WO2008004731A1 - 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
Purification method of crude naphthalene dicarboxylic acid using recombinated microorganism and 2,6-naphthalene dicarboxylic acid in crystalline form obtained by using the same Download PDFInfo
- Publication number
- WO2008004731A1 WO2008004731A1 PCT/KR2006/005729 KR2006005729W WO2008004731A1 WO 2008004731 A1 WO2008004731 A1 WO 2008004731A1 KR 2006005729 W KR2006005729 W KR 2006005729W WO 2008004731 A1 WO2008004731 A1 WO 2008004731A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- naphthalene dicarboxylic
- dicarboxylic acid
- acid
- cnda
- solution
- Prior art date
Links
- KYTZHLUVELPASH-UHFFFAOYSA-N naphthalene-1,2-dicarboxylic acid Chemical compound C1=CC=CC2=C(C(O)=O)C(C(=O)O)=CC=C21 KYTZHLUVELPASH-UHFFFAOYSA-N 0.000 title claims abstract description 86
- 238000000034 method Methods 0.000 title claims abstract description 79
- 244000005700 microbiome Species 0.000 title claims abstract description 76
- 238000000746 purification Methods 0.000 title claims abstract description 35
- RXOHFPCZGPKIRD-UHFFFAOYSA-N naphthalene-2,6-dicarboxylic acid Chemical compound C1=C(C(O)=O)C=CC2=CC(C(=O)O)=CC=C21 RXOHFPCZGPKIRD-UHFFFAOYSA-N 0.000 title claims abstract description 32
- 238000006243 chemical reaction Methods 0.000 claims abstract description 64
- 239000000243 solution Substances 0.000 claims abstract description 57
- 239000013078 crystal Substances 0.000 claims abstract description 53
- SHFLOIUZUDNHFB-UHFFFAOYSA-N 6-formylnaphthalene-2-carboxylic acid Chemical compound C1=C(C=O)C=CC2=CC(C(=O)O)=CC=C21 SHFLOIUZUDNHFB-UHFFFAOYSA-N 0.000 claims abstract description 32
- 238000003756 stirring Methods 0.000 claims abstract description 25
- 239000011259 mixed solution Substances 0.000 claims abstract description 18
- 238000001035 drying Methods 0.000 claims abstract description 12
- 239000003929 acidic solution Substances 0.000 claims abstract description 9
- 238000005406 washing Methods 0.000 claims abstract description 9
- HUMNYLRZRPPJDN-UHFFFAOYSA-N benzaldehyde Chemical compound O=CC1=CC=CC=C1 HUMNYLRZRPPJDN-UHFFFAOYSA-N 0.000 claims description 70
- 101100156993 Pseudomonas putida xylC gene Proteins 0.000 claims description 38
- 101710088194 Dehydrogenase Proteins 0.000 claims description 35
- 229940095076 benzaldehyde Drugs 0.000 claims description 35
- QNGNSVIICDLXHT-UHFFFAOYSA-N para-ethylbenzaldehyde Natural products CCC1=CC=C(C=O)C=C1 QNGNSVIICDLXHT-UHFFFAOYSA-N 0.000 claims description 35
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 33
- 239000002253 acid Substances 0.000 claims description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 24
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 24
- 108090000623 proteins and genes Proteins 0.000 claims description 23
- 239000013604 expression vector Substances 0.000 claims description 22
- 238000003259 recombinant expression Methods 0.000 claims description 18
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 17
- 239000000872 buffer Substances 0.000 claims description 16
- 239000012535 impurity Substances 0.000 claims description 15
- 238000001914 filtration Methods 0.000 claims description 14
- 241000233540 Novosphingobium aromaticivorans Species 0.000 claims description 10
- -1 polypropylene Polymers 0.000 claims description 10
- 241000588724 Escherichia coli Species 0.000 claims description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 8
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 8
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 8
- 241000589776 Pseudomonas putida Species 0.000 claims description 8
- 230000009466 transformation Effects 0.000 claims description 8
- 229960000583 acetic acid Drugs 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 7
- 239000003960 organic solvent Substances 0.000 claims description 7
- 229920000139 polyethylene terephthalate Polymers 0.000 claims description 7
- 239000005020 polyethylene terephthalate Substances 0.000 claims description 7
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 6
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 6
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 6
- 239000004327 boric acid Substances 0.000 claims description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 229910017604 nitric acid Inorganic materials 0.000 claims description 6
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 claims description 6
- 239000012670 alkaline solution Substances 0.000 claims description 5
- 239000002504 physiological saline solution Substances 0.000 claims description 5
- 239000004471 Glycine Substances 0.000 claims description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 4
- 229910021538 borax Inorganic materials 0.000 claims description 4
- 239000012362 glacial acetic acid Substances 0.000 claims description 4
- 239000011148 porous material Substances 0.000 claims description 4
- 235000010339 sodium tetraborate Nutrition 0.000 claims description 4
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 claims description 4
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 3
- FXHOOIRPVKKKFG-UHFFFAOYSA-N N,N-Dimethylacetamide Chemical compound CN(C)C(C)=O FXHOOIRPVKKKFG-UHFFFAOYSA-N 0.000 claims description 3
- 239000004743 Polypropylene Substances 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 229920001155 polypropylene Polymers 0.000 claims description 3
- 239000001384 succinic acid Substances 0.000 claims description 3
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 3
- 241000588722 Escherichia Species 0.000 claims description 2
- 241000589516 Pseudomonas Species 0.000 claims description 2
- 241000316848 Rhodococcus <scale insect> Species 0.000 claims description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 2
- 239000000919 ceramic Substances 0.000 claims description 2
- 229940069078 citric acid / sodium citrate Drugs 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000001632 sodium acetate Substances 0.000 claims description 2
- 235000017281 sodium acetate Nutrition 0.000 claims description 2
- 239000007974 sodium acetate buffer Substances 0.000 claims description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 2
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 claims description 2
- 239000012064 sodium phosphate buffer Substances 0.000 claims description 2
- 229940048086 sodium pyrophosphate Drugs 0.000 claims description 2
- 229910001220 stainless steel Inorganic materials 0.000 claims description 2
- 239000010935 stainless steel Substances 0.000 claims description 2
- 239000000758 substrate Substances 0.000 claims description 2
- 235000019818 tetrasodium diphosphate Nutrition 0.000 claims description 2
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 claims description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims 2
- 229960001760 dimethyl sulfoxide Drugs 0.000 claims 1
- 239000006185 dispersion Substances 0.000 claims 1
- 229910000160 potassium phosphate Inorganic materials 0.000 claims 1
- 235000011009 potassium phosphates Nutrition 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 15
- 238000002425 crystallisation Methods 0.000 description 32
- 230000008025 crystallization Effects 0.000 description 32
- 239000002245 particle Substances 0.000 description 29
- 239000002002 slurry Substances 0.000 description 18
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 15
- 238000004140 cleaning Methods 0.000 description 15
- 229920003207 poly(ethylene-2,6-naphthalate) Polymers 0.000 description 15
- 239000011112 polyethylene naphthalate Substances 0.000 description 15
- 108020004414 DNA Proteins 0.000 description 14
- 239000002585 base Substances 0.000 description 14
- 238000011084 recovery Methods 0.000 description 11
- 239000013598 vector Substances 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 10
- 239000000706 filtrate Substances 0.000 description 10
- 239000002904 solvent Substances 0.000 description 10
- 150000007513 acids Chemical class 0.000 description 9
- YGYNBBAUIYTWBF-UHFFFAOYSA-N 2,6-dimethylnaphthalene Chemical compound C1=C(C)C=CC2=CC(C)=CC=C21 YGYNBBAUIYTWBF-UHFFFAOYSA-N 0.000 description 8
- 239000012634 fragment Substances 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 7
- 239000007864 aqueous solution Substances 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 230000002776 aggregation Effects 0.000 description 6
- 238000004220 aggregation Methods 0.000 description 6
- 238000010438 heat treatment Methods 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- UOBYKYZJUGYBDK-UHFFFAOYSA-N 2-naphthoic acid Chemical compound C1=CC=CC2=CC(C(=O)O)=CC=C21 UOBYKYZJUGYBDK-UHFFFAOYSA-N 0.000 description 5
- 230000002068 genetic effect Effects 0.000 description 5
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 238000003752 polymerase chain reaction Methods 0.000 description 5
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000001000 micrograph Methods 0.000 description 4
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- 238000006116 polymerization reaction Methods 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 241000894007 species Species 0.000 description 4
- ARCGXLSVLAOJQL-UHFFFAOYSA-N trimellitic acid Chemical compound OC(=O)C1=CC=C(C(O)=O)C(C(O)=O)=C1 ARCGXLSVLAOJQL-UHFFFAOYSA-N 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
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- 238000004925 denaturation Methods 0.000 description 3
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- 239000012467 final product Substances 0.000 description 3
- 239000008057 potassium phosphate buffer Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
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- 238000011282 treatment Methods 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
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- 229910001385 heavy metal Inorganic materials 0.000 description 2
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- 230000001590 oxidative effect Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- FGDZQCVHDSGLHJ-UHFFFAOYSA-M rubidium chloride Chemical compound [Cl-].[Rb+] FGDZQCVHDSGLHJ-UHFFFAOYSA-M 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- ZSPDYGICHBLYSD-UHFFFAOYSA-N 2-methylnaphthalene-1-carboxylic acid Chemical compound C1=CC=CC2=C(C(O)=O)C(C)=CC=C21 ZSPDYGICHBLYSD-UHFFFAOYSA-N 0.000 description 1
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000588921 Enterobacteriaceae Species 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 101100346764 Mus musculus Mtln gene Proteins 0.000 description 1
- 241001655308 Nocardiaceae Species 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 229910003798 SPO2 Inorganic materials 0.000 description 1
- 101100478210 Schizosaccharomyces pombe (strain 972 / ATCC 24843) spo2 gene Proteins 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
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- 125000001931 aliphatic group Chemical group 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 229910000288 alkali metal carbonate Inorganic materials 0.000 description 1
- 150000008041 alkali metal carbonates Chemical class 0.000 description 1
- 150000001340 alkali metals Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000003637 basic solution Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 150000005690 diesters Chemical class 0.000 description 1
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- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000004880 explosion Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 230000000887 hydrating effect Effects 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
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- 230000008018 melting Effects 0.000 description 1
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- 239000000178 monomer Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
- C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
- C12Y102/01028—Benzaldehyde dehydrogenase (NAD+) (1.2.1.28)
Definitions
- the present invention relates to a method for purifying a crude naphthalene di- carboxylic acid using a recombinant microorganism and 2,6-naphthalene dicarboxylic acid in a crystalline form produced by the method.
- the present invention relates to a method for purifying a crude naphthalene dicarboxylic acid by reacting a recombinant microorganism having the ability to convert 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid with a crude naphthalene dicarboxylic acid to remove 2-formyl-6-naphthoic acid contained as an impurity in the crude naphthalene dicarboxylic acid, adding an acidic solution to the reaction solution under particular conditions, allowing the mixed solution to react with stirring to crystallize the crude naphthalene dicarboxylic acid, washing the crystal of the crude naphthalene dicarboxylic acid to remove other impurities contained therein, and drying the washed product to obtain 2,6-naphthalene dicarboxylic acid in a pure crystalline form.
- NDA 2,6-Naphthalene dicarboxylic acid
- NDC 2,6-naphthalene di- carboxylate
- PEN poly(ethylene 2,6-naphthalate)
- PET poly(ethylene terephthalate)
- PEN is highly resistant to the diffusion of gases, particularly carbon dioxide, oxygen and water vapor
- films made from PEN are useful for the manufacture of food containers, especially hot-fill food containers.
- PEN can also be used to produce reinforced fibers useful for the manufacture of tire cords.
