KR100851742B1 - 26- Purification method of crude naphthalene dicarboxylic acid using benzaldehyde dehydrogenase from recombinated microorganism and 26-naphthalene dicarboxylic acid in crystalline form obtained by using the same - Google Patents

Abstract

The present invention contains benzaldehyde dehydrogenase, which has the ability to convert 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid, to react with crude naphthalene dicarboxylic acid to contain in the crude naphthalene dicarboxylic acid. 2-formyl-6-naphthoic acid, which is an impurity, is converted to 2,6-naphthalene dicarboxylic acid and removed, and then, an acidic solution is added thereto under constant conditions, and the crude naphthalene dicarboxylic acid is crystallized while stirring. And a method for purifying crude naphthalene dicarboxylic acid characterized by obtaining 2,6-naphthalene dicarboxylic acid in a pure crystalline state by washing it to remove other impurities and drying the washing. . According to the present invention, there is an advantage in that environmentally friendly mass production of high purity crystalline 2,6-naphthalene dicarboxylic acid is possible.

Benzaldehyde dehydrogenase, recombinant microorganism, crude Naphthalene dicarboxylic acid, 2-formyl-6-naphthoic acid, 2,6-naphthalene dicarboxylic acid (2,6-naphthalene dicarboxylic acid), crystallization

Description

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

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,

Figure 4 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: enzyme purification reactor B: inert enzyme 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 provides a method for purifying crude naphthalene dicarboxylic acid using benzaldehyde dehydrogenase, which has been isolated and purified from recombinant microorganisms, and 2,6-naphthalene dicarboxylic acid in a crystalline state obtained by the method. More specifically, benzaldehyde dehydrogenase having the ability to convert 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid is reacted with crude naphthalene dicarboxylic acid to react the crude naphthalene dicarboxylic acid. 2-formyl-6-naphthoic acid, which is an impurity contained in the leric acid, was converted to 2,6-naphthalene dicarboxylic acid and removed, and then, an acidic solution was added thereto under constant conditions, followed by stirring. The acid is crystallized, washed to remove other impurities, and the wash is dried to give a pure crystalline 2,6-naphthalene di It relates to the purification of a crude naphthalene dicarboxylic acid, characterized in that for obtaining a 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 is prepared by contacting a carbon catalyst having no 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. It discloses how to.

Also in connection with the crystallization of NDA, U.S. Patent No. 6,087,531 discloses a poly (alkylene naphthalene dicarboxylate) [poly (alkylene naphthalene dicarboxylate)] at a basic solution (alkali metal basic solution, hydroxide) at 125 to 400 ° C. (hydroxide) or alkali metal carbonate) and neutralized with acid at 170-240 ° C. to recover NDA crystals, or the polyester material was dissolved in NaOH solution or KOH solution at 170-240 ° C. and then neutralized with acetic acid. To disclose how to recover an NDA 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, an object of the present invention by using a benzaldehyde dehydrogenase converting FNA to NDA to purify and crystallize cNDA at room temperature and atmospheric pressure conditions to obtain a high purity NDA crystals It is to provide a method for obtaining in high yield.

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 benzaldehyde dehydrogenase, which is expressed and purified from 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 a homology of 90% or more to the gene Benzaldehyde dehydrogenase isolated from and purified from a recombinant microorganism obtained by transformation with a vector; or

Pseudomonas footage as shown in SEQ ID NO: 4 is mt-2 (Pseudomonas Crude naphthalene was purified from benzaldehyde dehydrogenase isolated from benzaldehyde dehydrogenase gene xylC derived from putida ATCC 33015) or from a recombinant microorganism obtained by transformation with a recombinant expression vector containing a gene having a homology of 90% or more. Reacting with crude naphthalene dimethylcarboxylic acid to convert 2-formyl-6-naphthoic acid in the crude naphthalene dicarboxylic acid to 2,6-naphthalene dicarboxylic acid to remove it;

(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, the step of dispersing the crystallized crude naphthalene dimethylcarboxylic acid in water, followed by a pressure of 1 to 60 kg / cm ² Agitating for 10 minutes to 2 hours at a temperature of 120 to 270 ° C., followed by filtration to remove the water several times; And

(d) Purifying crude naphthalene dicarboxylic acid using benzaldehyde dehydrogenase comprising drying the wash to obtain 2,6-naphthalene dicarboxylic acid in pure crystalline state. It is about how.

