KR20050071188A - Expression vector for benzaldehyde dehydrogenase gene from pseudomonas putida, bacteria transformed with the same and method for preparing 2,6-naphthalene dicarboxylic acid with highly purified using the transformants - Google Patents

Abstract

The present invention relates to an expression vector of a benzaldehyde dehydrogenase gene derived from Pseudomonas putida, a microorganism transformed with the vector, and a method for producing high purity 2,6-naphthalene dicarboxylic acid using the transformant. Specifically, an expression vector of benzaldehyde dehydrogenase gene derived from Pseudomonas putida having the ability to convert 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid was prepared and expressed. The present invention relates to a method for increasing the purity of 2,6-naphthalene dicarboxylic acid using a microorganism transformed with a vector.

When using a transformed microorganism expressing benzaldehyde dehydrogenase derived from Pseudomonas putida of the present invention, 2-formyl-6-naph contained in crude naphthalene dicarboxylic acid produced by oxidizing 2,6-dimethylnaphthalene Tosan can be converted into high purity 2,6-naphthalene dicarboxylic acid economically and efficiently by biological method, so the industrial value is very high to produce purified 2,6-naphthalene dicarboxylic acid. There is an advantage.

Description

Expression vector of benzaldehyde dehydrogenase gene derived from Pseudomonas putida, microorganism transformed with this vector, and method for producing high purity 2,6-naphthalene dicarboxylic acid using the transformant {Expression vector for benzaldehyde dehydrogenase gene from Pseudomonas putida, bacteria transformed with the same and method for preparing 2,6-Naphthalene dicarboxylic acid with highly purified using the transformants}

The present invention relates to an expression vector of a benzaldehyde dehydrogenase gene derived from Pseudomonas putida, a microorganism transformed with the vector, and a method for producing 2,6-naphthalene dicarboxylic acid with high purity using the transformant. More specifically, a recombinant expression vector comprising a benzaldehyde dehydrogenase (hereinafter referred to as BZDH) gene ( xylC ) derived from Pseudomonas putida , a transformed microorganism transformed with the expression vector, and 2-formyl contained in crude naphthalene dicarboxylic acid (cNDA) produced by oxidizing 2,6-dimethyl naphthalene (2,6-DMN) using Eco-friendly and economical conversion of 6-naphthoic acid (FNA) to 2,6-Naphthalene dicarboxylic acid (NDA) The present invention relates to a method for obtaining high purity 2,6-naphthalene dicarboxylic acid.

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

NDC is currently produced by oxidizing 2,6-DMN to produce cNDA and then esterification, as shown in FIG. Currently, NDC is used as a main raw material when synthesizing PEN, but there are some problems compared to using NDA as a raw material. Firstly, water is produced during NDA condensation, whereas methanol is a by-product in the case of NDC, and there is a risk of explosiveness. Second, NDA is esterified to obtain pure NDC during the NDC manufacturing process, and then NDC is produced through purification process. Therefore, one more step is required compared to NDA, and thirdly, if you have an existing PET production facility, it is not suitable for the use of NDC in terms of using the existing facility.

Despite the disadvantages of NDC described above, the reason why NDC is used instead of NDA in PEN production is that it is still difficult to produce purified NDA having the purity necessary for polymerization. Oxidation of DMN produces cNDA containing various impurities such as FNA, 2-naphthoic acid, and trimellitic acid. Of these, in particular, the presence of FNA, the polymerization is stopped in the middle to adversely affect the physical properties of the polymer to remove the FNA, but there is a difficult problem to remove it.

Therefore, in order to remove FNA present in cNDA or to purify NDA, recrystallization, oxidation process is performed once more, cNDA is prepared by NDC using methanol and then hydrated to prepare NDA or by hydrogenation process. Methods of preparing purified NDA have been studied. In addition, various purification methods such as solvent treatment, melting crystals, high pressure crystals, and supercritical extraction have been used, but have not yet produced NDAs with satisfactory purity. In addition, even if the purity is increased, the yield is very difficult to apply to actual production.

