JP4582228B2 - polyester - Google Patents
polyester Download PDFInfo
- Publication number
- JP4582228B2 JP4582228B2 JP2008215686A JP2008215686A JP4582228B2 JP 4582228 B2 JP4582228 B2 JP 4582228B2 JP 2008215686 A JP2008215686 A JP 2008215686A JP 2008215686 A JP2008215686 A JP 2008215686A JP 4582228 B2 JP4582228 B2 JP 4582228B2
- Authority
- JP
- Japan
- Prior art keywords
- acid
- polyester
- dna
- dicarboxylic acid
- raw material
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 229920000728 polyester Polymers 0.000 title claims description 61
- 238000000034 method Methods 0.000 claims description 55
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 claims description 53
- 239000002994 raw material Substances 0.000 claims description 43
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 claims description 42
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 36
- 238000006243 chemical reaction Methods 0.000 claims description 36
- 238000000855 fermentation Methods 0.000 claims description 29
- 230000004151 fermentation Effects 0.000 claims description 29
- 239000001384 succinic acid Substances 0.000 claims description 24
- 150000002009 diols Chemical class 0.000 claims description 20
- 239000002253 acid Substances 0.000 claims description 17
- 229910052757 nitrogen Inorganic materials 0.000 claims description 17
- 230000002829 reductive effect Effects 0.000 claims description 17
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 15
- QPFMBZIOSGYJDE-UHFFFAOYSA-N 1,1,2,2-tetrachloroethane Chemical compound ClC(Cl)C(Cl)Cl QPFMBZIOSGYJDE-UHFFFAOYSA-N 0.000 claims description 12
- 238000007334 copolymerization reaction Methods 0.000 claims description 5
- 230000000379 polymerizing effect Effects 0.000 claims description 5
- 239000012046 mixed solvent Substances 0.000 claims description 4
- 108020004414 DNA Proteins 0.000 description 83
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 80
- 239000013612 plasmid Substances 0.000 description 46
- 239000012634 fragment Substances 0.000 description 42
- 239000002609 medium Substances 0.000 description 41
- 229910021529 ammonia Inorganic materials 0.000 description 39
- 239000000243 solution Substances 0.000 description 39
- 241000588724 Escherichia coli Species 0.000 description 34
- 108091008146 restriction endonucleases Proteins 0.000 description 25
- 239000000203 mixture Substances 0.000 description 23
- 241000319304 [Brevibacterium] flavum Species 0.000 description 20
- 108090000623 proteins and genes Proteins 0.000 description 19
- 230000000694 effects Effects 0.000 description 18
- 229930027917 kanamycin Natural products 0.000 description 17
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 17
- 229960000318 kanamycin Drugs 0.000 description 17
- 229930182823 kanamycin A Natural products 0.000 description 17
- 238000006116 polymerization reaction Methods 0.000 description 17
- WERYXYBDKMZEQL-UHFFFAOYSA-N butane-1,4-diol Chemical compound OCCCCO WERYXYBDKMZEQL-UHFFFAOYSA-N 0.000 description 16
- 239000013078 crystal Substances 0.000 description 16
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 14
- 239000008103 glucose Substances 0.000 description 14
- 239000006142 Luria-Bertani Agar Substances 0.000 description 13
- 238000007796 conventional method Methods 0.000 description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 239000003054 catalyst Substances 0.000 description 12
- 238000010276 construction Methods 0.000 description 12
- 239000012153 distilled water Substances 0.000 description 12
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 12
- 238000009630 liquid culture Methods 0.000 description 12
- 238000004519 manufacturing process Methods 0.000 description 12
- 229920000642 polymer Polymers 0.000 description 11
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 10
- 229920000936 Agarose Polymers 0.000 description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 10
- 108010053763 Pyruvate Carboxylase Proteins 0.000 description 10
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 10
- 229930006000 Sucrose Natural products 0.000 description 10
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 10
- 239000000047 product Substances 0.000 description 10
- 229960004793 sucrose Drugs 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 9
- 150000001413 amino acids Chemical class 0.000 description 9
- 229940088598 enzyme Drugs 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 9
- 239000005720 sucrose Substances 0.000 description 9
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Chemical compound OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 description 8
- 238000012790 confirmation Methods 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 229910052702 rhenium Inorganic materials 0.000 description 7
- 239000013598 vector Substances 0.000 description 7
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 6
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 6
- WNLRTRBMVRJNCN-UHFFFAOYSA-N adipic acid Chemical compound OC(=O)CCCCC(O)=O WNLRTRBMVRJNCN-UHFFFAOYSA-N 0.000 description 6
- 229960000723 ampicillin Drugs 0.000 description 6
- 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 6
- 239000000872 buffer Substances 0.000 description 6
- 229940041514 candida albicans extract Drugs 0.000 description 6
- 239000012535 impurity Substances 0.000 description 6
- 239000004310 lactic acid Substances 0.000 description 6
- 235000014655 lactic acid Nutrition 0.000 description 6
- 244000005700 microbiome Species 0.000 description 6
- -1 organic acid salt Chemical class 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 229910052707 ruthenium Inorganic materials 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 239000012138 yeast extract Substances 0.000 description 6
- YPFDHNVEDLHUCE-UHFFFAOYSA-N 1,3-propanediol Substances OCCCO YPFDHNVEDLHUCE-UHFFFAOYSA-N 0.000 description 5
- 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 5
- 229920001817 Agar Polymers 0.000 description 5
- 108010017826 DNA Polymerase I Proteins 0.000 description 5
- 102000004594 DNA Polymerase I Human genes 0.000 description 5
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 5
- 102100039895 Pyruvate carboxylase, mitochondrial Human genes 0.000 description 5
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 5
- 239000008272 agar Substances 0.000 description 5
- 238000000246 agarose gel electrophoresis Methods 0.000 description 5
- 125000001931 aliphatic group Chemical group 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 5
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 5
- 239000004202 carbamide Substances 0.000 description 5
- 229910052799 carbon Inorganic materials 0.000 description 5
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 5
- 229960005091 chloramphenicol Drugs 0.000 description 5
- 108010030074 endodeoxyribonuclease MluI Proteins 0.000 description 5
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 5
- 229960005542 ethidium bromide Drugs 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 238000001502 gel electrophoresis Methods 0.000 description 5
- 150000002291 germanium compounds Chemical class 0.000 description 5
- 230000000813 microbial effect Effects 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 150000003839 salts Chemical group 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 230000009466 transformation Effects 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 4
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 4
- KKEYFWRCBNTPAC-UHFFFAOYSA-N Terephthalic acid Chemical compound OC(=O)C1=CC=C(C(O)=O)C=C1 KKEYFWRCBNTPAC-UHFFFAOYSA-N 0.000 description 4
- 125000003118 aryl group Chemical group 0.000 description 4
- 239000002585 base Substances 0.000 description 4
- 150000001720 carbohydrates Chemical class 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 229910052802 copper Inorganic materials 0.000 description 4
- 150000001991 dicarboxylic acids Chemical class 0.000 description 4
- TVIDDXQYHWJXFK-UHFFFAOYSA-N dodecanedioic acid Chemical compound OC(=O)CCCCCCCCCCC(O)=O TVIDDXQYHWJXFK-UHFFFAOYSA-N 0.000 description 4
- YBMRDBCBODYGJE-UHFFFAOYSA-N germanium oxide Inorganic materials O=[Ge]=O YBMRDBCBODYGJE-UHFFFAOYSA-N 0.000 description 4
- 235000011187 glycerol Nutrition 0.000 description 4
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 4
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 4
- PVADDRMAFCOOPC-UHFFFAOYSA-N oxogermanium Chemical compound [Ge]=O PVADDRMAFCOOPC-UHFFFAOYSA-N 0.000 description 4
- 230000000704 physical effect Effects 0.000 description 4
- 239000004417 polycarbonate Substances 0.000 description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 4
- CXMXRPHRNRROMY-UHFFFAOYSA-N sebacic acid Chemical compound OC(=O)CCCCCCCCC(O)=O CXMXRPHRNRROMY-UHFFFAOYSA-N 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 239000012137 tryptone Substances 0.000 description 4
- DNIAPMSPPWPWGF-VKHMYHEASA-N (+)-propylene glycol Chemical compound C[C@H](O)CO DNIAPMSPPWPWGF-VKHMYHEASA-N 0.000 description 3
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 3
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 description 3
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 3
- 244000063299 Bacillus subtilis Species 0.000 description 3
- 235000014469 Bacillus subtilis Nutrition 0.000 description 3
- 241000186031 Corynebacteriaceae Species 0.000 description 3
- 235000000638 D-biotin Nutrition 0.000 description 3
- 239000011665 D-biotin Substances 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 239000001361 adipic acid Substances 0.000 description 3
- 235000011037 adipic acid Nutrition 0.000 description 3
- 239000001099 ammonium carbonate Substances 0.000 description 3
- 235000012501 ammonium carbonate Nutrition 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- 150000008064 anhydrides Chemical class 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
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- 238000010367 cloning Methods 0.000 description 3
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- 238000005520 cutting process Methods 0.000 description 3
- 125000001142 dicarboxylic acid group Chemical group 0.000 description 3
- 238000000909 electrodialysis Methods 0.000 description 3
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- 239000000284 extract Substances 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 230000006801 homologous recombination Effects 0.000 description 3
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- 239000003456 ion exchange resin Substances 0.000 description 3
- 229920003303 ion-exchange polymer Polymers 0.000 description 3
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 3
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 3
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 3
- 230000003472 neutralizing effect Effects 0.000 description 3
- 229910052759 nickel Inorganic materials 0.000 description 3
- 229910052763 palladium Inorganic materials 0.000 description 3
- 229920000166 polytrimethylene carbonate Polymers 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000012064 sodium phosphate buffer Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 229940014800 succinic anhydride Drugs 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 3
- 229960000344 thiamine hydrochloride Drugs 0.000 description 3
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 3
- 239000011747 thiamine hydrochloride Substances 0.000 description 3
- 229910052718 tin Inorganic materials 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- NGEWQZIDQIYUNV-UHFFFAOYSA-N 2-hydroxy-3-methylbutyric acid Chemical compound CC(C)C(O)C(O)=O NGEWQZIDQIYUNV-UHFFFAOYSA-N 0.000 description 2
- YEJRWHAVMIAJKC-UHFFFAOYSA-N 4-Butyrolactone Chemical compound O=C1CCCO1 YEJRWHAVMIAJKC-UHFFFAOYSA-N 0.000 description 2
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 241000186146 Brevibacterium Species 0.000 description 2
- KAKZBPTYRLMSJV-UHFFFAOYSA-N Butadiene Chemical compound C=CC=C KAKZBPTYRLMSJV-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 229930091371 Fructose Natural products 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
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- 229920002472 Starch Polymers 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
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- 229910001873 dinitrogen Inorganic materials 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 235000019797 dipotassium phosphate Nutrition 0.000 description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 2
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- QQVIHTHCMHWDBS-UHFFFAOYSA-N isophthalic acid Chemical compound OC(=O)C1=CC=CC(C(O)=O)=C1 QQVIHTHCMHWDBS-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000011572 manganese Substances 0.000 description 2
- VSEWZMQQQHLHQT-UHFFFAOYSA-N manganese;sulfuric acid;hydrate Chemical compound O.[Mn].OS(O)(=O)=O VSEWZMQQQHLHQT-UHFFFAOYSA-N 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
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- 238000000465 moulding Methods 0.000 description 2
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
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- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 239000008057 potassium phosphate buffer Substances 0.000 description 2
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- Polyesters Or Polycarbonates (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Description
本発明は、ポリエステルに関する。 The present invention relates to polyester.
従来ポリエステルは、芳香族ポリエステル、脂肪族ポリエステル、全芳香族ポリエステル、半芳香族ポリエステル、ポリ炭酸エステルのいずれのポリエステルも石油由来の原料を重縮合することにより製造されている。しかし、近年環境問題が重視されてきており、化石燃料枯渇問題、大気中の二酸化炭素増加という地球規模での環境負荷の問題に対する対策が必要となっている。これに対しポリエステルの原料として植物から誘導された原料を用いることにより、毎年再生可能な原料であるため原料供給が化石燃料枯渇とは無関係になり、植物の育成により二酸化炭素が吸収されるため二酸化炭素削減に大きく貢献することができる。 Conventional polyesters are produced by polycondensing petroleum-derived raw materials for any of aromatic polyesters, aliphatic polyesters, wholly aromatic polyesters, semi-aromatic polyesters, and polycarbonates. However, in recent years, environmental issues have been emphasized, and countermeasures against global environmental load problems such as fossil fuel depletion and increased carbon dioxide in the atmosphere are required. On the other hand, by using raw materials derived from plants as raw materials for polyester, the supply of raw materials is irrelevant to fossil fuel depletion because it is a renewable material every year. It can greatly contribute to carbon reduction.
コハク酸、アジピン酸などのジカルボン酸は、グルコースから発酵法を用いて製造する方法が各種知られている。しかしながら、これらの製法によれば、発酵法により得られたジカルボン酸は有機酸塩として得られるため、不純物としてアンモニアや金属カチオンが不純物として混入する。 Various methods for producing dicarboxylic acids such as succinic acid and adipic acid from glucose using a fermentation method are known. However, according to these production methods, since the dicarboxylic acid obtained by the fermentation method is obtained as an organic acid salt, ammonia or a metal cation is mixed as an impurity as an impurity.
ジオールの１，４−ブタンジオール、１，３−プロパンジオール、エチレングリコールなどは直接植物由来の原料から発酵法により得る方法と、相当するジカルボン酸を発酵法により製造した後、これを還元触媒により水添して得る方法がある（非特許文献１）。
精製法により精製した従来の技術で得られる通常の純度の原料を用いてジカルボン酸とジオールの重合を行おうとするとアンモニア塩や金属カチオンが重合反応を阻害し、十分な分子量のポリエステルが得られなかったり、得られたポリエステルが着色したりするという問題が生じており未だ実用化には至っていない。 When trying to polymerize dicarboxylic acids and diols using raw materials of ordinary purity obtained by conventional techniques purified by purification methods, ammonia salts and metal cations hinder the polymerization reaction and polyesters with sufficient molecular weight cannot be obtained. Or the obtained polyester is colored, and has not yet been put into practical use.
本発明の目的は、発酵法による原料を用いて高分子量で着色の少ない実用可能なポリエステルを提供することにある。 An object of the present invention is to provide a practical polyester having a high molecular weight and little coloring using a raw material obtained by fermentation.
従来から知られている発酵法のジカルボン酸原料の不純物に関して検討を行った結果、原料中に不純物として含まれるアンモニアの含有量が重合速度や樹脂の着色に大きく起因する知見が得られ本発明に到達した。 As a result of studying impurities of dicarboxylic acid raw materials of conventionally known fermentation methods, knowledge that the content of ammonia contained as impurities in the raw materials is largely attributed to the polymerization rate and coloration of the resin was obtained. Reached.
本発明は、ジカルボン酸原料及びジオール原料を含む反応液中でジカルボン酸とジオールとを重合することを含むポリエステルであって、ジカルボン酸原料及びジオール原料の少なくともいずれかが発酵法の工程を含む製造方法により得られたものであり、且つ製造されるポリエステルの窒素含有量が０より大きく１０００ｐｐｍ以下で、３０℃テトラクロロエタン／フェノール（５０／５０質量比）混合溶媒中で測定した還元粘度が０．５以上となるように反応液中のアンモニアの量を制御することを特徴とするポリエステルに存する。 The present invention relates to a polyester comprising polymerizing a dicarboxylic acid and a diol in a reaction solution containing a dicarboxylic acid raw material and a diol raw material, wherein at least one of the dicarboxylic acid raw material and the diol raw material includes a fermentation process. The reduced viscosity measured in a 30 ° C. tetrachloroethane / phenol (50/50 mass ratio) mixed solvent was obtained by the method, and the polyester produced had a nitrogen content of more than 0 and 1000 ppm or less. The polyester is characterized in that the amount of ammonia in the reaction solution is controlled to be 5 or more.
本発明のポリエステルにおいては、ジオール原料が発酵法により得られるジカルボン酸
原料を還元することにより得られるものであってもよい。
In the polyester of the present invention, the diol raw material may be obtained by reducing a dicarboxylic acid raw material obtained by a fermentation method.
