KR100475007B1 - Novel bacillus strains and method for purifying 2,6-naphthalene dicarboxylic acid thereby - Google Patents

Abstract

The present invention removes 2-formyl-6-naphthoic acid in crude naphthalene dicarboxylic acid produced by oxidizing 2,6-dimethylnaphthalene by using a novel Bacillus microorganism. A method for producing 6-naphthalene dicarboxylic acid, wherein the novel strains of the present invention Bacillus sp. F-1 and F-3 are 2-formyl contained in crude naphthalene dicarboxylic acid. Since there is an excellent effect of removing -6-naphthoic acid, it is expected that mass production of high purity 2,6-naphthalene dicarboxylic acid is realized by the method of the present invention.

Description

Novel bacillus microorganism and purification method of 2,6-naphthalene dicarboxylic acid using same {NOVEL BACILLUS STRAINS AND METHOD FOR PURIFYING 2,6-NAPHTHALENE DICARBOXYLIC ACID THEREBY}

The present invention relates to a novel Bacillus microorganism and a method for purifying 2,6-naphthalene dicarboxylic acid using the same, and more specifically, 2,6-dimethyl naphthalene (2,6-dimethylnaphthalene, hereinafter 2,6-). A novel Bacillus microorganism is produced from 2-Formyl-6-naphthoic acid (FNA) in crude Naphthalene dicarboxylic acid (cNDA) produced by oxidation of DMN). The present invention relates to a method of producing 2,6-naphthalene dicarboxylic acid (hereinafter referred to as NDA) of high purity by removing the same.

Diesters of naphthalene dicarboxylic acids are useful for preparing a variety of polymeric materials such as polyesters and polyamides. One example of a particularly useful diester is dimethyl-2,6-naphthalene dicarboxylate (hereinafter NDC). NDC can condense with ethylene glycol to produce poly (ethylene-2,6-naphthalene) (hereinafter PEN), a high performance polyester material. Fibers and films made from PEN have higher strength and better thermal properties than poly (ethylene terephthalate) (hereinafter PET). Because of this, PEN is widely used to produce commercial products such as thin films that can be used to make magnetic recording tapes and electronic components. Films made from 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. In addition, PEN can be used to make reinforcing fibers useful for the manufacture of tire cords.

Currently, NDC produces cNDA by oxidizing 2,6-DMN and then esterifying it, as shown in FIG. 3. NDC produced in this way is used as a main raw material when synthesizing PEN, but there are some problems compared to using NDA as a raw material. Firstly, water is produced during NDA condensation reaction, while methanol is a by-product in the case of NDC, and there is a risk of explosion. Second, in order to obtain pure NDC, NDC is esterified and purified through The production requires more one step process than 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, the reason why NDC is used instead of NDA in PEN production is that it has not yet produced a purified NDA having the purity necessary for polymerization. That is, cNDA containing various impurities such as FNA, 2-naphthoic acid, trimellitic acid, etc. is generated during oxidation of 2,6-DMN, and among these impurities, FNA is known to be difficult to remove. . The presence of FNA in the condensation reaction of NDA causes the polymerization reaction to stop in the middle, which adversely affects the physical properties of the polymer. Therefore, the removal of FNA from cNDA has become an important problem.

In the meantime, in order to remove FNA present in cNDA or to purify NDA, recrystallization, oxidation process is performed again, cNDA is reacted with methanol to make NDC, and then hydrated to produce NDA or purified by hydrogenation process. A method of producing NDA and the like have been proposed. In addition, various purification methods such as solvent treatment, melting crystals, high pressure crystals, supercritical extraction, etc. have been used, but NDAs with satisfactory purity have not yet been produced. In addition, if the purity is increased, the yield is very difficult to apply to actual production.

The present invention is to solve the problems of the prior art as described above, by identifying a novel microorganism having FNA degradation activity and purifying NDA by selectively removing the FNA contained in cNDA by a biological method using the same. The purpose.

That is, one aspect of the present invention provides Bacillus sp. F-1 (KCTC-10342BP) having 2-formyl-6-naphthoic acid degrading activity.

Another aspect of the present invention provides Bacillus sp. F-3 (KCTC-10335BP) having 2-formyl-6-naphthoic acid degradation activity.

