KR20060078799A - Method for preparing transformants expressing xanthine dehydrogenase and method for purification of 2,6-naphthalene dicarboxylic acid using the transformants - Google Patents

Abstract

The present invention provides a method for preparing a transformant by transforming a host cell with a gene encoding xanthine dehydrogenase derived from Bacillus subtilis and using the transformant obtained by the method. Method for purifying high purity 2,6-naphthalene dicarboxylic acid by removing 2-formyl-6-naphthoic acid in crude naphthalene dicarboxylic acid produced by oxidizing 2,6-dimethylnaphthalene It is about.

Xanthine dehydrogenase, 2-formyl-6-naphthoic acid, 2,6-naphthalene dicarboxylic acid

Description

       METHOD FOR PREPARING TRANSFORMANTS EXPRESSING XANTHINE DEHYDROGENASE AND METHOD FOR PURIFICATION OF 2,6-NAPHTHALENE DICARBOXYLIC ACID USING THE TRANSFORMANTS}             

1 is a reaction scheme illustrating a process of oxidizing 2,6-dimethyl naphthalene (DMN) according to the prior art to obtain naphthalene dicarboxylic acid (NDA) and naphthalene dicarboxylate (NDC),

FIG. 2 shows the products and by-products produced upon oxidation of 2,6-dimethyl naphthalene (DMN);

3 is a diagram showing a gene map and enzymes of Bacillus subtilis involved in purine degradation,

4 is a B. subtilis (Bcillus subtilis) is a gene map of the recombinant expression vector pFOREXT-pucABCDE containing a gene coding for xanthine-di dehydrogenase derived.

The present invention relates to a method for preparing a transformant expressing xanthine dehydrogenase and a method for purifying 2,6-naphthalene dicarboxylic acid using the same, and more specifically, from Bacillus subtilis A method for preparing a transformant expressing xanthine dehydrogenase comprising the step of transforming a host cell with a gene encoding xanthine dehydrogenase of and high purity of 2,6-naphthalene dicarboxyl using the same It relates to a purification method.

2,6-naphthalene dicarboxylic acid is a monomer useful in the preparation of high performance polymeric materials such as polyesters and polyamides. Among the representative high performance polymer materials are polyethylene 2,6-naphthalate (abbreviated as "PEN"), which is now abbreviated as 2,6-naphthalene dicarboxylate (hereinafter referred to as "NDC"). It is synthesize | combined by the polymerization reaction of and ethylene glycol. Since PEN is superior to polyethylene terephthalate (hereinafter, abbreviated as "PET") in strength, heat resistance, gas-barrier, etc., PEN is expected to be used as a large capacity magnetic tape, heat condenser, beverage container, etc. for a long time. have.

2,6-naphthalene dicarboxylic acid (abbreviated as "NDA") is most conveniently an oxygen source for oxidation reactions using molecular oxygen, in particular air, and liquid heavy metal catalysts. To 2,6-dimethyl naphthalene (hereinafter abbreviated as "DMN"). During this oxidation reaction, the methyl substituents present on the naphthalene ring of 2,6-DMN are oxidized to the carboxyl group. It is known to oxidize 2,6-DMN to 2,6-NDA by this liquid phase reaction. For example, US Pat. No. 5,183,933 to Harper et al. Describes a method for oxidizing 2,6-DMN to 2,6-NDA using large amounts of manganese and cobalt oxidation catalyst metals added to an oxidation reaction mixture. It is.

       Currently, 2,6-NDC is produced by oxidizing 2,6-dimethylnaphthalene to produce crude naphthalene dicarboxylic acid ("cNDA": crude naphthalene dicarboxylic acid) as shown in FIG. Doing. Currently, NDC is used as a main raw material when synthesizing PEN, but there are some problems compared to using NDA as a main raw material. Firstly, water is produced during NDA condensation reaction, whereas methanol produces as a by-product in the case of NDC, and there is a danger of explosion. Second, NDA is esterified to obtain pure NDC during NDC manufacturing process, and then NDC is produced through purification process. Therefore, one step process is needed compared to NDA, and thirdly, if you have existing PET production facilities, it is not appropriate to use NDC in terms of using existing facilities. Despite the disadvantages of NDC, the reason why NDC is used instead of NDA in PEN production is that it has not yet produced a purified NDA having the purity necessary for polymerization. Upon oxidation of dimethylnaphthalene, cNDA containing various impurities, such as 2,6-formylnaphthoic acid (2,6-FNA), trimellitic acid, is produced, as shown in FIG. Is difficult to remove. As such, when FNA is present in cNDA, the polymerization reaction is stopped in the middle, which adversely affects the physical properties of the polymer.                         