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

(Iii) step 1: from recombinant microorganisms Benzaldehyde Dehydrogenase  Purification process

First, a recombinant expression vector comprising a benzaldehyde dehydrogenase gene xylC derived from Sphingomonas aromaticivorans KCTC 2888 ( Sphingomonas aromaticivorans KCTC 2888) represented by SEQ ID NO: 1 or a gene having 90% or more homology with the gene. Benzaldehyde dehydrogenase isolated from and purified from a recombinant microorganism obtained by transformation; Or a benzaldehyde dehydrogenase gene xylC derived from Pseudomonas putida ATCC 33015 represented by SEQ ID NO: 4 or a recombinant expression vector comprising a gene having a homology of 90% or more to the gene. Benzaldehyde dehydrogenase isolated and purified from the obtained recombinant microorganism was reacted with crude naphthalene dimethylcarboxylic acid to react 2-formyl-6-naphthoic acid in the crude naphthalene dicarboxylic acid to 2,6-naphthalene. Remove by conversion to 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 the benzaldehyde dehydrogenase of the gene.

Specifically, the step 1) inoculates the recombinant microorganisms into a liquid medium to induce the expression of the benzaldehyde dehydrogenase, and then, the cells collected by centrifugation are suspended in buffer or physiological saline and destroyed by ultrasonic waves. Separating and purifying the supernatant obtained by centrifugation again to obtain the benzaldehyde dehydrogenase; 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 benzaldehyde dehydrogenase obtained in step 1) with the reaction solution prepared in step 2) to convert 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid. It comprises the step of increasing the purity of 6-naphthalene dicarboxylic acid (2,6-Naphthalene dicarboxylic acid).

Benzaldehyde dehydrogenase to be used in the present invention has a benzaldehyde dehydrogenase gene derived from Sphingomonas aromaticivorans KCTC 2888 ( Sphingomonas aromaticivorans KCTC 2888) represented by SEQ ID NO: 1 or more than 90% homology therewith. A gene or a benzaldehyde dehydrogenase gene derived from Pseudomonas putida ATCC 33015 represented by SEQ ID NO: 4, or a gene having a homology of 90% or more thereto, was cloned according to a generally known method. After introduction into an expression vector to prepare a recombinant expression vector, it may also be introduced into a microorganism and transformed according to a conventionally known general method to obtain a recombinant microorganism, and then separated and purified from the microorganism according to a conventional method. .

Specifically, it can be obtained by the following method:

First, in order to clone the respective benzaldehyde dehydrogenase gene ( xylC ), plasmid DNA or genomic DNA isolated from each microorganism is used as a template, and primers prepared from each gene are used. PCR 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 include, but are not particularly limited to, 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 Streptomyces , such as pIJ101, pIJ364, pIJ702 or pIJ361, and Bacillus , pUB110, pC194 or pBD214, and the like. 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 a gene xylC encoding a benzaldehyde dehydrogenase derived from sphingmonas aromatisiborance KCTC 2888 used in one embodiment of the present invention. 2 shows a genetic map of a recombinant expression vector pUC18- xylC comprising a gene xylC encoding benzaldehyde dehydrogenase derived from Pseudomonas putida used in another embodiment of the present invention.

Cloning of the enzyme gene into such a recombinant expression vector can be confirmed through restriction enzyme cleavage and sequence analysis.

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 Escherichia, Pseudomonas, and Rhodokocos , 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.

