JP2004194570A - Method for producing organic compound using coryneform bacterium - Google Patents
Method for producing organic compound using coryneform bacterium Download PDFInfo
- Publication number
- JP2004194570A JP2004194570A JP2002367331A JP2002367331A JP2004194570A JP 2004194570 A JP2004194570 A JP 2004194570A JP 2002367331 A JP2002367331 A JP 2002367331A JP 2002367331 A JP2002367331 A JP 2002367331A JP 2004194570 A JP2004194570 A JP 2004194570A
- Authority
- JP
- Japan
- Prior art keywords
- reaction
- organic compound
- reaction medium
- producing
- coryneform bacterium
- 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.)
- Granted
Links
- 150000002894 organic compounds Chemical class 0.000 title claims abstract description 55
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 26
- 241000186254 coryneform bacterium Species 0.000 title claims description 34
- 239000012429 reaction media Substances 0.000 claims abstract description 46
- 150000001720 carbohydrates Chemical class 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims description 41
- 230000033116 oxidation-reduction process Effects 0.000 claims description 22
- 235000019441 ethanol Nutrition 0.000 claims description 13
- 239000000126 substance Substances 0.000 claims description 9
- 235000001014 amino acid Nutrition 0.000 claims description 6
- 150000001413 amino acids Chemical class 0.000 claims description 6
- 229940088594 vitamin Drugs 0.000 claims description 6
- 235000013343 vitamin Nutrition 0.000 claims description 6
- 239000011782 vitamin Substances 0.000 claims description 6
- 229930003231 vitamin Natural products 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 5
- 150000007524 organic acids Chemical class 0.000 claims description 5
- 150000001298 alcohols Chemical class 0.000 claims description 3
- 235000005985 organic acids Nutrition 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 96
- 241000186031 Corynebacteriaceae Species 0.000 abstract description 15
- 230000000813 microbial effect Effects 0.000 abstract description 3
- 239000011368 organic material Substances 0.000 abstract 1
- 230000035755 proliferation Effects 0.000 abstract 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 26
- 239000000243 solution Substances 0.000 description 25
- 210000004027 cell Anatomy 0.000 description 20
- 239000000047 product Substances 0.000 description 20
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 16
- 239000001301 oxygen Substances 0.000 description 15
- 229910052760 oxygen Inorganic materials 0.000 description 15
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 14
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 8
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 8
- 239000007864 aqueous solution Substances 0.000 description 8
- 229910052799 carbon Inorganic materials 0.000 description 8
- 239000004310 lactic acid Substances 0.000 description 8
- 235000014655 lactic acid Nutrition 0.000 description 8
- 238000004811 liquid chromatography Methods 0.000 description 8
- 239000011550 stock solution Substances 0.000 description 8
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 7
- 210000004748 cultured cell Anatomy 0.000 description 7
- 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 6
- 241000894006 Bacteria Species 0.000 description 6
- 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 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
- 241000192041 Micrococcus Species 0.000 description 6
- 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 6
- 239000006227 byproduct Substances 0.000 description 6
- 239000007789 gas Substances 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 235000015097 nutrients Nutrition 0.000 description 6
- 238000006722 reduction reaction Methods 0.000 description 6
- 229940024606 amino acid Drugs 0.000 description 5
- 238000005842 biochemical reaction Methods 0.000 description 5
- 230000007102 metabolic function Effects 0.000 description 5
- 241000186226 Corynebacterium glutamicum Species 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 239000001569 carbon dioxide Substances 0.000 description 4
- 229910002092 carbon dioxide Inorganic materials 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 230000003248 secreting effect Effects 0.000 description 4
- 238000011218 seed culture Methods 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000001384 succinic acid Substances 0.000 description 4
- CWERGRDVMFNCDR-UHFFFAOYSA-N thioglycolic acid Chemical compound OC(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 238000005273 aeration Methods 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
- 239000012298 atmosphere Substances 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 229910001873 dinitrogen Inorganic materials 0.000 description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000009776 industrial production Methods 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 238000006479 redox reaction Methods 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 235000000346 sugar Nutrition 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 241000186146 Brevibacterium Species 0.000 description 2
- 241000186216 Corynebacterium Species 0.000 description 2
- 241000186145 Corynebacterium ammoniagenes Species 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- 241000191948 Kocuria rosea Species 0.000 description 2
- 241000191938 Micrococcus luteus Species 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 241000319304 [Brevibacterium] flavum Species 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- WERYXYBDKMZEQL-UHFFFAOYSA-N butane-1,4-diol Chemical compound OCCCCO WERYXYBDKMZEQL-UHFFFAOYSA-N 0.000 description 2