- NDC is currently produced by oxidizing 2,6-dimethylnaphthalene (2,6-DMN) to obtain a crude naphthalene dicarboxylic acid (cNDA) and esterifying the cNDA.
- 2,6-DMN 2,6-dimethylnaphthalene
- cNDA naphthalene dicarboxylic acid
- NDA 2,6-naphthalene dicarboxylic acid
- water is formed as a by-product during the condensation of NDA
- methanol is formed as a by-product in the case of NDC, thus risking the danger of explosion.
- NDC is preferentially used to produce PEN because it is still difficult to produce purified NDA having a purity necessary for the synthesis of PEN.
- 2,6-Dimethylnaphthalene (2,6-DMN) is oxidized to form a cNDA containing various impurities, such as 2-formyl-6-naphthoic acid (FNA), 2-naphthoic acid (NA) and trimellitic acid.
- FNA 2-formyl-6-naphthoic acid
- NA 2-naphthoic acid
- trimellitic acid trimellitic acid
- NDA is produced by i) recrystallizing a cNDA, ii) oxidizing a cNDA one more time, or iii) treating a cNDA with methanol to produce NDC and hydrating the NDC.
- purified NDA is produced by hydrogenation of a cNDA.
- many processes e.g., solvent treatment, melting/crystallization, high-pressure crystallization and supercritical extraction, have been employed to purify NDA.
- U.S. Patent No. 5,859,294 discloses a process for the production of a naphthalene dicarboxylic acid, which comprises dissolving a crude naphthalene dicarboxylic acid in an aqueous solution containing an aliphatic or alicyclic amine, removing heavy metal components contained as impurities until the content of the heavy metal components based on the crude naphthalene dicarboxylic acid is 100 ppm or less, and heating the aqueous solution containing a naphthalene dicarboxylic acid amine salt to distill off the amine.
- U.S. Patent No. 6,255,525 discloses a process for preparing an aromatic carboxylic acid having improved purity comprising the steps of contacting a mixture comprising an impure aromatic carboxylic acid and water at a pressure of 77 to 121 kg/cm and a temperature of 277 to 316 0 C in the presence of hydrogen with a carbon catalyst which is free of a hydrogenation metal component, cooling the mixture to form crystallized aromatic carboxylic acid, and recovering the crystallized aromatic carboxylic acid from the cooled mixture.
- U.S. Patent No. 6,087,531 teaches a process for recovering a naphthalene dicarboxylic acid (NDA) crystal comprising the steps of dissolving poly(alkylene naphthalene dicarboxylate) in an aqueous basic solution (e.g., an aqueous solution of an alkali metal base, an aqueous hydroxide solution or an aqueous solution of an alkali metal carbonate) at a temperature of 125 to 400 0 C, neutralizing the aqueous solution with an acid at 170 to 24O 0 C, and recovering the NDA.
- an NDA crystal is recovered by dissolving the polyester material in a NaOH or KOH solution at a temperature of 125 to 400 0 C, neutralizing the aqueous solution with acetic acid, and recovering the NDA.
- 2,6-NDA by as high as 45%, the method comprising treating an aqueous solution of K -NDA at a CO pressure of 0-200 psi and a temperature of 0-50 0 C to form KH-NDA, suspending the KH-NDA in water in a weight ratio higher than 1:8 (KH-NDA: water), and further treating the suspension at a temperature above 100 0 C (140-160 0 C) and at a CO pressure above 100 psi (175-250 psi).
- the present invention has been made in view of the problems of the prior art, and it is one object of the present invention to provide a method for efficiently producing high-purity NDA in high yield by purifying and crystallizing a crude naphthalene dicarboxylic acid at ambient pressure and temperature conditions using a recombinant microorganism capable of converting FNA to NDA.
- the purification method of the present invention comprises the following steps:
- step (b) adding an acidic solution to the reaction solution prepared in step (a) to adjust the pH of the reaction solution and reacting the mixed solution with stirring to crystallize the crude naphthalene dicarboxylic acid;
- a crude naphthalene dicarboxylic acid is reacted with at least one recombinant microorganism selected from the group consisting of microorganisms obtained by transformation with a recombinant expression vector carrying the benzaldehyde dehydrogenase gene (xylC) of SEQ ID NO: 1 derived from Sphingomonas aromaticivorans (KCTC 2888) or a gene having a homology of at least 90% with the xylC gene and microorganisms obtained by transformation with a recombinant expression vector carrying the benzaldehyde dehydrogenase gene (xylC) of SEQ ID NO: 4 derived from Pseudomonas putida mt-2 (ATCC 33015) or a gene having a homology of at least 90% with the xylC gene, to remove 2-formyl-6-naphthoic acid present in the crude naphthalene dicar
- the expression "gene having a homology of at least 90%” as used herein refers to a gene that has a homology of at least 90% with the benzaldehyde dehydrogenase gene used in the method of the present invention in the base or amino acid sequence level, and that shows the same activity as the benzaldehyde dehydrogenase despite the difference in base or amino acid sequence from the benzaldehyde dehydrogenase gene within 10%.
- the different sequence segments are intended to include all segments that may be modified by those skilled in the art without significantly affecting the inherent activity of the benzaldehyde dehydrogenase.
- this step includes the sub-steps of 1) inoculating the recombinant microorganism into a liquid medium, culturing the cells to induce the expression of benzaldehyde dehydrogenase, centrifuging the culture broth to collect the cells in which benzaldehyde dehydrogenase is expressed, and suspending the cells in physiological saline or distilled water, 2) mixing a crude naphthalene dicarboxylic acid (cNDA) as a substrate with a buffer solution and adjusting the pH of the mixed solution by the addition of an alkaline solution to prepare a reaction solution for subsequent purification, and 3) reacting the cells prepared in 1) with the reaction solution prepared in 2) to convert 2-formyl-6-naphthoic acid contained in the crude naphthalene dicarboxylic acid to 2,6-naphthalene dicarboxylic acid, so that the purity of the 2,6-naphthalene dicarboxylic acid is increased.
- cNDA
- Recombinant microorganisms that can be used in the present invention may be obtained by cloning the benzaldehyde dehydrogenase gene (xylC) of SEQ ID NO: 1 derived from Sphingomonas aromaticivorans (KCTC 2888), a gene having a homology of at least 90% with the xylC gene of SEQ ID NO: 1, the benzaldehyde dehydrogenase gene (xylC) of SEQ ID NO: 4 derived from Pseudomonas putida mt-2 (ATCC 33015) or a gene having a homology of at least 90% with the xylC gene of SEQ ID NO: 4 in a manner known in the art, introducing the cloned gene into an e xpression vector to construct a recombinant expression vector, and introducing the recombinant expression vector into a microorganism, and transforming the microorganism with the recombin
- the recombinant microorganisms can be obtained by the following procedure.
- each benzaldehyde dehydrogenase gene xylC
- plasmid DNA or genomic DNA is isolated from each of the microorganisms and polymerase chain reaction (PCR) using the plasmid DNA or genomic DNA as a template is performed using a set of primers synthesized on the basis of the xylC gene to obtain a gene encoding benzaldehyde dehydrogenase.
- PCR polymerase chain reaction
- the base sequence of the gene encoding benzaldehyde dehydrogenase may be partially modified by a common technique known in the art.
- the cloned xylC gene is functionally linked to a promoter of an expression vector by a conventional technique to construct a recombinant expression vector.
- any expression vector that can allow the optimum expression of the gene in a host may be available without any particular limitation.
- the gene can be inserted into a plasmid, phage or another DNA as the expression vector.
- suitable plasmids include, but are not specially limited to: known pForexT, pLG338, pACYC184, pBR322, pUC119, pUC18, pUC19, pKC30, P Rep4, pHSl, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III 138 -Bl, ⁇ gtl l and pBDCl vectors in E.
- promoters suitable for use in gram-negative bacteria such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacf, T7, T5, T3, gal, trc, ara, SP6, ⁇ -PR and ⁇ -PL promoters
- gram-positive promoters such as amy and SPO2 promoters
- fungi or yeast promoters such as ADCl, MFa, AC, P-60, CYCl, GAPDH, TEF, rp28 and ADH promoter.
- a regulatory sequence may be further included in the 3' end and/or 5' end of the gene.
- FIG. 1 shows a genetic map of a recombinant expression vector
- FIG. 2 shows a genetic map of a recombinant expression vector (pUC18-xy/C) carrying an xylC gene encoding benzaldehyde dehydrogenase derived from Pseudomonas putida used in Example 3 of the present invention.
- the recombinant expression vector thus constructed is introduced into a host microorganism by any conventional technique known in the art to transform the microorganism.
- a recombinant microorganism is prepared.
- the host microorganism any prokaryotic or eukaryotic organism may be used. Examples of preferred host microorganisms include, but are not necessarily limited to, bacteria, fungi and yeasts.
- the host microorganism may be typically a gram-positive or gram- negative bacterial species, preferably a species of Enterobacteriaceae or Nocardiaceae family, more preferably a bacterial species of the genus Escherichia, Pseudomonas, Rhodococcus or Bacillus, and most preferably a bacterial species of the genus E. coli.
- Specific examples of the host microorganism include MC 1061 (E. coli), JM 109 (E. coli), XLl -Blue (E. coli) and DH5 (E. coli).
- the transformation may be performed by any suitable technique, such as heat treatment, electric shock, microinjection, calcium chloride method, rubidium chloride method or pressure spraying, but the present invention is not necessarily limited to these techniques.
- 2-formyl-6-naphthoic acid is converted to 2,6-naphthalene dicarboxylic acid with the help of the recombinant microorganism transformed with the expression vector of benzaldehyde dehydrogenase.
- benzaldehyde dehydrogenase be expressed at a high level within the recombinant microorganism.
- IPTG isopropyl- ⁇ -D-thiogalactopyranoside
- the recombinant microorganism is sufficiently cultured in a typical culture temperature range (25-45 0 C), preferably at 37 0 C, and is then inoculated into a 100 ml of a liquid medium (e.g., an LB medium) in such an amount that the concentration is 1% (v/v).
- a liquid medium e.g., an LB medium
- the concentration is 0.1-2.0 mM and preferably 0.5 mM to induce the expression of the benzaldehyde dehydrogenase, followed by culture at 37 0 C. It is to be appreciated that the culture and the protein expression of the recombinant organism can be achieved, without any limitation to the foregoing techniques, by any technique known to those skilled in the art.
- the recombinant microorganism used in the method of the present invention enables the expression of benzaldehyde dehydrogenase. Therefore, the high- concentration culture of the recombinant microorganism is facilitated, which provides an economic advantage.
- the culture solution of the recombinant microorganism in which the protein is expressed at a high level is centrifuged to collect the cells, and then the cells are suspended in physiological saline or distilled water. The suspension is used as a reaction solution for the purification of a crude naphthalene dicarboxylic acid in the subsequent step.
- the kind of the buffer solution is not especially restricted.
- the buffer solution there can be used, for example, water, a sodium carbonate buffer (Na CO /NaHCO ), a glycine buffer (glycine/NaOH), a potassium phosphate buffer (KH PO /KOH), a sodium phosphate buffer (Na HPO /NaH PO ), a succinic acid buffer (succinic acid/ NaOH), a sodium acetate buffer (sodium acetate/acetic acid), a citric acid buffer (citric acid/sodium citrate), a sodium pyrophosphate buffer (Na P O /HCl), a boric acid buffer (boric acid/NaOH), or a sodium borate buffer (sodium borate/HCl).
- a sodium carbonate buffer Na CO /NaHCO
- glycine buffer glycine/NaOH
- KH PO /KOH potassium phosphate buffer
- KH PO -KOH potassium phosphate buffer
- boric acid buffer boric acid/NaOH
- concentration of the buffer solution be in the range of 0.01 to 100 mM.