       Various methods are known for removing FNA present in cNDA. For example, a method of recrystallization for NDA purification, a method of undergoing an oxidation process once more, a method of preparing cNDA using NDC using methanol, followed by hydration to produce NDA, or a method of preparing purified NDA by hydrogenation, etc. This has been proposed. 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, if the purity is increased, the yield is very difficult to apply to actual production.

As described above, the production of 2,6-NDA by the chemical method is a problem that the manufacturing process cost increases rapidly due to environmental pollution, the risk of explosion due to high temperature and high pressure, the size of equipment used and the excessive consumption of energy. Has In addition, in order to use 2,6-naphthalenedicarboxylic acid directly in the polymerization process, it has to maintain a high purity, but it is difficult to reach this purity by the conventional method, so it is subjected to a separate purification process. There is a problem that leads to complexity of the process and a decrease in productivity.

       Therefore, methods using microorganisms have been studied. The present applicant has proposed a method for removing FNA using a novel Bacillus strain in Korean Patent Application No. 2002-0087819, and Korean Patent Application No. 2002-7005344 discloses xylene mono Disclosed is a process for preparing aromatic aldehydes and carboxylic acids using Xylene monooxygenase. However, there is no known method of converting FNA to NDA by xanthine dehydrogenase.

The present invention is to solve the problems of the prior art as described above, an object of the present invention is a transformant expressing xanthine dehydrogenase that can be used in the high purity NDA purification process transformed by the pucABCDE gene It is to provide a manufacturing method.

Another object of the present invention is to provide a method for obtaining high purity NDA by inducing oxidation of FNA in cNDA to NDA using a transformant expressing xanthine dehydrogenase.

One aspect of the present invention for achieving the above object is recombinant expression of a gene ( pucABCDE ) encoding xanthine dehydrogenase derived from Bacillus subtilis HSB-21 (KFCC-11221) Inserting into a vector; And

It relates to a method for producing a transformant expressing xanthine dehydrogenase comprising the step of transforming a host cell with the expression vector.

Another aspect of the present invention for achieving the above object relates to a transformant expressing xanthine dehydrogenase produced by the method.

Another aspect of the present invention for achieving the above object is to react the transformant of claim 4 with crude naphthalene dimethylcarboxylic acid (crude Naphthalene dicarboxylic acid) in a buffer containing an organic solvent to the crude naphthalene dimethylcar A method for purifying 2,6-naphthalenedicarboxylic acid comprising the step of decomposing 2-formyl-6-naphthoic acid in an acid.

Hereinafter, with reference to the accompanying drawings will be described in more detail with respect to the present invention.

When preparing a transformant expressing xanthine dehydrogenase according to the present invention, first, xanthine dehydrogenase derived from Bacillus subtilis HSB-21 (KFCC-11221) A gene encoding puxABCDE (Xanthine dehydrogenase) is obtained and inserted into the recombinant expression vector. The host cell is then transformed with this recombinant expression vector.

3 is a view showing the genetic map and the enzyme of B. subtilis (Bcillus subtilis) which is involved in the purine degradation. Referring to Figure 3, B. subtilis (Bacillus subtilis) HSB-21 pucABCDE present on the puc operon play a key role in the purine degradation present in the chromosomal DNA of (KFCC-11221) is a hypoxanthine (hypoxanthine) It is a gene that encodes xanthine and xanthine dehydrogenase ("XDH"), an enzyme that converts xanthine to uric aicd. Adenosine monophosphate (AMP) is converted to adenosine by the 5'-nucleosidase enzyme, and then adenosine is converted to inosine by adenosine deamimidase. The converted inosine is hydrolyzed by nucleosidase to be decomposed into purine bases hypoxanthine and D-ribose, and then hypoxanthine is converted to uric acid by XDH and xanthine again by XDH. The uric acid thus formed is allantoin, allantoin is allantoic acid, and allantoic acid is converted back to urea and finally decomposed into ammonia (NH ₃ ) and CO ₂ . As shown in FIG. 3, the XDH consists of five subunits of pucABCDE . Here pucC is an enzyme consisting of a FAD binding domain, a pucD molybdenum (Mo) binding domain, and pucE is an iron-sulfur metal binding domain, which requires NAD ⁺ as an electron donor.