製造されるポリエステルは、黄色度（ＹＩ）が３０以下であることが好ましい。また、
製造されるポリエステルは共重合成分としてオキシカルボン酸単位を含んでもよい。
The produced polyester preferably has a yellowness (YI) of 30 or less. Also,
The produced polyester may contain an oxycarboxylic acid unit as a copolymerization component.
ジカルボン酸原料中のアンモニア含有量が４０００ｐｐｍ以下であることが好ましい。
ジカルボン酸の代表的な例はコハク酸である。
The ammonia content in the dicarboxylic acid raw material is preferably 4000 ppm or less.
A representative example of a dicarboxylic acid is succinic acid.
本発明は、発酵法の工程を含む製造方法により得られたジカルボン酸原料及び／又はジオール原料を含む反応液中でジカルボン酸とジオールとを重合することを含むポリエステルにおいて、反応液中のアンモニアの量を制御することにより実用的な物性を有するポリエステル、すなわち、窒素含有量が１０００ｐｐｍ以下で、３０℃テトラクロロエタン／フェノール（５０／５０質量比）混合溶媒中で測定した還元粘度が０．５以上であるポリエステルである。本発明は環境問題、化石資源の問題等の解決に貢献し、実用的な物性を有する樹脂を提供することが出来、産業上の利用価値は極めて大である。 The present invention relates to a polyester comprising polymerizing a dicarboxylic acid and a diol in a reaction liquid containing a dicarboxylic acid raw material and / or a diol raw material obtained by a production method including a fermentation process. Polyester having practical physical properties by controlling the amount, that is, the reduced viscosity measured in a 30 ° C. tetrachloroethane / phenol (50/50 mass ratio) mixed solvent having a nitrogen content of 1000 ppm or less and a nitrogen content of 0.5 or more Is a polyester. The present invention contributes to the solution of environmental problems, fossil resource problems, etc., can provide a resin having practical physical properties, and the industrial utility value is extremely large.
本発明において、「ジカルボン酸原料」及び「ジオール原料」とは、それぞれ、ポリエステルの製造における原料としてのジカルボン酸およびジオールを意味する。 In the present invention, “dicarboxylic acid raw material” and “diol raw material” mean dicarboxylic acid and diol as raw materials in the production of polyester, respectively.
本発明のポリエステルは、発酵法の工程を含む製造方法により得られたジカルボン酸原料及び／又はジオール原料を含む反応液中でジカルボン酸とジオールとを重合することを含むポリエステルにおいて、製造されるポリエステルの窒素含有量が１０００ｐｐｍ以下で、３０℃のテトラクロロエタン／フェノール（５０／５０質量比）混合溶媒中で測定した還元粘度が０．５以上となるように反応液中のアンモニアの量を制御することを特徴とするものである。 The polyester of the present invention is a polyester produced by polymerizing a dicarboxylic acid and a diol in a reaction solution containing a dicarboxylic acid raw material and / or a diol raw material obtained by a production method including a fermentation process. The amount of ammonia in the reaction solution is controlled so that the reduced viscosity measured in a tetrachloroethane / phenol (50/50 mass ratio) mixed solvent at 30 ° C. is not less than 0.5 when the nitrogen content of the solution is 1000 ppm or less. It is characterized by this.
本発明で発酵法の工程を含む製造方法により得られる原料のジカルボン酸としては芳香族ジカルボン酸でも脂肪族ジカルボン酸でも良い。芳香族ジカルボン酸としてはテレフタル酸、イソフタル酸等が挙げられるが、重合性の点でテレフタル酸が好ましい。脂肪族ジカルボン酸としてはシュウ酸、コハク酸、グルタル酸、アジピン酸、セバシン酸、ドデカン二酸等のジカルボン酸ならびにそれらの低級アルコールエステル及び無水物（例えば、無水コハク酸、無水アジピン酸）等が挙げられる。得られる共重合体の物性の面から、コハク酸、アジピン酸、セバシン酸、ドデカン二酸またはこれらの無水物もしくは低級アルコールエステルが好ましく、特にはコハク酸、無水コハク酸、またはこれらの混合物が好ましい。これらは単独でも２種以上混合して使用することもできる。なお、低級アルコールとは、通常、炭素数１〜４のアルコールを意味する。 The raw dicarboxylic acid obtained by the production method including the fermentation process in the present invention may be an aromatic dicarboxylic acid or an aliphatic dicarboxylic acid. Examples of the aromatic dicarboxylic acid include terephthalic acid and isophthalic acid, and terephthalic acid is preferred from the viewpoint of polymerizability. Aliphatic dicarboxylic acids include oxalic acid, succinic acid, glutaric acid, adipic acid, sebacic acid, dodecanedioic acid and the like, and lower alcohol esters and anhydrides thereof (for example, succinic anhydride, adipic anhydride), etc. Can be mentioned. From the viewpoint of the physical properties of the resulting copolymer, succinic acid, adipic acid, sebacic acid, dodecanedioic acid or anhydrides or lower alcohol esters thereof are preferable, and succinic acid, succinic anhydride, or a mixture thereof is particularly preferable. . These may be used alone or in combination of two or more. The lower alcohol usually means an alcohol having 1 to 4 carbon atoms.
これらのジカルボン酸は、例えば、炭素源を出発原料とした微生物変換により生成される。炭素源としては、通常、ガラクトース、ラクトース、グルコース、フルクトース、グリセロール、シュークロース、サッカロース、デンプン、セルロース等の炭水化物；グリセリン、マンニトール、キシリトール、リビトール等のポリアルコール類等の発酵性糖質が用いられ、このうちグルコース、フルクトース、グリセロールが好ましく、特にグルコースが好ましい。より広義の植物由来炭素源としては、紙の主成分であるセルロースが好ましい。また、上記発酵性糖質を含有する澱粉糖化液、糖蜜なども使用される。これらの発酵性糖質は、１種を単独で用いても２種以上を組み合わせて用いてもよい。 These dicarboxylic acids are produced, for example, by microbial conversion using a carbon source as a starting material. As the carbon source, carbohydrates such as galactose, lactose, glucose, fructose, glycerol, sucrose, saccharose, starch and cellulose; and fermentable carbohydrates such as polyalcohols such as glycerin, mannitol, xylitol and ribitol are usually used. Of these, glucose, fructose, and glycerol are preferable, and glucose is particularly preferable. As a broader plant-derived carbon source, cellulose which is the main component of paper is preferable. Moreover, the starch saccharified liquid, molasses, etc. containing the said fermentable saccharide | sugar are also used. These fermentable carbohydrates may be used alone or in combination of two or more.
微生物変換に用いる微生物としては、ジカルボン酸の生産能を有すれば特に限定されないが、例えば、Anaerobiospirillum属 （米国特許第５１４３８３３号明細書）等の嫌気
性細菌、Actinobacillus属（米国特許第５５０４００４号明細書）、Escherichia属（米国特許第５７７０４３５号明細書）等の通性嫌気性細菌、Corynebacterium属（特開平１１−１１３５８８）などの好気性細菌、Bacillus属、Rizobium属、Brevibacterium属、Arthrobacter属に属する好気性細菌（特開２００３−２３５５９３）、Bacteroides ruminicola、Bacteroides amylophilus等の嫌気性ルーメン細菌、Ｅ．ｃｏｌｉ（J.Bacteriol.,57:147-158）又はＥ．ｃｏｌｉの株の変異体（特表２０００−５００３３３、米国特許第６１５９７３８号明細書）を用いることができる。
The microorganism used for microbial conversion is not particularly limited as long as it has the ability to produce dicarboxylic acid. For example, anaerobic bacteria such as Anaerobiospirillum (US Pat. No. 5,143,833), Actinobacillus (US Pat. No. 5,504,004) ), Facultative anaerobes such as Escherichia (US Pat. No. 5,770,435), aerobic bacteria such as Corynebacterium (Japanese Patent Laid-Open No. 11-113588), Bacillus, Rizobium, Brevibacterium, Arthrobacter Anaerobic bacteria (JP 2003-235593), anaerobic rumen bacteria such as Bacteroides ruminicola, Bacteroides amylophilus, E. coli (J. Bacteriol., 57: 147-158) or E. coli. E. coli strains (Tokuyo 2000-500333, US Pat. No. 6,159,738) can be used.
微生物変換における反応温度、圧力等の反応条件は、選択される菌体、カビなど微生物の活性に依存することになるが、ジカルボン酸を得るための好適な条件を各々の場合に応じて選択すればよい。 Reaction conditions such as reaction temperature and pressure in microbial conversion will depend on the activity of microorganisms such as selected cells and molds, but suitable conditions for obtaining dicarboxylic acids should be selected in each case. That's fine.
上記微生物変換においては、ｐＨが低くなると微生物の代謝活性が低くなったり、或いは微生物が活動を停止するようになり、製造歩留まりが悪化したり、微生物が死滅するため、通常には中和剤を使用する。通常はｐＨセンサーによって反応系内のｐＨを計測し、所定のｐＨ範囲となるように中和剤の添加によりｐＨを調節する。中和剤の添加方法については特に制限はなく、連続添加であっても間欠添加であってもよい。 In the above-mentioned microbial conversion, when the pH is lowered, the metabolic activity of the microorganism is lowered, or the microorganism stops its activity, the production yield deteriorates, or the microorganism is killed. use. Usually, the pH in the reaction system is measured by a pH sensor, and the pH is adjusted by adding a neutralizing agent so as to be in a predetermined pH range. There is no restriction | limiting in particular about the addition method of a neutralizing agent, Continuous addition or intermittent addition may be sufficient.
中和剤としてはアンモニア、炭酸アンモニウム、尿素、アルカリ金属の水酸化物、アルカリ土類金属の水酸化物、アルカリ金属の炭酸塩、アルカリ土類金属の炭酸塩が挙げられる。好ましくはアンモニア、炭酸アンモニウム、尿素である。なお上記アルカリ（土類）金属の水酸化物としてはＮａＯＨ、ＫＯＨ、Ｃａ（ＯＨ）2、Ｍｇ（ＯＨ）2等、或いはこれらの混合物などが挙げられ、アルカリ（土類）金属の炭酸塩としては、Ｎａ2ＣＯ3、Ｋ2ＣＯ3、ＣａＣＯ3、ＭｇＣＯ3、ＮａＫＣＯ3等、或いはこれらの混合物などが挙げられる。 Examples of the neutralizing agent include ammonia, ammonium carbonate, urea, alkali metal hydroxide, alkaline earth metal hydroxide, alkali metal carbonate, and alkaline earth metal carbonate. Ammonia, ammonium carbonate and urea are preferred. Examples of the alkali (earth) metal hydroxide include NaOH, KOH, Ca (OH) 2 , Mg (OH) 2 , or a mixture thereof. is, Na 2 CO 3, K 2 CO 3, CaCO 3, MgCO 3, NaKCO 3 etc., or the like and mixtures thereof.
ｐＨ値は、用いる菌体、カビ等の微生物の種類に応じて、その活性が最も有効に発揮される範囲に調整されるが、一般的には、ｐＨ４〜１０、好ましくは６〜９程度の範囲である。 The pH value is adjusted to a range in which the activity is most effectively exhibited depending on the type of microorganisms such as fungus bodies and molds to be used. In general, the pH value is about 4 to 10, preferably about 6 to 9. It is a range.
発酵法を含む製造方法により得られるジカルボン酸の精製方法は電気透析を用いる方法、イオン交換樹脂を用いる方法、塩交換法等が知られている。例えばジカルボン酸塩を分離し純粋な酸を生成する電気透析および水分解工程を組み合わせて用いることによって製造し、更なる精製を、一連のイオン交換カラムに生成物ストリームを通すことによって達成しても良いし、ジカルボン酸の過飽和溶液に変換するための水分解電気透析を用いても良い（米国特許第５，０３４，１０５号明細書）。また、塩交換法は例えばジカルボン酸のアンモニア塩を硫酸水素アンモニウム及び／または硫酸と十分に低いｐＨで混合して反応させジカルボン酸及び硫酸アンモニウムを生成させても良い（特表２００１−５１４９００）。イオン交換樹脂を用いる具体的方法としては、ジカルボン酸の溶液から遠心分離、濾過等により菌体等の固形分を除去した後、イオン交換樹脂で脱塩し、その溶液から結晶化或いはカラムクロマトグラフィーによりジカルボン酸を分離精製する方法が挙げられる。精製方法はどのような方法を用いても良い。特に、コスト、効率の点でイオン交換法又は塩交換法が好ましく、工業的生産性の点で塩交換法が特に好ましい。 As a method for purifying a dicarboxylic acid obtained by a production method including a fermentation method, a method using electrodialysis, a method using an ion exchange resin, a salt exchange method and the like are known. For example, it can be produced by using a combination of electrodialysis and water splitting steps to separate the dicarboxylate salt to produce pure acid, and further purification can be achieved by passing the product stream through a series of ion exchange columns. Alternatively, hydrolytic electrodialysis for conversion to a supersaturated solution of dicarboxylic acid may be used (US Pat. No. 5,034,105). In the salt exchange method, for example, an ammonium salt of dicarboxylic acid may be mixed with ammonium hydrogen sulfate and / or sulfuric acid at a sufficiently low pH and reacted to generate dicarboxylic acid and ammonium sulfate (Japanese Patent Publication No. 2001-514900). As a specific method using an ion exchange resin, after removing solids such as cells from a dicarboxylic acid solution by centrifugation, filtration, etc., desalting with an ion exchange resin and crystallization or column chromatography from the solution. Can be used to separate and purify the dicarboxylic acid. Any purification method may be used. In particular, the ion exchange method or the salt exchange method is preferable in terms of cost and efficiency, and the salt exchange method is particularly preferable in terms of industrial productivity.
精製によりジカルボン酸原料中に含まれる不純物のアンモニアの量を減らすことが、通常、実用的な重合体を得るために必要である。ジカルボン酸原料中に含まれるアンモニア含有量は４０００ｐｐｍ以下であることが好ましい。より好ましくは２０００ｐｐｍ以下、さらに好ましくは５００ｐｐｍ以下、最も好ましくは１００ｐｐｍ以下である。原料中のアンモニア含有量が２０００ｐｐｍ以下であると重合反応が速やかに進行して十分に高分子量のポリエステルを得ることが出来る。アンモニア含有量は、アミノ酸分析計を用い
、生体アミノ酸分離条件にて試料中のアミノ酸やアンモニアを分離し、これらをニンヒドリン発色させて検出する方法により測定される値である。
It is usually necessary to reduce the amount of impurity ammonia contained in the dicarboxylic acid raw material by purification in order to obtain a practical polymer. The ammonia content contained in the dicarboxylic acid raw material is preferably 4000 ppm or less. More preferably, it is 2000 ppm or less, More preferably, it is 500 ppm or less, Most preferably, it is 100 ppm or less. When the ammonia content in the raw material is 2000 ppm or less, the polymerization reaction proceeds rapidly and a sufficiently high molecular weight polyester can be obtained. The ammonia content is a value measured by a method in which an amino acid analyzer is used to separate amino acids and ammonia in a sample under biological amino acid separation conditions, and these are detected by ninhydrin color development.
アンモニア含有量が上記の範囲にあるジカルボン酸原料を用いることで、通常には、重合反応液中のアンモニアの量を、ポリエステルの重合速度を低下させず、かつ、得られるポリエステルの窒素含有量を低下させる範囲に制御するのに有利になる。 By using a dicarboxylic acid raw material having an ammonia content in the above range, the amount of ammonia in the polymerization reaction solution is usually reduced without reducing the polymerization rate of the polyester, and the nitrogen content of the resulting polyester is reduced. It is advantageous to control the range to be lowered.
ジカルボン酸原料中に含まれる不純物のアンモニアの量を効率的に減らす具体的な方法として、目的とするジカルボン酸よりもｐＨの高い弱酸性の有機酸を使用した反応晶析により目的とするフリーのジカルボン酸をアンモニア塩から固体として分離する方法が挙げられる。 As a specific method for efficiently reducing the amount of impurity ammonia contained in the dicarboxylic acid raw material, the desired free of charge can be obtained by reaction crystallization using a weakly acidic organic acid having a pH higher than that of the target dicarboxylic acid. The method of isolate | separating dicarboxylic acid as a solid from ammonia salt is mentioned.