- -1 can be removed Substances 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 229940093915 gynecological organic acid Drugs 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 229910017053 inorganic salt Inorganic materials 0.000 description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 2
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 2
- 230000037353 metabolic pathway Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000027756 respiratory electron transport chain Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 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 2
- 229960000344 thiamine hydrochloride Drugs 0.000 description 2
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 2
- 239000011747 thiamine hydrochloride Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- DNIAPMSPPWPWGF-VKHMYHEASA-N (+)-propylene glycol Chemical compound C[C@H](O)CO DNIAPMSPPWPWGF-VKHMYHEASA-N 0.000 description 1
- QIJRTFXNRTXDIP-UHFFFAOYSA-N (1-carboxy-2-sulfanylethyl)azanium;chloride;hydrate Chemical compound O.Cl.SCC(N)C(O)=O QIJRTFXNRTXDIP-UHFFFAOYSA-N 0.000 description 1
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- YPFDHNVEDLHUCE-UHFFFAOYSA-N 1,3-propanediol Substances OCCCO YPFDHNVEDLHUCE-UHFFFAOYSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 description 1
- LLIANSAISVOLHR-GBCQHVBFSA-N 5-[(3as,4s,6ar)-2-oxidanylidene-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21.N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 LLIANSAISVOLHR-GBCQHVBFSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 241000186063 Arthrobacter Species 0.000 description 1
- 241000186074 Arthrobacter globiformis Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical class OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 241001485655 Corynebacterium glutamicum ATCC 13032 Species 0.000 description 1
- 241000671338 Corynebacterium glutamicum R Species 0.000 description 1
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- ODBLHEXUDAPZAU-ZAFYKAAXSA-N D-threo-isocitric acid Chemical compound OC(=O)[C@H](O)[C@@H](C(O)=O)CC(O)=O ODBLHEXUDAPZAU-ZAFYKAAXSA-N 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 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 1
- 239000005715 Fructose Substances 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- ODBLHEXUDAPZAU-FONMRSAGSA-N Isocitric acid Natural products OC(=O)[C@@H](O)[C@H](C(O)=O)CC(O)=O ODBLHEXUDAPZAU-FONMRSAGSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 1
- 241000186359 Mycobacterium Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 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 1
- 229930006000 Sucrose Natural products 0.000 description 1
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 150000004649 carbonic acid derivatives Chemical class 0.000 description 1
- 229920001525 carrageenan Polymers 0.000 description 1
- 239000000679 carrageenan Substances 0.000 description 1
- 235000010418 carrageenan Nutrition 0.000 description 1
- 229940113118 carrageenan Drugs 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- GTZCVFVGUGFEME-HNQUOIGGSA-N cis-Aconitic acid Natural products OC(=O)C\C(C(O)=O)=C/C(O)=O GTZCVFVGUGFEME-HNQUOIGGSA-N 0.000 description 1
- GTZCVFVGUGFEME-IWQZZHSRSA-N cis-aconitic acid Chemical compound OC(=O)C\C(C(O)=O)=C\C(O)=O GTZCVFVGUGFEME-IWQZZHSRSA-N 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000011437 continuous method Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 229960001305 cysteine hydrochloride Drugs 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
- 230000002542 deteriorative effect Effects 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000909 electrodialysis Methods 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- DUYAAUVXQSMXQP-UHFFFAOYSA-N ethanethioic S-acid Chemical compound CC(S)=O DUYAAUVXQSMXQP-UHFFFAOYSA-N 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 235000011087 fumaric acid Nutrition 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 210000001822 immobilized cell Anatomy 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 239000011261 inert gas Substances 0.000 description 1
- 229910052806 inorganic carbonate Inorganic materials 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- 239000011572 manganese Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000006241 metabolic reaction Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- KHPXUQMNIQBQEV-UHFFFAOYSA-N oxaloacetic acid Chemical compound OC(=O)CC(=O)C(O)=O KHPXUQMNIQBQEV-UHFFFAOYSA-N 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920000166 polytrimethylene carbonate Polymers 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000004886 process control Methods 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 229910052979 sodium sulfide Inorganic materials 0.000 description 1
- GRVFOGOEDUUMBP-UHFFFAOYSA-N sodium sulfide (anhydrous) Chemical compound [Na+].[Na+].[S-2] GRVFOGOEDUUMBP-UHFFFAOYSA-N 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 235000019157 thiamine Nutrition 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 1
- ODBLHEXUDAPZAU-UHFFFAOYSA-N threo-D-isocitric acid Natural products OC(=O)C(O)C(C(O)=O)CC(O)=O ODBLHEXUDAPZAU-UHFFFAOYSA-N 0.000 description 1
- 229910021654 trace metal Inorganic materials 0.000 description 1
- GTZCVFVGUGFEME-UHFFFAOYSA-N trans-aconitic acid Natural products OC(=O)CC(C(O)=O)=CC(O)=O GTZCVFVGUGFEME-UHFFFAOYSA-N 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【０００１】
【発明の属する技術分野】
