KR20080004041A - Purification method of crude naphthalene dicarboxylic acid using recombinated microorganism and 2,6-naphthalene dicarboxylic acid in crystalline form obtained by using the same - Google Patents

Abstract

A purification method of crude naphthalene dicarboxylic acid is provided to improve purification yield, purity and economic efficiency by reacting the crude naphthalene dicarboxylic acid with a recombinant microorganism under mild condition, so that high purity crystalline form of 2,6-naphthalene dicarboxylic acid is mass produced inexpensively in an environment-friendly manner. A purification method of crude naphthalene dicarboxylic acid comprises the steps of: (a) reacting the crude naphthalene dicarboxylic acid with a recombinant microorganism, which is obtained by transforming with a recombinant expression vector containing benzaldehyde dehydrogenase gene xylC of SEQ ID NO:1 derived from Sphingomonas aromaticivorans KCTC 2888 or benzaldehyde dehydrogenase gene xylC of SEQ ID NO:4 derived from Pseudomonas putida ATCC 33015, to remove 2-formyl-6-naphthoic acid; (b) regulating pH of the reaction solution obtained with acidic solution to crystallize the crude naphthalene dicarboxylic acid; (c) washing the crystalline crude naphthalene dicarboxylic acid to remove impurities; and (d) drying the washed product to obtain pure crystalline form of 2,6-naphthalene dicarboxylic acid.

Description

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

1 is a genetic map of a recombinant expression vector pET20- xylC comprising a gene xylC encoding a benzaldehyde dehydrogenase derived from sphingmonas aromatiborboranth of the present invention ,

2 is a genetic map of a recombinant expression vector pUC18- xylC comprising a gene xylC encoding benzaldehyde dehydrogenase derived from Pseudomonas putida of the present invention,

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

4 is a photomicrograph of crystallized cNDA according to Example 1 obtained by adding a sulfuric acid solution in Experimental Example 3 of the present invention after the reaction temperature is 80 ℃,

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

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

A: microbial purification reactor B: microbial removal device

C: Crystallization Reactor D: Preheater

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

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

I: Powder Separator J: Drying Machine

The present invention relates to a method for purifying crude naphthalene dicarboxylic acid using a recombinant microorganism and 2,6-naphthalene dicarboxylic acid in a crystalline state obtained by the above method, and more particularly, 2-formyl-6-naph Recombinant microorganisms having the ability to convert tomic acid into 2,6-naphthalene dicarboxylic acid were reacted with crude naphthalene dicarboxylic acid to remove 2-formyl-6-naphthoic acid, an impurity contained in the crude naphthalene dicarboxylic acid. Next, an acidic solution was added thereto under certain conditions, and then the crude naphthalene dicarboxylic acid was crystallized while stirring, washed to remove other impurities, and then the washings were dried to obtain pure crystals 2,6. It relates to a method for purifying crude naphthalene dicarboxylic acid, characterized by obtaining naphthalene dicarboxylic acid.

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

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

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

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

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

Other U.S. Patent No. 6,255,525 discloses that contains the aromatic carboxylic acid impurities (aromatic carboxylic acid) and a mixture solution of water 277 to ^{316 ℃, 77-121 kg / cm 2} , the hydrogenation metal component in the presence of hydrogen gas (hydrogenation metal a high purity aromatic carboxylic acid by contacting with a carbon catalyst without a component) and then cooling the mixed solution to crystallize the aromatic carboxylic acid, and recovering the crystallized aromatic carboxylic acid from the cooled mixed solution. The method is disclosed.

Also in connection with the crystallization of NDA, U.S. Pat. (hydroxide) or alkali metal carbonate) and neutralized with acid at 170-240 ° C. to recover NDA crystals, or the polyester material was dissolved with NaOH or KOH at 170-240 ° C. and then neutralized with acetic acid to NDA. Discloses how to recover the decision,

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

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

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

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

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

More specifically, the present invention

(a) Recombinant expression comprising a benzaldehyde dehydrogenase gene xylC from Sphingomonas aromaticivorans KCTC 2888 represented by SEQ ID NO: 1 or a gene having at least 90% homology with the gene A microorganism obtained by transformation with a vector and a benzaldehyde dehydrogenase gene xylC derived from Pseudomonas putida ATCC 33015 represented by SEQ ID NO: 4 or a gene having at least 90% homology with the gene 2-formyl in the crude naphthalene dicarboxylic acid by reacting at least one recombinant microorganism selected from the group consisting of microorganisms obtained by transformation with a recombinant expression vector comprising crude naphthalene dimethylcarboxylic acid. Removing 6-naphthoic acid;

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

(c) washing the crystallized crude naphthalene dicarboxylic acid to remove impurities contained therein; And

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

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

(Iii) First step: purification process using recombinant microorganisms

First, a recombinant expression vector comprising a benzaldehyde dehydrogenase gene xylC derived from Sphingomonas aromaticivorans KCTC 2888 represented by SEQ ID NO: 1 or a gene having a homology of 90% or more with the gene. A benzaldehyde dehydrogenase gene xylC derived from Pseudomonas putida ATCC 33015 or a gene having at least 90% homology with the gene. 2-formyl-6 in the crude naphthalene dicarboxylic acid by reacting at least one recombinant microorganism selected from the group consisting of microorganisms obtained by transformation with a recombinant expression vector to crude naphthalene dimethylcarboxylic acid -Naphthoic acid removed by conversion to 2,6-naphthalene dicarboxylic acid .