- the alkaline solution is preferably a NaOH or KOH solution, but is not limited thereto.
- the pH of the mixed solution is preferably adjusted to the range of 6 to 10, preferably 8 to 10.
- an organic solvent may be additionally added to the mixed solution for the purpose of dissolving the cNDA.
- preferred organic solvents include dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), and tetrahydrofuran (THF). Of these, dimethylsulfoxide is most preferred in terms of enzymatic activity.
- the organic solvent is preferably added at a concentration of 0.01 to 20% and more preferably 0.1 to 10%. It is most preferred that no organic solvent be added. The addition of the organic solvent at a concentration exceeding 20% causes lysis of the cell membranes of the microorganism, resulting in inhibition of the reaction.
- the reaction is preferably conducted at 25-7O 0 C for 1 min.-l hour and more preferably at 40-60 0 C for 10-40 minutes.
- the reaction temperature is lower than 25 0 C or higher than 7O 0 C, a marked decrease in the reactivity is undesirably caused.
- the concentration of the cNDA in the reaction solution is preferably between 0.001% and 20%, and the FNA content in the cNDA is between 0.001% and 10% and preferably 9%.
- the amount of the microorganism necessary to completely remove Ig of the FNA is approximately 5g.
- a microorganism e.g., Bacillus sp. F-I or F- 3 described in Korean Patent Application No. 2002-0087819, which was filed by the present inventors
- having the ability to convert FNA to NDA is generally used in an amount of about 1Og in order to completely remove Ig of FNA, it turns out that the purification of cNDA using the recombinant microorganism in accordance with the present invention is more efficient from an economic viewpoint.
- step (48) an acidic solution is added to the reaction solution prepared in step (a) to adjust the pH of the reaction solution, and is then reacted with the reaction solution with stirring to crystallize the crude naphthalene dicarboxylic acid.
- an appropriate amount of an acidic solution is added to the reaction solution prepared in step (a) in a reactor equipped with an stirrer to adjust the pH of the reaction solution to a desired range, and then the mixed solution is reacted with continuous stirring while maintaining the temperature of the mixed solution constant, so that the cNDA in an amorphous form present in the FNA-free reaction solution is crystallized at ambient pressure and temperature conditions.
- the purification method of the present invention is economically advantageous in terms of production cost and processing.
- the purification method of the present invention is suitable for actual use and is advantageous in terms of a high recovery rate of the final product.
- the acidic solution there may be used, for example, sulfuric acid, hydrochloric acid, glacial acetic acid or nitric acid.
- sulfuric acid or hydrochloric acid is preferred because it leads to large size and high yield of the cNDA crystal. That is, the addition of sulfuric acid or hydrochloric acid enables the production of a cNDA crystal having a uniform size of 100 D or more in high yield.
- the pH of the reaction solution is preferably adjusted to 1-4 to increase the recovery rate of the final product. As the pH of the reaction solution decreases, the recovery rate of the final product tends to increase from about 94% to about 99.9%.
- the crystallization is performed at about 4 0 C to about 15O 0 C, preferably 3O 0 C to
- the crystallization is performed for about 1 minute to about 10 hours, preferably 2 minutes to 5 hours and more preferably 10 minutes to one hour in terms of continuous processing.
- the most preferable conditions for the crystallization are 8O 0 C and 20-30 minutes. Too short crystallization time may cause a low degree of crystallization of the cNDA, resulting in aggregation between the individual cNDA crystal particles upon polymerization. Meanwhile, too long crystallization time may cause an unnecessary energy loss.
- the stirring required to perform the crystallization of the cNDA is typically performed at a rate of 0-1,000 rpm, preferably 0-400 rpm and more preferably 50-150 rpm.
- the temperature range for the separation is between 100 0 C and 27O 0 C and preferably between 15O 0 C and 24O 0 C.
- the separation of the solvent at a temperature higher than 27O 0 C causes increased loss of the purified NDA, leading to a considerable drop in yield.
- the separation of the solvent at a temperature lower than 100 0 C unfavorably causes low dissolution of the impurities, including NA, MNA and TLMA.
- the cNDA crystal is dispersed in water, stirred at a pressure of 1 to 60 kg/cm and a temperature of 100 to 27O 0 C for 10 minutes to 1 hour, and filtered to remove the water. This procedure may be repeated several times to remove the impurities. At this time, the solvent is preferably used in an amount of 5-20 times of the weight of the cNDA crystal.
- the NDA crystal, from which the impurities are removed by the washing is dried at a specified temperature to obtain NDA in a pure crystalline form.
- the drying is preferably performed at 30 to 200 0 C.
- the drying is performed, without limitation, by a conventional technique known in the art.
- the purification method of the present invention may further comprise the step of removing the recombinant microorganism used in step (a) after step (a) and prior to step (b).
- the removal of the recombinant microorganism is achieved, without limitation, by a conventional technique known in the art.
- a microfilter system a continuous type centrifugal separator or a decanter may be used to remove the recombinant microorganism.
- the microfilter system uses a filter having a pore size of 0.1-0.5 D and made of a material selected from ceramic, stainless steel, polypropylene and polyethylene terephthalate (PET).
- the present invention provides 2,6-naphthalene dicarboxylic acid in a pure crystalline form produced by the purification method.
- the purification method of the present invention enables the production of high- purity 2,6-naphthalene dicarboxylic acid in a high yield of 99.9% or more.
- the treatment conditions may be varied to produce 2,6-naphthalene dicarboxylic acid in a regular or random crystalline form.
- the 2,6-naphthalene dicarboxylic acid in a crystalline form may have a lattice structure.
- the 2,6-naphthalene dicarboxylic acid crystal of the present invention has an average particle diameter not smaller than 100 D and a uniform particle shape. Accordingly, the 2,6-naphthalene dicarboxylic acid crystal is very suitable for the formation of a low- viscosity slurry with ethylene glycol when it is polymerized with the ethylene glycol to produce PEN.
- a 2,6-naphthalene dicarboxylic acid crystal having an average particle diameter smaller than 100 D is difficult to treat and exhibits a poor ability to form a slurry with ethylene glycol when it is mixed with the ethylene glycol to prepare PEN, causing an increase in power consumption.
- the 2,6-naphthalene dicarboxylic acid crystal of the present invention preferably has an average particle diameter of 100-200 D and more preferably 110-170 D.
- FIG. 5 shows a purification system for implementing a method for purifying a crude naphthalene dicarboxylic acid according to an embodiment of the present invention.
- the purification method according to the embodiment of the present invention will be explained in more detail below.
- a specified amount of a buffer solution is introduced into a reactor A where a crude naphthalene dicarboxylic acid is purified using a microorganism.
- a cNDA is added to the reactor, and subsequently, an alkaline solution is added to the reactor with stirring to adjust the pH of the reaction solution.
- a specified amount of water is added to the reactor to prepare a reaction solution for subsequent purification.
- the cells are added to react with the reaction solution while maintaining the temperature of the reaction solution at a constant level. By this procedure, FNA contained in the cNDA is converted to NDA in the reactor A, and can be finally removed.
- reaction solution is passed through a unit B where the recombinant microorganism used to remove the FNA is removed.
- the removal of the recombinant microorganism using the unit B may be omitted, if needed, because the removal of the recombinant microorganism can be achieved in a downstream filtering/cleaning unit F.
- reaction solution from which the microorganism is removed, is transferred to a crystallization reactor C.
- An acidic solution is added to the crystallization reactor C with stirring to conduct a crystallization reaction.
- a slurry containing the cNDA crystal is obtained after the crystallization reaction.
- the slurry is heated to about 100 to about 27O 0 C using a preheater D, and is then fed into a filtering/cleaning unit F where a heater is provided to maintain the temperature of the slurry at 100-270 0 C.
- the pressure of the filtering/cleaning unit F is maintained at 1-60 kg/cm .
- the filtering/cleaning unit F is equipped with a filter (pore size: 10-100 D).
- the filtering/cleaning unit F is connected to a high-pressure filtrate collector G.
- a filtrate is discharged from the filtering/cleaning unit F into the high-pressure filtrate collector G and solid components are filtered by the filter included in the filtering/ cleaning unit F.
- Water (100-270 0 C) is preheated in a solvent heating/supply unit E and supplied to the filtering/cleaning unit F. After stirring is continued in the filtering/cleaning unit F for a given time, a secondary filtrate is discharged into the high-pressure filtrate collector G. If necessary, the cleaning step is repeated once or twice. Pure NDA remaining after the cleaning is mixed with preheated water (100-270 0 C) to form a slurry. The slurry is sent to a high-pressure slurry collector H via a slurry discharge line.
- the slurry containing the pure NDA is transferred to a powder separator I where the solvent is removed from the slurry, followed by drying in a dryer J to collect the pure NDA only.
- a crude naphthalene dicarboxylic acid is purified and crystallized under respective suitable conditions using a recombinant microorganism capable of converting FNA to NDA. Therefore, the purification method of the present invention enables the production of high-purity crystalline 2,6-naphthalene dicarboxylic acid on an industrial scale in an economically feasible and environmentally friendly manner.
- FIG. 1 is a genetic map of a recombinant expression vector (pET20-xy/C) carrying an xylC gene encoding benzaldehyde dehydrogenase derived from Sphingomonas aro- maticivorans;
- FIG. 2 is a genetic map of a recombinant expression vector (pUC18-xy/C) carrying an xylC gene encoding benzaldehyde dehydrogenase derived from Pseudomonas putida;
- FIG. 3 is a micrograph of a cNDA crystal obtained in the third step of Example 1 of the present invention.
- FIG. 4 is a micrograph of a cNDA crystal obtained by adding a sulfuric acid solution to a reaction solution of a purified cNDA prepared in Example 1 and allowing the mixed solution to react at 8O 0 C in Experimental Example 3 of the present invention.
- FIG. 5 shows a purification system for implementing a method for purifying a crude naphthalene dicarboxylic acid according to an embodiment of the present invention.
- the DNA fragment (about 1.5 kbp long) set forth in SEQ ID NO: 1 was isolated, cleaved with restriction enzymes Ndel and Sail, and cloned into the plasmid vector pET-20b(+), which was previously cleaved with the same restriction enzymes, to construct the recombinant expression vector pET20-xy/C shown in FIG. 1.
- Example 1 the recombinant vector was cleaved using various restriction enzymes on the basis of the genetic maps of two vectors M13mpl8 and M13mpl9 to obtain respective fragments.
- the fragments were subcloned into M13mpl8 and M13mpl9.
- the resulting subclones were subjected to base sequencing using an ABI PRISM BigDye primer cycle- sequencing kit (Perkin-Elmer, U.S.) with AmpliTaq DNA polymerase. In order to read the double-stranded DNA in both directions, nucleotide fragments were partially synthesized.
- the base sequence of the fragments of the cloned DNA was analyzed through the nucleotide fragments and compared with the base sequence available from GenBank (GenBank Accession Number: AF073917 or NC002030). As a result, the cloned DNA was confirmed to be xylC gene.
- E. coli XLl -Blue was transformed with the pET20-xy/C vector by a calcium chloride method (see, Sambrook et al., Molecular Cloning, A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and grown by culture in an LB plate medium (yeast extract 5 g/L, trypton 10 g/L and NaCl 1O g/ L) supplemented with ampicillin (100 mg/L), X-gal, IPTG and bacto-agar (15 g/L) to select transformed E. coli XLl-Blue (pET20-xy/C).
- the concentration was 0.5 mM to induce the expression of xylC, followed by culture at 37 0 C.
- Second step Purification of cNDA Using Recombinant Microorganism
- FIG. 3 shows a micrograph of the cNDA crystal.
- slurry containing the pure NDA was transferred to a high-pressure slurry collector H. After the pressure of the high-pressure slurry collector H dropped to ambient pressure, the slurry was transferred to a powder separator I (i.e. a decanter) where the water was removed from the slurry, followed by drying in a dryer J at 12O 0 C to collect the NDA in a pure crystalline form.
