1-3 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 to 99.99%.

[53] The crystallization is performed at about 10 ⁰C to about 110 ⁰C, preferably 30 ⁰C to 90 ⁰C and more preferably 50 ⁰C to 80 ⁰C. The crystallization is performed for about 1 minute to about 8 hours, preferably 5 minutes to 4 hours and more preferably 10 minutes to one hour in terms of continuous processing. The most preferable conditions for the crystallization are 70 ⁰C 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 50-100 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 benzaldehyde dehydrogenase 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 enzyme 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 120 ⁰C and 270 ⁰C and preferably between 180 ⁰C and 240 ⁰C. The separation of the solvent at a temperature higher than 270 ⁰C 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 120 ⁰C 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 120 to 270 ⁰C for 10 minutes to 2 hours, 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 50 to 250 ⁰C. 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 inactivated molecules of the enzyme used in step (a) after step (a) and prior to step (b).

[65] The previous removal of the inactivated enzyme molecules 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 inactivated enzyme molecules 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 inactivated enzyme molecules. The microfilter system uses a filter having a pore size of 0.01-1 μm 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, but is not necessarily limited thereto.

[69] The 2,6-naphthalene dicarboxylic acid crystal of the present invention has an average particle diameter not smaller than 100 μm 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 μm 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 μm and more preferably 110-180 μm.

[70] FIG. 4 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. 4, 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 benzaldehyde dehydrogenase as an enzyme. 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. Benzaldehyde dehydrogenase is 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 inactivated molecules of the enzyme used to remove the FNA are removed. The removal of the inactivated enzyme molecules using the unit B may be omitted, if needed, because the removal of the inactivated enzyme molecules and the remaining enzymes can be achieved in a downstream filtering/cleaning unit F.

[73] The reaction solution, from which the inactivated enzyme molecules are 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 120 ⁰C to about 270 ⁰C using a preheater D, and is then fed into a filtering/cleaning unit F provided with a heater to maintain the temperature of the slurry at 120-270 ⁰C. 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 μm).

[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 (120-270 ⁰C) 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 (120-270 ⁰C) 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 to remove the solvent 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 benzaldehyde dehydrogenase 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 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; and

[81] FIG. 4 shows a purification system for implementing a method for purifying a crude naphthalene dicarboxylic acid according to an embodiment of the present invention.

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

[83] A: Purification reactor using enzyme

[84] B: Unit for removing inactivated enzyme molecules

[85] C: Crystallization reactor D: Preheater

[86] E: Solvent heating/supply unit F: Filtering/cleaning unit

[87] G: High-pressure filtrate collector I: Powder separator

[88] J: Dryer

Mode for the Invention

[89] 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.

[90]

[91] EXAMPLES

[92] [Example 1]

[93] First Step: Separation of Pure Enzyme Solution from Recombinant Microorganism

[94] ( 1 ) Cloning of xylC Gene

[95] 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 ah, 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 carried out twice at 94 0C for 5 minutes (first denaturation) and at 94 ⁰C for one minute (second denaturation), annealing was carried out at 56 ⁰C for one minute, and extension was carried out at 72 0C for 1.5 minutes. This procedure was repeated forty times. Finally, extension was once more carried out at 72 ⁰C 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.

[96]

[97] (2) Analysis of Cloned Gene

[98] 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.

[99]

[100] (3) Preparation of Transformant

[101] 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).

[102]

[103] (4) Expression of xylC Gene and Purification of Benzaldehyde Dehydrogenase [104] 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 37 ⁰C, 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 37 ⁰C. The culture was centrifuged to collect the cells, suspended in a 50 mM potassium phosphate buffer (KH PO /KOH, pH 7.0), disrupted by sonication, and centrifuged to obtain a supernatant. The supernatant was purified by anion chromatography using a column containing Q-sepharose to isolate benzaldehyde dehydrogenase therefrom. The benzaldehyde dehydrogenase thus isolated was confirmed to have a molecular weight of about 55,000 Da, as determined by SDS-PAGE.