The gene encoding xanthine dehydrogenase ( pucABCDE) used in the present invention is derived from Bacillus subtilis HSB-21 (KFCC-11221), has a size of about 5.2 kb and has a sequence Nucleic acid sequence of 1. First, in order to clone a gene ( pucABCDE ) encoding Xanthine dehydrogenase from Bacillus subtilis HSB-21 (KFCC-11221), the genome DNA was used as a template DNA from pucABCDE . Xanthine dehydrogenase by performing polymerase chain reaction (PCR) using the prepared primer 1 (5'-ATGGGGAATTTTCACACGATGTTAGATGCG-3 ': SEQ ID NO: 2) and primer 2 (5'-TTAAAAGCCGGACTCCCGTCTACCCCCGTT-3': SEQ ID NO: 3). A gene encoding can be obtained.

When preparing a transformant expressing xanthine dehydrogenase by the method of the present invention, a recombinant expression vector comprising the pucABCDE gene (SEQ ID NO: 1) is first prepared. The recombinant expression vector into which the gene is inserted can be obtained by operably linking the pucABCDE gene cloned by the above method to a promoter.

4 is a B. subtilis (Bcillus subtilis) is a gene map of the recombinant expression vector pFOREXT-pucABCDE containing a gene coding for xanthine-di dehydrogenase derived. As shown in FIG. 4, the expression vector ForexT-pucABCDE shown in FIG. 4 may be prepared by recombining a DNA fragment of about 5.2 kb represented by the cloned sequence 1 into a Forex-T vector.

Regarding the pucABCDE gene derived from Bacillus subtilis HSB-21 (KFCC-11221), which is represented by SEQ ID NO: 1 of the present invention, not only the DNA sequence shown herein but also mutation through various methods It may also include genes that have new properties by introduction. In other words, in order to change the properties of the gene or to produce a gene that meets the purpose, the DNA sequence is changed through artificial mutation, chemical mutation, or random mutation introduction by an inducer. Genes having can be prepared. In addition, a gene having a new property can be produced by introducing a mutation by a technique using a polymerase chain reaction.

       The vector usable as an expression vector in the present invention is not particularly limited, and examples thereof include known pForexT vectors (TaKaRa), pUC119 (TaKaRa), pBluescript family vectors (Stratagene), pET family vectors (Merck), and the like. As the T7 promoter, T3 promoter, Sp6 promoter and the like can be used.

The process of preparing a transformant using the expression vector prepared in the above step can be carried out through a general method. In one example, a transformant expressing xanthine dehydrogenase by introducing a ForexT-pucABCDE expression vector prepared above into a host cell (eg, E. coli XL1-Blue) using a conventional transformation method. Can be obtained.

Examples of host cells for preparing transformants in the present invention include known MC1061 ( E. coli ), JM109 ( E. coli ), XL1-Blue ( E. coli ), and DH5αF '( E. coli ). For example, heat transformation, electroporation, microinjection, particle bombardment (pressure injection), etc. may be used as the transformation method, but are not limited thereto.

On the other hand, as a method for identifying a transformant expressing pucABCDE according to the culture of the transformant XL1-Blue (ForexT-pucABCDE), when cultured in a solid medium containing xanthine (Xanthine) xanthine dehydrogena The conversion of uric acid by the agent can be applied to the formation of hollow around the colony. Preferably, the transformant XL1-Blue (ForexT-pucABCDE) is inoculated into LB liquid medium containing ampicillin (100 mg / L) and shaken to culture, followed by 5% of Xanthine and empicillin ( 100 μl of the culture solution in LB solid medium containing ampicillin (100 mg / L) was incubated in a 37 ° C. incubator for 24 hours to finally select colonies in which hollows were formed.

In the present invention, each of the transformants were inoculated in LB liquid medium and incubated with stirring at 25-45 ° C., and then the cells were recovered by centrifugation. The recovered cells were suspended in physiological saline and used as enzyme solution for NDA purification. do.                     

When the transformant obtained by the method of the present invention is cultured in a medium containing cNDA, FNA is converted to NDA to remove FNA as an impurity. In the process of conversion from FNA to 2,6-NDA, expression of xanthine dehydrogenase results in the formation of NDA, a compound in which the aldehyde group (-COH) of FNA is converted to a carobox group (-COOH).

In the present invention, in order to remove the FNA which is an impurity contained in the cNDA, the cNDA to be purified is put in an enzyme reaction vessel containing a buffer solution, and the suspension (enzyme solution) of the transformant is added thereto, followed by stirring.