At this time, the "gene having a homology of 90% or more" is a gene having a homology of 90% or more at the base sequence or amino acid sequence level with the benzaldehyde dehydrogenase gene used in the present invention, the base sequence of the gene Or a gene having an amino acid sequence portion within 10% but exhibiting the same function as that of the benzaldehyde dehydrogenase. Such different sequence portions include all cases that can be modified by those skilled in the art to which the present invention pertains without significantly affecting the intrinsic function of benzaldehyde dehydrogenase.

Specifically, the step 1) inoculating the recombinant microorganisms in a liquid medium to induce the expression of benzaldehyde dehydrogenase, and then the cells recovered by centrifugation are suspended in physiological saline or distilled water to prepare the cells for reaction. ; 2) mixing the crude naphthalene dicarboxylic acid as a substrate with a buffer solution and then adding an alkaline solution to adjust the pH of the mixed solution to form a purification reaction solution; And 3) reacting the cells of the recombinant microorganism prepared in step 1) with the reaction solution prepared in step 2) to convert 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid to 2,6- The step of increasing the purity of naphthalene dicarboxylic acid.

Recombinant microorganisms used in the present invention, Sphingomonas aromatiboborance ( Sphingomonas) represented by SEQ ID NO: benzaldehyde dehydrogenase gene derived from aromaticivorans KCTC 2888) or gene having more than 90% homology thereto, or benzaldehyde dehydrogenase derived from Pseudomonas putida ATCC 33015 (SEQ ID NO: 4). The first gene or a gene having 90% or more homology thereto is cloned according to a commonly known general method, introduced into an expression vector to prepare a recombinant expression vector, and then introduced into a microorganism according to a conventionally known method. Can be obtained by switching.

Specifically, it can be obtained by the following method:

That is, in order to clone each of the benzaldehyde dehydrogenase genes ( xylC ), primers prepared from the respective genes are used by using plasmid DNA or genomic DNA isolated from each microorganism as a template. PCR is performed to obtain a gene encoding the benzaldehyde dehydrogenase. In this case, in some cases, according to a conventional method known in the art, it is also possible to obtain a gene in which some nucleotide sequences are modified.

Subsequently, the cloned xylC gene is operably linked to the promoter of the expression vector in a conventional manner to prepare a recombinant expression vector.

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

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

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

In this regard, Figure 1 shows a genetic map of the recombinant expression vector pET20- xylC comprising the gene xylC encoding a benzaldehyde dehydrogenase derived from sphingmonas aromatisiboranth used in one embodiment of the present invention, 2 shows a genetic map of the recombinant expression vector pUC18- xylC comprising the gene xylC encoding benzaldehyde dehydrogenase derived from Pseudomonas putida used in another embodiment of the present invention.

XylC into this recombinant expression vector Cloning of the gene can be confirmed through restriction enzyme digestion and sequencing.

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

Meanwhile, in order to convert 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid using a recombinant microorganism transformed with an expression vector of benzaldehyde dehydrogenase in the present invention, benzaldehyde in the recombinant microorganism. It is preferable that the dehydrogenase is highly expressed. As a specific example, pUC 18 used in the present invention is expressed under the control of the lock promoter, and the pET-20b (+) vector expresses the gene under the control of the T7 promoter. Propyl-β-D-thiogalactopyranoside (IPTG) can be used to induce the expression of benzaldehyde dehydrogenase.

Specifically, the recombinant microorganism is sufficiently incubated at a normal culture temperature of 25 to 45 ℃, preferably 37 ℃, it is again 1% (v / v) in 100 ml of liquid medium (eg LB medium) After the inoculation to the OD ₆₀₀ value of 0.4 to 0.5 when the IPTG concentration is added to 0.1 to 2.0 mM, preferably 0.5 mM to induce the expression of the benzaldehyde dehydrogenase and then again at 37 ℃ Incubate. Culture of such recombinant microorganisms and expression of proteins are not limited to the above methods, and any method known to those skilled in the art to which the present invention pertains may be used without limitation.

Since the recombinant microorganism of the present invention is capable of high expression of benzaldehyde dehydrogenase as described above, high concentration culture is easy and economically advantageous.

The recombinant microorganism with high expression of the protein is recovered as a cell only by centrifugation from the culture medium and then suspended in physiological saline or distilled water and used as a reaction cell for purifying crude naphthalene carboxylic acid.