本発明はコリネ型細菌を用いる有機化合物の製造方法に関する。さらに詳しくは、コリネ型細菌を用いて特定の反応状態下において有機酸、アルコール、アミノ酸およびビタミン類の有機化合物を生成せしめ、ついでこれを採取することからなる高効率な有機化合物の製造方法に関するものである。
【０００２】
【従来の技術】
従来より、好気性コリネ型細菌は通気攪拌培養法または振盪培養法等の好気的条件下でアミノ酸等有用物質生産に広く用いられてきた（特許文献１）。また、嫌気的条件下（微量酸素存在条件を含む）で炭酸イオンあるいは炭酸ガスを含有する反応液中で好気性コリネ型細菌またはその処理物を有機原料に作用させることにより含酸素化合物の製造にも用いられている（特許文献２）。
【０００３】
コリネ型細菌を好気的条件もしくは微酸素条件下、すなわち酸化条件下にて用いる従来技術の方法では、当然のことながらコリネ型細菌は酸素存在量に依存した分裂増殖が認められる。その分裂に要する時間は２時間程度以内の速い分裂速度の場合から、１０時間程度もしくはそれ以上の時間を要する場合など大きく変動する。
いずれにしても、分裂増殖する事により、コリネ型細菌に与えた栄養源は増殖に消費され、目的生産物の生産量が低下、すなわち栄養源からの目的生産物への変換率が低下するなどの工業的生産上重要な課題が指摘されている。さらに、増殖過程においては、これに起因する代謝物が分泌物として生成されることから、これら目的生産物以外の分泌副生成物と目的生産物の分離が生産物品質純度の観点より必要となり、特に微量で多様な分泌副生物の分離精製工程は工業的生産技術の経済性悪化の大きな要因となっている。
【０００４】
【特許文献１】
特開平０５−０１５３７７号公報（請求項３）
【特許文献２】
特開平１１−１１３５８８号公報（請求項１）
【０００５】
【発明が解決しようとする課題】
本発明は、好気性コリネ型細菌による増殖を伴う物質生産技術に関する上記技術的課題を解決することを目的とする。すなわち、本発明は、好気性コリネ型細菌またはその処理物を用いた有機化合物の製造方法において、糖などの有機炭素源から目的有機化合物への変換率、および得られる目的有機化合物の純度を向上させることができる方法を提供することを目的とする。
【０００６】
【課題を解決するための手段】
コリネ型細菌は従来より好気的条件下や嫌気的条件下（微量酸素存在条件を含む）で有機化合物の生産に用いられてきた。しかし、本発明者は今まで知られていなかった還元状態下でコリネ型細菌を用いることにより、より具体的には、好気性コリネ型細菌またはその処理物と糖類とを還元状態下の反応培地中で反応させることにより、従来技術が有していた、グルコース等糖類からの目的生産物への変換率が低いなどの問題点が克服され、有機化合物の製造をより効率的に行うことが出来ることを見出し、本発明に到達した。
【０００７】
すなわち、本発明は、
（１） 好気性コリネ型細菌を好気条件下で増殖培養し回収した菌体またはその菌体処理物と糖類とを還元条件下の反応培地中で反応させ、反応培地に生成する有機化合物を採取することを特徴とする有機化合物の製造方法、
（２） 還元状態下の反応培地の酸化還元電位が−２００ミリボルト乃至−５００ミリボルトであることを特徴とする前記（１）に記載の有機化合物の製造方法、
（３） 反応培地が、増殖培養過程で生成し、菌体内外に存在する生成物質を実質的に含有しないことを特徴とする前記（１）または（２）に記載の有機化合物の製造方法、
（４） 有機化合物が、有機酸、アルコール、アミノ酸およびビタミン類から選ばれることを特徴とする前記（１）〜（３）に記載の有機化合物の製造方法、に関する。
【０００８】
【発明の実施の形態】
本発明で用いられる好気性コリネ型細菌とは、バージーズ・マニュアル・デターミネイティブ・バクテリオロジー（Bargeys Manual of Determinative Bacteriology, 8, 599、1974）に定義されている一群の微生物であり、通常の好気的条件で増殖し、本発明の還元状態下で目的とする有機化合物を生成するものならば特に限定されるものではない。
具体例を挙げれば、コリネバクテリウム属菌、ブレビバクテリウム属菌、アースロバクター属菌、マイコバクテリューム属菌またはマイクロコッカス属菌等が挙げられる。
【０００９】
さらに具体的には、コリネバクテリウム属菌としては、コリネバクテリウム グルタミカム（Corynebacterium glutamicum）ＦＥＲＭ Ｐ−１８９７６、ＡＴＣＣ１３０３２、ＡＴＣＣ１３０５８、ＡＴＣＣ１３０５９、ＡＴＣＣ１３０６０、ＡＴＣＣ１３２３２、ＡＴＣＣ１３２８６、ＡＴＣＣ１３２８７、ＡＴＣＣ１３６５５、ＡＴＣＣ１３７４５、ＡＴＣＣ１３７４６、ＡＴＣＣ１３７６１、ＡＴＣＣ１４０２０またはＡＴＣＣ３１８３１等が挙げられる。
ブレビバクテリウム属菌としては、ブレビバクテリウム ラクトファーメンタム（Brevibacterium lactofermentum）ＡＴＣＣ１３８６９、ブレビバクテリウム フラバム（Brevibacterium flavum）ＭＪ−２３３(ＦＥＲＭ ＢＰ−１４９７)もしくはＭＪ−２３３ＡＢ−４１（ＦＥＲＭ ＢＰ−１４９８）、またはブレビバクテリウム アンモニアゲネス（Brevibacterium ammoniagenes）ＡＴＣＣ６８７２等があげられる。
アースロバクター属菌としては、アースロバクター グロビフォルミス（Arthrobacter globiformis）ＡＴＣＣ８０１０、ＡＴＣＣ４３３６、ＡＴＣＣ２１０５６、ＡＴＣＣ３１２５０、ＡＴＣＣ３１７３８またはＡＴＣＣ３５６９８等が挙げられる。
マイクロコッカス属菌としては、マイクロコッカス・フロイデンライヒ（Micrococcus freudenreichii）Ｎｏ．２３９（ＦＥＲＭ Ｐ−１３２２１）、マイクロコッカス・ルテウス（Micrococcus luteus）Ｎｏ．２４０（ＦＥＲＭ Ｐ−１３２２２）、マイクロコッカス ウレアエ（Micrococcus ureae）ＩＡＭ１０１０またはマイクロコッカス ロゼウス（Micrococcus roseus）ＩＦＯ３７６４等が挙げられる。
本発明で用いられる好気性コリネ型細菌としては、Corynebacterium glutamicum R (FERM P-18976)、Corynebacterium glutamicum ATCC13032またはCorynebacterium glutamicum ATCC13869などが好ましい。
【００１０】
本発明で用いられる好気性コリネ型細菌としては自然界に存在する野生株の変異株（例えば、ＦＥＲＭ Ｐ−１８９７７，ＦＥＲＭ Ｐ−１８９７８株など）であってもよく、また遺伝子組換え等のバイオテクノロジーを利用した人為株（例えば、ＦＥＲＭ Ｐ−１７８８７、ＦＥＲＭ Ｐ−１７８８８、ＦＥＲＭ Ｐ−１８９７９など）でもよい。
【００１１】
本発明に係る有機化合物の製造方法においては、まず上述した好気性コリネ型細菌を好気条件下で増殖培養する。
好気性コリネ型細菌の培養は、炭素源、窒素源および無機塩等を含む通常の栄養培地を用いて行うことが出来る。培養には、炭素源として、例えばグルコースまたは廃糖蜜等を、そして窒素源としては、例えばアンモニア、硫酸アンモニウム、塩化アンモニウム、硝酸アンモニウムまたは尿素等をそれぞれ単独もしくは混合して用いることが出来る。また、無機塩として、例えばリン酸一水素カリウム、リン酸ニ水素カリウムまたは硫酸マグネシウム等を使用することが出来る。この他にも必要に応じて、ペプトン、肉エキス、酵母エキス、コーンスティープリカー、カザミノ酸またはビオチンもしくはチアミン等の各種ビタミン等の栄養素を培地に適宜添加することも出来る。
【００１２】
培養は、通常、通気攪拌または振盪等の好気的条件下、約２０℃〜約４０℃、好ましくは約２５℃〜約３５℃の温度で行うことが出来る。培養時のｐＨは５〜１０付近、好ましくは７〜８付近の範囲がよく、培養中のｐＨ調整は酸またはアルカリを添加することにより行うことが出来る。培養開始時の炭素源濃度は、約１〜２０％（Ｗ／Ｖ）、好ましくは約２〜５％（Ｗ／Ｖ）である。また、培養期間は通常１〜７日間程度である。
【００１３】
ついで、好気性コリネ型細菌の培養菌体を回収する。上記の如くして得られる培養物から培養菌体を回収分離する方法としては、特に限定されず、例えば遠心分離や膜分離等の公知の方法を用いることができる。
回収された培養菌体に対して処理を加え、得られる菌体処理物を次工程に用いてもよい。前記菌体処理物としては、培養菌体に何らかの処理が加えられたものであればよく、例えば、菌体をアクリルアミドまたはカラギーナン等で固定化した固定化菌体等が挙げられる。
【００１４】
ついで、上記の如くして得られる培養物から回収分離された好気性コリネ型細菌の培養菌体またはその菌体処理物は還元状態下の反応培地での目的有機化合物の生成反応に供せられる。有機化合物生成方式は、回分式、連続式いずれの生成方式も可能である。
本発明の還元状態下の生化学反応に於いては、コリネ型細菌の増殖分裂が完全に抑制され、本発明の課題であるグルコース等糖類栄養源からの目的有機化合物への変換率が画期的に向上し、また増殖に伴う分泌副生物の実質的な完全抑制を実現することが出来る。この観点からは、培養回収されたコリネ型細菌またはその菌体処理物が反応培地に供せられるときには、コリネ型細菌細胞内外の培養時環境状態が反応培地にもたらされない方法や条件を用いることが推奨される。つまり、反応培地は、増殖培養過程で生成し、菌体内外に存在する生成物質を実質的に含有しないことが好ましい。より具体的には、増殖培養過程で生成し、菌体外に放出された分泌副生物、および培養菌体内の好気的代謝機能により生成し菌体内に残存する物質が、反応培地に実質的に存在しない状態であることが推奨される。このような状態は、例えば、増殖培養後の培養液の遠心分離、膜分離等の方法および／または培養後の菌体を還元状態下で２時間ないし１０時間程度放置することで実現される。
【００１５】
本工程においては、還元状態下の反応培地を用いる。反応培地は、還元状態下にあれば、固体状、半固体状または液体状等いずれの形状を有していてもよい。本発明の必須の要件は、還元状態下でコリネ型細菌の代謝機能による生化学反応を行わせしめ、目的とする有機化合物を生成することである。
本発明における還元状態とは、反応系の酸化還元電位で規定され、反応培地の酸化還元電位は、好ましくは約−２００ｍＶ〜−５００ｍＶ程度、より好ましくは約−２５０ｍＶ〜−５００ｍＶ程度である。反応培地の還元状態は簡便にはレサズリン指示薬（還元状態であれば、青色から無色への脱色）である程度推定できるが、正確には酸化還元電位差計（例えば、BROADLEY JAMES社製、ORP Electrodes）を用いる。本発明においては、反応培地に菌体またはその処理物を添加した直後から有機化合物を採取するまで、還元状態を維持していることが好ましいが、少なくとも有機化合物を採取する時点で反応培地が還元状態であればよい。反応時間の約５０％以上、より好ましくは約７０％以上、さらに好ましくは約９０％以上の時間、反応培地が還元状態に保たれていることが望ましい。なかでも、反応時間の約５０％以上、より好ましくは約７０％以上、さらに好ましくは約９０％以上の時間、反応培地の酸化還元電位が約−２００ｍＶ〜−５００ｍＶ程度に保たれていることがより望ましい。
【００１６】
このような還元状態の実現は具体的には、前記の培養後の培養菌体調製方法、反応培地の調整方法、または反応途中における還元状態の維持方法等によりなされる。
還元状態下の反応培地の調整方法は、公知の方法を用いてよい。例えば、反応培地用水溶液の調整方法は、例えば硫酸還元微生物などの絶対嫌気性微生物用の培養液調整方法（Pfennig, N et. al.(1981):
The dissimilatory sulfate-reducing bacteria, In The Prokaryotes,A Handbook on Habitats, Isolation and Identification of Bacteria,Ed. by Starr, M. P. et. al. p.926-940, Berlin, Springer Verlag.や「農芸化学実験書 第三巻、京都大学農学部 農芸化学教室編、1990年第２６刷、産業図書株式会社出版」)などが参考となり、所望する還元状態の水溶液を得ることが出来る。
【００１７】
反応培地用水溶液の調整方法として、より具体的には反応培地用水溶液を加熱処理や減圧処理することにより溶解ガスを除去する方法等が挙げられる。より具体的には、約１０ｍｍＨｇ以下、好ましくは約５ｍｍＨｇ以下、より好ましくは約３ｍｍＨｇ以下の減圧下で、約１〜６０分程度、好ましくは５〜４０分程度、反応培地用水溶液を処理することにより、溶解ガス、特に溶解酸素を除去し、還元条件下の反応培地用水溶液を作成することができる。また、適当な還元剤（例えば、チオグリコール酸、アスコルビン酸、システィン塩酸塩、メルカプト酢酸、チオール酢酸、グルタチオンそして硫化ソーダ等）を添加して還元状態の反応培地用水溶液を調整することも出来る。また、場合により、これらの方法を適宜組み合わせることも有効な還元状態の反応培地用水溶液を調整する方法となる。