Subsequently, after fully incubating the recombinant microorganism, the respective benzaldehyde dehydrogenases are separated and purified by conventional methods. In this case, in order to convert 2-formyl-6-naphthoic acid into 2,6-naphthalene dicarboxylic acid using benzaldehyde dehydrogenase obtained from the recombinant microorganism in the present invention, each benzaldehyde dehydrogena in the recombinant microorganism is used. Since it is preferable that I be highly expressed, it is preferable to induce the expression of each enzyme through shaking culture or addition of IPTG, and then purify it separately.

Specifically, the recombinant microorganism is sufficiently incubated at a normal culture temperature of 25 to 45 ℃, preferably 37 ℃, which is again 1% (v / v) in 100 ml of liquid medium (eg LB medium) After inoculation, the OD ₆₀₀ value was 0.4 to 0.5, and the IPTG concentration was added to 0.1 to 2.0 mM, preferably 0.5 mM to induce the expression of the benzaldehyde dehydrogenase, and then incubated again at 37 ° C. The cells were recovered by centrifugation of the culture solution, the cells were suspended in a buffer solution or saline solution, the suspension was sonicated, and the supernatant was recovered by centrifugation. Benzaldehyde dehydrogenase can be obtained by purification using the used chromatography. At this time, the centrifugation and suspension of the cells can be further repeated once or twice as necessary.

The method of culturing such recombinant microorganisms, expressing and purifying proteins is 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.

As described above, the present invention uses high-expression of benzaldehyde dehydrogenase in recombinant microorganisms, and then separates and uses them, thereby enabling high concentration culture to obtain a large amount of benzaldehyde dehydrogenase and having high recovery rate.

Furthermore, the benzaldehyde dehydrogenase of the present invention can be obtained using histidine tagging or the like. Specifically, a recombinant expression vector was prepared using a gene obtained by using a reverse primer prepared by attaching a histidine tag to each enzyme gene, and a transgenic microorganism was obtained, from which benzaldehyde dehydrogenase was attached. Can be separated and purified. Using histidine tagging in this way, when the enzyme is expressed later, there is an advantage that can be easily and easily separated and purified.

Meanwhile, the buffer solution used in the present invention is not particularly limited, but water, sodium carbonate buffer solution, glycine buffer solution, potassium phosphate buffer solution, sodium phosphate buffer solution, succinic acid buffer solution, sodium acetate buffer solution, citric acid buffer solution Sodium pyrophosphate buffer, boric acid buffer, sodium borate buffer, and the like can be used. Preferably, potassium phosphate buffer or boric acid buffer solution having a pH range of 6.0 to 9.0 is used. At this time, the concentration of the buffer solution is preferably used at a concentration of 0.01 to 100 mM.

Although not necessarily limited to the alkaline solution, it is preferable to use NaOH solution or KOH solution, 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% by weight, more preferably 0.1 to 10% by weight. Most preferably not added. On the other hand, the addition of more than 20% by weight of the organic solvent shows a result of inhibiting the reaction by reducing the activity of the enzyme.

The reaction temperature and reaction time in the step 3) is preferably 25 to 50 ℃, 1 minute to 1 hour, and more preferably 35 to 45 ℃, 10 minutes to 40 minutes to react. In particular, when the reaction temperature is less than 25 ° C or more than 50 ° C, the reactivity is significantly reduced and unsuitable.

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

Specifically, in the present invention, the concentration of cNDA in the purification reaction liquid is preferably 0.001 to 20% by weight, and the FNA content in cNDA is preferably 0.001 to 10% by weight, preferably 1% by weight.

(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 it is more preferable to use sulfuric acid or hydrochloric acid in terms of 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 3 in terms of recovery, but the lower the pH, the higher the recovery to 99.99%.

In addition, the crystallization temperature is about 10 to 110 ℃, preferably 30 to 90 ℃, more preferably 50 to 80 ℃ suitable for carrying out the crystallization reaction, the reaction time is about 1 minute to 8 hours, preferably 5 Minutes to 4 hours, more preferably 10 minutes to 1 hour range is good in terms of continuous process. Most preferred crystallization temperature and reaction time is 70 ℃, 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 which performs crystallization, it is preferable to carry out at more than 0 rpm and 1000 rpm or less, Preferably it is more than 0 rpm and 400 rpm or less, More preferably, it is more than 50 rpm and 100 rpm or less.