The buffer solution is not particularly limited, but water, sodium carbonate buffer solution (Na ₂ O ₃ / NaHCO ₃ ), glycine buffer solution (Glycine / NaOH), potassium phosphate buffer solution (KH ₂ PO ₄ / KOH), sodium phosphate Buffer solution (Na ₂ HPO ₄ / NaH ₂ PO ₄ ), Succinic acid buffer (Succinic acid / NaOH), Sodium acetate / Acetic acid, Citric acid buffer (Citric acid / Sodium citrate), Sodium pyrophosphate A buffer solution (Na ₄ P ₂ O ₇ / HCl), boric acid buffer (Boric acid / NaOH), sodium borate buffer (Sodium borate / HCl) and the like can be used. Preferably, potassium phosphate buffer (KH ₂ PO ₄ -KOH) or boric acid buffer (Boric acid-NaOH) having a buffer range of pH 6.0 to 9.0 may be used. At this time, the concentration of the buffer solution is preferably used at a concentration of 0.01 to 100 mM.

The alkali solution is not necessarily limited, but NaOH or KOH is preferably used, and the pH of the mixed solution is preferably adjusted to 6 to 10, further 8 to 10.

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

The reaction temperature and the reaction time in the step 3) is preferably 25 to 70 ℃, 1 minute to 1 hour, more preferably 40 to 60 ℃, 10 minutes to 40 minutes to react. In particular, when the reaction temperature is lower than 25 ℃ or higher than 70 ℃, the reactivity is significantly reduced and unsuitable.

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

Specifically, in the present invention, the concentration of cNDA in the purification reaction solution is preferably 0.001 to 20%, and the FNA content in cNDA is preferably 0.001 to 10%, preferably 9%.

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

(Ii) second step: crystallization process

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

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

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

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

The pH range is preferably adjusted to 1 to 4 in terms of recovery rate, and as the pH is lower, the recovery rate tends to increase from about 94% to 99.9%.

In addition, the crystallization temperature is about 4 to 150 ℃, preferably 30 to 130 ℃, more preferably 50 to 120 ℃ suitable for carrying out the crystallization reaction, the reaction time is about 1 minute to 10 hours, preferably 2 Minutes to 5 hours, more preferably 10 minutes to 1 hour range is preferable in terms of the continuous process. Most preferred crystallization temperature and reaction time is 80 ° C., 20 minutes to 30 minutes. If the crystallization time is too short, the degree of crystallinity is low, aggregation may occur between individual crystals during the polymerization reaction, and excessively long crystallization time may cause unnecessary energy loss.

As a suitable stirring speed for performing crystallization, it is preferable to carry out at 0 to 1000 rpm, preferably 0 to 400 rpm, more preferably 50 to 150 rpm.

(Iii) step 3: cleaning process

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

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

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

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

(Iii) 4th step: drying process

Finally, the NDA crystal from which impurities are removed through the washing process is dried at a constant temperature to obtain final pure NDA crystal. At this time, the drying treatment is preferably carried out at 30 to 200 ℃, the drying method can be used without limitation the conventional drying method known in the art to which the present invention belongs.

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

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

As a method for removing the recombinant microorganism, a conventionally known method may be used without limitation, and specifically, may be removed using a microfilter system, a continuous centrifuge or a decanter. In this case, the micro filter system has a pore size of 0.1 to 0.5 μm, and a filter made of a material such as ceramic, stainless, polypropylene, or PET may be used.

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

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

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

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

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

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

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

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

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

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

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

Example 1

Step 1: Preparation of Recombinant Microorganisms

(1) Cloning of the xylC Gene

In order to clone the xylC gene encoding benzaldehyde dehydrogenase derived from Sphingomonas aromaticivorans KCTC 2888, plasmid DNA (pNL1) containing the xylC gene sequence was first isolated (see Sambrook). et al ., Molecular Cloning, A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Primer 1, 5'-GGAGAATTCATATGGCTACGCAGT-3 '(SEQ ID NO: 2) prepared using the isolated plasmid DNA (pNL1) as a template DNA, based on the DNA base sequence of the xylC gene (GenBank Sequence Database, NC002033). Polymerase chain reaction (PCR) was performed using primers 2, 5'-GTCTTGCAGTGAGCTCGTTTCTCC-3 '(SEQ ID NO: 3). In the polymerase chain reaction, the first denaturation step was performed once at 94 ° C for 5 minutes, the second denaturation step 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 represented by SEQ ID NO: 1 having a size of about 1.5 kbp were separated from the fragments obtained in this manner, and were digested with restriction enzymes NdeI and SalI. This was cloned into the plasmid vector pET-20b (+) digested with the same restriction enzyme to prepare a recombinant expression vector pET20- xylC as shown in FIG. 1.

(2) Cloned Gene Analysis

For sequencing of the cloned gene of the recombinant vector (pET20- xylC ) prepared in (1), the recombinant vector was cleaved using various restriction enzymes based on a sequence map of two vectors M13mp18 and M13mp19, respectively. The fragments of were subcloned into M13mp18 and M13mp19, and they were sequenced using ABI PRISM BigDye primer cycle-sequencing kit (Perkin-Elmer, USA) using AmpliTaq DNA polymerase. At this time, in order to read both directions of both strands of DNA, a partially synthesized nucleotide was made, and the nucleotide sequence of the cloned DNA fragment gene was analyzed and the nucleotide sequence registered in GenBank (GenBank Accession Number, AF073917 or NC002030). As a result, the xylC gene was cloned.