【００１８】
反応途中における還元状態の維持方法としては、反応系外からの酸素の混入を可能な限り防止することが望ましく、反応系を窒素ガス等の不活性ガスや炭酸ガス等で封入する方法が通常用いられる。酸素混入をより効果的に防止する方法としては、反応途中においてコリネ型細菌の菌体内の代謝機能を効率よく機能させるために、反応系のｐＨ維持調整液の添加や各種栄養素溶解液を適宜添加する必要が生じる場合もあるが、このような場合には添加溶液から酸素を予め除去しておくことが有効である。
【００１９】
本発明の有機化合物生成反応において、生成反応系の酸化還元電位の規定が目的とする有機化合物の効率的な生産に関してなぜ有効であるかの理由は明らかではないが、下記にその推定理由を記す。ただし、本発明はその推定理由になんら限定されるものではない。
本発明の目的生産物である有機化合物はコリネ型細菌の代謝機能に基づく生化学反応により産生される化合物である。微生物細胞内の生化学反応には各種の酸化還元反応が関与しており、電子の授受移動が行われている。酸化還元電位は反応系での電子の受容性、供与性の難易度を示す尺度の一つであるが、この電位は微生物細胞内で起こっている代謝経路を構成する各種反応（酸化還元反応）の状態や細胞内外との電子授受の状態を反映している。電位差計により直接測定される酸化還元電位は反応溶液と電極との電位であるが反応溶液の電位は細胞膜を介してある電位勾配を持って細胞内で生じている反応と相関している。即ち、酸化還元電位は細胞内外を含む反応系全体の酸化還元反応の総和を反映（各種反応の内容やその頻度等も含めて）したものである。
【００２０】
反応系の酸化還元電位に影響する因子としては、反応系雰囲気ガスの種類と濃度、反応温度、反応溶液ｐＨ、反応液中に存在する目的有機化合物生成のために使用される無機および有機の各種化合物濃度と組成等が考えられる。本発明における反応培地の酸化還元電位とは上記各種影響因子が統合されて示されるものである。従って、本発明は、目的とする有機化合物への代謝経路には各種化学反応が関与し、これら化学反応は上記因子群の影響下にあるが、単一の酸化還元電位なる反応状態を規定する尺度により、効率的に目的有機化合物が生成されることを見出した結果、本発明に到達できたものである。
【００２１】
反応培地には、通常、有機化合物生成の原料となる有機炭素源が含まれている。有機炭素源としては、コリネ型細菌が生化学反応に利用できる物質が挙げられ、なかでもコリネ型細菌が代謝できる物質が好ましく、具体的には糖類や場合によりエタノールなどが挙げられる。特に、本発明で用いる反応培地には、糖類が含有されていることが好ましい。糖類としては、グルコース、ガラクトース、フルクトースもしくはマンノースなどの単糖類、セロビオース、ショ糖もしくはラクトース、マルトースなどの二糖類、またはデキストリンもしくは可溶性澱粉などの多糖類などが挙げられる。なかでも、グルコースが好ましい。
【００２２】
より好ましくは、有機化合物の生成反応に用いられる反応培地組成は、コリネ型細菌またはその処理物がその代謝機能を維持するために必要な成分、即ち、各種糖類等の炭素源、蛋白質合成に必要な窒素源、その他リン、カリウムまたはナトリウム等の塩類、さらに鉄、マンガンまたはカルシウム等の微量金属塩を含む。これらの添加量は所要反応時間、目的有機化合物生産物の種類または用いられるコリネ型細菌の種類等により適宜定めることが出来る。用いるコリネ型細菌によっては特定のビタミン類の添加が好ましい場合もある。また、前記の反応系の炭酸ガス封入法にも関連して、反応培地に二酸化炭素または各種の炭酸塩もしくは炭酸水素塩等の無機炭酸塩を糖類などの有機炭素源に加えて注入することが目的有機化合物によっては有効な場合もある。
【００２３】
好気性コリネ型細菌またはその菌体処理物と糖類との反応は、好気性コリネ型細菌またはその菌体処理物が活動できる温度条件下で行われることが好ましく、好気性コリネ型細菌またはその菌体処理物の種類などにより適宜選択することができる。
【００２４】
最後に、上述のようにして反応培地で生成した有機化合物を採取する。その方法はバイオプロセスで用いられる公知の方法を用いることが出来る。そのような公知の方法として、有機化合物生成液の塩析法、再結晶法、有機溶媒抽出法、エステル化蒸留分離法、クロマトグラフィー分離法または電気透析法等があり、生成有機化合物の特性に応じてその分離精製採取法は適宜定めることが出来る。
【００２５】
本発明で製造することができる有機化合物としては、有機酸、アルコール、アミノ酸またはビタミン類等が挙げられる。有機酸としては、例えば、乳酸、コハク酸、フマル酸、リンゴ酸、オキサロ酢酸、クエン酸、シスアコニット酸、イソクエン酸、２−オキソグルタル酸または酢酸などが挙げられ、なかでも、乳酸またはコハク酸が好ましい。アルコールとしては、例えば、エタノール、ブタノール、１，３−プロパンジオールまたは１，４−ブタンジオールなどが挙げられ、なかでもエタノールが好ましい。アミノ酸としては、例えば、バリン、ロイシン、アラニン、アスパラギン酸、リジン、イソロイシンまたはスレオニンなどが挙げられる。
【００２６】
【実施例】
以下、実施例でもって本発明を説明するが、本発明はこのような実施例に限定されるものではない。
【００２７】
〔実施例１〕
（１）コリネ型細菌Corynebacterium glutamicum R (FERM P-18976)の好気的条件による培養：
（培養基の調製）；尿素 ２ｇ、硫安 ７ｇ、ＫＨ２ＰＯ４ ０．５ｇ、Ｋ２ＨＰＯ４ ０．５ｇ、ＭｇＳＯ４・７Ｈ２Ｏ ０．５ｇ、ＦｅＳＯ４・７Ｈ２Ｏ ６ｍｇ、ＭｎＳＯ４・７Ｈ２Ｏ ４．２ｍｇ、Ｂｉｏｔｉｎ（ビオチン）２００μｇ、塩酸チアミン ２００μｇ、酵母エキス ２ｇ、カザミノ酸 ７ｇ、蒸留水１０００ｍｌからなる培地５００ｍｌを容量１Ｌフラスコに分注し、１２０℃で１０分間加熱滅菌後、室温に冷却した該フラスコを種培養基とした。同じく同組成の培地１０００ｍｌを２Ｌ容ガラス製ジャーファーメンターに入れ、１２０℃、１０分間加熱滅菌し、本培養基とした。
（培養）：上記種培養基１ケに、コリネ型細菌Corynebacterium glutamicum R (FERM P-18976) を無菌条件下にて接種し、３３℃にて１２時間好気的振盪培養を行い、種培養液とした。この種培養液５０ｍｌを上記ジャーファーメンターに接種し、通気量１ｖｖｍ（Volume/Volume/Minute）、温度３３℃で一昼夜、本培養を実施した。好気的培養に起因する影響を除去するため培養液を約３時間窒素ガス雰囲気下で静置した後、培養液２００ｍｌを遠心分離機にかけ（５０００回転、１５分）、上澄み液を除去した。このようにして得られたｗｅｔ菌体を、以下の反応に用いた。
【００２８】
（２）反応用還元状態反応培地溶液の調製：
硫安 ７ｇ、ＫＨ２ＰＯ４ ０．５ｇ、Ｋ２ＨＰＯ４ ０．５ｇ、ＭｇＳＯ４・７Ｈ２Ｏ ０．５ｇ、ＦｅＳＯ４・７Ｈ２Ｏ ６ｍｇ、ＭｎＳＯ４・７Ｈ２Ｏ４．２ｍｇ、Ｂｉｏｔｉｎ（ビオチン） ２００μｇ、塩酸チアミン ２００μｇ、蒸留水１０００ｍｌからなる反応原液を調製し、１２０℃で１０分加熱後、ただちに減圧条件（〜３ｍｍＨｇ）にて２０分間、溶解している酸素の除去を行った。反応原液の還元状態の確認は減圧開始時に反応原液に加えた還元状態指示薬レサズリンの色調変化（青色から無色への変化）にて行った。この反応原液５００ｍｌを容量１Ｌの窒素雰囲気下のガラス製反応容器に導入した。この反応容器はｐＨ調整装置、温度維持装置、容器内反応液攪拌装置および還元電位測定装置を備えている。
【００２９】
（３）反応の実施：
前記培養後調製されたコリネ型細菌菌体を窒素ガス雰囲気下にある反応容器内の反応原液５００ｍｌに加えた。グルコース２００ｍＭを加え、反応温度３３℃に維持し、有機化合物生成反応を行った。反応時の酸化還元電位は初期−２００ｍＶであったが反応開始後直ちに低下し、−４００ｍＶに維持して反応が継続された。３時間反応後、反応培地溶液を液体クロマトグラフィーを用いて分析したところ、乳酸１８６ｍＭ（１６．７ｇ／Ｌ）が生成していた。
【００３０】
〔実施例２〕
実施例１で使用したコリネ型細菌をCorynebacterium glutamicum ATCC13032に、培養温度を３０℃に変えた以外は、実施例１と同様の方法、条件にて有機化合物生成反応を行った。反応時の酸化還元電位は初期−１９０ｍＶであったが反応開始後直ちに低下し、−３９０ｍＶに維持して反応が継続された。３時間反応後、反応培地溶液を液体クロマトグラフィーを用いて分析したところ、乳酸６５ｍＭ（５．９ｇ／Ｌ）が生成していた。
【００３１】
〔実施例３〕
実施例１で使用したコリネ型細菌をCorynebacterium glutamicum ATCC13869に、培養温度を３０℃に変えた以外は、実施例１と同様の方法、条件にて有機化合物生成反応を行った。反応時の酸化還元電位は初期−１９５ｍＶであったが反応開始後直ちに低下し、−３９５ｍＶに維持して反応が継続された。３時間反応後、反応培地溶液を液体クロマトグラフィーを用いて分析したところ、乳酸６７ｍＭ（６．０ｇ／Ｌ）が生成していた。
【００３２】
〔実施例４〕
実施例１と同様にして得られた菌体および反応条件により、反応中に炭酸ナトリウム２００ｍＭを添加すること以外は実施例１と同様の反応を行い、得られた反応液を分析した。反応時の酸化還元電位は初期−２０５ｍＶであったが反応開始後直ちに低下し、−４０５ｍＶに維持して反応が継続された。３時間反応後、反応培地溶液を液体クロマトグラフィーを用いて分析したところ、乳酸２００ｍＭ（１８．０ｇ／Ｌ）、コハク酸８１ｍＭ（９．６ｇ／Ｌ）が生成していた。
【００３３】
〔実施例５〕
実施例１と同様にして得られた菌体および反応条件により、反応原液に炭酸ガスを１ｖｖｍ（Volume/Volume/Minute）で通気すること以外は実施例１と同様の反応を行い、得られた反応液を分析した。反応時の酸化還元電位は初期−２１０ｍＶであったが反応開始後直ちに低下し、−４１０ｍＶに維持して反応が継続された。３時間反応後、反応培地溶液を液体クロマトグラフィーを用いて分析したところ、乳酸２０２ｍＭ（１８．２ｇ／Ｌ）、コハク酸８５ｍＭ（１０ｇ／Ｌ）が生成していた。
【００３４】
〔比較例１〕
実施例１と同様の方法、条件にて反応を実施する際に、コリネ型細菌の培養後の静置時間を１５分間とし、減圧処理を施していない反応原液を使用し、そして反応時における還元状態を極微量の空気を導入することにより、酸化還元電位−１８０ｍＶに制御して、有機化合物生成反応を実施した。なお、このときの反応液溶存酸素濃度は０．０１ｐｐｍであった。溶存酸素濃度は、酸素膜電極電位と酸化還元電位との補正相関データより外挿して求めた。
得られた反応液を液体クロマトグラフィーを用いて分析したところ、乳酸２９ｍＭ（２．６ｇ／Ｌ）、コハク酸２ｍＭ（０．２４ｇ／Ｌ）が生成していた。
【００３５】
〔実施例６〕
実施例１で使用したコリネ型細菌をエタノール生産組換えコリネ型細菌（FERMP-17887）に、培養温度を３０℃に変えた以外は、実施例１と同様の方法、条件にて有機化合物生成反応を行った。反応時の酸化還元電位は初期−１９５ｍＶであったが反応開始後直ちに低下し、−３９５ｍＶに維持して反応が継続された３時間反応後、反応培地溶液を液体クロマトグラフィーを用いて分析したところ、エタノールが３．０（ｇエタノール／ｌ）の濃度で生成していた。
【００３６】
〔比較例２〕
実施例６と同様の方法、条件にて反応を実施する際に、コリネ型細菌の培養後の静置時間を１５分間とし、減圧処理を施していない反応原液を使用し、そして反応時における還元状態極微量の空気を導入することにより、酸化還元電位−１８０ｍＶに制御して、有機化合物生成反応を実施した。なお、このときの反応液溶存酸素濃度は０．０１ｐｐｍであった。溶存酸素濃度は、酸素膜電極電位と酸化還元電位との補正相関データより外挿して求めた。
得られた反応液を液体クロマトグラフィーを用いて分析したところ、エタノールが１．６（ｇエタノール／ｌ）の濃度で生成していた。
【００３７】
【発明の効果】