(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 benzaldehyde dehydrogenase, FNA is converted to NDA and removed, but impurities such as 2-naphthoic acid (NA), methylnaphthoic acid (MNA) and trimellitic acid (TLMA) are not removed. It exists as is. 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 enzyme used to purify the cNDA are removed together.

In this step, it is preferable that the reaction by-products are dissolved in a solvent and separated as much as possible from NDA, and the temperature range is 120 to 270 ° C, preferably 180 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 lowered, and when the separation at less than 120 ℃ temperature 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 2 hours at a pressure of 1 to 60 kg / cm ² , temperature 120 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 50 to 250 ℃, the drying method can be used without limitation the conventional drying method known in the art.

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

As such, if the crystallization reaction is performed by removing the enzyme previously inactivated after purification of cNDA, the purity and recovery of NDA crystals may be further improved.

As a method for removing the inactivated enzyme, a conventionally known method can be used without limitation, and specifically, at least one selected from the group consisting of a microfilter system, a continuous centrifuge, and a decanter is used. Can be removed. In this case, in the case of the microfilter system, a filter having a pore size of 0.01 to 1 μm and having at least one material selected from the group consisting of ceramic, stainless steel, polypropylene, and 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 in the range of 110 to 180 μm.

On the other hand, Figure 4 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 will be described in more detail with reference to FIG. 4 as follows.

First, a certain amount of buffer solution is injected into the enzyme purification reactor (A), and cNDA is added thereto, followed by stirring to adjust the pH of the reaction solution by adding an alkali solution, and then adding a certain amount of water to prepare a purification reaction solution. do. Reaction benzaldehyde dehydrogenase prepared in advance is added while the reaction solution is maintained at a constant temperature for reaction. Through this process, FNA in cNDA can be converted to NDA in the enzyme purification reactor (A) to remove FNA.

The reaction solution was passed through the inert enzyme removal device (B) to remove the inert enzyme used to remove the FNA. The inert enzyme removal process using this inert enzyme removal device B can be omitted as needed. Even if the inert enzyme removal step is omitted, the inactivated enzyme and remaining enzyme can be removed by the subsequent filtration and washing apparatus (F).

The purified reaction solution from which the inert enzyme has been removed is transferred to the crystallization reactor (C), and the acidification solution is added thereto while the crystallization reaction proceeds while stirring.

Then, the slurry containing the crystallized cNDA is heated to about 120 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 120 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 pre-heated at 120 to 270 ° C. from the solvent heating supply device (E) was added to the filtration and washing device (D) and stirred for a while, followed by secondary filtration with 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 preheated to 120 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  One]

Step 1: Isolation of Enzyme Purification Liquid from Recombinant Microorganism

(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) and The comparison confirmed that the xylC gene was cloned.

(3) Preparation of transformant

Using the calcium chloride method (see Sambrook et. al., Molecular Cloning, A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press, were transformed Cold Spring Harbor, NY, 1989) E. coli XL1-Blue by pET20- xylC vector. 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 and Purification of Benzaldehyde Dehydrogenase

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. Then, the cells were recovered by centrifugation of the culture solution, and the recovered cells were suspended in 50 mM potassium phosphate buffer solution (KH ₂ PO ₄ -KOH) at pH 7.0, and then destroyed by ultrasonication and centrifuged again. Was taken. Benzaldehyde dehydrogenase in the obtained supernatant was purified using anion chromatography using a column containing Q-Sepharose. SDS-PAGE was performed to confirm protein separation, and it was confirmed that about 55,000 Da of benzaldehyde dehydrogenase was separated and purified.