(3) Preparation of transformant

E. coli XL1-Blue was transformed with a pET20- 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 Strains grown in NaCl, 10g / L) were selected to prepare transformed Escherichia coli XL1-Blue (pET20- xylC ).

(4) expression of xylC gene

In order to express the xylC gene in the transformed microorganism, the E. coli obtained in (3) was inoculated in an LB test tube, then grown sufficiently at 37 ° C. and inoculated to 100% LB medium to 1% (v / v) again. When the OD ₆₀₀ value reached 0.4 to 0.5, IPTG concentration was added to 0.5 mM to induce the expression of xylC , and then cultured again at 37 ° C.

Second Step: Purification of cNDA Using Recombinant Microorganisms

Centrifugation of the culture medium of the benzaldehyde dehydrogenase-expressing recombinant microorganisms obtained in the first step to recover the cells, washed with 0.85% saline, and then suspended in 5 L of 0.85% saline again to the reaction cells Ready.

80 L of 50 mM potassium phosphate buffer solution was injected into the microbial purification reactor (A), 10 kg of cNDA was added thereto, and KOH was added with stirring to adjust the pH of the reaction solution to 8.0, and then water was added again. 95 L of a purification reaction solution (cNDA concentration: 10%, FNA content in cNDA: 0.58%) was prepared.

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

Step 3: Crystallization of Purified cNDA

100 L of the cNDA purification solution obtained in the second step was transferred to a crystallization reactor (C) equipped with a stirring device, and the sulfuric acid solution was added thereto to adjust the pH to 3.0, and then the stirring speed was 50 rpm and the temperature was 80 The crystallization reaction proceeded for 25 minutes while maintaining at ℃.

After the crystallization was completed, the crystals were analyzed using a microscope and a particle size analyzer. As a result, the crystallization proceeded well, and it was confirmed that entanglement between the individual crystals did not occur. In addition, the average crystal size of crystallized cNDA was analyzed to be about 162.5 ㎛. A micrograph of the crystallized cNDA is shown in FIG. 3.

Step 4: washing and drying the crystallized cNDA

The cNDA slurry solution crystallized in the third step was heated to 220 ° C. or higher through a preheater (D), and then charged into a filtration and washing apparatus (F) for 30 minutes at a pressure of 28 kg / cm ² and a temperature of 225 ° C. After stirring, the mixture was filtered to remove water with a high pressure filtrate recovery device (G). After adding 225 ° C. and 100 L of water from the solvent heating supply device (E), the mixture was stirred for 30 minutes under the same conditions and then filtered, and the process of removing water with the high-pressure filtrate recovery device (G) was repeated twice. It was. Then, 225 ° C. and 100 L of water were added from the solvent heating supply device (E) again, followed by stirring for 30 minutes or more to disperse evenly, and then transferred to a high pressure sludge recovery device (H) to depressurize to atmospheric pressure, and a powder separation device. Water was removed using a decanter (I), followed by drying at 120 ° C. in a drying apparatus (J) to obtain NDA in pure crystalline state.

[ Example  2]

Between the second step and the third step of Example 1, except that the cNDA purification solution obtained in the second step was used for the crystallization reaction after removing the recombinant microorganism using a polypropylene filter of 0.2 ㎛, The same procedure as in Example 1 was carried out to obtain NDA in pure crystalline state.

Example 3

A recombinant microorganism of the present invention is shown in FIG. 2 comprising a benzaldehyde dehydrogenase gene ( Pseudomonas putida ATCC 33015) derived from Pseudomonas putida ATCC 33015 represented by SEQ ID NO: 4 Except for using the microorganism JM109 (pUC18- xylC ) obtained by transforming with the recombinant expression vector pUC18- xylC was carried out in the same manner as in Example 1 to obtain a pure crystalline NDA. At this time, the detailed manufacturing method of the recombinant microorganism JM109 (pUC18- xylC ) is disclosed in Korean Patent Publication No. 2005-71188.

Example 4

Except for using JM109 (pUC18- xylC ) as a recombinant microorganism of the present invention was carried out in the same manner as in Example 2 to obtain a pure crystalline NDA.

Experimental Example  1: Component Analysis

The components were analyzed for the first cNDA used in Examples 1 and 3, the cNDA purification solution obtained after the second purification step, and the pure crystalline NDA obtained after the fourth step washing and drying step. The results are shown in Tables 1 and 2 below.