In addition, efforts have been made to remove FNA using a biological method. To date, there has been a report on a method for removing FNA using a strain of Bacillus genus (Korean Patent Application No. 02-0087819). A method for preparing aromatic aldehydes and carboxylic acids using XMO (Korean Patent Application No. 02-7005344) has been reported, but there is no known method for converting FNA to NDA.

On the other hand, Pseudomonas footage is (Pseudomonas putida) xylC present on the xyl operon of the TOL plasmid pWWO of mt-2 is a gene encoding benzaldehyde dehydrogenase of claim (BZDH) an enzyme which acts to oxidize an aromatic aldehyde. BZDH converts the aldehyde group of the aromatic ring to a carboxyl group by selective oxidation. This is the initial stage of the metabolic pathway that forms carboxylic acid derivatives that will be converted to substrates of the Krebs cycle via the meta-catabolic pathway. Figure 5 shows the stepwise oxidation of toluene to benzyl alcohol, benzaldehyde and benzoic acid by the organization of the xy genes of the early TOL metabolic pathway enzyme and early TOL operon. XMO stands for xylene monooxygenase, BADH stands for benzyl alcohol dehydrogenase and BZDH stands for benzaldehyde dehydrogenase. Pu indicates the upper TOL operon promoter, xylW and xylN are unknown function genes. XMO consists of two polypeptide subunits encoded by the genes xylM and xylA . xylA is an NADH receptor reductase, ie an electron transport protein that transports a reducing equivalent from NADH to xylM , a hydroxylase located on the membrane. BADH, the second enzyme of the metabolic pathway, is benzyl alcohol dehydrogenase, a homodimer member of the zinc-containing dehydrogenase family whose substrate is a long chain alcohol. This enzyme is encoded by the xylB gene.

BZDH, the third enzyme of the metabolic pathway, is also a homodimer, benzaldehyde dehydrogenase, an enzyme encoded by the xylC gene. It has been demonstrated that E. coli genetically engineered to express this xylC can oxidize benzaldehyde and 3- and 4-methyl-substituted benzaldehyde, 3-nitro-, chlorine-substituted benzaldehyde (Inoue et. al., J. Bacteriol., 177: 1196-1201, 1995). However, it is not known whether the BZDH converts FNA to NDA.

Therefore, in the present invention, as a result of research to solve the problem of removing FNA in cNDA, benzaldehyde dehydrogenase derived from Pseudomonas putida can be removed by oxidizing FNA in cNDA, thus high purity NDA can be obtained. It was confirmed, the present invention was completed based on this.

Accordingly, an object of the present invention is to provide a recombinant expression vector comprising an xylC gene encoding benzaldehyde dehydrogenase derived from Pseudomonas putida.

Another object of the present invention to provide a transformed microorganism transformed with the recombinant expression vector.

Still another object of the present invention is to provide a method for inducing high expression of benzaldehyde dehydrogenase in the transgenic microorganism.

Still another object of the present invention is to provide a transformed microorganism in which the benzaldehyde dehydrogenase is highly expressed.

Still another object of the present invention is to provide a method for producing 2,6-naphthalene dicarboxylic acid with high purity using the transformed microorganism.

In order to achieve the above object, the recombinant expression vector of the present invention is benzaldehyde dihydro derived from Pseudomonas putida having the ability to convert 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid Genease gene ( xylC ).

In order to achieve the above another object, the transformed microorganism of the present invention is transformed with the vector.

The high expression induction method of benzaldehyde dehydrogenase of the present invention for achieving the above another object is to incubate the transformed microorganism at a temperature of 25 to 45 ℃, add 0.1 ~ 2.0mM IPTG to the culture solution It is composed.

To achieve another object of the present invention, a transformed microorganism having high benzaldehyde dehydrogenase expression is prepared by the above method.

In order to achieve the above another object, the method for preparing high purity 2,6-naphthalene dicarboxylic acid of the present invention is a benzaldehyde dihydrogen in the presence of a buffer solution having a pH of 6.0 to 10.0, dimethyl sulfoxide of 0 to 20% The transformed microorganism which induced the expression of the genease was reacted at a temperature of 25 to 45 DEG C using crude naphthalene dimethylcarboxylic acid as a substrate to obtain 2-formyl-6-naphthoic acid contained in crude naphthalene dimethylcarboxylic acid 2, Conversion to 6-naphthalenedicarboxylic acid.