ジオールの具体例としては、エチレングリコール、トリメチレングリコール、１，３−プロパンジオール、１，４−ブタンジオール、１，５−ペンタンジオール、１，６−ヘキサンジオール、１，４−シクロヘキサンジオール、１，４−シクロヘキサンジメタノールが好適に挙げられる。得られる共重合体の物性の面から、エチレングリコール、１，３−プロパンジオール、１，４−ブタンジオールであることが好ましく、特に１，４−ブタンジオールが耐熱性の点で好ましい。これらは単独でも、二種以上の混合物として使用することもできる。ジオール化合物はグルコース等の植物由来物質から発酵法により直接製造してもよいし、発酵法により得られたジカルボン酸、ジカルボン酸無水物、環状エーテルを化学反応によりジオール化合物に変換しても良い。例えば１，４−ブタンジオールをコハク酸、コハク酸無水物、コハク酸エステル、マレイン酸、マレイン酸無水物、マレイン酸エステル、テトラヒドロフラン、γ−ブチロラクトン等から化学合成により１，４−ブタンジオールを製造しても良いし、発酵法により得られた１，３−ブタジエンから１，４−ブタンジオールを製造してもよい。この中でもコハク酸を還元触媒により水添して１，４−ブタンジオールを得る方法が効率的で好ましい。 Specific examples of the diol include ethylene glycol, trimethylene glycol, 1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, 1,4-cyclohexanediol, Suitable examples include 1,4-cyclohexanedimethanol. From the viewpoint of the physical properties of the obtained copolymer, ethylene glycol, 1,3-propanediol, and 1,4-butanediol are preferable, and 1,4-butanediol is particularly preferable in terms of heat resistance. These may be used alone or as a mixture of two or more. The diol compound may be directly produced from a plant-derived substance such as glucose by a fermentation method, or dicarboxylic acid, dicarboxylic acid anhydride and cyclic ether obtained by the fermentation method may be converted into a diol compound by a chemical reaction. For example, 1,4-butanediol is chemically synthesized from succinic acid, succinic anhydride, succinic ester, maleic acid, maleic anhydride, maleic ester, tetrahydrofuran, γ-butyrolactone, etc. to produce 1,4-butanediol. Alternatively, 1,4-butanediol may be produced from 1,3-butadiene obtained by a fermentation method. Among these, a method of hydrogenating succinic acid with a reduction catalyst to obtain 1,4-butanediol is efficient and preferable.
コハク酸を水添する触媒の例として、Ｐｄ、Ｒｕ、Ｒｅ、Ｒｈ、Ｎｉ、Ｃｕ、Ｃｏ及びその化合物が挙げられ、より具体的には、Ｐｄ／Ａｇ／Ｒｅ、Ｒｕ／Ｎｉ／Ｃｏ／ＺｎＯ、Ｃｕ／Ｚｎ酸化物、Ｃｕ／Ｚｎ／Ｃｒ酸化物、Ｒｕ／Ｒｅ、Ｒｅ／Ｃ、Ｒｕ／Ｓｎ、Ｒｕ／Ｐｔ／Ｓｎ、Ｐｔ／Ｒｅ／アルカリ、Ｐｔ／Ｒｅ、Ｐｄ／Ｃｏ／Ｒｅ、Ｃｕ／Ｓｉ、Ｃｕ／Ｃｒ／Ｍｎ、ＲｅＯ／ＣｕＯ／ＺｎＯ、ＣｕＯ／ＣｒＯ、Ｐｄ／Ｒｅ、Ｎｉ／Ｃｏ、Ｐｄ／ＣｕＯ／ＣｒＯ3、リン酸Ｒｕ、Ｎｉ／Ｃｏ、Ｃｏ／Ｒｕ／Ｍｎ、Ｃｕ／Ｐｄ／ＫＯＨ、Ｃｕ／Ｃｒ／Ｚｎが挙げられる。この中でもＲｕ／Ｓｎ又はＲｕ／Ｐｔ／Ｓｎが触媒活性の点で好ましい。 Examples of catalysts for hydrogenating succinic acid include Pd, Ru, Re, Rh, Ni, Cu, Co and compounds thereof, and more specifically, Pd / Ag / Re, Ru / Ni / Co / ZnO. Cu / Zn oxide, Cu / Zn / Cr oxide, Ru / Re, Re / C, Ru / Sn, Ru / Pt / Sn, Pt / Re / alkali, Pt / Re, Pd / Co / Re, Cu / Si, Cu / Cr / Mn, ReO / CuO / ZnO, CuO / CrO, Pd / Re, Ni / Co, Pd / CuO / CrO 3 , phosphoric acid Ru, Ni / Co, Co / Ru / Mn, Cu / Pd / KOH and Cu / Cr / Zn are mentioned. Among these, Ru / Sn or Ru / Pt / Sn is preferable in terms of catalytic activity.
本発明のポリエステルの構成単位の組成比は、ジオール単位とジカルボン酸単位のモル比が、実質的に等しいが、ポリエステルは共重合成分としてオキシカルボン酸単位を含んでもよい。オキシカルボン酸単位の量は、ジカルボン酸単位１００モルに対し、通常には、0.1〜３０モルである。 In the composition ratio of the structural unit of the polyester of the present invention, the molar ratio of the diol unit and the dicarboxylic acid unit is substantially equal, but the polyester may contain an oxycarboxylic acid unit as a copolymerization component. The amount of the oxycarboxylic acid unit is usually 0.1 to 30 mol per 100 mol of the dicarboxylic acid unit.
オキシカルボン酸の具体例としては、ｐ−ヒドロキシ安息香酸、乳酸、グリコール酸、２−ヒドロキシ−ｎ−酪酸、２−ヒドロキシカプロン酸、６−ヒドロキシカプロン酸、２−ヒドロキシ３，３−ジメチル酪酸、２−ヒドロキシ−３−メチル酪酸、２−ヒドロキシイソカプロン酸、あるいはこれらの混合物が挙げられる。これらに光学異性体が存在する場合には、Ｄ体、Ｌ体、またはラセミ体のいずれでもよく、形態としては固体、液体、または水溶液であってもよい。これらの中で好ましいのは、乳酸またはグリコール酸であり、特に好ましいのは、使用時の重合速度の増大が特に顕著で、かつ入手の容易な乳酸またはグリコール酸である。形態は、３０〜９５％の水溶液のものが容易に入手することができるので好ましい。これら脂肪族オキシカルボン酸は単独でも、二種以上の混合物として
使用することもできる。
Specific examples of the oxycarboxylic acid include p-hydroxybenzoic acid, lactic acid, glycolic acid, 2-hydroxy-n-butyric acid, 2-hydroxycaproic acid, 6-hydroxycaproic acid, 2-hydroxy3,3-dimethylbutyric acid, Examples include 2-hydroxy-3-methylbutyric acid, 2-hydroxyisocaproic acid, or a mixture thereof. When optical isomers exist in these, any of D-form, L-form, and racemic form may be sufficient, and a form may be a solid, a liquid, or aqueous solution. Of these, lactic acid or glycolic acid is preferred, and particularly preferred is lactic acid or glycolic acid, which is particularly prominent in increasing the polymerization rate during use and is readily available. The form is preferably 30 to 95% because an aqueous solution can be easily obtained. These aliphatic oxycarboxylic acids can be used alone or as a mixture of two or more.
本発明のジオール単位及びジカルボン酸単位を主体とするポリエステルの製造は、重合反応液中のアンモニアの量を制御する他は、ポリエステルを製造する公知技術で行うことができる。このポリエステルを製造する際の重合反応は、従来から採用されている適切な条件を設定することができ、特に制限されない。 The production of the polyester mainly composed of the diol unit and the dicarboxylic acid unit of the present invention can be carried out by a known technique for producing a polyester other than controlling the amount of ammonia in the polymerization reaction solution. The polymerization reaction at the time of producing this polyester can set appropriate conditions conventionally employed and is not particularly limited.
ポリエステルを製造する際に用いるジオールの使用量は、ジカルボン酸またはその誘導体１００モルに対し、実質的に等モルであるが、一般には、エステル化中の留出があることから、１〜２０モル％過剰に用いられる。添加される脂肪族オキシカルボン酸の量は、脂肪族ジカルボン酸またはその誘導体１００モルに対し好ましくは０〜６０モル、より好ましくは１．０〜４０モル、特に好ましくは２〜２０モルである。 The amount of the diol used in producing the polyester is substantially equimolar with respect to 100 mol of the dicarboxylic acid or derivative thereof, but generally 1 to 20 mol since there is distillation during esterification. % Excess is used. The amount of the aliphatic oxycarboxylic acid added is preferably 0 to 60 mol, more preferably 1.0 to 40 mol, particularly preferably 2 to 20 mol, per 100 mol of the aliphatic dicarboxylic acid or derivative thereof.
オキシカルボン酸の添加時期・方法は、重縮合反応以前であれば特に限定されず、例えば、(1) あらかじめ触媒を脂肪族オキシカルボン酸溶液に溶解させた状態で添加する方法、(2) 原料仕込み時触媒を添加すると同時に添加する方法、などが挙げられる。本発明のポリエステルは、好ましくは上記原料を重合触媒の存在下で重合することにより製造される。触媒としては、チタン化合物、ゲルマニウム化合物が好適である。特にゲルマニウム化合物が好ましい。ゲルマニウム化合物としては、特に制限されるものではなく、酸化ゲルマニウム、テトラアルコキシゲルマニウムなどの有機ゲルマニウム化合物、塩化ゲルマニウムなどの無機ゲルマニウム化合物が挙げられる。価格や入手の容易さなどから、酸化ゲルマニウム、テトラエトキシゲルマニウム、テトラブトキシゲルマニウムなどが好ましく、特には、酸化ゲルマニウムが好適である。また、本発明の目的を損なわない限り、他の触媒の併用を妨げない。 The timing and method of adding the oxycarboxylic acid is not particularly limited as long as it is before the polycondensation reaction. For example, (1) a method in which a catalyst is previously dissolved in an aliphatic oxycarboxylic acid solution, (2) a raw material The method of adding simultaneously with the catalyst at the time of preparation is mentioned. The polyester of the present invention is preferably produced by polymerizing the above raw materials in the presence of a polymerization catalyst. As a catalyst, a titanium compound and a germanium compound are suitable. In particular, a germanium compound is preferable. The germanium compound is not particularly limited, and examples thereof include organic germanium compounds such as germanium oxide and tetraalkoxygermanium, and inorganic germanium compounds such as germanium chloride. In view of price and availability, germanium oxide, tetraethoxygermanium, tetrabutoxygermanium and the like are preferable, and germanium oxide is particularly preferable. Moreover, unless the objective of this invention is impaired, combined use of another catalyst is not prevented.
触媒の使用量は、使用するモノマー量に対して通常には０．００１〜３重量％、より好ましくは０．００５〜１．５重量％である。触媒の添加時期は、重縮合以前であれば特に限定されないが、原料仕込み時に添加しておいてもよく、減圧開始時に添加してもよい。原料仕込み時に乳酸、グリコール酸等のオキシカルボン酸と同時に添加するか、またはオキシカルボン酸水溶液に触媒を溶解して添加する方法が好ましく、特には、触媒の保存性が良好となる点でオキシカルボン酸水溶液に触媒を溶解して添加する方法が好ましい。 The usage-amount of a catalyst is 0.001 to 3 weight% normally with respect to the amount of monomer to be used, More preferably, it is 0.005 to 1.5 weight%. The catalyst addition time is not particularly limited as long as it is before polycondensation, but it may be added when the raw materials are charged, or may be added at the start of pressure reduction. It is preferable to add the oxycarboxylic acid such as lactic acid or glycolic acid at the time of charging the raw material or to dissolve the catalyst in the oxycarboxylic acid aqueous solution and add the oxycarboxylic acid, particularly in terms of improving the storage stability of the catalyst. A method in which the catalyst is dissolved in an aqueous acid solution and added is preferred.
本発明のポリエステルを製造する際の温度、時間、圧力などの条件は、温度が１５０〜２６０℃、好ましくは１８０〜２３０℃の範囲で選ぶのがよく、重合時間は２時間以上、好ましくは４〜１５時間の範囲で選ぶのがよい。減圧度は１０ｍｍＨｇ以下、より好ましくは２ｍｍＨｇ以下で選ぶのがよい。 Conditions such as temperature, time, and pressure for producing the polyester of the present invention are selected in the range of 150 to 260 ° C., preferably 180 to 230 ° C., and the polymerization time is 2 hours or more, preferably 4 It is better to choose in the range of ~ 15 hours. The degree of vacuum should be 10 mmHg or less, more preferably 2 mmHg or less.
また、本発明のポリエステルの数平均分子量は通常には１万〜２０万、好ましくは３万〜２０万である。また本発明のポリエステル重合体を濃度０．５ｇ／ｄＬとなるようにフェノール／テトラクロロエタン（１／１（質量比）混合液）に溶解し、溶液が３０℃の恒温槽中で測定したときの還元粘度（実施例参照）は０．５以上、好ましくは１以上５以下、さらに好ましくは１．５以上４以下である。還元粘度が０．５未満であると機械的強度が不十分で実用的使用に耐えない。 The number average molecular weight of the polyester of the present invention is usually 10,000 to 200,000, preferably 30,000 to 200,000. Further, when the polyester polymer of the present invention was dissolved in phenol / tetrachloroethane (1/1 (mass ratio) mixed solution) so as to have a concentration of 0.5 g / dL, the solution was measured in a thermostat at 30 ° C. The reduced viscosity (see Examples) is 0.5 or more, preferably 1 or more and 5 or less, more preferably 1.5 or more and 4 or less. If the reduced viscosity is less than 0.5, the mechanical strength is insufficient and the practical use cannot be endured.
また、本発明の効果を損なわない限り、本発明のポリエステルに、他の共重合成分を導入することができる。他の共重合成分としては、トリメチロールプロパン、グリセリンなどの多価アルコール、多価カルボン酸またはその無水物、リンゴ酸などの多価オキシカルボン酸、ジフェニルカーボネート等が挙げられる。またポリエーテル成分、ポリアミド成分等を共重合しても良い。またジイソシアネート等で鎖延長剤を用いても良い。また末端基をカルボジイミド、エポキシ化合物、単官能性のアルコール又はカルボン酸で封止して
も良い。
Moreover, unless the effect of this invention is impaired, another copolymerization component can be introduce | transduced into the polyester of this invention. Examples of other copolymerization components include polyhydric alcohols such as trimethylolpropane and glycerin, polyhydric carboxylic acids or anhydrides thereof, polyhydric oxycarboxylic acids such as malic acid, and diphenyl carbonate. Moreover, you may copolymerize a polyether component, a polyamide component, etc. A chain extender may be used such as diisocyanate. Further, the end group may be sealed with carbodiimide, an epoxy compound, a monofunctional alcohol or carboxylic acid.
本発明のポリエステル中に含まれる窒素含有量は１０００ｐｐｍ以下である。窒素含有量は好ましくは５００ｐｐｍ以下、さらに好ましくは１００ｐｐｍ以下、最も好ましくは５０ｐｐｍ以下である。重合体中の窒素含有量は主に原料中のアンモニアに由来するものであるが、窒素含有量が１０００ｐｐｍ以下であると成型時の着色が少なく、成型後製品の熱または光等の劣化や加水分解が起こりにくく好ましい。また、ポリエステルの窒素含有量が１００ｐｐｍ以下であるとポリエステルの着色が少なくて用途によってはより好ましい。窒素含有量は、化学発光法により測定される値である。 The nitrogen content contained in the polyester of the present invention is 1000 ppm or less. The nitrogen content is preferably 500 ppm or less, more preferably 100 ppm or less, and most preferably 50 ppm or less. The nitrogen content in the polymer is mainly derived from ammonia in the raw material, but if the nitrogen content is 1000 ppm or less, there will be little coloration at the time of molding, and the molded product will be deteriorated by heat or light, etc. It is preferable that decomposition does not occur. Further, if the nitrogen content of the polyester is 100 ppm or less, the polyester is less colored and more preferable depending on the application. The nitrogen content is a value measured by a chemiluminescence method.
本発明のポリエステル中に含まれる窒素含有量と原料中に含まれるアンモニア含有量の比は０より大きく０．９より小さいことが好ましく、より好ましくは０より大きく０．６より小さい。 The ratio of the nitrogen content contained in the polyester of the present invention and the ammonia content contained in the raw material is preferably larger than 0 and smaller than 0.9, more preferably larger than 0 and smaller than 0.6.