Component Analysis Data of Example 1 cNDA After purification After washing, drying NA 0.037% 0.025% - MNA 0.112% 0.075% - FNA 0.580% - - TMLA 0.044% 0.028% - Etc 0.160% 0.124% 0.006% NDA 99.067% 99.748% 99.994%

Component Analysis Data of Example 3 cNDA After purification After washing, drying NA 0.037% 0.021% - MNA 0.112% 0.066% - FNA 0.580% - - TMLA 0.044% 0.025% - Etc 0.160% 0.120% 0.003% NDA 99.067% 99.768% 99.997%

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

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

Experimental Example 2: Crystallization Reaction According to Acid Type and Stirring Rate

100 ml of each cNDA purification solution obtained in the second step of Example 1 and Example 3 were placed in a reactor equipped with a stirring device, and sulfuric acid, hydrochloric acid, glacial acetic acid, and nitric acid solution were added thereto so that the pH was 3.0. After the adjustment, the stirring speed was maintained at 0, 50, 100, 200, 400, 800, 1000 rpm. The temperature was 80 ° C., and the crystallization reaction was performed for 25 minutes under the above conditions.

After the crystallization was completed, the crystals were analyzed using a microscope and a particle size analyzer. As a result, all crystallizations were confirmed, and it was confirmed that entanglement between individual crystals did not occur. In addition, the average size of cNDA crystals obtained under each of the above conditions is shown in Tables 3 and 4 below. As can be seen from the results of Tables 3 and 4, among the four acid solutions, sulfuric acid and hydrochloric acid showed the best results for the crystallization reaction, and the preferable stirring speed was 0 to 400 rpm, and more preferably 50 It was determined to be about 150 rpm.

Crystal size according to the acid type and stirring speed of Example 1 mountain Sulfuric acid Stirring speed 0 50 100 200 400 800 1000 Average Crystal Size (㎛) 116.5 135.8 125.8 118.7 110.6 42.7 25.1 mountain Hydrochloric acid Stirring speed 0 50 100 200 400 800 1000 Average Crystal Size (㎛) 117.2 135.4 126.2 119.4 99.8 40.3 20.0 mountain Glacial acetic acid Stirring speed 0 50 100 200 400 800 1000 Average Crystal Size (㎛) 73.4 91.2 81.5 74.3 56.6 15.1 4.9 mountain nitric acid Stirring speed 0 50 100 200 400 800 1000 Average Crystal Size (㎛) 92.1 110.8 99.1 94.7 80.6 23.1 17.3

Crystal size according to the acid type and stirring speed of Example 3 mountain Sulfuric acid Stirring speed 0 50 100 200 400 800 1000 Average Crystal Size (㎛) 118.5 136.8 127.7 120.2 111.8 45.7 28.1 mountain Hydrochloric acid Stirring speed 0 50 100 200 400 800 1000 Average Crystal Size (㎛) 121.1 137.2 129.5 120.3 101.7 48.5 30.0 mountain Glacial acetic acid Stirring speed 0 50 100 200 400 800 1000 Average Crystal Size (㎛) 76.4 99.7 86.5 79.3 61.7 32.1 10.9 mountain nitric acid Stirring speed 0 50 100 200 400 800 1000 Average Crystal Size (㎛) 92.1 119.7 113.3 95.7 81.1 25.1 18.6

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

The stirring rate was 50 rpm, and the reaction temperature was 4, 15, 50, 80, 120, 150 ℃ except that the experiment was carried out in the same manner as in Experiment 2.

After crystallization was completed, the crystals were analyzed using a microscope and a particle size analyzer. As a result, all crystallizations were confirmed, and it was confirmed that entanglement between individual crystals did not occur. The micrograph of the cNDA crystal | crystallization which concerns on Example 1 obtained by adding the sulfuric acid solution thereafter by making reaction temperature 80 degreeC is shown in FIG. In addition, the average size of cNDA crystals obtained under each of the above conditions is shown in Tables 5 and 6 below. As can be seen from the results of Tables 5 and 6, sulfuric acid and hydrochloric acid showed the best results for the crystallization reaction among the four acid solutions, and the preferred reaction temperature was 50 to 120 ° C., and the most preferable reaction temperature was It was judged that it was 80 degreeC.

Crystal size according to the acid type and crystallization temperature of Example 1 mountain Sulfuric acid Temperature 4 15 50 80 120 150 Average Crystal Size (㎛) 110.2 117.5 136.8 162.4 149.6 131.1 mountain Hydrochloric acid Temperature 4 15 50 80 120 150 Average Crystal Size (㎛) 110.3 115.0 139.3 159.7 145.3 132.7 mountain Glacial acetic acid Temperature 4 15 50 80 120 150 Average Crystal Size (㎛) 47.3 62.5 75.6 88.9 68.3 50.1 mountain nitric acid Temperature 4 15 50 80 120 150 Average Crystal Size (㎛) 65.9 90.3 114.3 140.5 113.9 97.2

Crystal size according to acid type and crystallization temperature of Example 3 mountain Sulfuric acid Temperature 4 15 50 80 120 150 Average Crystal Size (㎛) 112.4 121.9 149.8 166.1 150.8 135.1 mountain Hydrochloric acid Temperature 4 15 50 80 120 150 Average Crystal Size (㎛) 115.3 123.4 148.5 164.5 151.3 136.1 mountain Glacial acetic acid Temperature 4 15 50 80 120 150 Average Crystal Size (㎛) 49.1 65.5 80.6 92.7 72.3 52.3 mountain nitric acid Temperature 4 15 50 80 120 150 Average Crystal Size (㎛) 67.7 93.8 125.3 145.9 121.9 89.2