本発明によれば、好気性コリネ型細菌またはその処理物と糖類とを還元状態下で反応させることにより、コリネ型細菌の増殖分裂が抑制され代謝反応が主として行われることになるから、糖類からの目的有機化合物への変換率が画期的に向上する。また増殖に伴う分泌副生物の実質的な抑制を実現することができ、純度の高い目的有機化合物が得られる。その結果、分泌副生物と目的有機化合物との分離工程が事実上必要なくなり、工業的生産における工程管理が行いやすく、また安価な製品を提供することができるようになる。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for producing an organic compound using a coryneform bacterium. More specifically, the present invention relates to a highly efficient method for producing an organic compound, which comprises using a coryneform bacterium to produce an organic compound of an organic acid, an alcohol, an amino acid and a vitamin under a specific reaction state, and then collecting the organic compound. It is.
[0002]
[Prior art]
Conventionally, aerobic coryneform bacteria have been widely used for producing useful substances such as amino acids under aerobic conditions such as aeration and stirring culture methods or shaking culture methods (Patent Document 1). In addition, under the anaerobic conditions (including the presence of trace amounts of oxygen), aerobic coryneform bacteria or processed products thereof are allowed to act on organic raw materials in a reaction solution containing carbonate ions or carbon dioxide gas to produce oxygen-containing compounds. Is also used (Patent Document 2).
[0003]
In the prior art method using the coryneform bacterium under aerobic or micro-oxygen conditions, that is, under oxidizing conditions, the coryneform bacterium naturally undergoes division growth depending on the oxygen abundance. The time required for the division varies greatly, for example, when the division rate is as fast as about 2 hours or less, or when about 10 hours or more is required.
In any case, by dividing and multiplying, the nutrient source given to the coryneform bacterium is consumed for growth, and the production amount of the target product decreases, that is, the conversion rate from the nutrient source to the target product decreases. An important issue in industrial production has been pointed out. Furthermore, in the growth process, metabolites resulting therefrom are produced as secretions, so that it is necessary to separate secretory by-products other than these target products and target products from the viewpoint of product quality purity, In particular, the process of separating and purifying trace amounts of various secretory by-products is a major factor in deteriorating the economic efficiency of industrial production technology.
[0004]
[Patent Document 1]
Japanese Patent Application Laid-Open No. 05-015377 (Claim 3)
[Patent Document 2]
JP-A-11-113588 (Claim 1)
[0005]
[Problems to be solved by the invention]
An object of the present invention is to solve the above technical problems relating to a technique for producing a substance accompanied by growth by aerobic coryneform bacteria. That is, the present invention provides a method for producing an organic compound using an aerobic coryneform bacterium or a processed product thereof, in which the conversion rate from an organic carbon source such as sugar to the target organic compound, and the purity of the target organic compound obtained are improved. The aim is to provide a method by which this can be done.
[0006]
[Means for Solving the Problems]
Coryneform bacteria have been conventionally used for producing organic compounds under aerobic or anaerobic conditions (including the presence of trace amounts of oxygen). However, the present inventor has used a coryneform bacterium under a reducing condition that has not been known so far, more specifically, a reaction medium under reduced conditions of an aerobic coryneform bacterium or a processed product thereof and saccharides. By reacting in, the problems of the prior art, such as a low conversion rate from glucose and other saccharides to the target product, are overcome, and the production of organic compounds can be performed more efficiently. The inventors have found that the present invention has been achieved.
[0007]
That is, the present invention
(1) An aerobic coryneform bacterium is grown and cultured under aerobic conditions, and the recovered bacterial cells or the processed cells thereof are reacted with saccharides in a reaction medium under reducing conditions to remove organic compounds produced in the reaction medium. A method for producing an organic compound, characterized by collecting
(2) The method for producing an organic compound according to the above (1), wherein the oxidation-reduction potential of the reaction medium under a reduced state is from -200 mV to -500 mV.
(3) The method for producing an organic compound according to the above (1) or (2), wherein the reaction medium is produced during the growth and culture process and does not substantially contain a product substance existing inside and outside the cells.
(4) The method for producing an organic compound according to (1) to (3), wherein the organic compound is selected from organic acids, alcohols, amino acids, and vitamins.
[0008]
BEST MODE FOR CARRYING OUT THE INVENTION
The aerobic coryneform bacterium used in the present invention is a group of microorganisms defined in the Bargeys Manual of Determinative Bacteriology (8, 599, 1974), and is a common microorganism. There is no particular limitation as long as it grows under aerial conditions and produces the desired organic compound under the reduced state of the present invention.
Specific examples include Corynebacterium, Brevibacterium, Arthrobacter, Mycobacterium and Micrococcus.
[0009]
More specifically, the genus Corynebacterium includes Corynebacterium glutamicum (Corynebacterium glutamicum) FERM P-18976, ATCC13032, ATCC13058, ATCC13059, ATCC13060, ATCC13232, ATCC13286, ATCC13287, ATCC13655, ATCC13745, ATCC13746, ATCC13761, ATCC14020 or ATCC31831.