As a buffer solution for the enzyme reaction, sodium carbonate buffer (Na ₂ CO ₃ / NaHCO ₃ ), glycine buffer (Glycine / NaOH), potassium phosphate buffer (KH ₂ PO ₄ / KOH), sodium phosphate buffer (Na ₂ HPO _{4 /} NaH ₂ PO ₄ ), succinic acid buffer (Succinic acid / NaOH), sodium acetate buffer (Sodium acetate / Acetic acid), citric acid buffer (Citric acid / Sodium citrate), sodium pyrophosphate buffer (Na ₄ P ₂ O _{7 /} HCl), boric acid buffer (Boric acid / NaOH), sodium borate buffer (Sodium borate / HCl), etc. can be used, and among them, the use of phosphate buffer is most preferred in terms of enzyme activity And it is preferable that the final pH of a reaction liquid is 6-10.

In addition, an organic solvent may be added to the enzyme reaction solution for the purpose of dissolving cNDA. Examples of preferred organic solvents include dimethylsulfoxide (Dimethylsulfoxide, "DMSO"), and dimethylformamide (N, N-Dimethylformamide, "DMF." "), Dimethylacetamide (N, N-Dimethylacetamide," DMA "), tetrahydrofuran (Tetrahydrofuran," THF "), and the like. Among them, the use of dimethyl sulfoxide is most preferred in terms of enzymatic activity. The amount of the organic solvent added should not exceed 20% of the total reaction solution.

In the present invention, the reaction temperature is preferably 25 ~ 45 ℃, in terms of the reaction rate is most preferably reacted at 30 ℃. The reaction time is preferably about 6 hours to 48 hours.

The transformant XL1-Blue (ForexT-pucABCDE) was inoculated in an LB liquid medium containing 1,000 ppm cDMN and ampicillin (ampicillin, 100 mg / L), shaken and cultured at 37 ° C. for a sufficient time, and then concentrated by HPLC. Analysis shows that the expression of xanthine dehydrogenase ( pucABCDE ) is oxidized to produce 2,6-NDA.

Hereinafter, one or more exemplary embodiments will be described in more detail with reference to the following examples, which are intended to illustrate the present invention in more detail, and are not intended to limit the scope of the present invention.

Example 1: pucABCDE  Cloning of genes

To clone a gene encoding pucABCDE from Bacillus subtilis HSB-21 (KFCC-11221), genome DNA was first isolated (Sambrook et al., Molecular Cloning, A Laboratory). Manual 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Primer 1 (5'-ATGGGGAATTTTCACACGATGTTAGATGCG-3 ': SEQ ID NO: 2) and primer 2 (5'-TTAAAAGCCGGACTCCCGTGTACACCCCCGTT-3') prepared from pucABCDE (GenBank Sequence Database, Z99120) using the isolated genomic DNA as template DNA : Polymerase chain reaction was performed using SEQ ID NO: 3). In the polymerase chain reaction, the first denaturation was performed once at 94 ° C. for 5 minutes, the second denaturation was performed at 94 ° C. for 1 minute, and the annealing was performed at 60 ° C. for 1 minute and 30 seconds, extension. ) Was performed at 72 ° C. for 7 minutes and was repeated 35 times. The fragment obtained in this manner was separated from the DNA fragment of about 5.2kb in size represented by SEQ ID NO: 1 and recombined into a Forex-T vector to prepare an expression vector ForexT-pucABCDE as shown in Figure 4.

Example 2 Cloned Gene Analysis

For sequencing the cloned recombinant vector (ForexT-pucABCDE), the vector was cleaved using various restriction enzymes ( EcoR I, Kpn I, BamH 1, Pst I, Hind III) based on the cleavage map of the two vectors. Each fragment was subcloned into M13mp18 and M13mp19, and they were sequenced using an ABI PRISM BigDye primer cycle-sequencing kit (Perkin-Elmer, USA) using AmpliTaq DNA polymerase. At this time, in order to read both directions of both strands of DNA, partially synthesized nucleotides were made, and the sequence was analyzed and reconfirmed.

Example 3  : Preparation of transformant expressing XDH

ForexT-pucABCDE was introduced into E. coli XL1-Blue 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). Screened in LB plate medium (yeast extract, 5g / L; tryptone, 10g / L; NaCl, 10g / L) with acillin (ampicillin, 100mg / L) and bacto-agar (15g / L) The transformant XL1-Blue ( ForexTpucABCDE ) was prepared. The gene sequence of the cloned DNA fragment was analyzed and compared with the nucleotide sequence registered in GenBank. As a result, it was confirmed that the gene was pucABCDE .