反応液中のアンモニアの量を制御する方法は、製造されるポリエステルが必要な性質を有する限り制限されないが、例としては、原料の精製により原料に含まれるアンモニアを除く方法、重合中にアンモニアを除去する方法などが挙げられる。ポリエステルの重合工程でアンモニアが除かれることが、原料中のアンモニアを除くための精製にかかる負荷を減らすために好ましい。反応液中のアンモニアの量が制御されるべき範囲は、目的とするポリエステルの性質や重合の条件などによって異なるが、通常には、１０００ｐｐｍ以下である。 The method for controlling the amount of ammonia in the reaction solution is not limited as long as the produced polyester has the necessary properties, but examples include a method for removing ammonia contained in the raw material by purification of the raw material, and ammonia during polymerization. The method of removing is mentioned. It is preferable that ammonia is removed in the polyester polymerization step in order to reduce the load on purification for removing ammonia in the raw material. The range in which the amount of ammonia in the reaction solution should be controlled varies depending on the properties of the target polyester and the polymerization conditions, but is usually 1000 ppm or less.
ポリエステルの黄色度（ＹＩ）はＪＩＳ Ｋ７１０３で測定したときの値が３０以下であることが好ましく、より好ましくは２０以下、さらに好ましくは１０以下未満である。ＹＩが３０より大きいと着色しないフィルム、シート等の用途での使用が制限される。 The yellowness (YI) of the polyester is preferably 30 or less, more preferably 20 or less, and even more preferably less than 10 when measured according to JIS K7103. When YI is larger than 30, the use in non-colored films, sheets and the like is limited.
本発明に係るポリエステルは、射出成形法、中空成形法および押出成形法などの汎用プラスチック成形法などにより、フィルム、ラミネートフィルム、シート、板、延伸シート、モノフィラメント、マルチフィラメント、不織布、フラットヤーン、ステープル、捲縮繊維、筋付きテープ、スプリットヤーン、複合繊維、ブローボトル、発泡体などの成形品に利用可能である。その際、結晶核剤、酸化防止剤、滑剤、着色剤、離型剤、フィラー、他のポリマーなど、必要に応じ添加することができる。 The polyester according to the present invention can be produced by a general-purpose plastic molding method such as an injection molding method, a hollow molding method, and an extrusion molding method, etc. It can be used for molded products such as crimped fibers, striped tapes, split yarns, composite fibers, blow bottles and foams. At that time, a crystal nucleating agent, an antioxidant, a lubricant, a colorant, a release agent, a filler, other polymers, and the like can be added as necessary.
さらに、本発明に係るポリエステルは、従来の石油由来のポリマーに比較し環境に対する負荷が少ないため今後、ショッピングバッグ、ゴミ袋、農業用フィルム、化粧品容器、洗剤容器、漂白剤容器、釣り糸、漁網、ロープ、結束材、手術糸、衛生用カバーストック材、保冷箱、クッション材、医療材料、電気機器材料、家電筐体、自動車材料などの用途への使用が期待される。 Furthermore, since the polyester according to the present invention has less burden on the environment than conventional petroleum-derived polymers, in the future, shopping bags, garbage bags, agricultural films, cosmetic containers, detergent containers, bleach containers, fishing lines, fishing nets, Expected to be used in applications such as ropes, binding materials, surgical threads, sanitary cover stock materials, cold insulation boxes, cushion materials, medical materials, electrical equipment materials, home appliance housings, and automotive materials.
以下、本発明を実施例により具体的に説明するが、本発明はその要旨を超えない限り、これら実施例に限定されるものではない。なお、以下の例における特性値は、次の方法により測定した。 EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to these Examples, unless the summary is exceeded. The characteristic values in the following examples were measured by the following method.
希薄溶液粘度（還元粘度）：ポリエステルを濃度０．５ｇ／ｄＬとなるようにフェノール／テトラクロロエタン（１／１（質量比）混合液）に溶解し、溶液が３０℃の恒温槽中で粘度管を落下する時間ｔ（ｓｅｃ）を測定した。また溶媒のみの落下する時間ｔ0（ｓｅｃ）を測定し３０℃での還元粘度ηsp／Ｃ（＝（ｔ−ｔ0）／ｔ0・Ｃ）を算出した（Ｃは溶液の濃度）。
ＹＩ値：ＪＩＳ Ｋ７１０３の方法に基づいて測定した。
アンモニア含有量：日立L-8500型高速アミノ酸分析計を用い、生体アミノ酸分離条件に
て試料中のアミノ酸やアンモニアを分離し、これらをニンヒドリン発色させて検出した。
窒素含有量：化学発光法により定量分析をおこなった。
Dilute solution viscosity (reduced viscosity): Polyester is dissolved in phenol / tetrachloroethane (1/1 (mass ratio) mixed solution) to a concentration of 0.5 g / dL, and the solution is a viscosity tube in a thermostatic bath at 30 ° C. The time t (sec) for dropping was measured. Further, the time t 0 (sec) during which only the solvent falls was measured, and the reduced viscosity η sp / C (= (t−t 0 ) / t 0 · C) at 30 ° C. was calculated (C is the concentration of the solution).
YI value: Measured based on the method of JIS K7103.
Ammonia content: Using a Hitachi L-8500 type high-speed amino acid analyzer, amino acids and ammonia in the sample were separated under biological amino acid separation conditions, and these were detected by ninhydrin coloring.
Nitrogen content: Quantitative analysis was performed by chemiluminescence method.
［参考例１］
＜遺伝子破壊用ベクターの構築＞
（Ａ）枯草菌ゲノムDNAの抽出
ＬＢ培地［組成：トリプトン１０ｇ、イーストエキストラクト５ｇ、ＮａＣｌ ５ｇを蒸留水１Ｌに溶解］１０ｍＬに、枯草菌（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ＩＳＷ１２１４）を対数増殖期後期まで培養し、菌体を集めた。得られた菌体を１０ｍｇ／ｍＬの濃度にリゾチームを含む１０ｍＭ ＮａＣｌ／２０ｍＭトリス緩衝液（ｐＨ８．０）／１ｍＭ ＥＤＴＡ・２Ｎａ溶液０．１５ｍＬに懸濁した。
[Reference Example 1]
<Construction of vector for gene disruption>
(A) Extraction of Bacillus subtilis genomic DNA In 10 mL of LB medium [composition: tryptone 10 g, yeast extract 5 g, NaCl 5 g dissolved in 1 L of distilled water], Bacillus subtilis ISW1214 is cultured until the late logarithmic growth phase, The cells were collected. The obtained cells were suspended in 0.15 mL of a 10 mM NaCl / 20 mM Tris buffer solution (pH 8.0) / 1 mM EDTA · 2Na solution containing lysozyme at a concentration of 10 mg / mL.
次に、上記懸濁液にプロテナーゼＫを、最終濃度が１００μｇ／ｍＬになるように添加し、３７℃で１時間保温した。さらにドデシル硫酸ナトリウムを最終濃度が０．５％になるように添加し、５０℃で６時間保温して溶菌した。この溶菌液に、等量のフェノール／クロロフォルム溶液を添加し、室温で１０分間ゆるやかに振盪した後、全量を遠心分離（５，０００×ｇ、２０分間、１０〜１２℃）し、上清画分を分取し、酢酸ナトリウムを０．３Ｍとなるように添加した後、２倍量のエタノールを加え混合した。遠心分離（１５，０００×ｇ、２分）により回収した沈殿物を７０％エタノールで洗浄した後、風乾した。得られたＤＮＡに１０ｍＭトリス緩衝液（ｐＨ７．５）−１ｍＭ ＥＤＴＡ・２Ｎａ溶液５ｍＬを加え、４℃で一晩静置し、以後のＰＣＲの鋳型ＤＮＡに使用した。 Next, proteinase K was added to the suspension so that the final concentration was 100 μg / mL, and the mixture was incubated at 37 ° C. for 1 hour. Further, sodium dodecyl sulfate was added to a final concentration of 0.5%, and the mixture was incubated at 50 ° C. for 6 hours for lysis. To this lysate, add an equal amount of phenol / chloroform solution, shake gently at room temperature for 10 minutes, and then centrifuge (5,000 × g, 20 minutes, 10-12 ° C.) to obtain a supernatant. The fraction was collected and sodium acetate was added to a concentration of 0.3 M, and then twice as much ethanol was added and mixed. The precipitate collected by centrifugation (15,000 × g, 2 minutes) was washed with 70% ethanol and then air-dried. To the obtained DNA, 5 mL of a 10 mM Tris buffer (pH 7.5) -1 mM EDTA · 2Na solution was added and left overnight at 4 ° C., and used as template DNA for subsequent PCR.
（Ｂ）ＰＣＲによるＳａｃＢ遺伝子の増幅およびクローニング
枯草菌ＳａｃＢ遺伝子の取得は、上記（Ａ）で調製したＤＮＡを鋳型とし、既に報告されている該遺伝子の塩基配列（ＧｅｎＢａｎｋ Ｄａｔａｂａｓｅ Ａｃｃｅｓｓｉｏｎ
Ｎｏ．Ｘ０２７３０）を基に設計した合成ＤＮＡ（配列番号１および配列番号２）を用いたＰＣＲによって行った。
(B) Amplification and Cloning of SacB Gene by PCR Acquiring Bacillus subtilis SacB gene using the DNA prepared in (A) as a template and the nucleotide sequence of the gene already reported (GenBank Database Accession)
No. This was performed by PCR using synthetic DNA (SEQ ID NO: 1 and SEQ ID NO: 2) designed based on X02730).
反応液組成：鋳型ＤＮＡ１μＬ、ＰｆｘＤＮＡポリメラーゼ（インビトロジェン社製）
０．２μＬ、１倍濃度添付バッファー、０．３μＭ各々プライマー、１ｍＭ ＭｇＳＯ4、０．２５μＭｄＮＴＰｓを混合し、全量を２０μＬとした。
Reaction solution composition:
0.2 μL, 1 × concentration buffer, 0.3 μM each primer, 1 mM MgSO 4 and 0.25 μM dNTPs were mixed to make a total volume of 20 μL.
反応温度条件：ＤＮＡサーマルサイクラー ＰＴＣ−２００（ＭＪＲｅｓｅａｒｃｈ社製）を用い、９４℃で２０秒、６８℃で２分からなるサイクルを３５回繰り返した。但し、１サイクル目の９４℃での保温は１分２０秒、最終サイクルの６８℃での保温は５分とした。 Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds and 68 ° C. for 2 minutes was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 68 ° C. in the final cycle was 5 minutes.
増幅産物の確認は、０．７５％アガロース（ＳｅａＫｅｍ ＧＴＧ ａｇａｒｏｓｅ：ＦＭＣＢｉｏＰｒｏｄｕｃｔｓ製）ゲル電気泳動により分離後、臭化エチジウム染色により可視化することにより行い、約２ｋｂの断片を検出した。ゲルからの目的ＤＮＡ断片の回収は、ＱＩＡＱｕｉｃｋ Ｇｅｌ Ｅｘｔｒａｃｔｉｏｎ Ｋｉｔ（ＱＩＡＧＥＮ製）を用いて行った。 Confirmation of the amplified product was carried out by separating by 0.75% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and then visualized by ethidium bromide staining, and a fragment of about 2 kb was detected. The DNA fragment of interest was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN).
回収したＤＮＡ断片は、Ｔ４ ポリヌクレオチドキナーゼ（Ｔ４ Ｐｏｌｙｎｕｃｌｅｏｔｉｄｅ Ｋｉｎａｓｅ：宝酒造製）により５'末端をリン酸化した後、ライゲーションキットｖｅｒ．２（宝酒造製）を用いて大腸菌ベクター（ｐＢｌｕｅｓｃｒｉｐｔＩＩ：ＳＴＲＡＴＥＧＥＮＥ製）のＥｃｏＲＶ部位に結合し、得られたプラスミドＤＮＡで大腸菌（ＤＨ５α株）を形質転換した。この様にして得られた組換え大腸菌を５０μｇ／ｍＬアンピシリンおよび５０μｇ／ｍＬX-Galを含むＬＢ寒天培地［トリプトン１０ｇ、イーストエキストラクト５ｇ、ＮａＣｌ ５ｇ及び寒天１５ｇを蒸留水１Ｌに溶解］に塗抹した
。
The recovered DNA fragment was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Shuzo), and then ligated kit ver. 2 (manufactured by Takara Shuzo Co., Ltd.) was used to bind to the EcoRV site of the E. coli vector (pBluescript II: manufactured by STRATEGENE), and E. coli (DH5α strain) was transformed with the resulting plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium [10 g tryptone, 5 g yeast extract, 5 g NaCl, and 15 g agar dissolved in 1 L distilled water] containing 50 μg / mL ampicillin and 50 μg / mL X-Gal. .
この培地上で白色のコロニーを形成したクローンを、次に５０μｇ／ｍＬアンピシリンおよび１０％ショ糖を含むＬＢ寒天培地に移し３７℃で２４時間培養した。これらのクローンのうち、ショ糖を含む培地で生育できなかったものについて、常法により液体培養した後、プラスミドＤＮＡを精製した。ＳａｃＢ遺伝子が大腸菌内で機能的に発現する株は、ショ糖含有培地にて生育不能となるはずである。得られたプラスミドＤＮＡを制限酵素ＳａｌＩおよびＰｓｔＩで切断することにより、約２ｋｂの挿入断片が認められ、該プラスミドをｐＢＳ／ＳａｃＢと命名した。 Clones that formed white colonies on this medium were then transferred to LB agar medium containing 50 μg / mL ampicillin and 10% sucrose and cultured at 37 ° C. for 24 hours. Among these clones, those that could not grow on a medium containing sucrose were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. Strains in which the SacB gene is functionally expressed in E. coli should be unable to grow on sucrose-containing media. The obtained plasmid DNA was cleaved with restriction enzymes SalI and PstI, whereby an inserted fragment of about 2 kb was observed, and the plasmid was named pBS / SacB.
（Ｃ）クロラムフェニコール耐性ＳａｃＢベクターの構築
大腸菌プラスミドベクターｐＨＳＧ３９６（宝酒造：クロラムフェニコール耐性マーカー）５００ｎｇに制限酵素ＰｓｈＢＩ１０ユニットを３７℃で一時間反応させた後、フェノール／クロロフォルム抽出およびエタノール沈殿により回収した。クレノウフラグメント（Ｋｌｅｎｏｗ Ｆｒａｇｍｅｎｔ：宝酒造製）により両末端を平滑化した後、ライゲーションキットｖｅｒ．２（宝酒造製）を用いてＭｌｕＩリンカー（宝酒造）を連結、環状化させ、大腸菌（ＤＨ５α株）を形質転換した。この様にして得られた組換え大腸菌を３４μｇ／ｍＬクロラムフェニコールを含むＬＢ寒天培地に塗抹した。得られたクローンから常法によりプラスミドＤＮＡを調製し、制限酵素ＭｌｕＩの切断部位を有するクローンを選抜し、ｐＨＳＧ３９６Ｍｌｕと命名した。
(C) Construction of chloramphenicol resistant SacB vector 500 ng of E. coli plasmid vector pHSG396 (Takara Shuzo: Chloramphenicol resistant marker) was reacted with a restriction enzyme PshBI10 unit at 37 ° C. for 1 hour, followed by phenol / chloroform extraction and ethanol precipitation. It was collected by. After smoothing both ends with Klenow Fragment (manufactured by Takara Shuzo), ligation kit ver. MluI linker (Takara Shuzo) was ligated and circularized using 2 (Takara Shuzo), and Escherichia coli (DH5α strain) was transformed. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 34 μg / mL chloramphenicol. Plasmid DNA was prepared from the obtained clones by a conventional method, and a clone having a restriction enzyme MluI cleavage site was selected and named pHSG396Mlu.