- a powder separator I i.e. a decanter
- NDA in a pure crystalline form was collected in the same manner as in Example 1, except that the recombinant microorganism was previously removed from the solution of the purified cNDA, which was prepared in the second step, using a polypropylene filter (pore size: 0.2 D) after the second step and prior to the third step.
- Example 3 NDA in a pure crystalline form was collected in the same manner as in Example 1, except that a microorganism JM109 (pUC18-xy/C) obtained by transformation with the recombinant expression vector (pUC18-xy/C) shown in FIG. 2 carrying the ben- zaldehyde dehydrogenase gene (see, GenBank Sequence Database, D63341) of SEQ ID NO: 4 derived from Pseudomonas putida mt-2 (ATCC 33015) was used as the recombinant microorganism. Details of a method for preparing the recombinant microorganism JM109 (pUC18-xy/C) are found in Korean Patent Unexamined Publication No. 2005-71188.
- Example 4 NDA in a pure crystalline form was collected in the same manner as in Example 2, except that JM 109 (pUC18-xy/C) was used as the recombinant microorganism.
- Average particle size (/m) 116 .6 135. 8 125.8 118.7 110.6 42.7 25.1
- St irring rate (rpm) 0 50 100 200 400 800 1000
- Average particle size (AMI) 92. 1 110. 8 99.1 94.7 80.6 23.1 17.3
- St irring rate (rpm) 0 50 100 200 400 800 1000
- St irring rate (rpm) 0 50 100 200 400 800 1000
- Average particle size (/ail) 76. 4 99.7 86.. r 79.3 61.7 32.1 10.9
- Experimental Example 3 Crystallization Reactions at Different Kinds of Acids and Temperatures
- the procedure of Experimental Example 2 was repeated, except that the mixed solutions were stirred at a fixed rate of 50 rpm at different reaction temperatures of 4, 15, 50, 80, 120 and 15O 0 C.
- FIG. 4 is a micrograph of the cNDA crystal obtained by adding the sulfuric acid solution to the reaction solution of the purified cNDA prepared in Example 1 and allowing the mixed solution to react at 8O 0 C.
- the average particle sizes of the cNDA crystals obtained under the respective conditions were measured, and the results are shown in Tables 5 and 6.
- Tables 5 and 6 As can be seen from the data shown in Tables 5 and 6, the cNDA crystals obtained after the addition of the sulfuric acid or hydrochloric acid solution and reaction at a temperature of 50-120 0 C, particularly 8O 0 C, showed better results.
- Experimental Example 4 Recovery Rates at Different Kinds of Acids and pH Values
- the procedure of Experimental Example 2 was repeated, except that the mixed solutions were stirred at a fixed rate of 50 rpm, a fixed reaction temperature of 8O 0 C and varying pH values of 1, 2, 3, 4, 5 and 6. After the crystallization reactions were conducted for 25 minutes, equal amounts of the reaction solutions were taken out of the respective reactors.
- the recovery rates of the cNDA crystals were measured, and the results are shown in Tables 7 and 8.
- the results of Tables 7 and 8 show that the recovery rates of the cNDA crystals were not lower than 99% at a pH not higher than 3.
- the recovery rates of the cNDA crystals showed a tendency to increase with decreasing pH.
- a crude naphthalene dicarboxylic acid is purified and crystallized under respective suitable conditions using a recombinant microorganism capable of converting FNA to NDA. Therefore, the purification method of the present invention enables the production of high-purity crystalline 2,6-naphthalene dicarboxylic acid on an industrial scale in an economically feasible and environmentally friendly manner.
- SEQ ID NO: 1 is a base sequence of a benzaldehyde dehydrogenase gene (xylC) derived from Sphingomonas aromaticivorans (KCTC 2888).
- SEQ ID NO: 2 shows primer 1 synthesized on the basis of a DNA base sequence of a benzaldehyde dehydrogenase gene (xylC) derived from Sphingomonas aromaticivorans (KCTC 2888) to clone the xylC gene.
- xylC benzaldehyde dehydrogenase gene
- SEQ ID NO: 3 shows primer 2 synthesized on the basis of a DNA base sequence of a benzaldehyde dehydrogenase gene (xylC) derived from Sphingomonas aromaticivorans (KCTC 2888) to clone the xylC gene.
- xylC benzaldehyde dehydrogenase gene
- SEQ ID NO: 4 is a base sequence of a benzaldehyde dehydrogenase gene (xylC) derived from Pseudomonas putida mt-2 (ATCC 33015).
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Abstract
Disclosed is a method for purifying a crude naphthalene dicarboxylic acid using a recombinant microorganism. According to the purification method, a crude naphthalene dicarboxylic acid is purified by reacting a recombinant microorganism having the ability to convert 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid with a crude naphthalene dicarboxylic acid, adding an acidic solution to the reaction solution under particular conditions, allowing the mixed solution to react with stirring to crystallize the crude naphthalene dicarboxylic acid, washing the crystal of the crude naphthalene dicarboxylic acid, and drying the washed product to obtain 2,6-naphthalene dicarboxylic acid in a pure crystalline form. Advantageously, the purification method enables the production of high-purity crystalline 2,6-naphthalene dicarboxylic acid on an industrial scale in an economically feasible and environmentally friendly manner.
Description
Description PURIFICATION METHOD OF CRUDE NAPHTHALENE DI-
CARBOXYLIC ACID USING RECOMBINATED MICROORGANISM AND 2,6-NAPHTHALENE DICARBOXYLIC ACID IN CRYSTALLINE FORM OBTAINED BY USING THE
SAME Technical Field
[1] The present invention relates to a method for purifying a crude naphthalene di- carboxylic acid using a recombinant microorganism and 2,6-naphthalene dicarboxylic acid in a crystalline form produced by the method. More specifically, the present invention relates to a method for purifying a crude naphthalene dicarboxylic acid by reacting a recombinant microorganism having the ability to convert 2-formyl-6-naphthoic acid to 2,6-naphthalene dicarboxylic acid with a crude naphthalene dicarboxylic acid to remove 2-formyl-6-naphthoic acid contained as an impurity in the crude naphthalene dicarboxylic acid, adding an acidic solution to the reaction solution under particular conditions, allowing the mixed solution to react with stirring to crystallize the crude naphthalene dicarboxylic acid, washing the crystal of the crude naphthalene dicarboxylic acid to remove other impurities contained therein, and drying the washed product to obtain 2,6-naphthalene dicarboxylic acid in a pure crystalline form. Background Art
[2] 2,6-Naphthalene dicarboxylic acid (NDA) and its diester (i.e. 2,6-naphthalene di- carboxylate (NDC)) are useful monomers for the preparation of a variety of polymeric materials, such as polyesters and polyamides. For example, NDA and NDC can be condensed with ethylene glycol to form poly (ethylene 2,6-naphthalate) (PEN), which is a high-performance polyester material. Fibers and films made from PEN exhibit high strength and superior thermal properties, compared to those made from poly(ethylene terephthalate) (PET). Based on these advantages, PEN is highly suitable for use in the production of commercial articles, such as thin films, which can be used for the manufacture of magnetic recording tapes and electronic components. In addition, since PEN is highly resistant to the diffusion of gases, particularly carbon dioxide, oxygen and water vapor, films made from PEN are useful for the manufacture of food containers, especially hot-fill food containers. PEN can also be used to produce reinforced fibers useful for the manufacture of tire cords.
[3] NDC is currently produced by oxidizing 2,6-dimethylnaphthalene (2,6-DMN) to
obtain a crude naphthalene dicarboxylic acid (cNDA) and esterifying the cNDA. At present, NDC is used as a major raw material in the synthesis of PEN. However, some problems are presented when NDC is used as a raw material in the synthesis of PEN, compared to when 2,6-naphthalene dicarboxylic acid (NDA) is used. Firstly, water is formed as a by-product during the condensation of NDA, whereas methanol is formed as a by-product in the case of NDC, thus risking the danger of explosion. Secondly, since pure NDC is produced by esterifying NDA and purifying the esterification product, one additional processing step is involved, compared to the use of NDA. Thirdly, the use of NDC is not suitable in view of the use of existing PET production facilities. Despite the problems associated with the use of NDC, NDC is preferentially used to produce PEN because it is still difficult to produce purified NDA having a purity necessary for the synthesis of PEN.
[4] 2,6-Dimethylnaphthalene (2,6-DMN) is oxidized to form a cNDA containing various impurities, such as 2-formyl-6-naphthoic acid (FNA), 2-naphthoic acid (NA) and trimellitic acid. Particularly, the presence of FNA in a cNDA stops the polymerization for the production of PEN, resulting in a low degree of polymerization. Th is low degree of polymerization adversely affects the physical properties of the final polymer (i.e. PEN) and causes coloration of the polyester. It is thus essential to remove FNA present in a cNDA, but difficulties exist in removing FNA.
[5] Under these circumstances, research has been conducted on various chemical methods for the removal of FNA present in a cNDA or purification of NDA. For example, NDA is produced by i) recrystallizing a cNDA, ii) oxidizing a cNDA one more time, or iii) treating a cNDA with methanol to produce NDC and hydrating the NDC. Further, purified NDA is produced by hydrogenation of a cNDA. In addition to these chemical methods, many processes, e.g., solvent treatment, melting/crystallization, high-pressure crystallization and supercritical extraction, have been employed to purify NDA.
[6] For example, U.S. Patent No. 5,859,294 discloses a process for the production of a naphthalene dicarboxylic acid, which comprises dissolving a crude naphthalene dicarboxylic acid in an aqueous solution containing an aliphatic or alicyclic amine, removing heavy metal components contained as impurities until the content of the heavy metal components based on the crude naphthalene dicarboxylic acid is 100 ppm or less, and heating the aqueous solution containing a naphthalene dicarboxylic acid amine salt to distill off the amine.
[7] U.S. Patent No. 6,255,525 discloses a process for preparing an aromatic carboxylic acid having improved purity comprising the steps of contacting a mixture comprising an impure aromatic carboxylic acid and water at a pressure of 77 to 121 kg/cm and a temperature of 277 to 3160C in the presence of hydrogen with a carbon catalyst which
is free of a hydrogenation metal component, cooling the mixture to form crystallized aromatic carboxylic acid, and recovering the crystallized aromatic carboxylic acid from the cooled mixture.
[8] In connection with the crystallization reaction of NDA, U.S. Patent No. 6,087,531 teaches a process for recovering a naphthalene dicarboxylic acid (NDA) crystal comprising the steps of dissolving poly(alkylene naphthalene dicarboxylate) in an aqueous basic solution (e.g., an aqueous solution of an alkali metal base, an aqueous hydroxide solution or an aqueous solution of an alkali metal carbonate) at a temperature of 125 to 4000C, neutralizing the aqueous solution with an acid at 170 to 24O0C, and recovering the NDA. For example, an NDA crystal is recovered by dissolving the polyester material in a NaOH or KOH solution at a temperature of 125 to 4000C, neutralizing the aqueous solution with acetic acid, and recovering the NDA.
[9] U.S. Patent No. 6,426,431 teaches a method of increasing the purification yield of
2,6-NDA by as high as 45%, the method comprising treating an aqueous solution of K -NDA at a CO pressure of 0-200 psi and a temperature of 0-500C to form KH-NDA, suspending the KH-NDA in water in a weight ratio higher than 1:8 (KH-NDA: water), and further treating the suspension at a temperature above 1000C (140-1600C) and at a CO pressure above 100 psi (175-250 psi).