Another aspect of the present invention is Bacillus sp. F-1 (KCTC-10342BP) or at a temperature of 25 ~ 45 ℃ in a buffer solution of pH 6.0 ~ 10.0 containing 0 ~ 20% organic solvent or Bacillus sp. F-3 (KCTC-10335BP) was reacted with crude naphthalene dimethylcarboxylic acid (crude Naphthalene dicarboxylic acid) 2-formyl-6-naphthoic acid (2) -Formyl-6-naphthoic acid) provides a method for purifying 2,6-naphthalenedicarboxylic acid comprising the step of decomposing.

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

We have succeeded in separating two new strains from the soil with the ability to degrade FNA. The new strains of the present invention were identified as strains belonging to the genus Bacillus sp. By 16S rDNA partial sequencing method, and were named Bacillus sp. F-1 and F-3, respectively. 16S rDNA partial sequencing results of the Bacillus genus F-1, F-3 strains are shown in Figure 1 (SEQ ID NO: 1) and Figure 2 (SEQ ID NO: 2), respectively. Bacillus F-3 strain was deposited with KCTC-10335BP at the Korea Biotechnology Research Institute Genetic Bank of Korea on September 12, 2002, and Bacillus F-1 strain was deposited with the same institution on September 27, 2002. Deposited with KCTC-10342BP.

Bacillus genus F-1, F-3 strains of the present invention can be easily cultured using LB liquid medium, it is possible to culture at a wide range of temperature of 25 ~ 45 ℃. These strains have very similar morphological as well as biochemical properties (see Tables 3 and 4) and show high activity for FNA degradation but are not reactive at all with NDA.

Hereinafter, the NDA purification method of the present invention using the Bacillus genus F-1, F-3 strain will be described in more detail.

In the present invention, the microbial strains are inoculated in LB liquid medium, incubated with stirring at 25-45 ° C., and the cells are recovered by centrifugation, and the recovered cells are suspended in physiological saline and used as enzyme solution for NDA purification. .

In the present invention, in order to remove FNA, which is an impurity contained in cNDA, the cNDA to be purified is placed in an enzyme reaction tank containing a buffer solution, and the reaction is performed by adding the microbial suspension (ie, enzyme solution) and stirring.

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

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

The temperature of the enzyme reaction is preferably 25 ~ 45 ℃, in terms of reaction rate is most preferably reacted at 30 ℃.

Hereinafter, the present invention will be described in more detail with reference to examples and comparative examples, but these examples are for illustrative purposes only and should not be construed as limiting the present invention.

Example 1 Isolation of Strains

Soils were collected from wastewater treatment plants, oil depots and gas stations in Gyeonggi-do to isolate microorganisms with FNA degradation activity. 5 g of each soil sample was added to 50 ml of 0.85% saline, shaken, and the filtrate was diluted appropriately, plated in an LB solid medium containing a cNDA mixture, and incubated in a 30 ° C. incubator. Over 170 species of microorganisms were isolated from the culture.

Among them, the 170 microorganisms were inoculated into 5 ml of LB liquid medium to first screen microorganisms that decompose 2-naphthaldehyde having a formyl group at the same position as FNA. After incubation with shaking at 200 ° C. at 30 ° C. for 24 hours, the culture solution was centrifuged to suspend the recovered cells in 1 ml of 0.85% saline. 3 ml of β-NAD solution (50 mg / ml) was added to all glass cuvettes, 0.2 ml of 2-naphthaaldehyde solution was added to the sample cuvette, and 0.2 ml of DMSO was added to the blank cuvette. After stabilization for 3 minutes, 0.05 ml each of the microbial suspension was added and after 5 minutes the absorbance was measured at 340 nm to compare the absorbance difference with the blank. As a result, the absorbance of the six microbial samples increased by at least 0.1 or more than the absorbance of the blank, and it was confirmed that there was 2-naphthaaldehyde decomposition activity.

Thus, six kinds of microorganisms primary screened were inoculated in LB liquid medium again and cultured with shaking at 30 ° C. at 200 rpm for 24 hours. The cells were recovered by centrifuging the culture solution, suspended in 0.85% saline solution, and then prepared with a reaction solution having the composition shown in Table 1 below, followed by reaction for 6 hours in a reaction vessel at 30 ° C. ) Was finally selected for the strain having excellent FNA removal ability. At this time, HPLC analysis conditions were as shown in Table 2 below. As a result, two strains named F-1 and F-3 had high FNA resolution, and the F-3 strain was slightly better than the F-1 strain.