一方、上記（Ｂ）にて構築したｐＢＳ／ＳａｃＢを制限酵素ＳａｌＩおよびＰｓｔＩで切断した後、クレノウフラグメントにて末端を平滑化した。これにライゲーションキットｖｅｒ．２（宝酒造製）を用いてＭｌｕＩリンカーを連結したのち、０．７５％アガロースゲル電気泳動によりＳａｃＢ遺伝子を含む約２．０ｋｂのＤＮＡ断片を分離、回収した。このＳａｃＢ遺伝子断片を、制限酵素ＭｌｕＩ切断後、アルカリフォスファターゼ（Ａｌｋａｌｉｎｅ Ｐｈｏｓｐｈａｔａｓｅ Ｃａｌｆ ｉｎｔｅｓｔｉｎｅ：宝酒造）にて末端を脱リン酸化したｐＨＳＧ３９６Ｍｌｕ断片とライゲーションキットｖｅｒ．２（宝酒造製）を用いて連結させ、大腸菌（ＤＨ５α株）を形質転換した。この様にして得られた組換え大腸菌を３４μｇ／ｍＬクロラムフェニコールを含むＬＢ寒天培地に塗抹した。こうして得られたコロニーを、次に３４μｇ／ｍＬクロラムフェニコールおよび１０％ショ糖を含むＬＢ寒天培地に移し３７℃で２４時間培養した。これらのクローンのうち、ショ糖を含む培地で生育できなかったものについて、常法によりプラスミドＤＮＡを精製した。こうして得られたプラスミドＤＮＡをＭｌｕＩ切断により解析した結果、約２．０ｋｂの挿入断片を持つことが確認され、これをｐＣＭＢ１と命名した。 On the other hand, pBS / SacB constructed in (B) above was cleaved with restriction enzymes SalI and PstI, and the ends were blunted with Klenow fragment. Ligation kit ver. After linking the MluI linker using 2 (Takara Shuzo), a DNA fragment of about 2.0 kb containing the SacB gene was separated and recovered by 0.75% agarose gel electrophoresis. This SacB gene fragment was cleaved with the restriction enzyme MluI, and then the pHSG396Mlu fragment was dephosphorylated with alkaline phosphatase (Alkaline Phosphatase Calf intestine) and ligation kit ver. 2 (manufactured by Takara Shuzo) was used to transform E. coli (DH5α strain). The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 34 μg / mL chloramphenicol. The colonies thus obtained were then transferred to an LB agar medium containing 34 μg / mL chloramphenicol and 10% sucrose and cultured at 37 ° C. for 24 hours. Among these clones, plasmid DNA was purified by a conventional method for those that could not grow on a medium containing sucrose. As a result of analyzing the plasmid DNA thus obtained by MluI digestion, it was confirmed that it had an inserted fragment of about 2.0 kb, and this was named pCMB1.
（Ｄ）カナマイシン耐性遺伝子の取得
カナマイシン耐性遺伝子の取得は、大腸菌プラスミドベクターｐＨＳＧ２９９（宝酒造：カナマイシン耐性マーカー）のＤＮＡを鋳型とし、配列番号３および配列番号４で示した合成ＤＮＡをプライマーとしたＰＣＲ法によって行った。
(D) Acquisition of kanamycin resistance gene The kanamycin resistance gene is obtained by PCR using the DNA of E. coli plasmid vector pHSG299 (Takara Shuzo: Kanamycin resistance marker) as a template and the synthetic DNAs shown in SEQ ID NOs: 3 and 4 as primers. Went by.
反応液組成：鋳型ＤＮＡ１ｎｇ、ＰｙｒｏｂｅｓｔＤＮＡポリメラーゼ（宝酒造） ０．１μＬ、１倍濃度添付バッファー、０．５μＭ各々プライマー、０．２５μＭｄＮＴＰｓを混合し、全量を２０μＬとした。
Composition of reaction solution:
反応温度条件：ＤＮＡサーマルサイクラー ＰＴＣ−２００（ＭＪＲｅｓｅａｒｃｈ社製）を用い、９４℃で２０秒、６２℃で１５秒、７２℃で１分２０秒からなるサイクルを２０回繰り返した。但し、１サイクル目の９４℃での保温は１分２０秒、最終サイクルの７２℃での保温は５分とした。 Reaction temperature condition: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 62 ° C. for 15 seconds, and 72 ° C. for 1 minute 20 seconds was repeated 20 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 5 minutes.
増幅産物の確認は、０．７５％アガロース（ＳｅａＫｅｍ ＧＴＧ ａｇａｒｏｓｅ：ＦＭＣＢｉｏＰｒｏｄｕｃｔｓ製）ゲル電気泳動により分離後、臭化エチジウム染色により可視化することにより行い、約１．１ｋｂの断片を検出した。ゲルからの目的ＤＮＡ断片の回収は、ＱＩＡＱｕｉｃｋ Ｇｅｌ Ｅｘｔｒａｃｔｉｏｎ Ｋｉｔ（ＱＩＡＧＥＮ製）を用いて行った。回収したＤＮＡ断片は、Ｔ４ ポリヌクレオチドキナーゼ（Ｔ４ Ｐｏｌｙｎｕｃｌｅｏｔｉｄｅ Ｋｉｎａｓｅ：宝酒造製）により５'末端をリン酸化した。 Confirmation of the amplification product was carried out by separating by 0.75% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) gel electrophoresis and then visualized by ethidium bromide staining to detect a fragment of about 1.1 kb. The DNA fragment of interest was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN). The recovered DNA fragment was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Shuzo).
（Ｅ）カナマイシン耐性ＳａｃＢベクターの構築
上記（Ｃ）で構築したｐＣＭＢ１を制限酵素Ｖａｎ９１ＩおよびＳｃａＩで切断して得られた約３．５ｋｂのＤＮＡ断片を０．７５％アガロースゲル電気泳動により分離、回収した。これを上記（Ｄ）で調製したカナマイシン耐性遺伝子と混合し、ライゲーションキットｖｅｒ．２（宝酒造製）を用いて連結し、得られたプラスミドＤＮＡで大腸菌（ＤＨ５α株）を形質転換した。この様にして得られた組換え大腸菌を５０μｇ／ｍＬカナマイシンを含むＬＢ寒天培地に塗抹した。
(E) Construction of kanamycin-resistant SacB vector The approximately 3.5 kb DNA fragment obtained by cleaving pCMB1 constructed in (C) above with restriction enzymes Van91I and ScaI was separated and recovered by 0.75% agarose gel electrophoresis. did. This was mixed with the kanamycin resistance gene prepared in (D) above, and ligation kit ver. 2 (manufactured by Takara Shuzo), and Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin.
このカナマイシン含有培地上で生育した株は、ショ糖含有培地にて生育不能であることが確認された。また、同株から調製したプラスミドＤＮＡは、制限酵素ＨｉｎｄIII消化により３５４、４７３、１８０７、１９９７ｂｐの断片を生じたことから、図１に示した構造に間違いがないと判断し、該プラスミドをｐＫＭＢ１と命名した。 It was confirmed that the strain grown on this kanamycin-containing medium was unable to grow on the sucrose-containing medium. Further, the plasmid DNA prepared from the same strain produced fragments of 354, 473, 1807, and 1997 bp by digestion with the restriction enzyme HindIII. Therefore, it was determined that the structure shown in FIG. 1 was correct, and the plasmid was designated as pKMB1. Named.
［参考例２］
＜ＬＤＨ遺伝子破壊株の作製＞
（Ａ）ブレビバクテリウム・フラバムＭＪ２３３−ＥＳ株ゲノムＤＮＡの抽出
Ａ培地［尿素 ２ｇ、（ＮＨ4）2ＳＯ4 ７ｇ、ＫＨ2ＰＯ4 ０．５ｇ、Ｋ2ＨＰＯ4 ０．５ｇ、ＭｇＳＯ4・７Ｈ2Ｏ ０．５ｇ、ＦｅＳＯ4・７Ｈ2Ｏ ６ｍｇ、ＭｎＳＯ4・４−５Ｈ2Ｏ６ｍｇ、ビオチン ２００μｇ、チアミン １００μｇ、イーストエキストラクト １ｇ、カザミノ酸 １ｇ、グルコース ２０ｇ、蒸留水１Ｌに溶解］１０ｍＬに、ブレビバクテリウム・フラバムＭＪ−２３３株を対数増殖期後期まで培養し、得られた菌体から上記参考例１の（Ａ）に示す方法にてゲノムＤＮＡを調製した。
[Reference Example 2]
<Preparation of LDH gene disruption strain>
(A) Extraction of Brevibacterium flavum MJ233-ES strain genomic DNA A medium [urea 2 g, (NH 4 ) 2 SO 4 7 g, KH 2 PO 4 0.5 g, K 2 HPO 4 0.5 g, MgSO 4. 7H 2 O 0.5g, FeSO 4 · 7H 2 O 6mg, MnSO 4 · 4-5H 2 O6mg, biotin 200 [mu] g, thiamine 100 [mu] g, yeast extract 1g, casamino acid 1g, glucose 20g, dissolved in distilled water 1L] in 10mL Brevibacterium flavum MJ-233 strain was cultured until the late logarithmic growth phase, and genomic DNA was prepared from the obtained cells by the method shown in (A) of Reference Example 1 above.
（Ｂ）ラクテートデヒドロゲナーゼ遺伝子のクローニング
ＭＪ２３３株ラクテートデヒドロゲナーゼ遺伝子の取得は、上記（Ａ）で調製したＤＮＡを鋳型とし、特開平１１−２０６３８５に記載の該遺伝子の塩基配列を基に設計した合成ＤＮＡ（配列番号５および配列番号６）を用いたＰＣＲによって行った。
(B) Cloning of lactate dehydrogenase gene The MJ233 strain lactate dehydrogenase gene was obtained by using the DNA prepared in (A) above as a template and a synthetic DNA designed on the basis of the base sequence of the gene described in JP-A-11-206385 ( It was performed by PCR using SEQ ID NO: 5 and SEQ ID NO: 6).
反応液組成：鋳型ＤＮＡ １μＬ、ＴａｑＤＮＡポリメラーゼ（宝酒造） ０．２μＬ、１倍濃度添付バッファー、０．２μＭ各々プライマー、０．２５μＭｄＮＴＰｓを混合し、全量を２０μＬとした。
Composition of reaction solution:
反応温度条件：ＤＮＡサーマルサイクラー ＰＴＣ−２００（ＭＪＲｅｓｅａｒｃｈ社製）を用い、９４℃で２０秒、５５℃で２０秒、７２℃で１分からなるサイクルを３０回繰り返した。但し、１サイクル目の９４℃での保温は１分２０秒、最終サイクルの７２℃での保温は５分とした。 Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 55 ° C. for 20 seconds and 72 ° C. for 1 minute was repeated 30 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 5 minutes.
増幅産物の確認は、０．７５％アガロース（ＳｅａＫｅｍ ＧＴＧ ａｇａｒｏｓｅ：ＦＭＣＢｉｏＰｒｏｄｕｃｔｓ製）ゲル電気泳動により分離後、臭化エチジウム染色により可視化することにより行い、約０．９５ｋｂの断片を検出した。ゲルからの目的ＤＮＡ断片の回収は、ＱＩＡＱｕｉｃｋ Ｇｅｌ Ｅｘｔｒａｃｔｉｏｎ Ｋｉｔ（ＱＩＡＧＥＮ製）を用いて行った。 Confirmation of the amplification product was carried out by separating by 0.75% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) gel electrophoresis and then visualized by ethidium bromide staining to detect a fragment of about 0.95 kb. The DNA fragment of interest was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN).
回収したＤＮＡ断片を、ＰＣＲ産物クローニングベクターｐＧＥＭ−ＴＥａｓｙ（Ｐｒｏｍｅｇａ製）と混合し、ライゲーションキットｖｅｒ．２（宝酒造製）を用いて連結後、得られたプラスミドＤＮＡで大腸菌（ＤＨ５α株）を形質転換した。この様にして得られた組換え大腸菌を５０μｇ／ｍＬアンピシリンおよび５０μｇ／ｍＬX-Galを含むＬＢ寒天培地に塗抹した。 The recovered DNA fragment was mixed with a PCR product cloning vector pGEM-TEasy (Promega) and ligation kit ver. After ligation using 2 (Takara Shuzo), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL ampicillin and 50 μg / mL X-Gal.
この培地上で白色のコロニーを形成したクローンを、常法により液体培養した後、プラスミドＤＮＡを精製した。得られたプラスミドＤＮＡを制限酵素ＳａｃＩおよびＳｐｈＩで切断することにより、約１．０ｋｂの挿入断片が認められ、これをｐＧＥＭＴ／ＣｇＬＤＨと命名した。 Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. By cutting the obtained plasmid DNA with restriction enzymes SacI and SphI, an insert fragment of about 1.0 kb was recognized, and this was named pGEMT / CgLDH.
（Ｃ）ラクテートデヒドロゲナーゼ遺伝子破壊用プラスミドの構築
上記（Ｂ）で作製したｐＧＥＭＴ／ＣｇＬＤＨを制限酵素ＥｃｏＲＶおよびＸｂａＩで切断することにより約０．２５ｋｂからなるラクテートデヒドロゲナーゼのコーディング領域を切り出した。残った約３．７ｋｂのＤＮＡ断片の末端をクレノウフラグメントにて平滑化し、ライゲーションキットｖｅｒ．２（宝酒造製）を用いて環状化させ、大腸菌（ＤＨ５α株）を形質転換した。この様にして得られた組換え大腸菌を５０μｇ／ｍＬアンピシリンを含むＬＢ寒天培地に塗抹した。
(C) Construction of plasmid for disrupting lactate dehydrogenase gene The coding region of lactate dehydrogenase consisting of about 0.25 kb was excised by cutting the pGEMT / CgLDH prepared in (B) above with restriction enzymes EcoRV and XbaI. The ends of the remaining approximately 3.7 kb DNA fragment were blunted with Klenow fragment, and ligated kit ver. 2 (manufactured by Takara Shuzo) was cyclized to transform E. coli (DH5α strain). The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL ampicillin.
この培地上で生育した株を、常法により液体培養した後、プラスミドＤＮＡを精製した。得られたプラスミドＤＮＡを制限酵素ＳａｃＩおよびＳｐｈＩで切断することにより、約０．７５ｋｂの挿入断片が認められたクローンを選抜し、これをｐＧＥＭＴ／ΔＬＤＨと命名した。 The strain grown on this medium was subjected to liquid culture by a conventional method, and then the plasmid DNA was purified. By cutting the obtained plasmid DNA with restriction enzymes SacI and SphI, a clone in which an insert fragment of about 0.75 kb was recognized was selected and named pGEMT / ΔLDH.
次に、上記ｐＧＥＭＴ／ΔＬＤＨを制限酵素ＳａｃＩおよびＳｐｈＩにて切断して生じる約０．７５ｋｂのＤＮＡ断片を、０．７５％アガロースゲル電気泳動により分離、回収し、欠損領域を含むラクテートデヒドロゲナーゼ遺伝子断片を調製した。このＤＮＡ断片を、制限酵素ＳａｃＩおよびＳｐｈＩにて切断した参考例１にて構築したｐＫＭＢ１と混合し、ライゲーションキットｖｅｒ．２（宝酒造製）を用いて連結後、得られたプラスミドＤＮＡで大腸菌（ＤＨ５α株）を形質転換した。この様にして得られた組換え大腸菌を５０μｇ／ｍＬカナマイシンおよび５０μｇ／ｍＬX-Galを含むＬＢ寒天培地に塗抹した。 Next, a DNA fragment of about 0.75 kb generated by cleaving the above pGEMT / ΔLDH with restriction enzymes SacI and SphI is separated and recovered by 0.75% agarose gel electrophoresis, and a lactate dehydrogenase gene fragment containing a defective region Was prepared. This DNA fragment was mixed with pKMB1 constructed in Reference Example 1 cut with restriction enzymes SacI and SphI, and ligation kit ver. After ligation using 2 (Takara Shuzo), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin and 50 μg / mL X-Gal.
この培地上で白色のコロニーを形成したクローンを、常法により液体培養した後、プラスミドＤＮＡを精製した。得られたプラスミドＤＮＡを制限酵素ＳａｃＩおよびＳｐｈＩで切断することにより、約０．７５ｋｂの挿入断片が認められたものを選抜し、これをｐＫＭＢ１／ΔＬＤＨと命名した（図２）。 Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. The obtained plasmid DNA was cleaved with restriction enzymes SacI and SphI, and a plasmid with an inserted fragment of about 0.75 kb was selected and named pKMB1 / ΔLDH (FIG. 2).