Hereinafter, the present invention will be described in more detail.

As mentioned above, the present invention relates to a method for preparing high purity 2,6-naphthalene dicarboxylic acid using benzaldehyde dehydrogenase derived from Pseudomonas putida .

First, in the present invention, the plasmid DNA (pWW0) separated from Pseudomonas putida t-2 (ATCC 33015) by the conventional method is used as the xylC gene encoding the benzaldehyde dehydrogenase derived from Pseudomonas putida . Used as. Then, primers were prepared based on the gene bank's nucleotide sequence data (GenBank Sequence Database, D63341) and amplified by polymerase chain reaction. DNA fragments of the amplified xylC genes were purified by conventional methods and cloned into vectors. In the present invention, pUC18 is used as the vector, but various expression vectors are possible, and thus different expression vectors may be easily used depending on the purpose of expression.

According to the present invention, the xylC gene derived from Pseudomonas putida has been known in the past to selectively oxidize an aldehyde group of an aromatic ring to convert to a carboxyl group, but as in the present invention, 2-formyl-6-nap The function of converting tosan into 2,6-naphthalenedicarboxylic acid has not been confirmed. Therefore, in the present invention, Pseudomonas putida having an xylC gene encoding benzaldehyde dehydrogenase as a microorganism can be used, and Pseudomonas putida mt-2 (ATCC 33015) is preferable.

Meanwhile, cloning of the xylC gene into the prepared expression vector can be confirmed through restriction enzyme cleavage and sequencing.

The benzaldehyde dehydrogenase expression vector thus prepared is introduced into the host microorganism. In the present invention, E. coli is used as the host microorganism, and transformed by a conventional method such as calcium chloride method, rubidium chloride method, or electrophoresis (electroporation) method.

Meanwhile, in order to convert 2-formyl-6-naphthoic acid to 2,6-naphthalenedicarboxylic acid using the transformed microorganism transformed with the benzaldehyde dehydrogenase expression vector of the present invention, It is preferable that benzaldehyde dehydrogenase is highly expressed and pUC 18 used in the present invention is a gene expressed under the control of a lac promoter, thus isopropyl-β-D-thiogalactopyranoside (hereinafter referred to as "IPTG"). ) Induced the benzaldehyde dehydrogenase expression. The transformed microorganism is incubated at a temperature of 25 to 45 ° C., preferably 37 ° C., which is a normal culture temperature, and inoculated again to 1% (V / V) in 100 ml of LB medium, and an OD ₆₀₀ value of 0.4 to When it reaches 0.5, the IPTG concentration is added in an amount of 0.1 to 2.0 mM, preferably 0.5 mM, thereby inducing the expression of the protein, followed by incubation at 37 ° C. Cultivation of such transformants and expression of proteins are not limited to these methods, all methods known to those skilled in the art are possible.

Thus, the benzaldehyde dehydrogenase-transformed microorganism can be used to convert 2-formyl-6-naphthoic acid contained in crude naphthalene dimethylcarboxylic acid to 2,6-naphthalenedicarboxylic acid.

The transformed microorganism with high expression of the protein is used by recovering only cells from the culture medium and suspending them in physiological saline.

The reaction for converting 2-formyl-6-naphthoic acid contained in crude naphthalene dimethylcarboxylic acid to 2,6-naphthalenedicarboxylic acid is carried out at a buffer solution of pH 6.0 to 10, preferably pH 8 and 0 to 20%. , Preferably at a temperature of 25 to 45 ° C., preferably at 30 ° C., which is a typical reaction temperature in the presence of 5% of dimethylsulfoxide (DMSO).

As the buffer solution, KH ₂ PO ₄ , K ₂ HPO ₄ , or Na ₂ HPO ₄ may be used, and the DMSO is used as a solvent. In addition, in order to fully convert 2-formyl-6-naphthoic acid to 2,6-naphthalenedicarboxylic acid, reaction time is 1 to 20 hours, Preferably it is 6 hours.