Example 4  : Confirmation of pucABCDE expression

In order to confirm the expression of ForexT-pucABCDE according to the culture of the transformant, transformant XL1-Blue (ForexT-pucABCDE) transformed using ForexT-pucABCDE prepared in Example 1 and a recombinant expression vector as a control Wild lines XL1-Blue not included were cultured under the same conditions to determine the activity of the expressed XDH. As xanthine is converted to uric acid by xanthine dehydrogenase, the expression of pucABCDE was confirmed using hollow formation around colonies. Two types of E. coli were inoculated in 5 ml LB liquid medium containing ampicillin (100 mg / L), respectively, and then shaken for a sufficient time at a temperature of 25 ° C. to 45 ° C., particularly 37 ° C. 100 μl of the culture solution in an LB solid medium containing 5% Xanthine and ampicillin (100 mg / L), incubated in a 37 ° C. incubator for 24 hours, and then hollow formation around colonies. Was observed. As a result, it was confirmed that the hollow was not formed in the control group containing no recombinant expression vector, whereas in the transformant XL1-Blue (ForexT-pucABCDE) transformed using ForexT-pucABCDE, hollow was formed around the colony.

Example 5  : Conversion of FNA to NDA using Expressed XDH

      Transformants XL1-Blue (ForexT-pucABCDE) and wild strain XL1-Blue without a recombinant expression vector as a control were inoculated in 5 mL LB test tubes, and then grown sufficiently at 37 ° C., and then again in 100 mL LB medium. Inoculated to 1% (V / V) and incubated for 16 hours at 37 ℃. The cells were recovered by centrifugation of the culture medium, and then reacted with a reaction solution as shown in Table 1 washed with 0.85% physiological saline for 8 hours in a reaction vessel at a temperature of 25 to 45 ° C., particularly at 30 ° C., followed by high performance liquid chromatography (HPLC) It was. The pH of the buffer was pH 8.0 and the concentration of dimethyl sulfoxide (Dimethyl-sulfoxide) was 5%. The FNA content in NDA was 9%. As shown in Table 2, the transformant XL1-Blue (ForexT-pucABCDE) had a higher FNA conversion ability to NDA than the wild strain. HPLC analysis conditions were as shown in Table 3.                     

Reaction liquid composition Furtherance ratio Remarks 0.1 M KH ₂ PO _{4 /} KOH (pH 8.0) 42.5 ml Glocoose 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 of FNA to NDA (% Contrast Ratio) Experiment Reaction time FNA NDA 1 XL1-Blue ( ForexTpucABCDE ) 0 hr 100.0 100.0 2 hr 74.7 103.0 4 hr 53.5 104.6 8 hr 44.3 106.1 2 XL1-Blue 0 hr 100.0 100.0 2 hr 99.7 100.1 4 hr 99.5 100.0 8 hr 99.6 100.0

HPLC analysis conditions HPLC LC 10-AD VP (SHIMADZU) column ^{XTerra TM RP18 (4.6 × 50 mm} , Waters) Detector  UV 240 nm Column temperature  40 ℃ Flux  1 ml / min. Injection mouth  20 μl Mobile phase Time (min) 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

Example 6  : Conversion of FNA to NDA using Expressed XDH

       Transformant XL1-Blue (ForexT-pucABCDE) and wild strain XL1-Blue without a recombinant expression vector as a control were inoculated in 5 mL LB test tubes, and then shaken for 16 hours at 37 ° C. This was inoculated again with 1% of the culture solution to the medium of the composition shown in Table 4 of 20 ml shaking culture for 48 hours in a 37 ℃ incubator. The culture solution was placed in a round bottom flask, the culture solution was evaporated with a dry evaporator, and the remaining dry powder was dissolved in DMSO, and analyzed by liquid chromatography (HPLC) under the conditions of Example 5 to convert FNA to 2,6-NDA. Was measured. The concentration of dimethyl sulfoxide (dimethyl-sulfoxide) was 5%. The FNA content in NDA was 9%. As a result, the transformant XL1-Blue (ForexT-pucABCDE) had higher FNA conversion ability than NDA.