（Ｄ）ブレビバクテリウム・フラバムＭＪ２３３−ＥＳ株由来ラクテートデヒドロゲナーゼ遺伝子破壊株の作製
ブレビバクテリウム・フラバムＭＪ−２３３株の形質転換に用いるプラスミドＤＮＡは、ｐＫＭＢ１／ΔＬＤＨを用いて塩化カルシウム法（Ｊｏｕｒｎａｌ ｏｆ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ，５３，１５９，１９７０）により形質転換した大腸菌ＪＭ１１０株から調製した。
(D) Preparation of lactate dehydrogenase gene disruption strain derived from Brevibacterium flavum MJ233-ES strain Plasmid DNA used for transformation of Brevibacterium flavum MJ-233 strain was obtained by the calcium chloride method (Journal of using pKMB1 / ΔLDH). (Molecular Biology, 53, 159, 1970).
ブレビバクテリウム・フラバムＭＪ２３３−ＥＳ株の形質転換は、電気パルス法（Res.
Microbiol., Vol.144, p.181-185, 1993）によって行い、得られた形質転換体をカナマイシン ５０μｇ／ｍＬを含むＬＢＧ寒天培地［トリプトン１０ｇ、イーストエキストラ
Transformation of Brevibacterium flavum MJ233-ES strain is performed by electric pulse method (Res.
Microbiol., Vol. 144, p.181-185, 1993), and the obtained transformant was treated with LBG agar medium containing 50 μg / mL kanamycin [10 g tryptone, yeast extra.
クト５ｇ、ＮａＣｌ ５ｇ、グルコース ２０ｇ、及び寒天１５ｇを蒸留水１Ｌに溶解］に
塗抹した。
5 g of NaCl, 5 g of NaCl, 20 g of glucose, and 15 g of agar were dissolved in 1 L of distilled water.
この培地上に生育した株は、ｐＫＭＢ１／ΔＬＤＨがブレビバクテリウム・フラバムＭＪ２３３−ＥＳ株菌体内で複製不可能なプラスミドであるため、該プラスミドのラクテートデヒドロゲナーゼ遺伝子とブレビバクテリウム・フラバムＭＪ−２３３株ゲノム上の同遺伝子との間で相同組み換えを起こした結果、同ゲノム上に該プラスミドに由来するカナマイシン耐性遺伝子およびＳａｃＢ遺伝子が挿入されているはずである。 Since the strain grown on this medium is a plasmid in which pKMB1 / ΔLDH cannot be replicated in Brevibacterium flavum MJ233-ES, the lactate dehydrogenase gene of the plasmid and Brevibacterium flavum MJ-233 strain As a result of homologous recombination with the same gene on the genome, the kanamycin resistance gene and SacB gene derived from the plasmid should be inserted on the same genome.
次に、上記相同組み換え株をカナマイシン５０μｇ／ｍＬを含むＬＢＧ培地にて液体培養した。この培養液の菌体数約１００万相当分を１０％ショ糖含有ＬＢＧ培地に塗抹にした。結果、２回目の相同組み換えによりＳａｃＢ遺伝子が脱落しショ糖非感受性となったと考えられる株約１０個得た。 Next, the homologous recombination strain was subjected to liquid culture in an LBG medium containing kanamycin 50 μg / mL. A portion equivalent to about 1 million cells of this culture was smeared on a 10% sucrose-containing LBG medium. As a result, about 10 strains that were considered to be sucrose-insensitive due to elimination of the SacB gene by the second homologous recombination were obtained.
この様にして得られた株の中には、そのラクテートデヒドロゲナーゼ遺伝子がｐＫＭＢ１／ΔＬＤＨに由来する変異型に置き換わったものと野生型に戻ったものが含まれる。ラクテートデヒドロゲナーゼ遺伝子が変異型であるか野生型であるかの確認は、ＬＢＧ培地にて液体培養して得られた菌体を直接ＰＣＲ反応に供し、ラクテートデヒドロゲナーゼ遺伝子の検出を行うことによって容易に確認できる。ラクテートデヒドロゲナーゼ遺伝子をＰＣＲ増幅するためのプライマー（配列番号７および配列番号８）を用いて分析すると、野生型では７２０ｂｐ、欠失領域を持つ変異型では４７１ｂｐのＤＮＡ断片を認めるはずである。 Among the strains thus obtained, those in which the lactate dehydrogenase gene is replaced with a mutant derived from pKMB1 / ΔLDH and those in which the wild type is restored are included. Confirmation of whether the lactate dehydrogenase gene is a mutant type or a wild type is easily confirmed by subjecting the cells obtained by liquid culture in LBG medium to direct PCR reaction and detecting the lactate dehydrogenase gene. it can. When the lactate dehydrogenase gene is analyzed using primers (SEQ ID NO: 7 and SEQ ID NO: 8) for PCR amplification, a wild-type DNA fragment of 720 bp and a mutant type having a deletion region should have a DNA fragment of 471 bp.
上記方法にてショ糖非感受性となった菌株を分析した結果、変異型遺伝子のみを有する株を選抜し、該株をブレビバクテリウム・フラバムＭＪ２３３／ΔＬＤＨと命名した。 As a result of analyzing the strain that became insensitive to sucrose by the above method, a strain having only the mutant gene was selected, and the strain was named Brevibacterium flavum MJ233 / ΔLDH.
（Ｅ）ラクテートデヒドロゲナーゼ活性の確認
上記（Ｄ）で作製したブレビバクテリウム・フラバムＭＪ２３３／ΔＬＤＨ株をＡ培地に植菌し、３０℃で１５時間好気的に振とう培養した。得られた培養物を遠心分離（３，０００×ｇ、４℃、２０分間）して菌体を回収後、ナトリウム−リン酸緩衝液［組成：50mMリン酸ナトリウム緩衝液(pH7.3)］で洗浄した。
(E) Confirmation of lactate dehydrogenase activity The Brevibacterium flavum MJ233 / ΔLDH strain prepared in (D) above was inoculated in medium A and cultured with shaking aerobically at 30 ° C. for 15 hours. The obtained culture is centrifuged (3,000 × g, 4 ° C., 20 minutes) to recover the cells, and then sodium-phosphate buffer [composition: 50 mM sodium phosphate buffer (pH 7.3)] Washed with.
次いで、洗浄菌体０．５ｇ（湿重量）を上記ナトリウム−リン酸緩衝液２ｍＬに懸濁し、氷冷下で超音波破砕器（ブランソン社製）にかけ菌体破砕物を得た。該破砕物を遠心分離（１０，０００×ｇ，４℃，３０分間）し、上清を粗酵素液として得た。対照として、ブレビバクテリウム・フラバム ＭＪ２３３−ＥＳ株の粗酵素液を同様に調製し、以下の活性測定に供した。 Next, 0.5 g (wet weight) of washed cells was suspended in 2 mL of the sodium-phosphate buffer, and the cells were crushed by applying an ultrasonic crusher (manufactured by Branson) under ice cooling. The crushed material was centrifuged (10,000 × g, 4 ° C., 30 minutes), and the supernatant was obtained as a crude enzyme solution. As a control, a crude enzyme solution of Brevibacterium flavum MJ233-ES strain was similarly prepared and subjected to the following activity measurement.
ラクテートデヒドロゲナーゼ酵素活性の確認は、両粗酵素液について、ピルビン酸を基質とした乳酸の生成に伴い、補酵素ＮＡＤＨがＮＡＤ+に酸化されるのを、３４０ｎｍの吸光度変化として測定した［L. Kanarek and R. L. Hill, J. Biol. Chem. 239, 4202 (1964)］。反応は、５０ｍＭ カリウム−リン酸緩衝液(pH7.2)、１０ｍＭ ピルビン酸、０．４ｍＭＮＡＤＨ存在下、３７℃にて行った。その結果、ブレビバクテリウム・フラバム
ＭＪ２３３−ＥＳ株から調製された粗酵素液におけるラクテートデヒドロゲナーゼ活性に対し、ブレビバクテリウム・フラバムＭＪ２３３／ΔＬＤＨ株から調製された粗酵素液におけるラクテートデヒドロゲナーゼ活性は、１０分の１以下であった。
Confirmation of the enzyme activity of lactate dehydrogenase was carried out by measuring the oxidation of coenzyme NADH to NAD + as a change in absorbance at 340 nm with the production of lactic acid using pyruvate as a substrate for both crude enzyme solutions [L. Kanarek and RL Hill, J. Biol. Chem. 239, 4202 (1964)]. The reaction was performed at 37 ° C. in the presence of 50 mM potassium-phosphate buffer (pH 7.2), 10 mM pyruvic acid, 0.4 mM NADH. As a result, the lactate dehydrogenase activity in the crude enzyme solution prepared from Brevibacterium flavum MJ233 / ΔLDH was 10 minutes compared to the lactate dehydrogenase activity in the crude enzyme solution prepared from Brevibacterium flavum MJ233-ES strain. 1 or less.
［参考例３］
＜コリネ型細菌発現ベクターの構築＞
（Ａ）コリネ型細菌用プロモーター断片の調製
コリネ型細菌で強力なプロモーター活性を有することが報告された特開平７−９５８９
１の配列番号４に記載のＤＮＡ断片（以降ＴＺ４プロモーターと称する）を利用することとした。本プロモーター断片の取得は、参考例２の（Ａ）で調製したブレビバクテリウム・フラバムＭＪ２３３ゲノムＤＮＡを鋳型とし、特開平７−９５８９１の配列番号４に記載の配列を基に設計した合成ＤＮＡ（配列番号９および配列番号１０）を用いたＰＣＲによって行った。
[Reference Example 3]
<Construction of Coryneform Bacterial Expression Vector>
(A) Preparation of Promoter Fragment for Coryneform Bacteria JP-A-7-9589 reported to have a strong promoter activity in coryneform bacteria
The DNA fragment described in SEQ ID NO: 4 (hereinafter referred to as TZ4 promoter) was used. This promoter fragment was obtained by using the Brevibacterium flavum MJ233 genomic DNA prepared in (A) of Reference Example 2 as a template and a synthetic DNA designed based on the sequence described in SEQ ID NO: 4 of JP-A-7-95891 ( It was carried out by PCR using SEQ ID NO: 9 and SEQ ID NO: 10).
反応液組成：鋳型ＤＮＡ1μＬ、ＰｆｘＤＮＡポリメラーゼ（インビトロジェン社製） ０．２μＬ、１倍濃度添付バッファー、０．３μＭ各々プライマー、１ｍＭ ＭｇＳＯ4、０．２５μＭｄＮＴＰｓを混合し、全量を２０μＬとした。 Composition of reaction solution: 1 μL of template DNA, 0.2 μL of Pfx DNA polymerase (manufactured by Invitrogen), 1 × concentration attached buffer, 0.3 μM each primer, 1 mM MgSO 4 and 0.25 μM dNTPs were mixed to make a total volume of 20 μL.
反応温度条件：ＤＮＡサーマルサイクラー ＰＴＣ−２００（ＭＪＲｅｓｅａｒｃｈ社製）を用い、９４℃で２０秒、６０℃で２０秒、７２℃で３０秒からなるサイクルを３５回繰り返した。但し、１サイクル目の９４℃での保温は１分２０秒、最終サイクルの７２℃での保温は２分とした。 Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds, and 72 ° C. for 30 seconds was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute and 20 seconds, and the heat retention at 72 ° C. in the final cycle was 2 minutes.
増幅産物の確認は、２．０％アガロース（ＳｅａＫｅｍ ＧＴＧ ａｇａｒｏｓｅ：ＦＭＣＢｉｏＰｒｏｄｕｃｔｓ製）ゲル電気泳動により分離後、臭化エチジウム染色により可視化することにより行い、約０．２５ｋｂの断片を検出した。ゲルからの目的ＤＮＡ断片の回収は、ＱＩＡＱｕｉｃｋ Ｇｅｌ Ｅｘｔｒａｃｔｉｏｎ Ｋｉｔ（ＱＩＡＧＥＮ製）を用いて行った。 The amplification product was confirmed by separation by 2.0% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.25 kb was detected. The DNA fragment of interest was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN).
回収したＤＮＡ断片は、Ｔ４ ポリヌクレオチドキナーゼ（Ｔ４ Ｐｏｌｙｎｕｃｌｅｏｔｉｄｅ Ｋｉｎａｓｅ：宝酒造製）により５'末端をリン酸化した後、ライゲーションキットｖｅｒ．２（宝酒造製）を用いて大腸菌ベクターｐＵＣ１９（宝酒造）のＳｍａI部位に結合し、得られたプラスミドＤＮＡで大腸菌（ＤＨ５α株）を形質転換した。この様にして得られた組換え大腸菌を５０μｇ／ｍＬアンピシリンおよび５０μｇ／ｍＬX-Galを含むＬＢ寒天培地に塗抹した。 The recovered DNA fragment was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Shuzo), and then ligated kit ver. 2 (Takara Shuzo) was used to bind to the SmaI site of E. coli vector pUC19 (Takara Shuzo), and Escherichia coli (DH5α strain) was transformed with the resulting plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL ampicillin and 50 μg / mL X-Gal.
この培地上で白色のコロニーを形成した６クローンについて、常法により液体培養した後、プラスミドＤＮＡを精製し、塩基配列を決定した。これ中でＴＺ４プロモーターがｐＵＣ１９のｌａｃプロモーターと逆方向に転写活性を有するように挿入されたクローンを選抜し、これをｐＵＣ／ＴＺ４と命名した。 Six clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then the plasmid DNA was purified and the nucleotide sequence was determined. Among them, a clone in which the TZ4 promoter was inserted so as to have a transcriptional activity in the reverse direction to the lac promoter of pUC19 was selected and named pUC / TZ4.
次に、ｐＵＣ／ＴＺ４を制限酵素ＢａｍＨＩおよびＰｓｔＩで切断して調製したＤＮＡ断片に、５’末端がリン酸化された合成ＤＮＡ（配列番号１１および配列番号１２）から成り、両末端にそれぞれＢａｍＨＩとＰｓｔＩに対する粘着末端を有するＤＮＡリンカーを混合し、ライゲーションキットｖｅｒ．２（宝酒造製）を用いて連結後、得られたプラスミドＤＮＡで大腸菌（ＤＨ５α株）を形質転換した。本ＤＮＡリンカーには、リボソーム結合配列（ＡＧＧＡＧＧ）およびその下流に配したクローニングサイト（上流から順に、ＰａｃＩ、ＮｏｔＩ、ＡｐａＩ）が含まれている。 Next, a DNA fragment prepared by cleaving pUC / TZ4 with restriction enzymes BamHI and PstI consists of synthetic DNA (SEQ ID NO: 11 and SEQ ID NO: 12) phosphorylated at the 5 ′ end, and BamHI and A DNA linker having a sticky end against PstI was mixed, and ligation kit ver. After ligation using 2 (Takara Shuzo), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. This DNA linker contains a ribosome binding sequence (AGGAGG) and a cloning site (PacI, NotI, ApaI in this order from the upstream).
この培地上で白色のコロニーを形成したクローンを、常法により液体培養した後、プラスミドＤＮＡを精製した。得られたプラスミドＤＮＡの中から制限酵素ＮｏｔＩによって切断されるものを選抜し、これをｐＵＣ／ＴＺ４−ＳＤと命名した。 Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. From the obtained plasmid DNA, one that was cleaved by the restriction enzyme NotI was selected and named pUC / TZ4-SD.
この様にして構築したｐＵＣ／ＴＺ４−ＳＤを制限酵素ＰｓｔＩで切断後、クレノウフラグメントにて末端を平滑化し、次いで制限酵素ＫｐｎＩで切断することにより生じた約０．３ｋｂのプロモーター断片を、２．０％アガロースゲル電気泳動により分離、回収した。 The thus constructed pUC / TZ4-SD was digested with the restriction enzyme PstI, blunted with Klenow fragment, and then cleaved with the restriction enzyme KpnI to obtain a promoter fragment of about 0.3 kb. Separated and collected by 0% agarose gel electrophoresis.