[10] However, since these processes require high-temperature and high-pressure conditions, time-consuming treatments and use of large amounts of expensive materials to produce a high-purity NDA crystal, they are economically disadvantageous. Other disadvantages of the processes are very low purification yield and purity of NDA and occurrence of aggregation between individual NDA crystal particles, which makes the processes difficult to practice industrially. Disclosure of Invention Technical Problem
[11] Therefore, the present invention has been made in view of the problems of the prior art, and it is one object of the present invention to provide a method for efficiently producing high-purity NDA in high yield by purifying and crystallizing a crude naphthalene dicarboxylic acid at ambient pressure and temperature conditions using a recombinant microorganism capable of converting FNA to NDA.
[12] It is another object of the present invention to provide a high-purity NDA crystal with a uniform size produced by the method. Technical Solution
[13] In accordance with one aspect of the present invention for achieving the above obj ects, there is provided a method for purifying a crude naphthalene dicarboxylic acid using a recombinant microorganism.
[14] Specifically, the purification method of the present invention comprises the following steps:
[15] (a) reacting a crude naphthalene dicarboxylic acid with at least one recombinant microorganism selected from the group consisting of microorganisms obtained by transformation with a recombinant expression vector carrying the benzaldehyde dehydrogenase gene (xylC) of SEQ ID NO: 1 derived from Sphingomonas aromaticivorans (KCTC 2888) or a gene having a homology of at least 90% with the xylC gene and microorganisms obtained by transformation with a recombinant expression vector carrying the benzaldehyde dehydrogenase gene (xylC) of SEQ ID NO: 4 derived from Pseudomonas putida mt-2 (ATCC 33015) or a gene having a homology of at least 90% with the xylC gene, to remove 2-formyl-6-naphthoic acid present in the crude naphthalene dicarboxylic acid;
[16] (b) adding an acidic solution to the reaction solution prepared in step (a) to adjust the pH of the reaction solution and reacting the mixed solution with stirring to crystallize the crude naphthalene dicarboxylic acid;
[17] (c) washing the crystal of the crude naphthalene dicarboxylic acid to remove impurities contained therein; and
[18] (d) drying the washed product to obtain 2,6-naphthalene dicarboxylic acid in a pure crystalline form.
[19] A more detailed explanation of the respective steps of the purification method according to the present invention will be provided below.
[20]
[21] (a) First step: Purification using recombinant microorganism
[22] First, a crude naphthalene dicarboxylic acid is reacted with at least one recombinant microorganism selected from the group consisting of microorganisms obtained by transformation with a recombinant expression vector carrying the benzaldehyde dehydrogenase gene (xylC) of SEQ ID NO: 1 derived from Sphingomonas aromaticivorans (KCTC 2888) or a gene having a homology of at least 90% with the xylC gene and microorganisms obtained by transformation with a recombinant expression vector carrying the benzaldehyde dehydrogenase gene (xylC) of SEQ ID NO: 4 derived from Pseudomonas putida mt-2 (ATCC 33015) or a gene having a homology of at least 90% with the xylC gene, to remove 2-formyl-6-naphthoic acid present in the crude naphthalene dicarboxylic acid;
[23] The expression "gene having a homology of at least 90%" as used herein refers to a gene that has a homology of at least 90% with the benzaldehyde dehydrogenase gene used in the method of the present invention in the base or amino acid sequence level, and that shows the same activity as the benzaldehyde dehydrogenase despite the difference in base or amino acid sequence from the benzaldehyde dehydrogenase gene
within 10%. The different sequence segments are intended to include all segments that may be modified by those skilled in the art without significantly affecting the inherent activity of the benzaldehyde dehydrogenase.
[24] Specifically, this step includes the sub-steps of 1) inoculating the recombinant microorganism into a liquid medium, culturing the cells to induce the expression of benzaldehyde dehydrogenase, centrifuging the culture broth to collect the cells in which benzaldehyde dehydrogenase is expressed, and suspending the cells in physiological saline or distilled water, 2) mixing a crude naphthalene dicarboxylic acid (cNDA) as a substrate with a buffer solution and adjusting the pH of the mixed solution by the addition of an alkaline solution to prepare a reaction solution for subsequent purification, and 3) reacting the cells prepared in 1) with the reaction solution prepared in 2) to convert 2-formyl-6-naphthoic acid contained in the crude naphthalene dicarboxylic acid to 2,6-naphthalene dicarboxylic acid, so that the purity of the 2,6-naphthalene dicarboxylic acid is increased.
[25] Recombinant microorganisms that can be used in the present invention may be obtained by cloning the benzaldehyde dehydrogenase gene (xylC) of SEQ ID NO: 1 derived from Sphingomonas aromaticivorans (KCTC 2888), a gene having a homology of at least 90% with the xylC gene of SEQ ID NO: 1, the benzaldehyde dehydrogenase gene (xylC) of SEQ ID NO: 4 derived from Pseudomonas putida mt-2 (ATCC 33015) or a gene having a homology of at least 90% with the xylC gene of SEQ ID NO: 4 in a manner known in the art, introducing the cloned gene into an e xpression vector to construct a recombinant expression vector, and introducing the recombinant expression vector into a microorganism, and transforming the microorganism with the recombinant expression vector by a conventional known technique.
[26] Specifically, the recombinant microorganisms can be obtained by the following procedure.
[27] In order to clone each benzaldehyde dehydrogenase gene (xylC), plasmid DNA or genomic DNA is isolated from each of the microorganisms and polymerase chain reaction (PCR) using the plasmid DNA or genomic DNA as a template is performed using a set of primers synthesized on the basis of the xylC gene to obtain a gene encoding benzaldehyde dehydrogenase. If needed, the base sequence of the gene encoding benzaldehyde dehydrogenase may be partially modified by a common technique known in the art.
[28] Subsequently, the cloned xylC gene is functionally linked to a promoter of an expression vector by a conventional technique to construct a recombinant expression vector.
[29] Any expression vector that can allow the optimum expression of the gene in a host
may be available without any particular limitation. For example, the gene can be inserted into a plasmid, phage or another DNA as the expression vector. Examples of suitable plasmids include, but are not specially limited to: known pForexT, pLG338, pACYC184, pBR322, pUC119, pUC18, pUC19, pKC30, PRep4, pHSl, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III138-Bl, λgtl l and pBDCl vectors in E. colϊ, pIJlOl, pIJ364, pIJ702 and pIJ361 vectors in Streptomyces; pUBl lO, pC194 and pBD214 vectors in Bacillus; and 2D, pAG-1. YEp6, YEpl3 and pEMBLYe23 vectors in yeast.
[30] Examples of promoters that can be used in the present invention include, but are not limited to: promoters suitable for use in gram-negative bacteria, such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacf, T7, T5, T3, gal, trc, ara, SP6, λ-PR and λ-PL promoters; gram-positive promoters, such as amy and SPO2 promoters; and fungi or yeast promoters, such as ADCl, MFa, AC, P-60, CYCl, GAPDH, TEF, rp28 and ADH promoter.
[31] To ensure the optimum expression of the gene depending on the type of the selected host organism, a regulatory sequence may be further included in the 3' end and/or 5' end of the gene.
[32] In this connection, FIG. 1 shows a genetic map of a recombinant expression vector
(pET20-xy/C) carrying an xylC gene encoding benzaldehyde dehydrogenase derived from Sphingomonas aromaticivorans used in Example 1 of the present invention, and FIG. 2 shows a genetic map of a recombinant expression vector (pUC18-xy/C) carrying an xylC gene encoding benzaldehyde dehydrogenase derived from Pseudomonas putida used in Example 3 of the present invention.
[33] The cloning of the xylC gene into the corresponding recombinant expression vector may be confirmed by various techniques, such as restriction enzyme cleavage and base sequencing.
[34] Then, the recombinant expression vector thus constructed is introduced into a host microorganism by any conventional technique known in the art to transform the microorganism. As a result, a recombinant microorganism is prepared. As the host microorganism, any prokaryotic or eukaryotic organism may be used. Examples of preferred host microorganisms include, but are not necessarily limited to, bacteria, fungi and yeasts. The host microorganism may be typically a gram-positive or gram- negative bacterial species, preferably a species of Enterobacteriaceae or Nocardiaceae family, more preferably a bacterial species of the genus Escherichia, Pseudomonas, Rhodococcus or Bacillus, and most preferably a bacterial species of the genus E. coli. Specific examples of the host microorganism include MC 1061 (E. coli), JM 109 (E. coli), XLl -Blue (E. coli) and DH5 (E. coli). The transformation may be performed by any suitable technique, such as heat treatment, electric shock, microinjection, calcium
chloride method, rubidium chloride method or pressure spraying, but the present invention is not necessarily limited to these techniques.
[35] According to the method of the present invention, 2-formyl-6-naphthoic acid is converted to 2,6-naphthalene dicarboxylic acid with the help of the recombinant microorganism transformed with the expression vector of benzaldehyde dehydrogenase. To this end, it is preferred that benzaldehyde dehydrogenase be expressed at a high level within the recombinant microorganism. For example, since the pUC 18 vector used in the present invention is used to express the gene under the control of a lac promoter and the vector pET-20b(+) is used to express the gene under the control of a T7 promoter, isopropyl-β-D-thiogalactopyranoside (IPTG) can be used to induce the expression of benzaldehyde dehydrogenase.
[36] Specifically, the recombinant microorganism is sufficiently cultured in a typical culture temperature range (25-450C), preferably at 370C, and is then inoculated into a 100 ml of a liquid medium (e.g., an LB medium) in such an amount that the concentration is 1% (v/v). When the OD value reaches 0.4 to 0.5, IPTG is added until
600 the concentration is 0.1-2.0 mM and preferably 0.5 mM to induce the expression of the benzaldehyde dehydrogenase, followed by culture at 370C. It is to be appreciated that the culture and the protein expression of the recombinant organism can be achieved, without any limitation to the foregoing techniques, by any technique known to those skilled in the art.
[37] The recombinant microorganism used in the method of the present invention enables the expression of benzaldehyde dehydrogenase. Therefore, the high- concentration culture of the recombinant microorganism is facilitated, which provides an economic advantage.
[38] The culture solution of the recombinant microorganism in which the protein is expressed at a high level is centrifuged to collect the cells, and then the cells are suspended in physiological saline or distilled water. The suspension is used as a reaction solution for the purification of a crude naphthalene dicarboxylic acid in the subsequent step.
[39] The kind of the buffer solution is not especially restricted. As the buffer solution, there can be used, for example, water, a sodium carbonate buffer (Na CO /NaHCO ), a glycine buffer (glycine/NaOH), a potassium phosphate buffer (KH PO /KOH), a sodium phosphate buffer (Na HPO /NaH PO ), a succinic acid buffer (succinic acid/ NaOH), a sodium acetate buffer (sodium acetate/acetic acid), a citric acid buffer (citric acid/sodium citrate), a sodium pyrophosphate buffer (Na P O /HCl), a boric acid buffer (boric acid/NaOH), or a sodium borate buffer (sodium borate/HCl). Preferred is a potassium phosphate buffer (KH PO -KOH) or a boric acid buffer (boric acid/NaOH), which has a pH range of 6.0-9.0. It is preferred that the concentration of the buffer
solution be in the range of 0.01 to 100 mM.
[40] The alkaline solution is preferably a NaOH or KOH solution, but is not limited thereto. The pH of the mixed solution is preferably adjusted to the range of 6 to 10, preferably 8 to 10.
[41] In the second sub-step, an organic solvent may be additionally added to the mixed solution for the purpose of dissolving the cNDA. Examples of preferred organic solvents include dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), and tetrahydrofuran (THF). Of these, dimethylsulfoxide is most preferred in terms of enzymatic activity. The organic solvent is preferably added at a concentration of 0.01 to 20% and more preferably 0.1 to 10%. It is most preferred that no organic solvent be added. The addition of the organic solvent at a concentration exceeding 20% causes lysis of the cell membranes of the microorganism, resulting in inhibition of the reaction.