Reaction solution for final screening ingredient Volume (ml) Remarks 0.1 M KH ₂ PO ₄ / KOH (pH 8.0) 39 cNDA solution 4 cNDA solution concentration: 50 mg / ml DMSO One Final concentration in the reaction solution: 10% β-NAD solution One β-NAD solution concentration: 50 mg / ml Microbial suspension 5 Microbial Suspension Concentration: 1 × 10 ⁹ cfu / ml Sum 50

HPLC analysis conditions HPLC LC 10-AD VP (SHIMADZU) column ^{XTerra TM RP18 (4.6 × 50 mm} , Waters) Detector  UV 240 nm Column temperature  40 ℃ Flow rate  1 ml / min. Injection volume  20 μl Mobile phase Time (min.) 0.3% phosphoric acid Acetonitrile 0 98 2 5 92 8 28 52 48 30 20 80 35 5 95 36 98 2 49 98 2

Example 2: Identification of Isolated Microorganisms

In order to identify two strains obtained in Example 1, 16S rDNA partial sequencing was performed, and the results are shown in FIGS. 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2). According to the results, both strains of the present invention were found to belong to the genus Bacillus ( Bacillus sp.), Respectively, Bacillus ( Bacillus sp.) F-1 (KCTC-10342BP), F-3 (KCTC-10335BP) Was named.

As a result of testing the morphological and biochemical properties of the two strains F-1, F-3 it was as shown in Table 3 and Table 4, it was confirmed that the two strains have very similar characteristics.

Characteristics of Bacillus sp. F-1 and F-3 Item Characteristics Gram Dyeing positivity Cell morphology Bacillus Growth temperature 25 ~ 45 ℃ Catalase production positivity Nitrate reduction positivity Voges-Proskauer Reaction positivity Casein decomposition positivity Gelatin breakdown positivity Indole generation voice NaCl concentration 7% less than Acid production (substrate)  D-glucose D-arabinose D-mannitol Yang Sungyang Sungyang Castle

Carbohydrates of Bacillus sp. F-1 and F-3 carbohydrate Ability carbohydrate Ability Fructose + Sucrose + Glucose + Trehalose + Inositol - Rhamnose - Lactose - N-acetyl-D-glucosamine - maltose + Cyclodextrin + Maltotrios + dextrin + Mannitol - Ribose + Mannose +

Example 3: Effect of β-nicotinamide (β-NAD) on enzyme reaction

In order to confirm whether β-nicotinamide (β-NAD) included in the microbial final screening reaction solution affects the reaction, β-NAD was expressed in the reaction solution composition of Table 1 for each strain of F-1 and F-3. The control group included and the experimental group not included were reacted in a 30 ° C. reactor for 6 hours, and each reaction solution was separated and analyzed and compared with HPLC. HPLC analysis conditions were the same as in Table 2 above. As a result, as shown in Table 5, both strains maintained a similar level of FNA reduction regardless of the addition of β-NAD. Therefore, it was confirmed that there is no problem in the reaction even without adding β-NAD in the enzyme reaction of the present invention.

Reduction of FNA with or without β-NAD (FNA remaining ratio,%) microbe Reaction time β-NAD added ○ β-NAD addition × Bacillus F-1 0 hr 100 100 6 hr 30.6 30.8 Bacillus F-3 0 hr 100 100 6 hr 23.1 23.8

Example 4: Reactivity to NDA Example 4: Reactivity to NDA

Since Bacillus F-1 or F-3 reduces NDA in cNDA, it is not suitable for use in NDA purification, so to determine whether these two strains reduce NDA, the composition of Table 6 below using NDA as a reductant After preparing the reaction solution, the enzyme reaction was carried out under the same reaction conditions as in Example 1, and the reaction product was analyzed by HPLC. As a result, as shown in Table 7 below, even after 24 hours of reaction, the amount of NDA in the reaction solution did not change, and these results demonstrate that neither Bacillus F-1 or F-3 reacted with NDA.