[105]

[106] (5) Measurement of Titer of Enzyme

[107] Benzaldehyde is converted to benzoic acid by nicotinamide- adenine dinucleotide

(NAD⁺) in the presence of benzaldehyde dehydrogenase. The amount of NADH produced is determined by measuring the absorbance at 340 nm. The titer of the enzyme was determined by adding 0.5 ml of the enzyme solution to 3 ml of a 50 mM potassium phosphate buffer (KH PO /KOH, pH 7.0) containing 5 mM NAD⁺ and 10 mM benzaldehyde, allowing the mixture to react at 20 ⁰C for 5 minutes, and measuring the absorbance of the reaction mixture at 340 nm. One unit of the enzyme activity is defined as the amount of the enzyme producing 1 μmole of NDAH per minute.

[108]

[109] Second step: Purification of cNDA Using Benzaldehvde Dehydrogenase

Isolated from Recombinant Microorganism

[110] 50 L of a 50 mM potassium phosphate buffer was injected into a reactor A where a cNDA is purified by the enzyme. 10 kg of a cNDA was added to the reactor A and then KOH 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%).

[I l l] 5L of the benzaldehyde dehydrogenase solution (500 units) prepared in the first step was fed to the reaction solution to react with the reaction solution at 40 ⁰C for 30 minutes while maintaining the temperature of the reaction solution at 40 ⁰C. As a result of the reaction, FNA contained in the cNDA was converted to NDA in the reactor A.

[112]

[113] Third step: Crystallization of Purified cNDA

[114] 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 20 minutes while maintaining the temperature at 70 ⁰C.

[115] 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 163.5 μm. FIG. 3 shows a micrograph of the cNDA crystal.

[116]

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

[118] The slurry of the cNDA crystal prepared in the third step was heated to above 220

⁰C using a preheater D, fed into a filtering/cleaning unit F, stirred at a pressure of 35 kg/cm and a temperature of 230 ⁰C for 30 minutes, and filtered. The filtrate (i.e. water) was discharged into a high-pressure filtrate collector G. 100 L of water at 230 ⁰C 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 230 ⁰C 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 180 ⁰C to collect the NDA in a pure crystalline form.

[119]

[120] rExample 21

[121] NDA in a pure crystalline form was collected in the same manner as in Example 1, except that benzaldehyde dehydrogenase isolated and purified from a microorganism JM109 (pUC18-xy/C) obtained by transformation with the recombinant expression vector (pUC18-xy/C) shown in FIG. 2 carrying the benzaldehyde dehydrogenase gene (see, GenBank Sequence Database, D63341) of SEQ ID NO: 4 derived from Pseudomonas putida mt-2 (ATCC 33015) was used as the pure enzyme solution.

[122] Details of a method for preparing the recombinant microorganism JM109 (pUC18- xylC) are found in Korean Patent Unexamined Publication No. 2005-71188. The isolation and purification of benzaldehyde dehydrogenase from the recombinant microorganism JM109 (pUC18-xy/C) were carried out in accordance with the following procedure.

[123] The recombinant microorganism JM109 (pUC18-xy/C) was inoculated into an LB test tube, allowed to sufficiently grow at 37 ⁰C, and inoculated into another 100 ml LB medium in such an amount that the concentration is 1% (v/v). When the OD value

600 reached 0.4-0.5, IPTG was added until the concentration was 0.5 mM to induce the expression of xylC, followed by culture at 37 ⁰C. The culture was centrifuged to collect the cells, suspended in a 50 mM potassium phosphate buffer (KH PO /KOH, pH 7.0), disrupted by sonication, and centrifuged to obtain a supernatant. The supernatant was purified by anion chromatography using a column containing Q-sepharose to isolate benzaldehyde dehydrogenase therefrom. The benzaldehyde dehydrogenase thus isolated was confirmed to have a molecular weight of about 53,500 Da, as determined by SDS-PAGE.

[124]

[125] rExample 31

[126] NDA in a pure crystalline form was collected in the same manner as in Example 1, except that inactivated molecules of the enzyme were previously removed from the solution of the purified cNDA, which was prepared in the second step, using a polypropylene filter (pore size: 0.10 μm) after the second step and prior to the third step.

[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 2 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 Obtained in Example 1

[131] cNDA (⁰Ai) Alter purification (⁰Ai) Afler washing and drving (⁰Ai)

NA 0.037 0.020

MNA 0 112 0.070 -

FNA 0 580 - -

TMLA 0 044 0.023 .