Brevibacterium species include Brevibacterium lactofermentum (Brevibacterium lactofermentum) ATCC 13869, Brevibacterium flavum (Brevibacterium flavum) MJ-233 (FERM BP-1497) or MJ-233AB-41 (FERM BP-1498), or Brevibacterium ammoniagenes (Brevibacterium ammoniagenes) ATCC6872 and the like.
As the earth bacterium, there are earth bacterium gloviformis (Arthrobacter globiformis) ATCC8010, ATCC4336, ATCC21056, ATCC31250, ATCC31738 or ATCC35698.
Micrococcus spp. Include Micrococcus Freudenreich (Micrococcus freudenreichii) No. 239 (FERM P-13221), Micrococcus luteus (Micrococcus luteus) No. 240 (FERM P-13222), Micrococcus ureae (Micrococcus ureae) IAM1010 or Micrococcus Roseus (Micrococcus roseus) IFO3764 and the like.
The aerobic coryneform bacteria used in the present invention include:Corynebacterium glutamicum R (FERM P-18976),Corynebacterium glutamicum ATCC13032 orCorynebacterium glutamicum ATCC13869 is preferred.
[0010]
The aerobic coryneform bacterium used in the present invention may be a mutant strain of a wild strain existing in nature (for example, FERM P-18977, FERM P-18978), or a biotechnology such as genetic recombination. (For example, FERM P-17887, FERM P-17888, FERM P-18979, etc.).
[0011]
In the method for producing an organic compound according to the present invention, first, the aerobic coryneform bacterium described above is grown and cultured under aerobic conditions.
The cultivation of the aerobic coryneform bacterium can be performed using an ordinary nutrient medium containing a carbon source, a nitrogen source, an inorganic salt and the like. For the culture, glucose or molasses or the like can be used as a carbon source, and ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate or urea can be used alone or in combination as a nitrogen source. In addition, as the inorganic salt, for example, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, or the like can be used. In addition, if necessary, nutrients such as peptone, meat extract, yeast extract, corn steep liquor, casamino acid, or various vitamins such as biotin or thiamine can be appropriately added to the medium.
[0012]
The cultivation can be carried out usually at a temperature of about 20 ° C to about 40 ° C, preferably about 25 ° C to about 35 ° C under aerobic conditions such as aeration and stirring or shaking. The pH during culturing is preferably in the range of about 5 to 10, preferably about 7 to 8, and the pH during culturing can be adjusted by adding an acid or an alkali. The carbon source concentration at the start of the culture is about 1 to 20% (W / V), preferably about 2 to 5% (W / V). The culture period is usually about 1 to 7 days.
[0013]
Next, the cultured cells of the aerobic coryneform bacteria are collected. The method for collecting and separating cultured cells from the culture obtained as described above is not particularly limited, and for example, a known method such as centrifugation or membrane separation can be used.
The recovered cultured cells may be treated, and the resulting treated cells may be used in the next step. The treated cells may be those obtained by subjecting cultured cells to some treatment, and include, for example, immobilized cells obtained by immobilizing cells with acrylamide, carrageenan, or the like.
[0014]
Next, the cultured cells of the aerobic coryneform bacterium recovered or separated from the culture obtained as described above or the treated cells thereof are subjected to a production reaction of the target organic compound in the reaction medium under a reduced state. . As a method for producing an organic compound, either a batch method or a continuous method can be used.
In the biochemical reaction under the reducing state of the present invention, the growth and division of coryneform bacteria are completely suppressed, and the conversion rate of glucose and other saccharide nutrients to the target organic compound, which is the object of the present invention, is a breakthrough. And substantially complete suppression of secretory by-products accompanying growth. From this point of view, when the coryneform bacterium or the treated product of the cultured coryneform bacterium is provided to the reaction medium, a method and conditions that do not bring the environmental conditions inside and outside the coryneform bacterium during the culture to the reaction medium should be used. Is recommended. That is, it is preferable that the reaction medium is produced substantially during the growth culture process and does not substantially contain a product substance existing inside and outside the cells. More specifically, secretion by-products generated during the growth culture process and released outside the cells, and substances generated by the aerobic metabolic function in the cultured cells and remaining in the cells, are substantially contained in the reaction medium. It is recommended that it not be present. Such a state is realized, for example, by a method such as centrifugation and membrane separation of a culture solution after growth and culture, and / or by leaving the cells after culture for about 2 to 10 hours under a reduced state.
[0015]
In this step, a reaction medium under a reduced state is used. The reaction medium may have any shape such as solid, semi-solid or liquid as long as it is under a reducing condition. An essential requirement of the present invention is to produce a target organic compound by causing a biochemical reaction by a metabolic function of a coryneform bacterium under a reducing condition.
The reduced state in the present invention is defined by the oxidation-reduction potential of the reaction system, and the oxidation-reduction potential of the reaction medium is preferably about -200 mV to -500 mV, more preferably about -250 mV to -500 mV. The reduction state of the reaction medium can be easily estimated to some extent with a resazurin indicator (if it is in a reduced state, decolorization from blue to colorless), but more precisely, an oxidation-reduction potentiometer (for example, ORP Electrodes manufactured by BROADLEY JAMES) is used. Used. In the present invention, it is preferable that the reduced state is maintained from immediately after the addition of the cells or the processed product thereof to the reaction medium until the organic compound is collected, but the reaction medium is reduced at least when the organic compound is collected. Any condition is acceptable. It is desirable that the reaction medium is kept in a reduced state for about 50% or more of the reaction time, more preferably about 70% or more, and even more preferably about 90% or more. Among them, the oxidation-reduction potential of the reaction medium is maintained at about -200 mV to -500 mV for about 50% or more, more preferably about 70% or more, and still more preferably about 90% or more of the reaction time. More desirable.
[0016]
Specifically, such a reduced state is achieved by a method for preparing cultured cells after the above-mentioned culture, a method for adjusting the reaction medium, or a method for maintaining the reduced state during the reaction.
A known method may be used for adjusting the reaction medium under the reducing condition. For example, a method for preparing an aqueous solution for a reaction medium is, for example, a method for preparing a culture solution for anaerobic microorganisms such as sulfate-reducing microorganisms (Pfennig, N et. Al. (1981):
The dissimilatory sulfate-reducing bacteria, In The Prokaryotes, A Handbook on Habitats, Isolation and Identification of Bacteria, Ed. By Starr, MP et.al. p.926-940, Berlin, Springer Verlag. The third volume, Kyoto University Faculty of Agriculture, Department of Agricultural Chemistry, edited by Agricultural Chemistry, 1990, 26th edition, published by Sangyo Tosho Co., Ltd.) can be used to obtain an aqueous solution in a desired reduced state.
[0017]
As a method of adjusting the aqueous solution for the reaction medium, more specifically, a method of removing the dissolved gas by subjecting the aqueous solution for the reaction medium to a heat treatment or a reduced pressure treatment, or the like can be mentioned. More specifically, treating the aqueous solution for the reaction medium for about 1 to 60 minutes, preferably about 5 to 40 minutes, under a reduced pressure of about 10 mmHg or less, preferably about 5 mmHg or less, more preferably about 3 mmHg or less. Thereby, dissolved gas, especially dissolved oxygen, can be removed, and an aqueous solution for reaction medium under reducing conditions can be prepared. An aqueous solution for a reaction medium in a reduced state can also be prepared by adding an appropriate reducing agent (for example, thioglycolic acid, ascorbic acid, cysteine hydrochloride, mercaptoacetic acid, thiolacetic acid, glutathione, sodium sulfide, and the like). In some cases, an appropriate combination of these methods is also an effective method for preparing an aqueous solution for a reaction medium in a reduced state.
[0018]
As a method of maintaining the reduced state during the reaction, it is desirable to prevent mixing of oxygen from outside the reaction system as much as possible, and a method of sealing the reaction system with an inert gas such as nitrogen gas or carbon dioxide gas is usually used. Can be As a method for more effectively preventing oxygen contamination, in order to efficiently function the metabolic function of the coryneform bacterium in the course of the reaction, the addition of a pH maintaining and adjusting solution for the reaction system and the addition of various nutrient dissolving solutions are appropriately performed. In some cases, it is effective to remove oxygen from the additive solution in advance.