Reaction liquid composition containing NDA ingredient Volume (ml) Remarks 0.1 M KH ₂ PO ₄ / KOH (pH 8.0) 40 NDA solution 4  NDA solution concentration: 50 mg / ml DMSO One Final concentration in the reaction solution: 10% Microbial suspension 5 Microbial Suspension Concentration: 1 × 10 ⁹ cfu / ml Sum 50

NDA Reduction by Bacillus F-1 and F-3 (NDA Remaining Ratio,%) Reaction time Bacillus F-1 Bacillus F-3 0 hr 100.0 100.0 6 hr 100.1 100.5 24 hr 100.3 99.7

Examples 5-18 and Comparative Example 1: Purification efficiency according to the reaction conditions

In order to find the optimum reaction conditions, the FNA content of cNDA used as a substrate, the kind of buffer solution, the pH, the type and concentration of the organic solvent, the reaction temperature, etc. were changed as shown in Table 8. The reduction rate was compared. In this case, Bacillus F-3 was cultured in 300 ml of LB broth and the recovered cells were suspended in 5 ml of 0.85% physiological saline. The concentration of cells in each reaction solution was 1 × 10 ⁹ cfu / ml. Unified to a concentration of. After 6 hours, each reaction solution was aliquoted and analyzed using HPLC under the same conditions as in Table 2. The analysis results are shown in Table 9 below.

Changes in Reaction Conditions for NDA Purification Using Bacillus F-3 # FNA content in cNDA Buffer Organic solvent Reaction temperature Kinds pH Kinds density Example 5 0.01% KH ₂ PO ₄ / KOH 8.0 DMSO 5% 30 ℃ Example 6 10% KH ₂ PO ₄ / KOH 8.0 DMSO 10% 30 ℃ Example 7 10% KH ₂ PO ₄ / NaOH 8.0 DMSO 10% 30 ℃ Example 8 10% Na ₄ P ₂ O ₇ / HCl 8.0 DMSO 10% 30 ℃ Example 9 10% H ₃ BO ₃ / NaOH 8.0 DMSO 10% 30 ℃ Example 10 10% Na ₂ B ₄ O ₇ / HCl 8.0 DMSO 10% 30 ℃ Example 11 10% Na ₂ CO ₃ / NaHCO ₃ 10.0 DMSO 10% 30 ℃ Example 12 10% Glycine / NaOH 9.0 DMSO 10% 30 ℃ Example 13 10% Na ₂ HPO ₄ / NaH ₂ PO ₄ 7.0 DMSO 10% 30 ℃ Example 14 10% KH ₂ PO ₄ / KOH 8.0 DMSO 0% 30 ℃ Example 15 10% KH ₂ PO ₄ / KOH 8.0 DMSO 10% 25 ℃ Example 16 10% KH ₂ PO ₄ / KOH 8.0 DMSO 10% 45 ℃ Example 17 10% KH ₂ PO ₄ / KOH 8.0 DMF 10% 30 ℃ Example 18 10% KH ₂ PO ₄ / KOH 8.0 DMA 10% 30 ℃ Comparative Example 1 10% Distilled water - DMSO 10% 30 ℃

Reduction of FNA after 6 hours of purification # FNA Reduction (%) Example 5 95.1 Example 6 76.5 Example 7 76.8 Example 8 61.2 Example 9 59.6 Example 10 59.0 Example 11 59.8 Example 12 69.8 Example 13 76.7 Example 14 59.5 Example 15 71.4 Example 16 67.3 Example 17 59.1 Example 18 56.7 Comparative Example 1 3.8

As can be seen in Table 9, when distilled water was used, the FNA reduction rate was only 3.8%, and the reaction time was reduced by only 4% even if the reaction time was extended to 24 hours. That is, when distilled water was used, the reaction hardly proceeded, and thus, it would be difficult to substitute distilled water for the buffer solution. In addition, although not described in Tables 8 and 9, when ethyl ether, chloroform, acetone, ethanol, methanol, propanol, and the like were used as the organic solvent, cNDA did not dissolve and the reaction did not proceed at all.

As described in detail above, the novel strains of the present invention Bacillus sp. F-1 and F-3 remove 2-formyl-6-naphthoic acid contained in crude naphthalene dicarboxylic acid. Since there is an excellent effect, it is expected that mass production of high purity 2,6-naphthalene dicarboxylic acid is realized by the method of the present invention.

1 shows a partial sequence of 16S rDNA of Bacillus sp. F-1 strain (SEQ ID NO: 1);

2 shows a partial sequence of 16S rDNA of Bacillus sp. F-3 strain (SEQ ID NO: 2);

3 is a schematic diagram of a process for synthesizing NDA and NDC from 2,6-DMN; And

4 shows a product resulting from the oxidation of 2,6-DMN.