On the other hand, the content of 2-formyl-6-naphthoic acid present in the crude naphthalene dimethylcarboxylic acid used as the substrate was used 0.01 to 10%, preferably 9%.

As such, the conversion of 2-formyl-6-naphthoic acid to 2,6-naphthalenedicarboxylic acid using a transformed microorganism expressing benzaldehyde dehydrogenase according to the method of the present invention results in a purity of 99% or more. Highly purified 2,6-naphthalenedicarboxylic acid can be obtained, which is economical and industrially useful.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited thereto.

Example 1

xylC  Cloning of genes

In order to clone the xylC gene (SEQ ID NO: 1) encoding benzaldehyde dehydrogenase derived from Pseudomonas putida mt-2 (ATCC 33015), plasmid DNA (pWW0) comprising the xylC gene sequence was first prepared. Isolated from Pseudomonas putida mt-2 (ATCC 33015) (Sambrook et al., Molecular Cloning, A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Primer 1,5'-GCTGCAGAGGATGCGTTCGAAATG-3 '(SEQ ID NO: 2) prepared using the isolated plasmid DNA (pWW0) as a template DNA, based on the DNA base sequence of the xylC gene (GenBank Sequence Database, D63341); Polymerase chain reaction was performed using primers 2,5'-GATCAGCCCGCAAGCTGCAGCAAC-3 '(SEQ ID NO: 3). In the polymerase chain reaction, the first denaturation step was performed once at 94 ° C. for 5 minutes, then the second denaturation step was at 94 ° C. for 1 minute, and the annealing step at 56 ° C. for 1 minute, extension ) Step was performed at 72 ° C. for 1.5 minutes, and this was repeated 40 times, after which the last extension step was performed once at 72 ° C. for 10 minutes. DNA fragments of about 1.6kbp size were isolated from the fragments obtained in this manner, and digested with restriction enzyme Pst I. This was cloned into plasmid pUC18 digested with the same restriction enzyme to prepare a recombinant expression vector pUC18- xylC . Figure 2 shows a gene map of the produced pUC18- xylC .

Example 2

Analysis of Cloned Genes

For sequencing the cloned gene of the recombinant vector (pUC18- xylC ) prepared in Example 1, the recombinant vector was cut using various restriction enzymes based on a cleavage map of two vectors M13mp18 and M13mp19, and each fragment Were subcloned into M13mp18 and M13mp19, and the ABI PRISM BigDye primer cycle-sequencing kit (Perkin-Elmer, USA) using AmpliTaq DNA polymerase. Were sequenced. At this time, in order to read both directions of both strands of DNA, synthetic nucleotides were partially made. Through this analysis, the gene sequences of the cloned DNA fragments were analyzed and compared with the base sequences registered in GenBank , and the xylC gene was cloned. It was confirmed.

Example 3

pUC18- xylC Introduced Preparation of Transgenic Microorganisms

E. coli JM109 was transformed with the pUC18- xylC vector using the calcium chloride method (Sambrook et al., Molecular Cloning, A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). LB plate medium (yeast extract, 5 g / L; tryptone, 10 g / L) to which ampicillin (100 mg / L), X-gal, IPTG and bacto-agar (15 g / L) was added Transformed E. coli JM109 (pUC18- xylC ) was prepared by selecting strains grown by incubation in NaCl, 10g / L).

Example 4

pUC18- xylC Through the cultivation of the xylC   Gene expression

To express and confirm the xylC gene in a transformed microorganism, E. coli transformant JM109 prepared in Example 3 (pUC18- xylC ) and wild line JM109 without a recombinant expression vector as a control were cultured under the same conditions. Activity of the benzaldehyde dehydrogenase was measured. Each of the two strains of E. coli was inoculated in an LB test tube, then grown sufficiently at 37 ° C., and inoculated again to 1% (V / V) in 100 ml of LB medium, and when the OD ₆₀₀ value reached 0.4 to 0.5, IPTG concentration was added to 0.5 mM to induce the expression of benzaldehyde dehydrogenase, and then incubated at 37 ° C.