(5) enzyme titer measurement

Benzaldehyde is converted to benzoic acid by NAD ⁺ (nicotinamide-adenine dinucleotide) in the presence of benzaldehyde dehydrogenase. The amount of NADH produced at this time is determined by measuring the absorbance at 340 mm. Enzyme titer was added to 0.5 ml of enzyme solution to 3 ml of 50 mM potassium phosphate buffer (KH ₂ PO ₄ -KOH) at pH 7.0 containing 5 mM NAD ⁺ and 10 mM benzaldehyde, and reacted at 20 ° C. for 5 minutes. It was determined by measuring absorbance at nm. One unit of enzyme activity is defined as the amount of enzyme that produces 1 umole of NDAH per minute.

Second step: isolated from recombinant microorganisms Benzaldehyde Dehydrogenase  Used cNDA Tablets

Inject 50 L of 50 mM potassium phosphate buffer solution into the enzyme purification reactor (A), add 10 kg of cNDA thereto, add KOH solution while stirring to adjust the pH of the reaction solution to 8.0, and then add water again. 100 L of a purification reaction solution (cNDA concentration: 10%, FNA content in cNDA: 0.58%) was prepared.

While maintaining the reaction solution at 40 ° C., 5 L of the benzaldehyde dehydrogenase enzyme solution (500 units) obtained in the first step was added thereto, followed by reaction at 40 ° C. for 30 minutes to convert FNA in the cNDA into NDA.

Step 3: Refined cNDA Crystallization

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 adjusted to a pH of 3.0 by adding sulfuric acid solution thereto, and then the stirring speed was 50 rpm and the temperature was 70 The crystallization reaction proceeded for 20 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 163.5 ㎛. A micrograph of the crystallized cNDA is shown in FIG. 3.

Step 4: Crystallize cNDA Washing and drying

The cNDA slurry solution crystallized in the third step is heated to 220 ° C. or more through a preheater (D), and then put into a filtration and washing apparatus (F), for 30 minutes at a pressure of 35 kg / cm ² and a temperature of 230 ° C. After stirring, the mixture was filtered to remove water with a high pressure filtrate recovery device (G). After adding 230 L 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. Here, again, 230 ° C. and 100 L of water were added from the solvent heating supply device (E), followed by stirring for 30 minutes or more, and then evenly dispersed. Water was removed using a decanter (I), followed by drying at 180 ° C. in a drying apparatus (J) to obtain NDA in pure crystalline state.

[ Example  2]

Pseudomonas footage shown by Sequence Listing SEQ ID NO: 4 with purified enzyme solution of the present invention is mt-2 (Pseudomonas Isolation and purification from microorganism JM109 (pUC18- xylC ) obtained by transformation with the recombinant expression vector pUC18- xylC shown in FIG. 2 including a benzaldehyde dehydrogenase gene (put GenBank Sequence Database, D63341) derived from putida ATCC 33015) NDA in pure crystalline state was obtained in the same manner as in Example 1, except that one benzaldehyde dehydrogenase was used.

In this case, a detailed method for preparing the recombinant microorganism JM109 (pUC18- xylC ) is disclosed in Korean Patent Publication No. 2005-71188, and the separation and purification of benzaldehyde dehydrogenase from the recombinant microorganism JM109 (pUC18- xylC ) It was performed by the following method:

The recombinant microorganism JM109 (pUC18- xylC ) 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, with an OD ₆₀₀ of 0.4 to 0.5. When reached, IPTG concentration was added to 0.5 mM to induce the expression of xylC , and then cultured again at 37 ° C. Then, the culture solution was centrifuged to recover the cells, and the recovered cells were suspended in 50 mM potassium phosphate buffer (KH ₂ PO ₄ -KOH) at pH 7.0, then disrupted by ultrasonication, and centrifuged again. Supernatant was taken. Benzaldehyde dehydrogenase in the obtained supernatant was purified using anion chromatography using a column containing Q-Sepharose. SDS-PAGE was performed to confirm protein separation, and it was confirmed that about 53,500 Da of benzaldehyde dehydrogenase was separated and purified.