（Ｂ）コリネ型細菌発現ベクターの構築
コリネ型細菌にて安定的に自立複製可能なプラスミドとして、特開平１２−９３１８３記載のｐＨＳＧ２９８ｐａｒ−ｒｅｐを利用する。本プラスミドは、ブレビバクテリウム・スタチオニスＩＦＯ１２１４４株が保有する天然型プラスミドｐＢＹ５０３の複製領域および安定化機能を有する領域と大腸菌ベクターｐＨＳＧ２９８（宝酒造）に由来するカナマイシン耐性遺伝子および大腸菌の複製領域を備える。ｐＨＳＧ２９８ｐａｒ−ｒｅｐを制限酵素ＳｓｅＩで切断後、クレノウフラグメントにて末端を平滑化し、次いで制限酵素ＫｐｎＩで切断することによって調製したＤＮＡを、上記（Ａ）で調製したＴＺ４プロモーター断片と混合し、ライゲーションキットｖｅｒ．２（宝酒造製）を用いて連結後、得られたプラスミドＤＮＡで大腸菌（ＤＨ５α株）を形質転換した。この様にして得られた組換え大腸菌を５０μｇ／ｍＬカナマイシンを含むＬＢ寒天培地に塗抹した。
(B) Construction of Coryneform Bacterial Expression Vector pHSG298par-rep described in JP-A-12-93183 is used as a plasmid that can stably replicate independently in coryneform bacteria. This plasmid comprises a replication region and a region having a stabilizing function of the natural plasmid pBY503 possessed by Brevibacterium stachynis IFO12144, a kanamycin resistance gene derived from E. coli vector pHSG298 (Takara Shuzo), and a replication region of E. coli. The DNA prepared by cleaving pHSG298par-rep with the restriction enzyme SseI, blunting the ends with Klenow fragment, and then cleaving with the restriction enzyme KpnI is mixed with the TZ4 promoter fragment prepared in (A) above and ligated. Kit ver. After ligation using 2 (Takara Shuzo), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin.
この培地上で生育した株を、常法により液体培養した後、プラスミドＤＮＡを精製した。得られたプラスミドＤＮＡの中から制限酵素ＮｏｔＩによって切断されるものを選抜し、該プラスミドをｐＴＺ４と命名した（図３に構築手順を示した）。 The strain grown on this medium was subjected to liquid culture by a conventional method, and then the plasmid DNA was purified. From the obtained plasmid DNA, one that was cleaved by the restriction enzyme NotI was selected, and the plasmid was named pTZ4 (construction procedure is shown in FIG. 3).
［参考例４］
＜ピルベートカルボキシラーゼ活性増強株の作製＞
（Ａ）ピルベートカルボキシラーゼ遺伝子の取得
ブレビバクテリウム・フラバムＭＪ２３３株由来ピルベートカルボキシラーゼ遺伝子の取得は、参考例２の（Ａ）で調製したＤＮＡを鋳型とし、全ゲノム配列が報告されているコリネバクテリウム・グルタミカム ＡＴＣＣ１３０３２株の該遺伝子の配列（ＧｅｎＢａｎｋ Ｄａｔａｂａｓｅ Ａｃｃｅｓｓｉｏｎ Ｎｏ．ＡＰ００５２７６）を基に設計した合成ＤＮＡ（配列番号１３および配列番号１４）を用いたＰＣＲによって行った。
[Reference Example 4]
<Preparation of a strain with enhanced pyruvate carboxylase activity>
(A) Acquisition of pyruvate carboxylase gene The acquisition of pyruvate carboxylase gene derived from Brevibacterium flavum MJ233 strain is a corynebacteria in which the entire genome sequence has been reported using the DNA prepared in (A) of Reference Example 2 as a template. Um Glutamicum It was carried out by PCR using synthetic DNA (SEQ ID NO: 13 and SEQ ID NO: 14) designed based on the sequence of the gene of ATCC13032 strain (GenBank Database Accession No. AP005276).
反応液組成：鋳型ＤＮＡ1μＬ、ＰｆｘＤＮＡポリメラーゼ（インビトロジェン社製） ０．２μＬ、１倍濃度添付バッファー、０．３μＭ各々プライマー、１ｍＭ ＭｇＳＯ4、
０．２５μＭｄＮＴＰｓを混合し、全量を２０μＬとした。
Composition of reaction solution:
0.25 μM dNTPs was mixed to make a total volume of 20 μL.
反応温度条件：ＤＮＡサーマルサイクラー ＰＴＣ−２００（ＭＪＲｅｓｅａｒｃｈ社製）を用い、９４℃で２０秒、６８℃で４分からなるサイクルを３５回繰り返した。但し、１サイクル目の９４℃での保温は１分２０秒、最終サイクルの６８℃での保温は１０分とした。ＰＣＲ反応終了後、Ｔａｋａｒａ Ｅｘ Ｔａｑ（宝酒造）を０．１μＬ加え、さらに７２℃で３０分保温した。 Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds and 68 ° C. for 4 minutes was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute and 20 seconds, and the heat retention at 68 ° C. in the final cycle was 10 minutes. After completion of the PCR reaction, 0.1 μL of Takara Ex Taq (Takara Shuzo) was added, and the mixture was further incubated at 72 ° C. for 30 minutes.
増幅産物の確認は、０．７５％アガロース（ＳｅａＫｅｍ ＧＴＧ ａｇａｒｏｓｅ：ＦＭＣＢｉｏＰｒｏｄｕｃｔｓ製）ゲル電気泳動により分離後、臭化エチジウム染色により可視化することにより行い、約３．７ｋｂの断片を検出した。ゲルからの目的ＤＮＡ断片の回収は、ＱＩＡＱｕｉｃｋ Ｇｅｌ Ｅｘｔｒａｃｔｉｏｎ Ｋｉｔ（ＱＩＡＧＥＮ製）を用いて行った。 Confirmation of the amplification product was carried out by separating by 0.75% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and then visualized by ethidium bromide staining, and a fragment of about 3.7 kb was detected. The DNA fragment of interest was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN).
回収したＤＮＡ断片を、ＰＣＲ産物クローニングベクターｐＧＥＭ−ＴＥａｓｙ（Ｐｒｏｍｅｇａ製）と混合し、ライゲーションキットｖｅｒ．２（宝酒造製）を用いて連結後、得られたプラスミドＤＮＡで大腸菌（ＤＨ５α株）を形質転換した。この様にして得られた組換え大腸菌を５０μｇ／ｍＬアンピシリンおよび５０μｇ／ｍＬX-Galを含むＬＢ寒天培地に塗抹した。 The recovered DNA fragment was mixed with a PCR product cloning vector pGEM-TEasy (Promega) and ligation kit ver. After ligation using 2 (Takara Shuzo), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL ampicillin and 50 μg / mL X-Gal.
この培地上で白色のコロニーを形成したクローンを、常法により液体培養した後、プラスミドＤＮＡを精製した。得られたプラスミドＤＮＡを制限酵素ＰａｃＩおよびＡｐａＩで切断することにより、約３．７ｋｂの挿入断片が認められ、これをｐＧＥＭ／ＭＪＰＣ
と命名した。
Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. By cleaving the obtained plasmid DNA with restriction enzymes PacI and ApaI, an insertion fragment of about 3.7 kb was observed, which was identified as pGEM / MJPC.
Named.
ｐＧＥＭ／ＭＪＰＣの挿入断片の塩基配列は、アプライドバイオシステム社製塩基配列解読装置（モデル３７７ＸＬ）およびビックダイターミネーターサイクルシークエンスキットｖｅｒ３を用いて決定した。その結果得られたＤＮＡ塩基配列および推測されるアミノ酸配列を配列番号１５に記載する。また、アミノ酸配列のみを配列番号１６に記載する。本アミノ酸配列はコリネバクテリウム・グルタミカムＡＴＣＣ１３０３２株由来のそれと極めて高い相同性（９９．４％）を示すことから、ｐＧＥＭ／ＭＪＰＣの挿入断片がブレビバクテリウム・フラバムＭＪ２３３株由来のピルベートカルボキシラーゼ遺伝子であると断定した。 The base sequence of the insert fragment of pGEM / MJPC was determined using a base sequencing device (Model 377XL) manufactured by Applied Biosystems and a Big Dye Terminator cycle sequence kit ver3. The resulting DNA base sequence and deduced amino acid sequence are set forth in SEQ ID NO: 15. Only the amino acid sequence is shown in SEQ ID NO: 16. Since this amino acid sequence shows extremely high homology (99.4%) with that derived from Corynebacterium glutamicum ATCC13032, the insert of pGEM / MJPC is a pyruvate carboxylase gene derived from Brevibacterium flavum MJ233. I determined that there was.
（Ｂ）ピルベートカルボキシラーゼ活性増強用プラスミドの構築
上記（Ａ）で作製したｐＧＥＭ／ＭＪＰＣを制限酵素ＰａｃＩおよびＡｐａＩで切断することにより生じる約３．７ｋｂからなるピルベートカルボキシラーゼ遺伝子断片を、０．７５％アガロースゲル電気泳動により分離、回収した。
(B) Construction of Pyruvate Carboxylase Activity Enhancement Plasmid A pyruvate carboxylase gene fragment consisting of about 3.7 kb generated by cleaving the pGEM / MJPC prepared in (A) above with restriction enzymes PacI and ApaI It was separated and collected by% agarose gel electrophoresis.
このＤＮＡ断片を、制限酵素ＰａｃＩおよびＡｐａＩにて切断した、参考例３にて構築したｐＴＺ４と混合し、ライゲーションキットｖｅｒ．２（宝酒造製）を用いて連結後、得られたプラスミドＤＮＡで大腸菌（ＤＨ５α株）を形質転換した。この様にして得られた組換え大腸菌を５０μｇ／ｍＬカナマイシンを含むＬＢ寒天培地に塗抹した。 This DNA fragment was mixed with pTZ4 constructed in Reference Example 3 cleaved with restriction enzymes PacI and ApaI, and ligation kit ver. After ligation using 2 (Takara Shuzo), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin.
この培地上で生育した株を、常法により液体培養した後、プラスミドＤＮＡを精製した。得られたプラスミドＤＮＡを制限酵素ＰａｃＩおよびＡｐａＩで切断することにより、約３．７ｋｂの挿入断片が認められたものを選抜し、これをｐＭＪＰＣ１と命名した（図４）。 The strain grown on this medium was subjected to liquid culture by a conventional method, and then the plasmid DNA was purified. The obtained plasmid DNA was cleaved with restriction enzymes PacI and ApaI, and a plasmid in which an insertion fragment of about 3.7 kb was recognized was selected and named pMJPC1 (FIG. 4).
（Ｃ）ブレビバクテリウム・フラバムＭＪ２３３／ΔＬＤＨ株への形質転換
ブレビバクテリウム・フラバムＭＪ２３３株内で複製可能なｐＭＪＰＣ１による形質転換用のプラスミドＤＮＡは、上記（Ｂ）で形質転換した大腸菌（ＤＨ５α株）から調製した。
(C) Transformation into Brevibacterium flavum MJ233 / ΔLDH strain Plasmid DNA for transformation with pMJPC1 capable of replicating in Brevibacterium flavum MJ233 strain is the Escherichia coli (DH5α strain transformed with (B) above. ).
ブレビバクテリウム・フラバムＭＪ２３３／ΔＬＤＨ株への形質転換は、電気パルス法（Res. Microbiol., Vol.144, p.181-185, 1993）によって行い、得られた形質転換体をカナマイシン ５０μｇ／ｍＬを含むＬＢＧ寒天培地［トリプトン１０ｇ、イーストエキストラクト５ｇ、ＮａＣｌ ５ｇ、グルコース ２０ｇ、及び寒天１５ｇを蒸留水１Ｌに溶解］に塗抹した。 Transformation into Brevibacterium flavum MJ233 / ΔLDH strain was performed by the electric pulse method (Res. Microbiol., Vol. 144, p.181-185, 1993), and the obtained transformant was kanamycin 50 μg / mL. In an LBG agar medium [tryptone 10 g, yeast extract 5 g, NaCl 5 g, glucose 20 g, and agar 15 g dissolved in 1 L of distilled water].
この培地上に生育した株から、常法により液体培養した後、プラスミドＤＮＡを抽出、制限酵素切断による解析を行った結果、同株がｐＭＪＰＣ１を保持していることを確認し、該株をブレビバクテリウム・フラバムＭＪ２３３／ＰＣ／ΔＬＤＨ株と命名した。 From the strain grown on this medium, liquid culture was performed by a conventional method, and then plasmid DNA was extracted and analyzed by restriction enzyme digestion. As a result, it was confirmed that the strain retained pMJPC1. The strain was named as bacterial flavum MJ233 / PC / ΔLDH strain.
（Ｄ）ピルベートカルボキシラーゼ酵素活性
上記（Ｃ）で得られた形質転換株ブレビバクテリウム・フラバムＭＪ２３３／ＰＣ／ΔＬＤＨ株をグルコース２％、カナマイシン25mg/Lを含むＡ培地１００ｍｌで終夜培養を行った。得られた菌体を集菌後、５０ｍＭ リン酸カリウム緩衝液（pH7.5）50mlで洗浄し、同組成の緩衝液２０ｍｌに再度懸濁させた。懸濁液にをＳＯＮＩＦＩＥＲ ３５０（ＢＲＡＮＳＯＮ製）で破砕し、遠心分離した上清を無細胞抽出液とした。得られた無細胞抽出液を用いピルベートカルボキシラーゼ活性を測定した。酵素活性の測定は100mM Tris/HCl緩衝液（pH7.5）、 0.1mg/10mlビオチン、5mM 塩化マグネシウム、50mM 炭酸水素ナトリウム、5mM ピルビン酸ナトリウム 、5mM アデノシン三リン酸ナトリウム、0.32 mM NADH
、20units/1.5mlリンゴ酸デヒドロゲナーゼ（WAKO製、酵母由来）及び酵素を含む反応液中で２５℃で反応させることにより行った。1Uは１分間に１μmolのNADHの減少を触媒する酵素量とした。ピルベートカルボキシラーゼを発現させた無細胞抽出液における比活性は 0.2U/mg蛋白質であった。なお親株であるMJ233/△ＬＤＨ株をＡ培地を用いて同様に培養した菌体では、本活性測定方法によりピルベートカルボキシラーゼ活性は検出されなかった。
(D) Pyruvate carboxylase enzyme activity The transformant Brevibacterium flavum MJ233 / PC / ΔLDH obtained in (C) above was cultured overnight in 100 ml of A medium containing 2% glucose and 25 mg / L kanamycin. . The obtained cells were collected, washed with 50 ml of 50 mM potassium phosphate buffer (pH 7.5), and resuspended in 20 ml of the same composition. The suspension was crushed with SONIFIER 350 (manufactured by BRANSON), and the centrifuged supernatant was used as a cell-free extract. The pyruvate carboxylase activity was measured using the obtained cell-free extract. Measurement of enzyme activity is 100 mM Tris / HCl buffer (pH 7.5), 0.1 mg / 10 ml biotin, 5 mM magnesium chloride, 50 mM sodium bicarbonate, 5 mM sodium pyruvate, 5 mM sodium adenosine triphosphate, 0.32 mM NADH
The reaction was carried out at 25 ° C. in a reaction solution containing 20 units / 1.5 ml malate dehydrogenase (manufactured by WAKO, derived from yeast) and the enzyme. 1 U was defined as the amount of enzyme that catalyzes the decrease of 1 μmol of NADH per minute. The specific activity in the cell-free extract in which pyruvate carboxylase was expressed was 0.2 U / mg protein. It should be noted that pyruvate carboxylase activity was not detected by this activity measurement method in cells obtained by similarly culturing the parent strain MJ233 / ΔLDH strain using A medium.
［参考例５］
＜発酵液の調製＞
尿素：４ｇ、硫酸アンモニウム：１４ｇ、リン酸１カリウム：０．５ｇ、リン酸２カリウム０．５ｇ、硫酸マグネシウム・７水和物：０．５ｇ、硫酸第一鉄・７水和物：２０ｍｇ、硫酸マンガン・水和物：２０ｍｇ、Ｄ−ビオチン：２００μｇ、塩酸チアミン：２００μｇ、酵母エキス：１ｇ、カザミノ酸：１ｇ、及び蒸留水：１０００ｍＬの培地１００ｍＬを５００ｍＬの三角フラスコにいれ、１２０℃、２０分加熱滅菌した。これを室温まで冷やし、あらかじめ滅菌した５０％グルコース水溶液を４ｍＬ、無菌濾過した５％カナマイシン水溶液を５０μＬ添加し、参考例４（Ｃ）で作製したブレビバクテリウム・フラバムＭＪ２３３／ＰＣ／ΔＬＤＨ株を接種して２４時間３０℃にて種培養した。
[Reference Example 5]
<Preparation of fermentation broth>
Urea: 4 g, ammonium sulfate: 14 g, monopotassium phosphate: 0.5 g, dipotassium phosphate 0.5 g, magnesium sulfate heptahydrate: 0.5 g, ferrous sulfate heptahydrate: 20 mg, sulfuric acid Manganese hydrate: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 200 μg, yeast extract: 1 g, casamino acid: 1 g, and distilled water: 1000 mL of a medium of 100 mL is placed in a 500 mL Erlenmeyer flask at 120 ° C. for 20 minutes. Heat sterilized. This was cooled to room temperature, 4 mL of 50% glucose aqueous solution sterilized in advance and 50 μL of sterile 5% kanamycin aqueous solution were added, and inoculated with Brevibacterium flavum MJ233 / PC / ΔLDH prepared in Reference Example 4 (C). The seed culture was performed at 30 ° C. for 24 hours.