Culture composition Furtherance content Yeast extract 5 g / L Trypton 10 g / L NaCl 10 g / L 2,6-DMN 1 g / L Empicillin 100 mg / L DMSO 10 mL

Since the transformant obtained by the method of the present invention has an excellent effect of removing 2-formyl-6-naphthoic acid contained in the crude naphthalene dicarboxylic acid, it is highly purified 2,6- by the method of the present invention. Mass production of naphthalene dicarboxylic acid is expected to be realized. In addition, according to the present invention it is possible to overcome problems such as environmental pollution, the risk of explosion, the enlargement of equipment and excessive consumption of energy by applying the chemical method.

<110> HYOSUNG CORPORATION <120> METHOD FOR PREPARING RECOMBINANT MOCROORGANISM AND PURIFICATION          METHOD OF 2,6-NAPHTHALENE DICARBOXYLIC ACIT USING THE          MICROORGANISM <130> 04-TNC-29 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 5188 <212> DNA <213> Bacillus subtilis <400> 1 atggggaatt ttcacacgat gttagatgcg ctcctggaag accaggagga ggctgtactg 60 gcgacaattg ttcaagttga aggctccgcg tatcgaaaag caggcgcctc tatgctcttt 120 aagaaaaaag gcagacggat tggcttgctg agcggcgggt gtgtggaaga agatgtattc 180 caaagaatca gtgcgctggg cgatcagctc acatcaacgc tgattcccta cgacatgcgg 240 tcggaggacg atctttcttg gggcatggga gccgggtgca atggcattat tcatgttcat 300 gcagagcgaa tcactcagga aaaaaggcgg cactatgaga aagtgaggga ctgccttcat 360 tcaggcaaag ctgtcacttc cgttataaag attgaatcct cccattattt atttctgacg 420 gagaacggac attttgggaa ctggccggac gcgcctctgc aagatattca acgtactgtt 480 tcaacgcttc atttaccgca tttcgatcaa accactaaca tgtttattca gcgaattgaa 540 ccgaagccgc gtctcattct gtttggggcg ggaccggata atgtaccgct ggccaatttg 600 gcggctgaca cagggttttc tgttatcgtg acggattggc ggcccgctta ttgtacatcc 660 tctctttttc caaaagcgga tcagctgatc accgcttttc cggaacaaat gcttagcgaa 720 tttcaatttt ttccgcatga tgcagctgtt gtggcaaccc atcattatca gcacgatcaa 780 acgatcatca acttcttatt ttctcaaaac ctccattata ttggactgtt gggttcggca 840 aaccgcacga aacggctgct gagcggaaaa catcctccgt ctcactttta cagtccggtc 900 gggctgaaaa tcggtgcgga aggtcccgag gaaattgcgg taagtgtagt tgcagaaatc 960 attcaaacaa gaaaacgggt ggcggttgta tgagaagccc ctatctgatc ggcgtgtttc 1020 tcgctgccgg caaaagcaga agaatgggac aaaacaagct ggctttgccg ctaaaaggag 1080 aaaacatagg ctcactttcc ttgaaaactg ctctgtcgtc ccgccttgat cacgtgctgg 1140 tcgtcgagcg gacagaacat gcctcccttg agtggatagg agcgccgtat cacgctccgc 1200 cttttcaaaa acgctggagc ctgcacgttt gccaagacgc agaaaagggt caggggcatt 1260 cagtcagcag cggggtgaga aaagcagaaa gcatgggggc ggacggcatt gtcattttgt 1320 tagccgacca gcctcagctt tccgtcgatc atctcaatgc ccttgtggct ctggctcctg 1380 agtcatttgc cgtgtcatcg tttttagggg cgttcacccc gccaatctac ttctcgtcga 1440 catgttttcc ttatgtaaag ggattaaagg gagatgaagg ggctcgcagg ctcctgaaaa 1500 gcggacagct tggagcagga gcggttttgg aggcaaagga tagcggtgag ttggacgata 1560 ttgatacacc ccgaggaata tgacatggta aggagggcga tgtcttgaac ggccaagtga 1620 caaaagcaag gatgaatatt caattatgga gaccggcagc gcttgatgag gcctattcgc 1680 ttttagagaa gcttgctccg gatgtgtgtg ccgcatcggg aagcacgctc cttcagcttc 1740 agtgggacaa aggaactcta ccgaaacagc accttgtcag ccttgaaggg attgacgaaa 1800 tgcgagggat cagcaccagt gacacccacg tgtcgatcgg ggggctcaca tcactgaatg 1860 aatgcagaaa gaaccctttg attaagagag cgcttagctg tttcagtgac gctgcttccg 1920 ctgtcgctgc accggggatt cgcagccgtg ccacgattgg cggcaacata gcgagtaaaa 1980 tcggtgattt cattccgctt ctgcttgtgc ttggtgctga gcttatcgta tatcaaaagg 2040 agctaatcag gctgccgctt ggcgcttggc tcagtgagga agactttaga acggctatcg 2100 tcacgcgtgt catcatcccg cgggcggagg gagagcgtgt attttatcac aagcttggaa 2160 ggcgccaggc ttttaccgga gcggctgctg ttgcagctgg acgttttttg aaagacggca 2220 gcattcgcct cgccgctggc catgccgata tcactcctag gagattattg