[ Example  3]

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 inactivated enzyme using a polypropylene filter of 0.10 μm, The same procedure as in Example 1 was carried out to obtain NDA in pure crystalline state.

Experimental Example  1: Component Analysis

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

Using the cNDA purification method using the benzaldehyde dehydrogenase of the present invention 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 99.99% or more. It was confirmed that a, 6-naphthalene dicarboxylic acid crystal could be obtained.

On the other hand, in order to find the optimum reaction conditions of the crystallization step of the cNDA purification solution of cNDA using the benzaldehyde dehydrogenase of the present invention, in particular, experiments by varying the reaction conditions as follows and the crystallization reaction and recovery rate accordingly Was analyzed comparatively.

Experimental Example  2: acid type and At the stirring speed  Crystallization reaction

100 ml of each cNDA purification solution obtained in the second step of Examples 1 and 2 were placed in a reactor equipped with a stirring device, and the sulfuric acid, hydrochloric acid, glacial acetic acid, and nitric acid solutions were added thereto to adjust the pH to 3.0. Next, the stirring speed was maintained at 0, 50, 100, 200, 400, 800, 1000 rpm. The temperature was 70 ° C., and the crystallization reaction was performed for 20 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, respectively. As can be seen from the results of Tables 3 and 4, sulfuric acid and hydrochloric acid showed the best results for the crystallization reaction among the four acid solutions, and the preferred stirring speed was more than 0 rpm and 400 rpm or less, and more preferable stirring speed. Was determined to be about 50 rpm or more and 100 rpm or less.

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

Experiment was carried out in the same manner as in Experiment 2 except that the stirring speed was 50 rpm and the reaction temperature was 10, 30, 50, 70, 90, 110 ° C.

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. 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 in the crystallization reaction among the four acid solutions, and the most preferable reaction temperature was determined to be 70 ° C.

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 70 ℃, pH range was adjusted to 1, 2, 3, 4, 5, 6. That is, after performing the crystallization reaction for 20 minutes, a certain amount was taken to compare the recovery rate. 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 the recovery rate was 99.9% or more under the condition of pH 3 or less. At this time, the lower the pH was, the higher the recovery was.

As described in detail above, the present invention provides a high-purity crystal form by purifying and crystallizing crude naphthalene dicarboxylic acid under suitable conditions using benzaldehyde dehydrogenase, which is expressed and purified from recombinant microorganisms converting FNA into NDA. There is an advantage that can mass-produce 2,6-naphthalene dicarboxylic acid environmentally friendly.

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Claims (18)