Others 0.160 0.120 0.002

NDA 99 067 99.767 99 998

[132] TABLE 2 Component Analysis Data Obtained in Example 2

[133] cNDA (%) After purification (%) After washing and diving (%)

NA 0 037 0 020 -

MNA 0 il2 0.065 -

FNA 0.580 - -

TMLA 0 044 0.025 -

Others 0 i60 0 120 0.002

NDA 99 067 99.770 99.998

[134] From the results of Tables 1 and 2, it could be confirmed that the purification method using the benzaldehyde dehydrogenase 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 benzaldehyde dehydrogenase, 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 2 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 70 0C. Under these reaction conditions, crystallization reactions were conducted for 20 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 100 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 rale (rpm) 0 50 100 200 400 800 1,000

A\ erage particle size (μm) 121 9 128 9 122 5 1 15 7 1 13 7 56 4 30 5

Acid H\drochloπc <icid

Stirring rate (rpm) 0 50 100 200 400 800 1 000

A\cragc particle size (μm) 123 7 129 S 124 1 114 9 110 9 47 7 30 2

Acid Glacial acetic acid

Stirring rate (rpm) 0 50 100 200 400 800 1,000

A\ erage particle size (μm) 80 1 96 1 83 4 75 1 59 9 22 5 6 8

Acid Nitric acid

Stirring rate (rpm) 0 50 100 200 400 800 1 000

A\erage particle size (μm) 95 4 111 2 100 6 90 4 81 8 33 2 21 4

[142] TABLE 4 Crystal sizes at different kinds of acids and stirring rates in Example 2 [143]

Acid Sulfuric acid Stirring rate (rpm) 0 50 100 200 400 800 1 000 A\cragc particle size (μm) 118 9 135 7 131 0 120 7 113 8 56 1 35 8

Acid HΛ drochloric acid Stirring rate (rpm) 0 50 100 200 400 800 I 000 A\er,ige particle size (μm) 128 1 139 7 135 2 122 4 115 7 50 4 32 8

Acid Glacial acetic acid Stirring rate (rpm) 0 50 100 200 400 800 1 000 Av erage particle size (μm) 80 9 96 1 90 3 79 7 61 1 28 3 8 5

Acid Nitric acid S Luring rale (rpm) 0 50 100 200 400 800 1 000 A\cragc particle size (μm) 96 8 111 8 106 5 90 3 77 3 32 1 22 5

[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 10, 30, 50, 70, 90 and HO ⁰C.

[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. 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 showed better results and the most preferred reaction temperature was 70 ⁰C.

[147] TABLE 5 Crystal sizes at different kinds of acids and stirring rates in Example 1 [148] Acid Sulfuric acid Temp (⁰C) 10 30 50 70 90 110 Average particle size (μm) 115 8 140 1 158 9 165 6 147 9 132 1

Acid H\ drocliloπc acid Temp (⁰C) 10 30 50 70 90 110 Average particle size (μm) 1 17 5 140 7 156 0 162 9 147 9 120 5

Acid Glacial acetic acid Temp (⁰C) 10 30 50 70 90 110 Average particle size (μm) Ϊ1 4 73 8 89 5 93 1 75 8 53 1

Acid Nitric acid Temp (⁰C) 10 30 50 70 90 1 10 Average particle size (μm) 70 1 114 7 128 3 139 4 121 3 95 2

[149] TABLE 6 Crystal sizes at different kinds of acids and stirrin

[150]

Acid Sulfuric acid Temp (⁰C) 10 30 50 70 90 110 Average particle size (μm) 114 9 147 1 157 7 163 8 151 5 132 1

Acid Hvdrochloπc acid Temp (⁰C) 10 30 50 70 90 no Average particle size (μm) 122 3 145 4 161 8 166 5 148 7 130 1

Acid Glacral acetic acrd Temp (⁰C) 10 30 50 70 90 110 Average particle size (μm) 51 3 80 4 89 9 106 4 82 3 59 7

Acid Nitric dcid Temp (⁰C) 10 30 50 70 90 110 Average particle size (μm) 69 8 117 1 125 7 140 7 120 9 94 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 70 ⁰C and varying pH values of 1, 2, 3, 4, 5 and 6. After the crystallization reactions were conducted for 20 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 99.9% 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] Acid Sulfuric dcid pH 1 2 3 4 5 6 Rcco-vcrv rate (%) 99 99 99 98 99 96 98 00 85 00 60 50