gacagcgaag 2280 cgaaatggat ggcaccaggg tgggacccgc atgagctgta caaaacactt atccatgagc 2340 tgccgttttc ctcagatgtt tttatgtcag cggcatacag aaaaaaagcc gctgccaacg 2400 tcatcatggc agagctgatg gctgagggag gggaataaat gatcatcaac aaaccgtcaa 2460 gagtcaggcc ggatggaagg ggtaaggtca caggggaatt gaaatatatg accgatctca 2520 gctttccagg aatgctgtat ggcaaggtgc tgagaagtgc ttatcctcat gcagagattg 2580 tgtcggtttg caccataaaa gctgagaaaa tggaaggcgt gcaggccgtc gtcacacata 2640 aagacgttcc cggcttaaac cgatttggca ttgtcatccc tgatcagccg gttttatgtg 2700 aggacagggt gcggtatgtc ggggatgcga tcgccgcagt tgctgcggaa acggaagaaa 2760 tcgcggaagc ggcgctggag ctgattcaag ttgagtataa agagctggaa gtcatggatt 2820 caccagaaaa agcgcttcgg ccgaatgcgc aaagactgca tgaggacggg aatatcctgc 2880 accgcgcatt tttttcaaac ggtgatgtgg aagaagggtt tcaagcatct gacacagtct 2940 ttgaagaaac ctatgagctg ccgcggcaga tgcatacgta tatggaaacg gagggaggtg 3000 tagccgttcc ggaagacgac gggggtttta cgatgtatgc aggaacccag cacggctata 3060 aagaccgttt ccagcttgcc cgtatttttg atattcctga agagaagatc agaattgtgt 3120 caagtccgat gggcggctcg ttcggaggga aggatgagct gaatattcag ccgtatgctg 3180 cgcttttggc cctgaaaagc ggacgccctg tcaaaattca tcagacacga aaggaatctg 3240 tacgttctgg cattaagcgc catccgatga agattacgat aaaaacgggt gcagatcatt 3300 cgggaaatct attggcgcac gacgtgaaaa ttgtggcgga cacaggcgct tacgctacgc 3360 tcggaccggc tgttcttgat ttttctgtcg agcatgccgc ggggccgtac cgtattccga 3420 atatccggac tgaaggaata tcagtattta cgaacaacgg ggtggcgggg gaatttcggg 3480 gctttggcgg caaccaaatc acatttgcac tcgaaaccca tctcgatcgt ctcagcggca 3540 tgcttggcat cgacccgctc gagctgagaa gaaaaaatat cagaaaaccg catgacttgg 3600 ggccgcttga gcatcgaatt gcaccaactg acggagcggc acaggtgttg aatgccatat 3660 ctaaatctcc cattcttaaa aaaacaagcc ggaactgcgg gtatctgcaa agaggcacgg 3720 gagcggcaat tacaatgcac ggcggcgggc tggggtttgg ccgaatggat gcggcaggag 3780 gccgtttgtc tctttcgagt gaaggcaaaa tcactgcttc gtttggattt gaagaatgcg 3840 gacaggggat tctagcagcg attgaacaga ttgtcatgga ggagctgggt tgtgccgcag 3900 aggatatttc cattgttatt ggggataccg caaaagtgcc gaagtcaggc tcctccacag 3960 catcccgcgg cacaagcatg gtgtggcacg cgatccagcg tttgaagaag ccgtttctag 4020 ctcagttgaa aaaacgggcg gcagaatgga gcggctgttc agcggaaaat cttattcccg 4080 gcgctgcagg cttgcgtgac aaaaacacaa aggcgctcgt ggtgacgtat aaggagctgg 4140 ctgaaaaagg ccctttggca gaggaaacgg cctttgactt tccaacaacg cctgatcctg 4200 tggtgggcgg ccatttcctc tactcatttg gagcggctgc cgttgaggtg gaggtagatc 4260 tgttaacagg cgatgtcaaa ttgatagatt gtgagcacgc tattgcggca ggtccggttg 4320 tcagtccgca gggatatcga ggtcaaattg aaggcggcgc tgccatggca ctcggctaca 4380 cactgatgga ggaagcgaaa atgacggatg gccgttatgc tgcggaaaat ctcgatcact 4440 atttgattcc cggaatcaaa gatgttcctg acatgaagct gattgcaata gaagacttaa 4500 tgaagggaga cgtatatgga ccgcgcggtg ttggtgaaat cggaacaatc gccatcactc 4560 cggcgattgt aaaggcagta catgatgcgg tcggatgctg gataaacaag ctgccaattt 4620 caagagaaga gttgcttgaa gcgatcgaca gaaaggggct gaagcaatgg acataaaaga 4680 ggccgggcca tttcctgtaa aaaaggaaca gttccggatg accgtgaatg gtcaggcgtg 4740 ggaggttgct gccgttccta cgacacatct gagtgacctg cttagaaagg aatttcagct 4800 gaccgggaca aaggtgtcct gcggaatcgg ccgctgcgga gcctgctcta ttttaatcga 4860 cggaaaactg gccaatgcgt gcatgacgat ggcctatcaa gcagacggcc attccatcac 4920 aaccatcgag gggcttcaaa aagaagagct ggatatgtgt caaaccgctt tcctggaaga 4980 aggcggcttc caatgcggct actgtacgcc gggaatgatc attgcgctta aagcattgtt 5040 tcgggaaacc cctcaacctt ctgacaaaga tatagaagaa gggctggcgg ggaatttgtg 5100 ccggtgtacc gggtatggcg ggattatgcg gtcagcttgc aggattagaa gagagttgaa 5160 cgggggtaga cgggagtccg gcttttaa 5188 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> PRIMER <400> 2 atggggaatt ttcacacgat gttagatgcg 30 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> PRIMER <400> 3 ttaaaagccg gactcccgtc tacccccgtt 30  