尿素：１２ｇ、硫酸アンモニウム：４２ｇ、リン酸１カリウム：１．５ｇ、リン酸２カリウム１．５ｇ、硫酸マグネシウム・７水和物：１．５ｇ、硫酸第一鉄・７水和物：６０ｍｇ、硫酸マンガン・水和物：６０ｍｇ、Ｄ−ビオチン：６００μｇ、塩酸チアミン：６００μｇ、酵母エキス３ｇ、カザミノ酸３ｇ、消泡剤（アデカノールＬＧ２９４：旭電化製）：１ｍＬ及び蒸留水：２５００ｍＬの培地を５Ｌの発酵糟に入れ、１２０℃、２０分加熱滅菌した。これを室温まで冷やした後、あらかじめ滅菌した１２％グルコース水溶液を５００ｍＬ添加し、これに前述の種培養液を全量加えて、３０℃に保温した。通気は毎分５００ｍＬ、攪拌は毎分５００回転で本培養を行った。１２時間後にグルコースがほぼ消費されていた。 Urea: 12 g, ammonium sulfate: 42 g, monopotassium phosphate: 1.5 g, dipotassium phosphate 1.5 g, magnesium sulfate heptahydrate: 1.5 g, ferrous sulfate heptahydrate: 60 mg, sulfuric acid Manganese hydrate: 60 mg, D-biotin: 600 μg, thiamine hydrochloride: 600 μg, yeast extract 3 g, casamino acid 3 g, antifoaming agent (Adecanol LG294: manufactured by Asahi Denka): 1 mL and distilled water: 2500 mL of medium is 5 L The mixture was placed in a fermenter and sterilized by heating at 120 ° C. for 20 minutes. After cooling this to room temperature, 500 mL of a 12% aqueous glucose solution sterilized in advance was added, and the whole amount of the above-mentioned seed culture solution was added thereto, and the mixture was kept at 30 ° C. Main culture was performed at aeration of 500 mL / min and stirring at 500 rpm. Glucose was almost consumed after 12 hours.
硫酸マグネシウム・７水和物：1.5ｇ、硫酸第一鉄・７水和物：60ｍｇ、硫酸マンガン・水和物：60ｍｇ、Ｄ−ビオチン：600μｇ、塩酸チアミン：600μｇ、消泡剤（アデカノールＬＧ２９４：旭電化製）：５ｍｌ及び蒸留水：1.5Ｌの培地を３Ｌの三角フラスコに入れ、１２０℃、２０分加熱滅菌した。室温まで冷やした後、上記の本培養により得られた培養液を１００００ｇ、５分の遠心分離により集菌した菌体を添加して、Ｏ．Ｄ．（６６０ｎｍ）が６０になるように再懸濁した。この懸濁液1.5Ｌとあらかじめ滅菌した２０％グルコース溶液1.5Ｌを５Ｌのジャーファーメンターに入れて混合し、３５℃に保温した。ｐＨは２Ｍ炭酸アンモニウムを用いて７．６に保ち、毎分５００ｍＬで通気、毎分３００回転で攪拌しながら反応を行った。反応開始後約５０時間でグルコースがほぼ消費されていた。コハク酸が４８ｇ蓄積されていた。この発酵液を１００００ｇ、５分間の遠心分離、限外濾過（日東電工（株）製 NTU-3000-C1R）により菌体と上清に分離した。以上の操作を６回行うことにより、コハク酸発酵液上清を２１Ｌ得ることが出来た。 Magnesium sulfate heptahydrate: 1.5 g, ferrous sulfate heptahydrate: 60 mg, manganese sulfate hydrate: 60 mg, D-biotin: 600 μg, thiamine hydrochloride: 600 μg, defoamer (Adecanol LG294: Asahi Denka): 5 ml and distilled water: 1.5 L of medium was placed in a 3 L Erlenmeyer flask and sterilized by heating at 120 ° C. for 20 minutes. After cooling to room temperature, microbial cells collected by centrifugation at 10,000 g for 5 minutes were added to the culture solution obtained by the above main culture. D. It was resuspended so that (660 nm) was 60. 1.5 L of this suspension and 1.5 L of a 20% glucose solution sterilized in advance were mixed in a 5 L jar fermenter and kept at 35 ° C. The pH was kept at 7.6 using 2M ammonium carbonate, and the reaction was carried out with aeration at 500 mL / min and stirring at 300 rpm. In about 50 hours after the start of the reaction, glucose was almost consumed. 48 g of succinic acid was accumulated. This fermentation broth was separated into cells and supernatant by centrifugation at 10,000 g for 5 minutes and ultrafiltration (NTU-3000-C1R manufactured by Nitto Denko Corporation). By performing the above operation 6 times, 21 L of a succinic acid fermentation broth supernatant could be obtained.
［参考例６］
＜発酵液からコハク酸の調製＞
参考例５で得られた発酵液を80℃のウォーターバスを用いて濃縮した。圧力は成り行きで、留出が少なくなったら減圧を繰り返し、約２．５Lに濃縮した液を得た。
[Reference Example 6]
<Preparation of succinic acid from fermentation broth>
The fermentation broth obtained in Reference Example 5 was concentrated using a water bath at 80 ° C. The pressure was steady, and when distilling decreased, the pressure reduction was repeated to obtain a liquid concentrated to about 2.5 L.
この発酵液１２００ｇを晶析装置に入れ、酢酸４００ｇを加え、２０℃にて晶析した。種晶としてコハク酸アンモニウムの結晶を２ｇ入れた。種晶を入れてから３時間攪拌し、濾過した。これを２回繰り返した 1200 g of this fermentation broth was put into a crystallizer, 400 g of acetic acid was added, and crystallized at 20 ° C. 2 g of ammonium succinate crystals were added as seed crystals. After adding seed crystals, the mixture was stirred for 3 hours and filtered. This was repeated twice
析出した固体は１２６９ｇであり、分析すると、コハク酸５３ｗｔ％、アンモニア１１．５ｗｔ％、酢酸１４ｗｔ％であった。 The precipitated solid was 1269 g, and analyzed to be 53 wt% succinic acid, 11.5 wt% ammonia, and 14 wt% acetic acid.
この結晶を２日間、デシケータ中にて乾燥した。 The crystals were dried for 2 days in a desiccator.
この結晶１０００ｇ、酢酸１２００ｇを晶析装置に入れ、８５℃にて溶解した後、２５℃に冷却した。２５℃になってから１０分後、コハク酸試薬を種晶として１ｇ追加した。種晶を入れてから３時間攪拌し、濾過を行った。 1000 g of this crystal and 1200 g of acetic acid were put in a crystallizer, dissolved at 85 ° C., and then cooled to 25 ° C. Ten minutes after reaching 25 ° C., 1 g of a succinic acid reagent was added as a seed crystal. After adding seed crystals, the mixture was stirred for 3 hours and filtered.
コハク酸結晶が３２０ｇ得られた。分析すると、コハク酸８５ｗｔ％、アンモニア２．６ｗｔ％、酢酸１１ｗｔ％であった。これを更に５℃に調整した蒸留水３００ｇを用いてリンスし、得られた結晶をデシケータ中にて２日乾燥後分析すると、結晶は２８０ｇ得られ、アンモニアは５０００ｐｐｍになった。 320 g of succinic acid crystals were obtained. As a result of analysis, succinic acid was 85 wt%, ammonia was 2.6 wt%, and acetic acid was 11 wt%. This was further rinsed with 300 g of distilled water adjusted to 5 ° C., and the obtained crystals were dried in a desiccator for 2 days and analyzed. As a result, 280 g of crystals were obtained and ammonia was 5000 ppm.
［参考例７］
＜高純度コハク酸の調製＞
参考例６で得られたコハク酸１００ｇを更に５℃に調整した蒸留水１００ｇを２回、合計２００ｇ用いてリンスし、得られた結晶をデシケータ中にて２日乾燥後分析すると、結晶は７５ｇ得られ、アンモニアは３００ｐｐｍになった。
[Reference Example 7]
<Preparation of high purity succinic acid>
When 100 g of succinic acid obtained in Reference Example 6 was further rinsed with 100 g of distilled water adjusted to 5 ° C. twice for a total of 200 g, the obtained crystal was dried in a desiccator for 2 days and analyzed. The resulting ammonia was 300 ppm.
［参考例８］
＜高純度コハク酸の調製＞
参考例６で得られたコハク酸１００ｇ、蒸留水１５０ｇを晶析装置に入れ、８５℃にて溶解した後、２５℃に冷却した。２５℃になってから１０分後、コハク酸試薬を種晶として１ｇ追加した。種晶を入れてから３時間攪拌し、濾過を行った。得られた結晶７３ｇを５℃に調整した蒸留水５０ｇを２回、合計１００ｇ用いてリンスし、得られた結晶をデシケータ中にて２日乾燥後分析すると、結晶は６２ｇ得られ、アンモニアは８７ｐｐｍになった。
[Reference Example 8]
<Preparation of high purity succinic acid>
100 g of succinic acid obtained in Reference Example 6 and 150 g of distilled water were put into a crystallizer, dissolved at 85 ° C., and then cooled to 25 ° C. Ten minutes after reaching 25 ° C., 1 g of a succinic acid reagent was added as a seed crystal. After adding seed crystals, the mixture was stirred for 3 hours and filtered. The obtained crystals (73 g) were rinsed twice with 50 g of distilled water adjusted to 5 ° C. using a total of 100 g. The obtained crystals were dried in a desiccator for 2 days and analyzed to obtain 62 g of crystals and ammonia of 87 ppm. Became.
［実施例１］
＜アンモニア含有量 ３００ｐｐｍの発酵法コハク酸を用いたポリエステルの製造＞
攪拌装置、窒素導入管、加熱装置、温度計、助剤添加口を備えた容量１００ｍｌの反応容器に、参考例７で得られた発酵法コハク酸（アンモニア含有量３００ｐｐｍ）を３５．４ｇ、１，４−ブタンジオールを２８．４ｇ、酸化ゲルマニウムをあらかじめ１重量％溶解させた９０％乳酸水溶液２．９ｇを仕込んだ。容器内容物を攪拌下、窒素ガスを導入し、窒素ガス雰囲気下１６０℃に昇温し、さらにこの温度から１時間で２２０℃まで昇温し、２２０℃で１時間保持した。その後３０分で２３０℃まで昇温すると同時に、１時間３０分間かけて反応系内の圧力を真空ポンプで０，５ｍｍＨｇまで減圧し、０．５mmHgの減圧下において４時間１０分重合を行った。このポリマーの還元粘度は１．６であった。またこのポリマーは淡黄色であり、黄色度ＹＩは４０であった。またポリマーの窒素含有量は５５ｐｐｍであった。
[Example 1]
<Production of polyester using fermentation method succinic acid with ammonia content of 300 ppm>
35.4 g of the fermentation method succinic acid (ammonia content 300 ppm) obtained in Reference Example 7 in a reaction vessel having a capacity of 100 ml equipped with a stirrer, a nitrogen introducing tube, a heating device, a thermometer, and an auxiliary agent addition port, , 4-butanediol 28.4 g and 90% aqueous lactic acid solution in which 1% by weight of germanium oxide was dissolved in advance were charged. While stirring the contents of the vessel, nitrogen gas was introduced, and the temperature was raised to 160 ° C. in a nitrogen gas atmosphere. Thereafter, the temperature was raised to 230 ° C. in 30 minutes, and simultaneously, the pressure in the reaction system was reduced to 0.5 mmHg with a vacuum pump over 1 hour and 30 minutes, and polymerization was carried out for 4 hours and 10 minutes under a reduced pressure of 0.5 mmHg. The reduced viscosity of this polymer was 1.6. Moreover, this polymer was light yellow and yellow degree YI was 40. The polymer nitrogen content was 55 ppm.
［比較例１］
（アンモニア含有量 〜５０００ｐｐｍの発酵法コハク酸を用いたポリエステルの製造）
実施例１においてアンモニア含有量３００ｐｐｍの発酵法コハク酸に替えて、参考例６で得られた発酵法コハク酸（アンモニア含有量５０００ｐｐｍ）を用いた以外は実施例１と同様にポリエステルの重合反応を行った。その結果、重合度が上がらず低粘度のオリゴマーしか得られなかった。このオリゴマーの還元粘度は０．３であった。
[Comparative Example 1]
(Manufacture of polyester using fermentation method succinic acid having ammonia content of ~ 5000 ppm)
A polyester polymerization reaction was carried out in the same manner as in Example 1 except that the fermentation method succinic acid (ammonia content 5000 ppm) obtained in Reference Example 6 was used in place of the fermentation method succinic acid having an ammonia content of 300 ppm in Example 1. went. As a result, the degree of polymerization did not increase and only low viscosity oligomers were obtained. The reduced viscosity of this oligomer was 0.3.
［実施例２］
（アンモニア含有量 ８７ｐｐｍの発酵法コハク酸を用いたポリエステルの製造）
実施例１においてアンモニア含有量３００ｐｐｍの発酵法コハク酸に替えて、参考例８で得られた発酵法コハク酸（アンモニア含有量８７ｐｐｍ）を用いたこと、及び、０．５ｍｍＨｇの減圧下において２．８時間重合を行ったこと以外は実施例１と同様にポリエステルの重合反応を行った。その結果、このポリマーの還元粘度は２．６であった。またこのポリマーは白色であり、黄色度ＹＩは１２であった。またポリマーの窒素含有量は４４ｐｐｍであった。
[Example 2]
(Production of polyester using fermentation method succinic acid having an ammonia content of 87 ppm)
In Example 1, the fermented succinic acid (ammonia content 87 ppm) obtained in Reference Example 8 was used instead of the fermented succinic acid having an ammonia content of 300 ppm, and 2. under a reduced pressure of 0.5 mmHg. A polyester polymerization reaction was carried out in the same manner as in Example 1 except that the polymerization was carried out for 8 hours. As a result, the reduced viscosity of this polymer was 2.6. Moreover, this polymer was white and yellowness YI was 12. The nitrogen content of the polymer was 44 ppm.
Claims (5)
A polyester obtained by polymerizing dicarboxylic acid and diol in a reaction solution containing a dicarboxylic acid raw material and a diol raw material, wherein at least one of the dicarboxylic acid raw material and the diol raw material includes a fermentation process step. The obtained polyester has a nitrogen content of greater than 0 and less than or equal to 1000 ppm, and a reduced viscosity measured in a tetrachloroethane / phenol (50/50 mass ratio) mixed solvent at 30 ° C. is 0.5. That is the polyester.
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003206337A (en) * | 1994-08-31 | 2003-07-22 | Mitsubishi Chemicals Corp | Manufacturing method of aliphatic polymer copolymer |
| JP2003206338A (en) * | 1994-08-31 | 2003-07-22 | Mitsubishi Chemicals Corp | Film comprising aliphatic polyester copolymer |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9598388B2 (en) | 2011-07-04 | 2017-03-21 | Mitsubishi Chemical Corporation | Method for producing tetrahydrofuran |
| US9434706B2 (en) | 2011-07-08 | 2016-09-06 | Mitsubishi Chemical Corporation | 1,4-butanediol-containing composition |
| US10472311B2 (en) | 2011-07-08 | 2019-11-12 | Mitsubishi Chemical Corporation | 1,4-butanediol-containing composition |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2009024179A (en) | 2009-02-05 |
| JP2009062366A (en) | 2009-03-26 |
| JP2005139287A (en) | 2005-06-02 |
| JP4301918B2 (en) | 2009-07-22 |
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