[0019]
In the organic compound production reaction of the present invention, it is not clear why the definition of the oxidation-reduction potential of the production reaction system is effective for the efficient production of the target organic compound, but the reason for the estimation is described below. . However, the present invention is not limited to the presumed reasons.
The organic compound as the target product of the present invention is a compound produced by a biochemical reaction based on the metabolic function of coryneform bacteria. Various redox reactions are involved in biochemical reactions in microbial cells, and electron transfer is performed. The oxidation-reduction potential is one of the measures of the difficulty of accepting and donating electrons in the reaction system, and this potential is used for various reactions that constitute metabolic pathways occurring in microbial cells (redox reaction). And the state of electron transfer between the inside and outside of the cell. The oxidation-reduction potential directly measured by a potentiometer is the potential between the reaction solution and the electrode, but the potential of the reaction solution correlates with the reaction occurring inside the cell with a certain potential gradient through the cell membrane. That is, the oxidation-reduction potential reflects the total of the oxidation-reduction reactions of the whole reaction system including inside and outside of the cell (including the contents of various reactions and the frequency thereof).
[0020]
Factors affecting the oxidation-reduction potential of the reaction system include the type and concentration of the reaction system atmosphere gas, the reaction temperature, the pH of the reaction solution, and various types of inorganic and organic substances used to produce the target organic compound present in the reaction solution. Compound concentration and composition can be considered. The oxidation-reduction potential of the reaction medium in the present invention is a value obtained by integrating the above various influencing factors. Therefore, in the present invention, various chemical reactions are involved in the metabolic pathway to the target organic compound, and these chemical reactions are under the influence of the above-described factors, but define a single redox potential reaction state. As a result of finding that the target organic compound is efficiently produced by the scale, the present invention has been achieved.
[0021]
The reaction medium usually contains an organic carbon source as a raw material for producing an organic compound. Examples of the organic carbon source include substances that coryneform bacteria can utilize for biochemical reactions. Among them, substances capable of metabolizing coryneform bacteria are preferable, and specific examples include sugars and, in some cases, ethanol. In particular, the reaction medium used in the present invention preferably contains saccharides. The saccharides include monosaccharides such as glucose, galactose, fructose and mannose, disaccharides such as cellobiose, sucrose or lactose, maltose, and polysaccharides such as dextrin or soluble starch. Of these, glucose is preferred.
[0022]
More preferably, the reaction medium composition used for the production reaction of the organic compound is a component necessary for the coryneform bacterium or a processed product thereof to maintain its metabolic function, that is, a carbon source such as various sugars and a protein necessary for protein synthesis. And various salts such as phosphorus, potassium or sodium, and trace metal salts such as iron, manganese or calcium. The amount of these additives can be appropriately determined depending on the required reaction time, the type of the target organic compound product, the type of coryneform bacterium used, and the like. Depending on the coryneform bacterium used, it may be preferable to add specific vitamins. In addition, in connection with the carbon dioxide gas encapsulation method of the above-mentioned reaction system, it is possible to inject carbon dioxide or an inorganic carbonate such as various carbonates or bicarbonates into an organic carbon source such as a saccharide into a reaction medium. It may be effective depending on the target organic compound.
[0023]
The reaction between the aerobic coryneform bacterium or the processed product thereof and the saccharide is preferably performed under a temperature condition at which the aerobic coryneform bacterium or the processed product thereof can be activated, and the aerobic coryneform bacterium or the bacterium thereof is preferably used. It can be appropriately selected depending on the type of the body treatment.
[0024]
Finally, the organic compounds generated in the reaction medium as described above are collected. As the method, a known method used in a bioprocess can be used. As such known methods, there are a salting-out method, a recrystallization method, an organic solvent extraction method, an esterification distillation separation method, a chromatographic separation method, an electrodialysis method, and the like of an organic compound production liquid, and the properties of the produced organic compound are determined. Accordingly, the method of separation and purification can be determined as appropriate.
[0025]
Examples of the organic compound that can be produced in the present invention include organic acids, alcohols, amino acids, and vitamins. Examples of the organic acid include lactic acid, succinic acid, fumaric acid, malic acid, oxaloacetic acid, citric acid, cisaconitic acid, isocitric acid, 2-oxoglutaric acid or acetic acid, among which lactic acid or succinic acid is preferred. preferable. Examples of the alcohol include ethanol, butanol, 1,3-propanediol, and 1,4-butanediol, and among them, ethanol is preferable. Examples of the amino acid include valine, leucine, alanine, aspartic acid, lysine, isoleucine, threonine and the like.
[0026]
【Example】
Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to such examples.
[0027]
[Example 1]
(1) Coryneform bacteriaCorynebacterium glutamicum Culture of R (FERM P-18976) under aerobic conditions:
(Preparation of culture medium); 2 g of urea, 7 g of ammonium sulfate, KH2PO4 0.5g, K2HPO4 0.5g, MgSO4・ 7H2O 0.5 g, FeSO4・ 7H2O 6mg, MnSO4・ 7H2O 4.2 mg, Biotin (biotin) 200 µg, thiamine hydrochloride 200 µg, yeast extract 2 g, casamino acid 7 g, distilled water 500 ml of medium 500 ml is dispensed into a 1 L flask, and heat sterilized at 120 ° C for 10 minutes, and then room temperature. The cooled flask was used as a seed culture medium. Similarly, 1000 ml of a medium having the same composition was placed in a 2 L glass jar fermenter and sterilized by heating at 120 ° C. for 10 minutes to obtain a main culture medium.
(Culture): Coryneform bacteria per seed cultureCorynebacterium glutamicum R (FERM P-18976) was inoculated under aseptic conditions and subjected to aerobic shaking culture at 33 ° C. for 12 hours to obtain a seed culture. 50 ml of this seed culture was inoculated into the above jar fermenter, and main culture was carried out at 33 ° C. all day and night at an aeration rate of 1 vvm (Volume / Volume / Minute). After removing the influence of the aerobic culture from the culture solution for about 3 hours in a nitrogen gas atmosphere, 200 ml of the culture solution was centrifuged (5000 rpm, 15 minutes) to remove the supernatant. The wet cells thus obtained were used in the following reaction.
[0028]
(2) Preparation of reduced state reaction medium solution for reaction:
Ammonium sulfate 7g, KH2PO4 0.5g, K2HPO4 0.5g, MgSO4・ 7H2O 0.5 g, FeSO4・ 7H2O 6mg, MnSO4・ 7H2A reaction stock solution consisting of 4.2 mg of O, 200 μg of Biotin (biotin), 200 μg of thiamine hydrochloride and 1000 ml of distilled water was prepared, heated at 120 ° C. for 10 minutes, and immediately dissolved under reduced pressure (減 圧 3 mmHg) for 20 minutes. Was removed. The reduction state of the reaction stock solution was confirmed by the color change (change from blue to colorless) of the resazurin, a reduction state indicator, added to the reaction stock solution at the start of decompression. 500 ml of this reaction stock solution was introduced into a glass reactor under a nitrogen atmosphere having a volume of 1 L. This reaction vessel is provided with a pH adjusting device, a temperature maintaining device, a reaction solution stirring device in the container, and a reduction potential measuring device.
[0029]
(3) Performing the reaction:
The coryneform bacterium prepared after the culture was added to 500 ml of the reaction stock solution in a reaction vessel under a nitrogen gas atmosphere. 200 mM of glucose was added, and the reaction temperature was maintained at 33 ° C. to perform an organic compound generation reaction. The oxidation-reduction potential during the reaction was -200 mV at the initial stage, but dropped immediately after the start of the reaction, and was maintained at -400 mV to continue the reaction. After the reaction for 3 hours, the reaction medium solution was analyzed using liquid chromatography, and it was found that 186 mM lactic acid (16.7 g / L) was produced.
[0030]
[Example 2]
Coryneform bacteria used in Example 1Corynebacterium glutamicum An organic compound production reaction was performed on ATCC 13032 in the same manner and under the same conditions as in Example 1 except that the culture temperature was changed to 30 ° C. The oxidation-reduction potential during the reaction was -190 mV in the initial stage, but immediately decreased after the start of the reaction, and was maintained at -390 mV to continue the reaction. After the reaction for 3 hours, the reaction medium solution was analyzed using liquid chromatography, and it was found that 65 mM lactic acid (5.9 g / L) was produced.