[42] In the third sub-step, the reaction is preferably conducted at 25-7O0C for 1 min.-l hour and more preferably at 40-600C for 10-40 minutes. When the reaction temperature is lower than 250C or higher than 7O0C, a marked decrease in the reactivity is undesirably caused.
[43] On the other hand, there is an intimate relationship among the FNA content in the cNDA, the concentration of the cNDA in the reaction solution and the amount of the microorganism necessary to completely remove the FNA. That is, as the FNA content in the cNDA and the concentration of the cNDA in the reaction solution increase, a larger amount of the microorganism is required to remove the FNA.
[44] Specifically, the concentration of the cNDA in the reaction solution is preferably between 0.001% and 20%, and the FNA content in the cNDA is between 0.001% and 10% and preferably 9%.
[45] The amount of the microorganism necessary to completely remove Ig of the FNA is approximately 5g. In view of the fact that a microorganism (e.g., Bacillus sp. F-I or F- 3 described in Korean Patent Application No. 2002-0087819, which was filed by the present inventors) having the ability to convert FNA to NDA is generally used in an amount of about 1Og in order to completely remove Ig of FNA, it turns out that the purification of cNDA using the recombinant microorganism in accordance with the present invention is more efficient from an economic viewpoint.
[46]
[47] (b) Second step: Crystallization
[48] In this step, an acidic solution is added to the reaction solution prepared in step (a) to adjust the pH of the reaction solution, and is then reacted with the reaction solution with stirring to crystallize the crude naphthalene dicarboxylic acid.
[49] More specifically, an appropriate amount of an acidic solution is added to the
reaction solution prepared in step (a) in a reactor equipped with an stirrer to adjust the pH of the reaction solution to a desired range, and then the mixed solution is reacted with continuous stirring while maintaining the temperature of the mixed solution constant, so that the cNDA in an amorphous form present in the FNA-free reaction solution is crystallized at ambient pressure and temperature conditions.
[50] Since this crystallization enables production of a cNDA crystal having a uniform size of 100 D or more at ambient pressure and temperature conditions, the purification method of the present invention is economically advantageous in terms of production cost and processing. In addition, since no aggregation between the individual cNDA crystal particles occurs, the purification method of the present invention is suitable for actual use and is advantageous in terms of a high recovery rate of the final product.
[51] As the acidic solution, there may be used, for example, sulfuric acid, hydrochloric acid, glacial acetic acid or nitric acid. The use of sulfuric acid or hydrochloric acid is preferred because it leads to large size and high yield of the cNDA crystal. That is, the addition of sulfuric acid or hydrochloric acid enables the production of a cNDA crystal having a uniform size of 100 D or more in high yield.
[52] The pH of the reaction solution is preferably adjusted to 1-4 to increase the recovery rate of the final product. As the pH of the reaction solution decreases, the recovery rate of the final product tends to increase from about 94% to about 99.9%.
[53] The crystallization is performed at about 40C to about 15O0C, preferably 3O0C to
13O0C and more preferably 5O0C to 12O0C. The crystallization is performed for about 1 minute to about 10 hours, preferably 2 minutes to 5 hours and more preferably 10 minutes to one hour in terms of continuous processing. The most preferable conditions for the crystallization are 8O0C and 20-30 minutes. Too short crystallization time may cause a low degree of crystallization of the cNDA, resulting in aggregation between the individual cNDA crystal particles upon polymerization. Meanwhile, too long crystallization time may cause an unnecessary energy loss.
[54] The stirring required to perform the crystallization of the cNDA is typically performed at a rate of 0-1,000 rpm, preferably 0-400 rpm and more preferably 50-150 rpm.
[55]
[56] (c) Third step: Washing
[57] In this step, the crystal of the crude naphthalene dicarboxylic acid obtained in step
(b) is washed to remove impurities contained therein.
[58] Although the FNA is converted to NDA and is completely removed through the purification reaction using the recombinant microorganism and the crystallization reaction, other impurities, including 2-naphthoic acid (NA), methylnaphthoic acid (MNA) and trimellitic acid (TLMA), remain unremoved. When the cNDA crystal is
dispersed in water and washed several times under given conditions to completely remove the remaining impurities. The complete removal of the impurities is based on a difference in solubility of the cNDA and the impurities in water. At this time, the recombinant microorganism used to purify the cNDA is also removed by the washing.
[59] In this step, it is desirable to practice the separation in a state in which the reaction by-products are dissolved in the solvent and NDA is precipitated as much as possible. The temperature range for the separation is between 1000C and 27O0C and preferably between 15O0C and 24O0C. The separation of the solvent at a temperature higher than 27O0C causes increased loss of the purified NDA, leading to a considerable drop in yield. Meanwhile, the separation of the solvent at a temperature lower than 1000C unfavorably causes low dissolution of the impurities, including NA, MNA and TLMA.
[60] More specifically, the cNDA crystal is dispersed in water, stirred at a pressure of 1 to 60 kg/cm and a temperature of 100 to 27O0C for 10 minutes to 1 hour, and filtered to remove the water. This procedure may be repeated several times to remove the impurities. At this time, the solvent is preferably used in an amount of 5-20 times of the weight of the cNDA crystal.
[61]
[62] (d) Fourth step: Drying
[63] In this step, the NDA crystal, from which the impurities are removed by the washing, is dried at a specified temperature to obtain NDA in a pure crystalline form. At this time, the drying is preferably performed at 30 to 2000C. The drying is performed, without limitation, by a conventional technique known in the art.
[64] The purification method of the present invention may further comprise the step of removing the recombinant microorganism used in step (a) after step (a) and prior to step (b).
[65] The previous removal of the recombinant microorganism after the purification of the cNDA and prior to the crystallization of the cNDA can contribute to improvements in purity and recovery rate of the NDA crystal.
[66] The removal of the recombinant microorganism is achieved, without limitation, by a conventional technique known in the art. For example, a microfilter system, a continuous type centrifugal separator or a decanter may be used to remove the recombinant microorganism. The microfilter system uses a filter having a pore size of 0.1-0.5 D and made of a material selected from ceramic, stainless steel, polypropylene and polyethylene terephthalate (PET).
[67] In another aspect, the present invention provides 2,6-naphthalene dicarboxylic acid in a pure crystalline form produced by the purification method.
[68] The purification method of the present invention enables the production of high- purity 2,6-naphthalene dicarboxylic acid in a high yield of 99.9% or more. Depending
on the intended applications and needs, the treatment conditions may be varied to produce 2,6-naphthalene dicarboxylic acid in a regular or random crystalline form. The 2,6-naphthalene dicarboxylic acid in a crystalline form may have a lattice structure.
[69] The 2,6-naphthalene dicarboxylic acid crystal of the present invention has an average particle diameter not smaller than 100 D and a uniform particle shape. Accordingly, the 2,6-naphthalene dicarboxylic acid crystal is very suitable for the formation of a low- viscosity slurry with ethylene glycol when it is polymerized with the ethylene glycol to produce PEN. A 2,6-naphthalene dicarboxylic acid crystal having an average particle diameter smaller than 100 D is difficult to treat and exhibits a poor ability to form a slurry with ethylene glycol when it is mixed with the ethylene glycol to prepare PEN, causing an increase in power consumption. The 2,6-naphthalene dicarboxylic acid crystal of the present invention preferably has an average particle diameter of 100-200 D and more preferably 110-170 D.
[70] FIG. 5 shows a purification system for implementing a method for purifying a crude naphthalene dicarboxylic acid according to an embodiment of the present invention. With reference to FIG. 5, the purification method according to the embodiment of the present invention will be explained in more detail below.
[71] First, a specified amount of a buffer solution is introduced into a reactor A where a crude naphthalene dicarboxylic acid is purified using a microorganism. A cNDA is added to the reactor, and subsequently, an alkaline solution is added to the reactor with stirring to adjust the pH of the reaction solution. A specified amount of water is added to the reactor to prepare a reaction solution for subsequent purification. The cells are added to react with the reaction solution while maintaining the temperature of the reaction solution at a constant level. By this procedure, FNA contained in the cNDA is converted to NDA in the reactor A, and can be finally removed.
[72] After completion of the reaction, the reaction solution is passed through a unit B where the recombinant microorganism used to remove the FNA is removed. The removal of the recombinant microorganism using the unit B may be omitted, if needed, because the removal of the recombinant microorganism can be achieved in a downstream filtering/cleaning unit F.
[73] The reaction solution, from which the microorganism is removed, is transferred to a crystallization reactor C. An acidic solution is added to the crystallization reactor C with stirring to conduct a crystallization reaction.
[74] A slurry containing the cNDA crystal is obtained after the crystallization reaction.
The slurry is heated to about 100 to about 27O0C using a preheater D, and is then fed into a filtering/cleaning unit F where a heater is provided to maintain the temperature of the slurry at 100-2700C. The pressure of the filtering/cleaning unit F is maintained at 1-60 kg/cm . The filtering/cleaning unit F is equipped with a filter (pore size: 10-100
D).
[75] The filtering/cleaning unit F is connected to a high-pressure filtrate collector G. A filtrate is discharged from the filtering/cleaning unit F into the high-pressure filtrate collector G and solid components are filtered by the filter included in the filtering/ cleaning unit F.
[76] Water (100-2700C) is preheated in a solvent heating/supply unit E and supplied to the filtering/cleaning unit F. After stirring is continued in the filtering/cleaning unit F for a given time, a secondary filtrate is discharged into the high-pressure filtrate collector G. If necessary, the cleaning step is repeated once or twice. Pure NDA remaining after the cleaning is mixed with preheated water (100-2700C) to form a slurry. The slurry is sent to a high-pressure slurry collector H via a slurry discharge line. After the pressure of the high-pressure slurry collector H drops to ambient pressure, the slurry containing the pure NDA is transferred to a powder separator I where the solvent is removed from the slurry, followed by drying in a dryer J to collect the pure NDA only.
Advantageous Effects
[77] According to the purification method of the present invention, a crude naphthalene dicarboxylic acid is purified and crystallized under respective suitable conditions using a recombinant microorganism capable of converting FNA to NDA. Therefore, the purification method of the present invention enables the production of high-purity crystalline 2,6-naphthalene dicarboxylic acid on an industrial scale in an economically feasible and environmentally friendly manner. Brief Description of the Drawings
[78] FIG. 1 is a genetic map of a recombinant expression vector (pET20-xy/C) carrying an xylC gene encoding benzaldehyde dehydrogenase derived from Sphingomonas aro- maticivorans;
[79] FIG. 2 is a genetic map of a recombinant expression vector (pUC18-xy/C) carrying an xylC gene encoding benzaldehyde dehydrogenase derived from Pseudomonas putida;
[80] FIG. 3 is a micrograph of a cNDA crystal obtained in the third step of Example 1 of the present invention;
[81] FIG. 4 is a micrograph of a cNDA crystal obtained by adding a sulfuric acid solution to a reaction solution of a purified cNDA prepared in Example 1 and allowing the mixed solution to react at 8O0C in Experimental Example 3 of the present invention; and
[82] FIG. 5 shows a purification system for implementing a method for purifying a crude naphthalene dicarboxylic acid according to an embodiment of the present invention.
[83]
[84] * Brief explanation of essential parts of the drawings *
[85] A: Purification reactor using microorganism
[86] B: Microorganism removing unit C: Crystallization reactor
[87] D: Preheater E: Solvent heating/supply unit
[88] F: Filtering/cleaning unit G: High-pressure filtrate collector
[89] I: Powder separator J: Dryer
Mode for the Invention
[90] Hereinafter, the constitutions and effects of the present invention will be concretely explained in detail with reference to the following examples. However, these examples are given for the purpose of illustration and are not to be construed as limiting the scope of the invention.