Example 5

pUC18- xylC Conversion from 2-formyl-6-naphthoic acid (FNA) to 2,6-naphthalene dicarboxylic acid (NDA) using benzaldehyde dehydrogenase expressed from transgenic microorganisms

The culture medium of the transformed microorganism expressing the benzaldehyde dehydrogenase obtained in Example 4 was recovered by centrifugation, washed with 0.85% saline, and then suspended in the saline again. To prepare a reaction solution in the composition of Table 1 and after reacting for 6 hours in a reaction vessel of 30 ℃ the reaction solution was analyzed by high performance liquid chromatography (HPLC).

The pH of the buffer solution used in the reaction solution was 8.0 and the concentration of dimethyl sulfoxide was 5%. The FNA content in NDA was 9%. The experimental results are shown in Table 2 below, and JM109 (pUC18- xylC ) strain had higher FNA conversion to NDA than wild strains. HPLC analysis conditions were as shown in Table 3 below.

Reaction liquid composition Furtherance usage Remarks 0.1 M KH ₂ PO ₄ / KOH (pH 8.0) 42.5 ml glucose 0.25 g  Final concentration. 0.5% DMSO 0.5 ml NDA solution (100 mg / ml DMSO)  2 ml  DMSO final concentration: FNA content in 5% NDA: 9% Cell suspension 5 ml Sum 50 ml

Conversion rate of FNA to NDA (%) Experiment Response time (hr) % Conversion FNA NDA JM109 (pUC18- xylC ) 0 100.0 100.0 One 85.4 101.3 3 72.8 103.0 6 51.6 104.2 JM109 0 100.0 100.0 One 99.5 100.1 3 99.3 100.0 6 99.3 100.0

HPLC analysis conditions HPLC LC 10-AD VP (Shimatsu) column XTerra ^TM RP18 (4.6 ㅧ 250㎜, Waters) Detector  UV 240nm Column temperature  40 ℃ Flow rate  1 ml / min Injection volume  20 μl Mobile phase Minutes 0.3% phosphoric acid Acetonitrile 0 98 2 5 92 8 28 52 48 30 20 80 35 5 95 36 98 2 49 98 2

When using a microorganism transformed with an expression vector containing the xylC gene derived from Pseudomonas putida prepared according to the present invention, 2,6-dimethyl naphthalene is included in the crude naphthalene dicarboxylic acid produced. It is excellent in converting 2-formyl-6-naphthoic acid into 2,6-naphthalene dicarboxylic acid. Therefore, when the method according to the present invention is used, it is possible to produce high purity 2,6-naphthalene dicarboxylic acid in an economical and environmentally friendly way, which is industrially useful.

1 is a genetic map of a recombinant expression vector pUC18- xylC containing a gene encoding benzaldehyde dehydrogenase derived from Pseudomonas putida of the present invention.

2 is a diagram illustrating a process of synthesizing NDA and NDC by oxidizing DMN.

3 shows a product upon oxidation of DMN.

4 is a diagram showing the step-by-step oxidation mechanism by the upper TOL metabolic pathway enzyme and the upper TOL operon xyl genes.