<110> HYOSUNG CORPORATION <120> NOVEL BACILLUS STRAINS AND METHOD FOR PURIFYING 2,6-NAPHTHALENE DICARBOXYLIC ACID THEREBY <130> 02-TNC-41 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 705 <212> DNA <213> Bacillus sp. F-1 <400> 1 agtcgagcgg acagatggga gcttgctccc tgatgttagc ggcggacggg tgagtaacac 60 gtgggtaacc tgcctgtaag actgggataa ctccgggaaa ccggggctaa taccggatgg 120 ttgtttgaac cgcatggttc aaacataaaa ggtggcttcg gctaccactt acagatggac 180 ccgcggcgca ttagctagtt ggtgaggtaa cggctcacca aggcgacgat gcgtagccga 240 cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct acgggaggca 300 gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg 360 aaggttttcg gatcgtaaag ctctgttgtt agggaagaac aagtaccgtt cgaatagggc 420 ggtnccttga cggtacctaa ccagaaagcc acggctaact acgtgccagc agccgcggta 480 atacgtaggt ggcaagcgtt gtccggaatt attgggcgta aagggctcgc aggcggtttc 540 ttaagtctga tgtgaaagcc cccggctcaa ccgggagggt cattggaaac tggggaactt 600 gagtgcagaa gaggagagtg gaattccacg tgtagcggtg aaatgcgtaa agatgtggag 660 gaacaccagt ggcgaagcga ctctctggtc tgtaactgac gctga 705 <210> 2 <211> 715 <212> DNA <213> Bacillus sp. F-3 <400> 2 acatgcaagt cgagcggaca gatgggagct tgctccctga tgttagcggc ggacgggtga 60 gtaacacgtg ggtaacctgc ctgtaagact gggataactc cgggaaaccg gggctaatac 120 cggatggttg tttgaaccgc atggttcaaa cataaaaggt ggcttcggct accacttaca 180 gatggacccg cggcgcatta gctagttggt gaggtaacgg ctcaccaagg cgacgatgcg 240 tagccgacct gagagggtga tcggccacac tgggactgag acacggccca nactcctacg 300 ggaggcagca gtagggaatc ttccgcaatg gacgaaagtc tgacggagca acgcccgcgt 360 gagtgatgaa ggttttcgga tcgtaaagct ctgttgttag ggaagaacaa gtaccgttcg 420 aatagggcgg tnccttgacg gtacctaacc agaaagccac ggctaactac gtgccagcag 480 ccgcggtaat acgtaggtgg caagcgttgt ccggaattat tgggcgtaaa gggctcgcag 540 gcggtttctt aagtctgatg tgaaagcccc cggctcaacc ggggagggtc attggaaact 600 ggggaacttg agtgcagaag aggagagtgg aattccacgt gtagcggtga aatgcgtaaa 660 gatgtggagg aacaccagtg gcgaaggcga ctctctggtc tgtaactgac gctga 715

Claims (5)

Bacillus sp. F-1 (KCTC-10342BP). delete Bacillus sp. F-1 (KCTC-10342BP) was treated with crude naphthalene dimethylcarboxylic acid (crude) at a temperature of 25-45 ° C. in a buffer solution of pH 6.0-10.0 containing 0-20% organic solvent. Naphthalene dicarboxylic acid) to decompose the 2-formyl-6-naphthoic acid in the crude naphthalene dimethylcarboxylic acid (2,6-naphthalenedicarboxylic acid) Purification method. 4. The method according to claim 3, wherein the organic solvent is one selected from the group consisting of dimethyl sulfoxide, dimethylformamide, dimethylacetamide and tetrahydrofuran. The method of claim 3, wherein the buffer solution is sodium carbonate buffer (Na ₂ CO ₃ / NaHCO ₃ ), glycine buffer (Glycine / NaOH), potassium phosphate buffer (KH ₂ PO ₄ / KOH), sodium phosphate buffer ( Na ₂ HPO ₄ / NaH ₂ PO ₄ ), succinic acid buffer (Succinic acid / NaOH), sodium acetate buffer (Sodium acetate / Acetic acid), citric acid buffer (Citric acid / Sodium citrate), sodium pyrophosphate buffer ( Na ₄ P ₂ O ₇ / HCl), boric acid buffer (Boric acid / NaOH) and sodium borate buffer (Sodium borate / HCl) method characterized in that one selected from the group consisting of.