Claims (9)

Preparing a recombinant expression vector comprising the nucleic acid sequence of SEQ ID NO: 1; And Method for producing a transformant expressing xanthine dehydrogenase comprising the step of transforming the host cell with the recombinant expression vector.        The method for preparing a transformant expressing xanthine dehydrogenase according to claim 1, wherein the recombinant expression vector is a vector selected from the group consisting of Forex-T, pUC119, pBluscript, and pET. The method of claim 1, wherein the host cell is characterized in that is selected from the group consisting of MC1061 ( E. coli ), JM109 ( E. coli ), XL1-Blue ( E. coli ), and DH5αF '( E. coli ). A method for producing a transformant expressing xanthine dehydrogenase. A transformant prepared by the method of claim 1. The transformant of claim 4 is reacted with crude naphthalene dimethylcarboxylic acid in a buffer containing an organic solvent to form 2-formyl-6-naphthoic acid (2- in the crude naphthalene dimethylcarboxylic acid. Method for purifying 2,6-naphthalenedicarboxylic acid comprising the step of decomposing Formyl-6-naphthoic acid). The method according to claim 5, wherein the organic solvent is one selected from the group consisting of dimethyl sulfoxide, dimethylformamide, dimethylacetamide and tetrahydrofuran and the concentration is 0 to 20%. The method according to claim 5, wherein the organic solvent is one selected from the group consisting of dimethyl sulfoxide, dimethylformamide, dimethylacetamide and tetrahydrofuran and the concentration is 0 to 20%. The method of claim 5, wherein the reaction is carried out at a temperature of 25 ~ 45 ℃, 6 hours to 48 hours. The method of claim 5, wherein the buffer solution is sodium carbonate buffer (Na ₂ CO ₃ / NaHCO ₃ ), glycine buffer (Glycine / NaOH), potassium phosphate buffer (KH ₂ PO ₄ / KOH), sodium phosphate buffer ( Na ₂ HPO ₄ / NaH ₂ PO ₄ ), succinic acid buffer (Succinic acid / NaOH), sodium acetate buffer (Sodium acetate / Acetic acid), citric acid buffer (Citric acid / Sodium citrate), sodium pyrophosphate buffer ( Na ₄ P ₂ O ₇ / HCl), boric acid buffer (Boric acid / NaOH) and sodium borate buffer (Sodium borate / HCl) is one selected from the group consisting of, the pH of the buffer solution is 6.0 to 10.0 How to feature.

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