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

Experiment was carried out in the same manner as in Experiment 2 except that the stirring speed was 50 rpm, the reaction temperature was 80 ℃, the pH range was adjusted to 1, 2, 3, 4, 5, 6. That is, after performing the crystallization reaction for 25 minutes, a certain amount was taken to compare the recovery. The results are shown in Tables 7 and 8 below. As can be seen from the results of Tables 7 and 8, sulfuric acid and hydrochloric acid showed the best results in the recovery rate, and a recovery rate of 99% or more was obtained under the condition of pH 3 or less. At this time, the lower the pH was, the higher the recovery was.

Recovery rate according to the acid type and pH conditions of Example 1 mountain Sulfuric acid pH One 2 3 4 5 6 % Recovery 99.9 99.9 99.9 97.5 78.6 55.5 mountain Hydrochloric acid pH One 2 3 4 5 6 % Recovery 99.9 99.9 99.9 96.1 73.4 51.1 mountain Glacial acetic acid pH One 2 3 4 5 6 % Recovery 99.9 99.9 99.8 94.0 72.6 50.5 mountain nitric acid pH One 2 3 4 5 6 % Recovery 99.9 99.9 99.8 93.6 68.5 44.1

Recovery rate according to the acid type and pH conditions of Example 3 mountain Sulfuric acid pH One 2 3 4 5 6 % Recovery 99.9 99.9 99.9 97.7 79.1 58.5 mountain Hydrochloric acid pH One 2 3 4 5 6 % Recovery 99.9 99.9 99.9 96.9 75.9 53.1 mountain Glacial acetic acid pH One 2 3 4 5 6 % Recovery 99.9 99.9 99.8 95.1 76.6 52.5 mountain nitric acid pH One 2 3 4 5 6 % Recovery 99.9 99.9 99.8 94.2 69.3 45.7

As described in detail through the above examples, the present invention purifies and crystallizes crude naphthalene dicarboxylic acid under suitable conditions using a recombinant microorganism which converts FNA into NDA, thereby purifying crystalline 2,6-naphthalene dicarboxylic acid of high purity. There is an advantage to mass production of acid economically and environmentally friendly.