(a) isolated and purified from a recombinant microorganism obtained by transformation with a recombinant expression vector comprising a benzaldehyde dehydrogenase gene xylC derived from Spingomonas aromaticivorans KCTC 2888 (SEQ ID NO: 1). Benzaldehyde dehydrogenase; Or benzaldehyde dehydrogenase isolated and purified from a recombinant microorganism obtained by transformation with a recombinant expression vector comprising a benzaldehyde dehydrogenase gene xylC derived from Pseudomonas putida ATCC 33015 (SEQ ID NO: 4). Reacting with crude naphthalene dimethylcarboxylic acid to convert 2-formyl-6-naphthoic acid in the crude naphthalene dicarboxylic acid to 2,6-naphthalene dicarboxylic acid to remove it; (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, the step of dispersing the crystallized crude naphthalene dimethylcarboxylic acid in water, followed by a pressure of 1 to 60 kg / cm ² Agitating for 10 minutes to 2 hours at a temperature of 120 to 270 ° C., followed by filtration to remove the water several times; And (d) drying the wash to obtain crude Naphthalene dicarboxylic acid using benzaldehyde dehydrogenase, comprising the step of obtaining 2,6-naphthalene dicarboxylic acid in pure crystalline state. Purification method. The method of claim 1, wherein step (a) 1) The supernatant obtained by inoculating the recombinant microorganisms in a liquid medium to induce the expression of the benzaldehyde dehydrogenase, and then the cells recovered by centrifugation were suspended in buffer or physiological saline, destroyed by ultrasound and centrifuged again. Separating and purifying to obtain the benzaldehyde dehydrogenase; 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) 2,6 by converting 2-formyl-6-naphthoic acid into 2,6-naphthalene dicarboxylic acid by reacting the benzaldehyde dehydrogenase obtained in step 1) with the reaction solution prepared in step 2). -Purifying crude naphthalene dicarboxylic acid using benzaldehyde dehydrogenase, comprising the step of increasing the purity of naphthalene dicarboxylic acid (2,6-Naphthalene dicarboxylic acid). The method of claim 2, wherein the buffer solution is water, sodium carbonate buffer solution, glycine buffer solution, potassium phosphate buffer solution, sodium phosphate buffer solution, succinic acid buffer solution, sodium acetate buffer solution, citric acid buffer solution, sodium pyrophosphate buffer solution, boric acid Purification method of crude naphthalene dicarboxylic acid using benzaldehyde dehydrogenase, characterized in that at least one selected from the group consisting of a buffer solution and sodium borate buffer solution, the concentration is 0.01 to 100 mM. The method for purifying crude naphthalene dicarboxylic acid using benzaldehyde dehydrogenase according to claim 2, wherein the alkali solution is a NaOH solution or a KOH solution. The method for purifying crude naphthalene dicarboxylic acid using benzaldehyde dehydrogenase according to claim 2, wherein the mixed solution of step 2) further comprises an organic solvent. The method of claim 5, wherein the organic solvent is selected from the group consisting of dimethylsulfoxide, dimethylformamide (N, N-Dimethylformamide), dimethylacetamide (N, N-Dimethylacetamide) and tetrahydrofuran (Tetrahydrofuran). Purification method of crude naphthalene dicarboxylic acid using benzaldehyde dehydrogenase, characterized in that at least one, and the addition concentration is 0.01 to 20% by weight. The method for purifying crude naphthalene dicarboxylic acid using benzaldehyde dehydrogenase according to claim 2, wherein the reaction temperature in step 3) is 25 to 50 ° C and the reaction time is 1 minute to 1 hour. . The purification of crude naphthalene dicarboxylic acid using benzaldehyde dehydrogenase according to claim 2, wherein the concentration of crude naphthalene dicarboxylic acid in the purification reaction liquid in step 2) is 0.001 to 20% by weight. Way. 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 3. A method for purifying crude naphthalene dicarboxylic acid using benzaldehyde dehydrogenase. The crude naphthalene dicarboxylic acid using benzaldehyde dehydrogenase according to claim 1, wherein the reaction temperature in the step (b) is in the range of 10 to 110 ° C, and the reaction time is in the range of 1 minute to 8 hours. Purification method. The method of claim 1, wherein the stirring speed in the step (b) is in the range of more than 0 rpm and less than 1000 rpm range of crude naphthalene dicarboxylic acid using benzaldehyde dehydrogenase. delete The method for purifying crude naphthalene dicarboxylic acid using benzaldehyde dehydrogenase according to claim 1, wherein the drying temperature of step (d) is in the range of 50 to 250 ° C. The crude naphthalene using benzaldehyde dehydrogenase according to claim 1, further comprising the step of removing the inactivated enzyme used in step (a) between step (a) and step (b). Method for Purifying Dicarboxylic Acid. The benzaldehyde dehydrogenase according to claim 15, wherein the removal of the inactivated enzyme is performed using at least one selected from the group consisting of a microfilter system, a continuous centrifuge and a decanter. Purification method of crude naphthalene dicarboxylic acid. 17. The method of claim 16, wherein the microfilter system has a pore size of 0.01 to 1 [mu] m and uses a filter made of at least one material selected from the group consisting of ceramic, stainless, polypropylene and PET. A method for purifying crude naphthalene dicarboxylic acid using benzaldehyde dehydrogenase. delete

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