Acid H\ drocliloric acid pH 1 2 3 4 5 6 Reco\er\ rate (%) 99 99 99 97 99 95 97 95 79 50 57 80

Acid Glacial acetic acid pH 1 2 3 4 5 6 Reco\erv rate (%) 99 99 99 91 99 91 96 20 76 00 53 70

Acid Nitric acid pH 1 2 3 4 5

Reco-verv rate (%) 99 99 99 91 99 90 96 30 72 00 50 50

[155] TABLE 8 Recovery rates at different kinds of acids and pH values in Example 2 [156]

Acid Sulfuric acid pH 1 2 3 4 5 6

Recovery rate (%) 99 99 99 99 99 96 98 00 80 00 62 50

Acid Hydrochloric <iαd pH 1 2 3 4 5 6

Recovery rate (%) 99 99 99 99 99 96 97 90 77 00 58 50

Acid Glacial acetic acid pH 1 2 3 4 5 6

Recovery rate (%) 99 99 99 96 99 92 96 90 75 90 55 00

Acid Nitric acid pH 1 2 3 4 5 6

Recovery rate (%) 99 99 99 94 99 91 96 50 75 50 49 80

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 benzaldehyde dehydrogenase expressed in a recombinant microorganism capable of converting FNA to NDA and isolated and purified from the recombinant microorganism. 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 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 ben- zaldehyde dehydrogenase, the method comprising the steps of:

(a) reacting a crude naphthalene dicarboxylic acid with benzaldehyde dehydrogenase isolated and purified from a recombinant microorganism obtained by transformation with a recombinant expression vector carrying the benzaldehyde dehydrogenase gene (xylC) of SEQ ID NO: 1 derived from Sphingomonas aro- maticivorans (KCTC 2888) or a gene having a homology of at least 90% with the xylC gene, or benzaldehyde dehydrogenase isolated and purified from a recombinant microorganism 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 convert 2-formyl-6-naphthoic acid present in the crude naphthalene dicarboxylic acid to 2,6-naphthalene dicarboxylic acid, thereby removing the 2-formyl-6-naphthoic acid from 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 allowing the mixed solution to react 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 the benzaldehyde dehydrogenase, cen- trifuging the culture broth to collect the cells in which the benzaldehyde dehydrogenase is expressed, suspending the cells in a buffer solution or physiological saline, disrupting the cells by sonication, centrifuging the resulting suspension to obtain a supernatant, and isolating and purifying the benzaldehyde dehydrogenase from the supernatant;

2) mixing a crude naphthalene dicarboxylic acid 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 benzaldehyde dehydrogenase obtained 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 buffer solution is selected from the group consisting of water, sodium carbonate buffers (Na O /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. [4] The method according to claim 2, wherein the alkaline solution is a NaOH or

KOH solution. [5] The method according to claim 2, wherein the mixed solution further contains an organic solvent. [6] The method according to claim 5, 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%. [7] The method according to claim 2, wherein the reaction is conducted at 25 to 50

⁰C for 1 minute to 1 hour. [8] 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. [9] 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. [10] The method according to claim 1, wherein the pH of the reaction solution is adjusted to the range of 1 to 3. [11] The method according to claim 1, wherein, in step (b), the reaction is carried out at 10 ⁰C to 110 ⁰C for 1 minute to 8 hours. [12] The method according to claim 1, wherein the stirring is performed at a rate of 0 to 1,000 rpm. [13] 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 120 to 270 ⁰C for 10 minutes to 2 hours, filtering the cNDA crystal to remove the water, and repeating the above procedure. [14] The method according to claim 1, wherein the drying is performed at 50 to 250

⁰C. [15] The method according to claim 1, further comprising the step of removing in- activated molecules of the enzyme used in step (a) after step (a) and prior to step

(b). [16] The method according to claim 15, wherein the removal of the inactivated enzyme molecules is achieved using a microfilter system, a continuous type centrifugal separator or a decanter. [17] The method according to claim 16, wherein the microfilter system uses a filter having a pore size of 0.01 to 1 μm and made of ceramic, stainless steel, polypropylene or polyethylene terephthalate (PET). [18] 2,6-Naphthalene dicarboxylic acid in a pure crystalline form produced by the method according to any one of claims 1 to 17.