<110> HYOSUNG CORPORATION <120> Purification method of crude naphthalene dicarboxylic acid using          recombinated microorganism and 2,6-naphthalene dicarboxylic acid          in crystalline form obtained by using the same <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 1506 <212> DNA <213> Sphingomonas aromaticivorans <400> 1 atggctacgc agttgagaag tgcagaaaac gaatacggga tcaagtccga gtacggccac 60 tatatcggcg gcgagtggat cgccggggac agcggcaaga ccatcgatct gctcaatccc 120 tcgaccggca aggtgctgac caagattcag gccggcaacg ccaaggatat cgaacgcgcg 180 attgccgccg ccaaggcggc gtttcccaag tggtcgcaga gcctgcccgg cgagcgccaa 240 gaaatcctga tcgaggttgc gcgtcgtctg aaggcacgcc attcgcacta tgccaccctc 300 gaaacgctca acaacggcaa gccgatgcgc gaatcgatgt atttcgatat gccgcagacg 360 atcgggcagt ttgagctgtt cgccggtgcc gcctatggcc tgcacggcca gacgctcgat 420 tatcccgacg cgatcggcat cgtccaccgc gaaccgctcg gcgtctgcgc gcagattatc 480 ccatggaacg tgccgatgtt gatgatggcg tgcaagatcg cgcccgcgct ggcctcgggc 540 aacactgtcg ttctgaagcc ggccgaaacg gtctgccttt cggtgattga attcttcgtg 600 gaaatggctg atctgttgcc gccgggtgtg atcaacgtcg tcaccggcta tggcgcggac 660 gtgggcgagg cgctggtcac cagccccgat gtcgccaagg tggccttcac cggttcgatc 720 gccaccgcgc gccggatcat ccagtatgcc tcggccaaca tcatccccca gacgctcgag 780 ttgggcggca agtcggcgca catcgtgtgt ggcgatgccg acatcgacgc ggcggtggaa 840 agcgcgacta tgtcgaccgt gctcaacaag ggcgaagtct gtctggccgg ttcgcgcctg 900 ttcctgcacc agtcgatcca ggacgagttc ctggccaagt tcaagaccgc gcttgaaggc 960 atccgccagg gcgacccgct cgacatggcg acccagcttg gcgcccaggc atcgaagatg 1020 cagtttgaca aggtgcaaag ctacctgcgc ctggccaccg aggaaggggc cgaggtcctg 1080 accggcggca gccgctcgga tgccgcagat ctggccgatg gcaattttat caagccgacc 1140 gtgttcacca acgtcaacaa ctccatgcgg atcgcgcagg aagagatctt cggaccggtt 1200 accagcgtca tcacctggag cgacgaagac gacatgatga agcaggccaa caatacaact 1260 tacggcctcg ctggcggcgt ctggaccaag gacatcgccc gagcccaccg gattgcgcgc 1320 aagctcgaaa ctggcacggt ctggatcaat cgctactaca acctgaaggc caacatgccg 1380 ctgggcggtt acaagcaaag cggcttcggg cgtgaattca gccatgaagt gctgaatcac 1440 tacacccaga ccaagtcggt ggtggtcaac ctccaggaag gtcgcaccgg aatgttcgat 1500 cagtga 1506 <210> 2 <211> 24 <212> DNA <213> Sphingomonas aromaticivorans <400> 2 ggagaattca tatggctacg cagt 24 <210> 3 <211> 24 <212> DNA <213> Sphingomonas aromaticivorans <400> 3 gtcttgcagt gagctcgttt ctcc 24 <210> 4 <211> 1464 <212> DNA (213) Pseudomonas putida <400> 4 atgcgggaaa caaaagagca gcctatctgg tacgggaagg tgtttagttc taattgggta 60 gaggcgcggg gaggtgttgc caatgttgtc gatccgtcca atggagacat tcttggcatt 120 acgggtgttg ctaacggcga agatgtcgat gctgctgtga acgcagctaa gagagcgcaa 180 aaggaatggg ccgcaatacc atttagtgaa agagccgcca ttgtccgcaa ggctgccgaa 240 aaactaaagg agcgcgagta tgaattcgcc gattggaacg tacgggaatg cggcgcaatt 300 cgtccgaagg gcttatggga ggccggaatt gcgtatgagc aaatgcatca agctgcgggt 360 ctagcttctt tgcctaacgg tacattgttt ccatcggcag ttccagggcg catgaatctt 420 tgtcagcgcg ttccagttgg cgtggtcggc gtaattgcac cttggaattt cccgttgttt 480 ctagcaatgc gttcggtagc accagcctta gcgttgggta atgcggtgat cttaaagccc 540 gaccttcaga ctgctgtcac cgggggggcg ctcattgccg aaatcttttc cgacgctggc 600 atgccggacg gtgttcttca cgttcttcct ggtggagcgg acgtaggaga gtcaatggtt 660 gcgaactccg gaattaacat gatttctttt accgggtcca cacaggtggg ccggttgatc 720 ggagagaaat gcgggagaat gctgaaaaag gttgcgcttg aactgggtgg taataatgtc 780 cacatcgtgt tgcctgacgc cgatttagaa ggggctgtca gctgcgctgc ttggggtacg 840 tttttgcatc agggccaagt gtgcatggcc gccggacgtc atttagtaca tagggacgtt 900 gctcagcaat atgcagagaa actggcgcta cgtgccaaga acttagtggt gggggatcca 960 aactcggatc aagtgcatct cggcccgctt atcaatgaga aacaggtagt tcgcgtccac 1020 gcgctcgttg aatctgcgca aagggccggt gctcaggttt tggcgggagg tacgtatcaa 1080 gatcgctact accaagctac cgtaatcatg gatgtgaagc cggagatgga ggttttcaaa 1140 tctgaaattt tcggcccggt ggctccgatc actgtatttg acagtattga agaggcgatt 1200 gaattggcaa actgttcgga gtatgggttg gccgcatcta tccatactag ggcgttggcg 1260 actggtctag acatcgcaaa gcgtctaaat accggtatgg tccatattaa tgaccagcca 1320 attaactgtg agccgcatgt tcccttcgga ggaatgggtg cctcgggtag cggaggccgg 1380 tttggcggac ctgcaagtat tgaagaattt actcaatctc aatggattag tatggttgag 1440 aagccagcta attacccatt ttga 1464

Claims (21)