[91]
[92] EXAMPLES
[93] Example 1
[94] First Step: Preparation of Recombinant Microorganism
[95] (1) Cloning of xylC Gene
[96] First, in order to clone an xylC gene encoding benzaldehyde dehydrogenase derived from Sphingomonas aromaticivorans (KCTC 2888), a plasmid DNA (pNLl) carrying a sequence of the xylC gene was isolated (see, Sambrook et al., Molecular Cloning, A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Polymerase chain reaction (PCR) using the isolated plasmid DNA (pNLl) as a template was performed using primer 1,
5'-GGAGAATTCATATGGCTACGCAGT-S' (SEQ ID NO: 2) and primer 2, 5'-GTCTTGCAGTGAGCTCGTTTCTCC-S' (SEQ ID NO: 3), which were synthesized on the basis of the DNA base sequence of the xylC gene (available from GenBank Sequence Database, NC002033). In the PCR, denaturation was twice carried out at 940C for 5 minutes (first denaturation) and at 940C for one minute (second denaturation), annealing was carried out at 560C for one minute, and extension was carried out at 720C for 1.5 minutes. This procedure was repeated forty times. Finally, extension was once more carried out at 720C for 10 minutes to obtain a fragment. From the fragment, the DNA fragment (about 1.5 kbp long) set forth in SEQ ID NO: 1 was isolated, cleaved with restriction enzymes Ndel and Sail, and cloned into the plasmid vector pET-20b(+), which was previously cleaved with the same restriction enzymes, to construct the recombinant expression vector pET20-xy/C shown in FIG. 1.
[97]
[98] (2) Analysis of Cloned Gene
[99] For base sequencing of the cloned gene of the recombinant vector pET20-xy/C in
Example 1, the recombinant vector was cleaved using various restriction enzymes on the basis of the genetic maps of two vectors M13mpl8 and M13mpl9 to obtain respective fragments. The fragments were subcloned into M13mpl8 and M13mpl9. The resulting subclones were subjected to base sequencing using an ABI PRISM BigDye primer cycle- sequencing kit (Perkin-Elmer, U.S.) with AmpliTaq DNA polymerase. In order to read the double-stranded DNA in both directions, nucleotide fragments were partially synthesized. The base sequence of the fragments of the cloned DNA was analyzed through the nucleotide fragments and compared with the base sequence available from GenBank (GenBank Accession Number: AF073917 or NC002030). As a result, the cloned DNA was confirmed to be xylC gene.
[100]
[101] (3) Preparation of Transformant
[102] E. coli XLl -Blue was transformed with the pET20-xy/C vector by a calcium chloride method (see, Sambrook et al., Molecular Cloning, A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and grown by culture in an LB plate medium (yeast extract 5 g/L, trypton 10 g/L and NaCl 1O g/ L) supplemented with ampicillin (100 mg/L), X-gal, IPTG and bacto-agar (15 g/L) to select transformed E. coli XLl-Blue (pET20-xy/C).
[103]
[ 104] (4) Expression of xylC Gene
[105] To express the xylC gene in the transformed microorganism, the E. coli obtained in
(3) above was inoculated into an LB test tube, allowed to sufficiently grow at 370C, and inoculated into another 100 ml LB medium in such an amount that the concentration is 1% (v/v). When the OD value reached 0.4-0.5, IPTG was added until
600 the concentration was 0.5 mM to induce the expression of xylC, followed by culture at 370C.
[106]
[107] Second step: Purification of cNDA Using Recombinant Microorganism
[108] The culture of the recombinant microorganism in which benzaldehyde dehydrogenase was expressed, which was obtained in the first step, was centrifuged to collect the cells. The collected cells were washed with a 0.85% physiological saline solution and suspended in 5 L of a 0.85% physiological saline solution.
[109] 80 L of a 50 mM potassium phosphate buffer was injected into a reactor A where a cNDA is purified by the recombinant microorganism. 10 kg of a cNDA was added to the reactor A and then KOH or NaOH was added thereto with stirring to adjust the pH of the reaction solution to 8.0. Water was added to the mixed solution to prepare a reaction solution for subsequent purification (cNDA concentration: 10%, FNA content
in the cNDA: 0.58%).
[110] 0.29 kg/5L of the cells was fed to the reaction solution to react with the reaction solution at 5O0C for 30 minutes while maintaining the temperature of the reaction solution at 5O0C. As a result of the reaction, FNA contained in the cNDA was converted to NDA in the reactor A.
[I l l]
[112] Third step: Crystallization of Purified cNDA
[113] 100 L of the solution of the purified cNDA prepared in the second step was transferred to a crystallization reactor C equipped with an agitator. After a sulfuric acid solution was added to the crystallization reactor to adjust the pH of the solution of the purified cNDA to 3.0, a crystallization reaction was conducted with stirring at a rate of 50 rpm for 25 minutes while maintaining the temperature at 8O0C.
[114] After completion of the crystallization, analysis of the cNDA crystal was conducted using a microscope and a particle size analyzer. The analytical results reveal that the cNDA was satisfactorily crystallized without any aggregation between the individual crystal particles. The cNDA crystal was measured to have an average particle size of about 162.5 D. FIG. 3 shows a micrograph of the cNDA crystal.
[115]
[116] Fourth step: Washing and Drying of cNDA Crystal
[117] The slurry of the cNDA crystal prepared in the third step was heated to above 22O0C using a preheater D, fed into a filtering/cleaning unit F, stirred at a pressure of 28 kg/ cm and a temperature of 2250C for 30 minutes, and filtered. The filtrate (i.e. water) was discharged into a high-pressure filtrate collector G. 100 L of water at 2250C from a solvent heating/supply unit E was added to the filtering/cleaning unit F, stirred under the same conditions as above for 30 minutes, and filtered. The filtrate (i.e. water) was discharged into the high-pressure filtrate collector G. This procedure was repeated a total of two times. 100 L of water at 2250C from the solvent heating/supply unit E was added to the filtering/cleaning unit F and stirred for at least 30 minutes to homogeneously disperse pure NDA. The slurry containing the pure NDA was transferred to a high-pressure slurry collector H. After the pressure of the high-pressure slurry collector H dropped to ambient pressure, the slurry was transferred to a powder separator I (i.e. a decanter) where the water was removed from the slurry, followed by drying in a dryer J at 12O0C to collect the NDA in a pure crystalline form.
[118]
[119] Example 2
[120] NDA in a pure crystalline form was collected in the same manner as in Example 1, except that the recombinant microorganism was previously removed from the solution of the purified cNDA, which was prepared in the second step, using a polypropylene
filter (pore size: 0.2 D) after the second step and prior to the third step.
[121] [122] Example 3 [123] NDA in a pure crystalline form was collected in the same manner as in Example 1, except that a microorganism JM109 (pUC18-xy/C) obtained by transformation with the recombinant expression vector (pUC18-xy/C) shown in FIG. 2 carrying the ben- zaldehyde dehydrogenase gene (see, GenBank Sequence Database, D63341) of SEQ ID NO: 4 derived from Pseudomonas putida mt-2 (ATCC 33015) was used as the recombinant microorganism. Details of a method for preparing the recombinant microorganism JM109 (pUC18-xy/C) are found in Korean Patent Unexamined Publication No. 2005-71188.
[124] [125] Example 4 [126] NDA in a pure crystalline form was collected in the same manner as in Example 2, except that JM 109 (pUC18-xy/C) was used as the recombinant microorganism.
[127] [128] Experimental Example 1 : Component Analysis [129] The unpurified cNDA, the solution of the purified cNDA obtained after the second step (purification step), and the NDA in a pure crystalline form obtained after the fourth step (washing/drying step) of each of Examples 1 and 3 were analyzed for the contents of the components therein. The respective analytical results are shown in Tables 1 and 2.
[130] TABLE 1: Component Analysis Data in Example 1 [131]
[134] From the results of Tables 1 and 2, it could be confirmed that the purification method using the recombinant microorganism enabled substantially complete removal of impurities contained in cNDA. Particularly, FNA was converted to NDA in a yield of 100%. As a result, 2,6-naphthalene dicarboxylic acid was produced with a purity of 99.99% or higher.
[135] To determine optimal reaction conditions for the purification of the cNDA using the recombinant microorganism, particularly those for the crystallization of the solution of the purified cNDA, a series of experiments was conducted by varying reaction conditions as follows, and the average particle sizes and recovery rates of cNDA crystals obtained after respective crystallization reactions according to the different reaction conditions were compared and analyzed.
[136] [137] Experimental Example 2: Crystallization Reactions at Different Kinds of Acids and Stirring Rates
[138] 100 ml of the solution of the purified cNDA prepared in the second step of each of Examples 1 and 3 was added to reactors, each of which was equipped with an agitator, and then sulfuric acid, hydrochloric acid, glacial acetic acid and nitric acid solutions were added to the reactors to adjust the pH of the solutions of the purified cNDA to 3.0. The mixed solutions were stirred at different rates of 0, 50, 100, 200, 400, 800 and 1,000 rpm. At this time, the temperature of the mixed solutions was maintained at 8O0C. Under these reaction conditions, crystallization reactions were conducted for 25 minutes to obtain cNDA crystals.
[139] After completion of the crystallization, analysis of the cNDA crystals was conducted using a microscope and a particle size analyzer. The analytical results reveal that the cNDA was satisfactorily crystallized without any aggregation between the individual crystal particles. The average particle sizes of the cNDA crystals obtained under the respective conditions were measured, and the results are shown in Tables 3 and 4. As can be seen from the data shown in Tables 3 and 4, the cNDA crystals obtained after the addition of the sulfuric acid or hydrochloric acid solution and stirring at a rate of 0 to 400 rpm, particularly 50 to 150 rpm, showed better results.
[140] TABLE 3: Crystal sizes at different kinds of acids and stirring rates in Example 1
[141]
Acid Sulfuric acid
Stirring rate (rpm) 0 50 100 200 400 800 1000
Average particle size (/m) 116 .6 135. 8 125.8 118.7 110.6 42.7 25.1
Acid Hydrochloric acid
St irring rate (rpm) 0 50 100 200 400 800 1000
Average particle size (/an) 117 .2 135. 4 126.2 119.4 99.8 40.3 20.0
Acid Glacial acetic acid
Stirring rate (rpm) 0 50 100 200 100 800 1000
Average particle size (jean) ( O . 4 91. 2 81.5 74.3 56.6 15.1 4.9
Acid Nitric acid
Stirring rate (rpm) 0 50 100 200 400 800 1000
Average particle size (AMI) 92. 1 110. 8 99.1 94.7 80.6 23.1 17.3
[142] TABLE 4: Crystal sizes at different kinds of acids and stirring rates in Example 3 [143]
Acid Sulfuric tacid
Stirring rate (rpm) 0 50 100 200 100 800 1000
Average part icle size (jaw) 118 .5 136.8 127. I 120.2 118.8 45.7 28.1
Acid Hydrochloric acid
St irring rate (rpm) 0 50 100 200 400 800 1000
Average particle size (jum) 121 .1 137.2 129. 5 120.3 101.7 48.5 30.0
Acid Gl acial acet c acid
St irring rate (rpm) 0 50 100 200 400 800 1000
Average particle size (/ail) 76. 4 99.7 86..r 79.3 61.7 32.1 10.9
Acid Nitric acid
Stirring rate (rpm) 0 50 100 200 400 800 1000
Average particle size (μm) 92. 1 119.7 113. ') 95.7 81.1 25.1 18.6
[144] Experimental Example 3: Crystallization Reactions at Different Kinds of Acids and Temperatures [145] The procedure of Experimental Example 2 was repeated, except that the mixed solutions were stirred at a fixed rate of 50 rpm at different reaction temperatures of 4, 15, 50, 80, 120 and 15O0C.