<110> hyosung <120> Expression vector for benzaldehyde dehydrogenase gene from Pseudomonas putida, bacteria transformed with the same and method for preparing 2,6-Naphthalene dicarboxylic acid with highly purified using the transformants <130> lee03-488 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 1578 <212> DNA (213) Pseudomonas putida <400> 1 agttaaggag gcataattat gcgggaaaca aaagagcagc ctatctggta cgggaaggtg 60 tttagttcta attgggtaga ggcgcgggga ggtgttgcca atgttgtcga tccgtccaat 120 ggagacattc ttggcattac gggtgttgct aacggcgaag atgtcgatgc tgctgtgaac 180 gcagctaaga gagcgcaaaa ggaatgggcc gcaataccat ttagtgaaag agccgccatt 240 gtccgcaagg ctgccgaaaa actaaaggag cgcgagtatg aattcgccga ttggaacgta 300 cgggaatgcg gcgcaattcg tccgaagggc ttatgggagg ccggaattgc gtatgagcaa 360 atgcatcaag ctgcgggtct agcttctttg cctaacggta cattgtttcc atcggcagtt 420 ccagggcgca tgaatctttg tcagcgcgtt ccagttggcg tggtcggcgt aattgcacct 480 tggaatttcc cgttgtttct agcaatgcgt tcggtagcac cagccttagc gttgggtaat 540 gcggtgatct taaagcccga ccttcagact gctgtcaccg ggggggcgct cattgccgaa 600 atcttttccg acgctggcat gccggacggt gttcttcacg ttcttcctgg tggagcggac 660 gtaggagagt caatggttgc gaactccgga attaacatga tttcttttac cgggtccaca 720 caggtgggcc ggttgatcgg agagaaatgc gggagaatgc tgaaaaaggt tgcgcttgaa 780 ctgggtggta ataatgtcca catcgtgttg cctgacgccg atttagaagg ggctgtcagc 840 tgcgctgctt ggggtacgtt tttgcatcag ggccaagtgt gcatggccgc cggacgtcat 900 ttagtacata gggacgttgc tcagcaatat gcagagaaac tggcgctacg tgccaagaac 960 ttagtggtgg gggatccaaa ctcggatcaa gtgcatctcg gcccgcttat caatgagaaa 1020 caggtagttc gcgtccacgc gctcgttgaa tctgcgcaaa gggccggtgc tcaggttttg 1080 gcgggaggta cgtatcaaga tcgctactac caagctaccg taatcatgga tgtgaagccg 1140 gagatggagg ttttcaaatc tgaaattttc ggcccggtgg ctccgatcac tgtatttgac 1200 agtattgaag aggcgattga attggcaaac tgttcggagt atgggttggc cgcatctatc 1260 catactaggg cgttggcgac tggtctagac atcgcaaagc gtctaaatac cggtatggtc 1320 catattaatg accagccaat taactgtgag ccgcatgttc ccttcggagg aatgggtgcc 1380 tcgggtagcg gaggccggtt tggcggacct gcaagtattg aagaatttac tcaatctcaa 1440 tggattagta tggttgagaa gccagctaat tacccatttt gagtcgacaa taataaagta 1500 ggtggatata tggacacgct tcgttattac ctgattcctg ttgttactgc ttgcgggctg 1560 atcggatttt actatggt 1578 <210> 2 <211> 24 <212> DNA (213) Pseudomonas putida <400> 2 gctgcagagg atgcgttcga aatg 24 <210> 3 <211> 24 <212> DNA (213) Pseudomonas putida <400> 3 gatcagcccg caagctgcag caac 24

Claims (8)

Recombinant comprising a benzaldehyde dehydrogenase gene ( xylC ) from Pseudomonas putida having the ability to convert 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid Expression vector. The recombinant expression vector of claim 1, wherein the Pseudomonas putida is Pseudomonas putida mt-2 (ATCC 33015). A transformed microorganism characterized by transforming by introducing the expression vector of claim 1 or 2 into a microorganism. The method of claim 3, wherein the microorganism is E. coli. The transformed microorganism according to claim 3 is incubated at a temperature of 25 to 45 ℃, and benzaldehyde dehydrogenase, characterized in that to induce the expression of benzaldehyde dehydrogenase by adding 0.1 to 2.0 mM IPTG to the culture medium Method of inducing expression of. A transformed microorganism characterized in that benzaldehyde dehydrogenase is highly expressed by the method according to claim 5. In the presence of a buffer solution having a pH of 6.0 to 10.0 and 0 to 20% of dimethyl sulfoxide, the transformed microorganism according to claim 6 is reacted at a temperature of 25 to 45 DEG C using crude naphthalene dimethylcarboxylic acid as a substrate. A process for producing high purity 2,6-naphthalenedicarboxylic acid, characterized by converting 2-formyl-6-naphthoic acid contained in crude naphthalene dimethylcarboxylic acid to 2,6-naphthalenedicarboxylic acid. 8. The process for producing 2,6-naphthalenedicarboxylic acid according to claim 7, wherein the crude naphthalene dimethylcarboxylic acid comprises 0.01 to 10% 2-formyl-6-naphthoic acid.