(a) Recombinant expression comprising a benzaldehyde dehydrogenase gene xylC from Sphingomonas aromaticivorans KCTC 2888 represented by SEQ ID NO: 1 or a gene having at least 90% homology with the gene A microorganism obtained by transformation with a vector and a benzaldehyde dehydrogenase gene xylC derived from Pseudomonas putida ATCC 33015 represented by SEQ ID NO: 4 or a gene having at least 90% homology with the gene 2-formyl in the crude naphthalene dicarboxylic acid by reacting at least one recombinant microorganism selected from the group consisting of microorganisms obtained by transformation with a recombinant expression vector comprising crude naphthalene dimethylcarboxylic acid. Removing 6-naphthoic acid; (b) crystallizing the crude naphthalene dicarboxylic acid by adding an acidic solution to the reaction solution obtained in step (a) to adjust pH and then reacting with stirring; (c) washing the crystallized crude naphthalene dicarboxylic acid to remove impurities contained therein; And (d) Purifying crude naphthalene dicarboxylic acid using a recombinant microorganism comprising the step of drying the wash to obtain 2,6-naphthalene dicarboxylic acid in a pure crystalline state. The method of claim 1, wherein step (a) 1) inoculating the recombinant microorganism in a liquid medium to induce the expression of benzaldehyde dehydrogenase and then suspending the cells recovered through centrifugation in physiological saline or distilled water to prepare the cells for reaction; 2) mixing the crude naphthalene dicarboxylic acid as a substrate with a buffer solution and then adding an alkaline solution to adjust the pH of the mixed solution to form a purification reaction solution; And 3) Reacting the cells of the recombinant microorganism prepared in step 1) and the reaction solution prepared in step 2) converts 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid to 2,6-naphthalene Purifying crude naphthalene dicarboxylic acid using a recombinant microorganism comprising the step of increasing the purity of dicarboxylic acid (2,6-Naphthalene dicarboxylic acid). The method for purifying crude naphthalene dicarboxylic acid using a recombinant microorganism according to claim 2, wherein the microorganism is a bacterium of the genus Escheriachia, Pseudomonas, Rhodococcus or Bacillus. The method for purifying crude naphthalene dicarboxylic acid using a recombinant microorganism according to claim 2, wherein the microorganism is Escherichia coli. The method of claim 2, wherein the buffer solution is water, sodium carbonate buffer (Na ₂ O ₃ / NaHCO ₃ ), glycine buffer (Glycine / NaOH), potassium phosphate buffer (KH ₂ PO ₄ / KOH), sodium phosphate buffer Solution (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 At least one selected from the group consisting of a solution (Na ₄ P ₂ O ₇ / HCl), a boric acid buffer solution (Boric acid / NaOH), and a sodium borate buffer solution (Sodium borate / HCl), and the concentration is 0.01 to 100 mM. A method for purifying crude naphthalene dicarboxylic acid using a recombinant microorganism, characterized in that. The method for purifying crude naphthalene dicarboxylic acid using a recombinant microorganism according to claim 2, wherein the alkaline solution is NaOH or KOH. The method of claim 2, wherein the mixed solution of step 2) further comprises an organic solvent. The method of claim 7, wherein the organic solvent is dimethyl sulfoxide (Dimethylsulfoxide), dimethylformamide (N, N-Dimethylformamide), dimethylacetamide (N, N-Dimethylacetamide), tetrahydrofuran (Tetrahydrofuran) and dimethyl sulfoxide ( Dimethylsulfoxide) is one or more selected from the group consisting of, the concentration of the addition method of the crude naphthalene dicarboxylic acid using a recombinant microorganism, characterized in that 0.01 to 20%. The method for purifying crude naphthalene dicarboxylic acid using a recombinant microorganism according to claim 2, wherein the reaction temperature is 25 to 70 ° C and the reaction time is 1 minute to 1 hour. The method for purifying crude naphthalene dicarboxylic acid according to claim 2, wherein the concentration of crude naphthalene dicarboxylic acid in the reaction solution is 0.001 to 20%. The method of claim 1, wherein the acid solution in step (b) is at least one selected from the group consisting of sulfuric acid, hydrochloric acid, glacial acetic acid and nitric acid. The method of claim 1, wherein in step (b), the pH is adjusted to a range of 1 to 4. The method for purifying crude naphthalene carboxylic acid using recombinant microorganisms according to claim 1. The method of claim 1, wherein the reaction temperature in the step (b) is in the range of 4 to 150 ℃, the reaction time range of 1 minute to 10 hours, characterized in that the purification method of crude naphthalene dicarboxylic acid using a recombinant microorganism. The method of claim 1, wherein the stirring speed in the step (b) is in the range of 0 to 1000 rpm, the method for purifying crude naphthalene dicarboxylic acid using a recombinant microorganism. The method of claim 1, wherein step (c) comprises dispersing the crystallized crude naphthalene dimethylcarboxylic acid in water and then stirring it for 10 minutes to 1 hour at a pressure of 1 to 60 kg / cm ² and a temperature of 100 to 270 ° C. Purification of crude naphthalene dicarboxylic acid using a recombinant microorganism, characterized in that it is carried out by repeating the process of removing water by filtration several times. The method of claim 1, wherein the drying temperature of step (d) is in the range of 30 to 200 ° C. 3. The crude naphthalene dicarboxylic acid using a recombinant microorganism according to claim 1, further comprising the step of removing the recombinant microorganism used in the step (a) between the steps (a) and (b). Purification method. 18. The method for purifying crude naphthalene dicarboxylic acid using recombinant microorganisms according to claim 17, wherein the removal of the recombinant microorganism is performed using a microfilter system, a continuous centrifuge or a decanter. 19. The method of claim 18, wherein the microfilter system has a pore size of 0.1 to 0.5㎛, coarse naphthalene using recombinant microorganisms, characterized in that using a filter made of ceramic, stainless, polypropylene or PET material Method for Purifying Dicarboxylic Acid. 20. 2,6-naphthalene dicarboxylic acid in pure crystalline state obtained using the method of any one of claims 1-19. 21. The 2,6-naphthalene dicarboxylic acid in a crystalline state of claim 20, wherein the crystal form is amorphous or amorphous.

Images