[146] After completion of the crystallization, analysis of the cNDA crystals was conducted using a microscope and a particle size analyzer. The analytical results reveal that the cNDA was satisfactorily crystallized without any aggregation between the
individual crystal particles. FIG. 4 is a micrograph of the cNDA crystal obtained by adding the sulfuric acid solution to the reaction solution of the purified cNDA prepared in Example 1 and allowing the mixed solution to react at 8O0C. The average particle sizes of the cNDA crystals obtained under the respective conditions were measured, and the results are shown in Tables 5 and 6. As can be seen from the data shown in Tables 5 and 6, the cNDA crystals obtained after the addition of the sulfuric acid or hydrochloric acid solution and reaction at a temperature of 50-1200C, particularly 8O0C, showed better results.
[147] TABLE 5: Crystal sizes at different kinds of acids and stirring rates in Example 1 [148]
[149] TABLE 6: Crystal sizes at different kinds of acids and stirring rates in Example 3 [150]
Acid Sulfuric acid
Temp. (0C) 4 15 50 80 120 150
'\vcragc particle size (μm) 112 .4 121.9 149.8 166.1 150.8 135.1
Acid Hydrochloric acid lemp. CC) 4 15 50 80 120 150
Average particle size OiITl) 115. O 12.1.4 118.5 164.5 151.3 136.1
Acid Glacial icetic acid
Temp. (0C) 4 15 50 80 120 150
Average particle size (AMI) 49. 1 65.5 80.6 92.7 72.3 52.3
Acid Nitric acid
Temp. CC) 4 15 50 80 120 150
Average particle size OiITl) 67. 7 93.8 125..3 145.9 121.9 89.2
[151] Experimental Example 4: Recovery Rates at Different Kinds of Acids and pH Values [152] The procedure of Experimental Example 2 was repeated, except that the mixed solutions were stirred at a fixed rate of 50 rpm, a fixed reaction temperature of 8O0C and varying pH values of 1, 2, 3, 4, 5 and 6. After the crystallization reactions were conducted for 25 minutes, equal amounts of the reaction solutions were taken out of the respective reactors. The recovery rates of the cNDA crystals were measured, and the results are shown in Tables 7 and 8. The results of Tables 7 and 8 show that the recovery rates of the cNDA crystals were not lower than 99% at a pH not higher than 3. The recovery rates of the cNDA crystals showed a tendency to increase with decreasing pH.
[153] TABLE 7:Recovery rates at different kinds of acids and pH values in Example 1 [154]
[155] TABLE 8:Recovery rates at different kinds of acids and pH values in Example 3 [156]
Industrial Applicability
[157] According to the purification method of the present invention, a crude naphthalene dicarboxylic acid is purified and crystallized under respective suitable conditions using a recombinant microorganism capable of converting FNA to NDA. Therefore, the purification method of the present invention enables the production of high-purity crystalline 2,6-naphthalene dicarboxylic acid on an industrial scale in an economically feasible and environmentally friendly manner. Sequence Listing
[158] SEQ ID NO: 1 is a base sequence of a benzaldehyde dehydrogenase gene (xylC)
derived from Sphingomonas aromaticivorans (KCTC 2888).
[159] SEQ ID NO: 2 shows primer 1 synthesized on the basis of a DNA base sequence of a benzaldehyde dehydrogenase gene (xylC) derived from Sphingomonas aromaticivorans (KCTC 2888) to clone the xylC gene.
[160] SEQ ID NO: 3 shows primer 2 synthesized on the basis of a DNA base sequence of a benzaldehyde dehydrogenase gene (xylC) derived from Sphingomonas aromaticivorans (KCTC 2888) to clone the xylC gene.
[161] SEQ ID NO: 4 is a base sequence of a benzaldehyde dehydrogenase gene (xylC) derived from Pseudomonas putida mt-2 (ATCC 33015).
Claims
[1] A method for purifying a crude naphthalene dicarboxylic acid using a recombinant microorganism, the method comprising the steps of:
(a) reacting a crude naphthalene dicarboxylic acid with at least one recombinant microorganism selected from the group consisting of microorganisms obtained by transformation with a recombinant expression vector carrying the ben- zaldehyde dehydrogenase gene (xylC) of SEQ ID NO: 1 derived from Sph- ingomonas aromaticivorans (KCTC 2888) or a gene having a homology of at least 90% with the xylC gene and microorganisms obtained by transformation with a recombinant expression vector carrying the benzaldehyde dehydrogenase gene (xylC) of SEQ ID NO: 4 derived from Pseudomonas putida mt-2 (ATCC 33015) or a gene having a homology of at least 90% with the xylC gene, to remove 2-formyl-6-naphthoic acid present in the crude naphthalene dicarboxylic acid;
(b) adding an acidic solution to the reaction solution prepared in step (a) to adjust the pH of the reaction solution and reacting the mixed solution with stirring to crystallize the crude naphthalene dicarboxylic acid;
(c) washing the crystal of the crude naphthalene dicarboxylic acid to remove impurities contained therein; and
(d) drying the washed product to obtain 2,6-naphthalene dicarboxylic acid in a pure crystalline form.
[2] The method according to claim 1, wherein step (a) includes the sub-steps of:
1) inoculating the recombinant microorganism into a liquid medium, culturing the cells to induce the expression of benzaldehyde dehydrogenase, centrifuging the culture broth to collect the cells in which benzaldehyde dehydrogenase is expressed, and suspending the cells in physiological saline or distilled water;
2) mixing a crude naphthalene dicarboxylic acid (cNDA) as a substrate with a buffer solution and adjusting the pH of the mixed solution by the addition of an alkaline solution to prepare a reaction solution for subsequent purification; and
3) reacting the cells prepared in 1) with the reaction solution prepared in 2) to convert 2-formyl-6-naphthoic acid contained in the crude naphthalene dicarboxylic acid to 2,6-naphthalene dicarboxylic acid, so that the purity of the 2,6-naphthalene dicarboxylic acid is increased.
[3] The method according to claim 2, wherein the microorganism is a bacterium belonging to the genus Escherichia, Pseudomonas, Rhodococcus or Bacillus.
[4] The method according to claim 2, wherein the microorganism is E. coli.
[5] The method according to claim 2, wherein the buffer solution is selected from
the group consisting of water, sodium carbonate buffers (Na CO /NaHCO ), glycine buffers (glycine/NaOH), potassium phosphate buffers (KH PO /KOH),
2 4 sodium phosphate buffers (Na HPO /NaH PO ), succinic acid buffers (succinic
2 4 2 4 acid/NaOH), sodium acetate buffers (sodium acetate/acetic acid), citric acid buffers (citric acid/sodium citrate), sodium pyrophosphate buffers (Na P O / HCl), boric acid buffers (boric acid/NaOH), sodium borate buffers (sodium borate/HCl), and mixtures thereof; and has a concentration of 0.01 to 100 mM.
[6] The method according to claim 2, wherein the alkaline solution is a NaOH or
KOH solution.
[7] The method according to claim 2, wherein the mixed solution further contains an organic solvent.
[8] The method according to claim 7, wherein the organic solvent is selected from the group consisting of dimethylsulf oxide, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, and mixtures thereof; and is added at a concentration of 0.01 to 20%.
[9] The method according to claim 2, wherein the reaction is conducted at 25 to
7O0C for 1 minute to 1 hour.
[10] The method according to claim 2, wherein the crude naphthalene dicarboxylic acid is present at a concentration of between 0.001% and 20% in the reaction solution.
[11] The method according to claim 1, wherein the acidic solution is selected from the group consisting of sulfuric acid, hydrochloric acid, glacial acetic acid, nitric acid, and mixtures thereof.
[12] The method according to claim 1, wherein the pH of the reaction solution is adjusted to the range of 1 to 4.
[13] The method according to claim 1, wherein the reaction is carried out at 40C to
15O0C for 1 minute to 10 hours.
[14] The method according to claim 1, wherein the stirring is performed at a rate of 0 to 1,000 rpm.
[15] The method according to claim 1, wherein step (c) is carried out by dispersing the cNDA crystal in water, stirring the dispersion at a pressure of 1 to 60 kg/cm and a temperature of 100 to 27O0C for 10 minutes to 1 hour, filtering the cNDA crystal to remove the water, and repeating the above procedure.
[16] The method according to claim 1, wherein the drying is performed at 30 to
2000C.
[17] The method according to claim 1, further comprising the step of removing the recombinant microorganism used in step (a) after step (a) and prior to step (b).
[18] The method according to claim 17, wherein the removal of the microorganism is
achieved using a microfilter system, a continuous type centrifugal separator or a decanter. [19] The method according to claim 18, wherein the microfilter system uses a filter having a pore size of 0.1 to 0.5 D and made of ceramic, stainless steel, polypropylene or polyethylene terephthalate (PET). [20] 2,6-Naphthalene dicarboxylic acid in a pure crystalline form produced by the method according to any one of claims 1 to 19. [21] The 2,6-naphthalene dicarboxylic acid according to claim 20, wherein the
2,6-naphthalene dicarboxylic acid is in a regular crystalline form.
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CN108753673A (en) * | 2018-06-14 | 2018-11-06 | 迪沙药业集团有限公司 | A kind of recombinant pseudomonas putida and application |
CN114290454A (en) * | 2022-01-06 | 2022-04-08 | 江西省林业科学院 | Modified oil-tea camellia fruit shell powder and preparation method thereof, oil-tea camellia fruit shell powder filler and application thereof |
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KR20200085086A (en) | 2019-01-04 | 2020-07-14 | 김영관 | Time stamp generating system and the generating method thereof for an image format data |
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JPH07313176A (en) * | 1994-05-24 | 1995-12-05 | Sekiyu Sangyo Kasseika Center | New microorganism and production of 2, 6-naphthalene dicarboxylic acid using the same |
US5859294A (en) * | 1996-02-05 | 1999-01-12 | Mitsubishi Gas Chemical Corporation, Inc. | Process for the production of high-purity naphthalenedicarboxylic acid |
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KR20040060351A (en) * | 2002-12-30 | 2004-07-06 | 주식회사 효성 | A refining process of 2,6-Naphtalene Dicarboxylic Acid using a microorganism |
JP2005278549A (en) * | 2004-03-30 | 2005-10-13 | Yoshihiro Katayama | Gene for producing 2-pyrone-4,6-dicarboxylic acid by fermentation, plasmid containing gene, transformant containing plasmid and method for producing 2-pyrone-4,6-dicarboxylic acid |
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- 2006-07-04 KR KR1020060062495A patent/KR20080004041A/en not_active Application Discontinuation
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US5256817A (en) * | 1992-06-18 | 1993-10-26 | Amoco Corporation | Method for purifying a naphthalenedicarboxylic acid |
JPH07313176A (en) * | 1994-05-24 | 1995-12-05 | Sekiyu Sangyo Kasseika Center | New microorganism and production of 2, 6-naphthalene dicarboxylic acid using the same |
US5859294A (en) * | 1996-02-05 | 1999-01-12 | Mitsubishi Gas Chemical Corporation, Inc. | Process for the production of high-purity naphthalenedicarboxylic acid |
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JP2005278549A (en) * | 2004-03-30 | 2005-10-13 | Yoshihiro Katayama | Gene for producing 2-pyrone-4,6-dicarboxylic acid by fermentation, plasmid containing gene, transformant containing plasmid and method for producing 2-pyrone-4,6-dicarboxylic acid |
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CN108753673A (en) * | 2018-06-14 | 2018-11-06 | 迪沙药业集团有限公司 | A kind of recombinant pseudomonas putida and application |
CN114290454A (en) * | 2022-01-06 | 2022-04-08 | 江西省林业科学院 | Modified oil-tea camellia fruit shell powder and preparation method thereof, oil-tea camellia fruit shell powder filler and application thereof |
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