[0031]
[Example 3]
Coryneform bacteria used in Example 1Corynebacterium glutamicum An organic compound production reaction was performed on ATCC 13869 under the same method and conditions as in Example 1 except that the culture temperature was changed to 30 ° C. The oxidation-reduction potential during the reaction was -195 mV at the initial stage, but immediately decreased after the start of the reaction, and the reaction was continued at -395 mV. After the reaction for 3 hours, the reaction medium solution was analyzed using liquid chromatography, and it was found that 67 mM of lactic acid (6.0 g / L) had been produced.
[0032]
[Example 4]
According to the cells and reaction conditions obtained in the same manner as in Example 1, the same reaction as in Example 1 was performed except that 200 mM sodium carbonate was added during the reaction, and the obtained reaction solution was analyzed. The oxidation-reduction potential during the reaction was -205 mV at the initial stage, but immediately decreased after the start of the reaction, and the reaction was continued at -405 mV. After the reaction for 3 hours, the reaction medium solution was analyzed using liquid chromatography. As a result, 200 mM of lactic acid (18.0 g / L) and 81 mM of succinic acid (9.6 g / L) were produced.
[0033]
[Example 5]
The reaction was carried out in the same manner as in Example 1, except that the reaction stock solution was aerated with 1 vvm (Volume / Volume / Minute) of the bacterial cells and reaction conditions obtained in the same manner as in Example 1. The reaction was analyzed. The oxidation-reduction potential during the reaction was -210 mV at the initial stage, but immediately decreased after the start of the reaction, and the reaction was continued at -410 mV. After the reaction for 3 hours, the reaction medium solution was analyzed using liquid chromatography. As a result, it was found that 202 mM (18.2 g / L) of lactic acid and 85 mM (10 g / L) of succinic acid were produced.
[0034]
[Comparative Example 1]
When carrying out the reaction under the same method and conditions as in Example 1, the standing time after culturing the coryneform bacterium was set to 15 minutes, a reaction stock solution not subjected to reduced pressure treatment was used, and reduction during the reaction was performed. The state was controlled to an oxidation-reduction potential of −180 mV by introducing a very small amount of air to carry out an organic compound generation reaction. At this time, the concentration of dissolved oxygen in the reaction solution was 0.01 ppm. The dissolved oxygen concentration was determined by extrapolating from the corrected correlation data between the oxygen membrane electrode potential and the oxidation-reduction potential.
When the obtained reaction solution was analyzed using liquid chromatography, 29 mM of lactic acid (2.6 g / L) and 2 mM of succinic acid (0.24 g / L) were generated.
[0035]
[Example 6]
The coryneform bacterium used in Example 1 was replaced with ethanol-producing recombinant coryneform bacterium (FERMP-17887) by the same method and under the same conditions as in Example 1 except that the culture temperature was changed to 30 ° C. Was done. The oxidation-reduction potential during the reaction was -195 mV in the initial stage, but dropped immediately after the start of the reaction. After the reaction was continued for 3 hours while maintaining the reaction at -395 mV, the reaction medium solution was analyzed using liquid chromatography. , Ethanol was produced at a concentration of 3.0 (g ethanol / l).
[0036]
[Comparative Example 2]
When carrying out the reaction under the same method and conditions as in Example 6, the standing time after culturing the coryneform bacterium was 15 minutes, a reaction stock solution not subjected to a reduced pressure treatment was used, and reduction during the reaction was performed. The state was controlled to an oxidation-reduction potential of -180 mV by introducing a very small amount of air to carry out an organic compound generation reaction. At this time, the concentration of dissolved oxygen in the reaction solution was 0.01 ppm. The dissolved oxygen concentration was determined by extrapolating from the corrected correlation data between the oxygen membrane electrode potential and the oxidation-reduction potential.
When the obtained reaction solution was analyzed using liquid chromatography, ethanol was produced at a concentration of 1.6 (g ethanol / l).
[0037]
【The invention's effect】
According to the present invention, by reacting the aerobic coryneform bacterium or a processed product thereof with a saccharide under a reducing state, the growth and division of the coryneform bacterium is suppressed and the metabolic reaction is mainly performed, so Conversion rate to the target organic compound is remarkably improved. In addition, it is possible to substantially suppress secretion by-products accompanying the growth, and to obtain a target organic compound having high purity. As a result, a step of separating the secretory by-product and the target organic compound is practically unnecessary, and the process control in industrial production can be easily performed, and an inexpensive product can be provided.
Claims (4)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2002367331A JP3869788B2 (en) | 2002-12-18 | 2002-12-18 | Process for producing organic compounds using coryneform bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2002367331A JP3869788B2 (en) | 2002-12-18 | 2002-12-18 | Process for producing organic compounds using coryneform bacteria |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2004194570A true JP2004194570A (en) | 2004-07-15 |
| JP3869788B2 JP3869788B2 (en) | 2007-01-17 |
Family
ID=32764266
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2002367331A Expired - Fee Related JP3869788B2 (en) | 2002-12-18 | 2002-12-18 | Process for producing organic compounds using coryneform bacteria |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3869788B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011111693A1 (en) | 2010-03-09 | 2011-09-15 | 三菱化学株式会社 | Succinic acid manufacturing method |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8216820B2 (en) | 2008-04-25 | 2012-07-10 | Research Institute Of Innovative Technology For The Earth | Transformant of coryneform bacteria capable of producing isopropanol |
| EP2415860A4 (en) | 2009-03-30 | 2012-09-05 | Res Inst Innovative Tech Earth | Coryneform bacterium transformant, and process for producing isobutanol using same |
-
2002
- 2002-12-18 JP JP2002367331A patent/JP3869788B2/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011111693A1 (en) | 2010-03-09 | 2011-09-15 | 三菱化学株式会社 | Succinic acid manufacturing method |
| CN102812127A (en) * | 2010-03-09 | 2012-12-05 | 三菱化学株式会社 | Method of producing succinic acid |
| US8647843B2 (en) | 2010-03-09 | 2014-02-11 | Mitsubishi Chemical Corporation | Method of producing succinic acid |
| CN102812127B (en) * | 2010-03-09 | 2014-12-03 | 三菱化学株式会社 | Method of producing succinic acid |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3869788B2 (en) | 2007-01-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7156594B2 (en) | Preparation of (R)-3-hydroxybutyric acid or its salts by one-step fermentation | |
| JP4275666B2 (en) | Highly efficient hydrogen production method using microorganisms | |
| JP4197754B2 (en) | Method for producing lactic acid or succinic acid | |
| JP5333213B2 (en) | Method for producing succinic acid and ammonium succinate solution | |
| Sugimoto et al. | Control of acetic acid concentration by pH‐stat continuous substrate feeding in heterotrophic culture phase of two‐stage cultivation of Alcaligenes eutrophus for production of P (3HB) from CO2, H2, and O2 under non‐explosive conditions | |
| JPH0357756B2 (en) | ||
| KR910002857B1 (en) | Process for the biotechnological preparation of poly -d- (-) -3- hydroxybutyric acid | |
| JP4745753B2 (en) | Method for producing amino acids under reducing conditions using coryneform bacteria | |
| EP1616018A1 (en) | METHOD OF PRODUCING 1,2-PROPANEDIOL USING i KLEBSIELLA PNEUMONIAE /i | |
| JP3959403B2 (en) | Purification method of organic acid | |
| JPH0346118B2 (en) | ||
| JP3869788B2 (en) | Process for producing organic compounds using coryneform bacteria | |
| JP4742610B2 (en) | Process for producing fumaric acid | |
| JP4647391B2 (en) | Highly efficient production method of dicarboxylic acid by coryneform bacteria | |
| CA2021869C (en) | Process for microbiological batch production of l-carnitine | |
| JP4537862B2 (en) | Highly efficient production method of organic acid by aerobic bacteria | |
| JP4198147B2 (en) | Method for producing organic acid | |
| WO2006001054A1 (en) | Method of producing organic compound with the use of coryneform bacterium | |
| JPS58201992A (en) | Preparation of beta-substituted propionic acid or amide thereof by microorganism | |
| JPS62111693A (en) | Production of l-amino acid from alpha-ketocarboxilic acid byfermentation | |
| JP2003159091A (en) | Method for producing lactic acid | |
| JPH0523748B2 (en) | ||
| JP2721975B2 (en) | Method for producing L-lysine | |
| KR830001260B1 (en) | Method for preparing L-glutamine by fermentation | |
| JP3163362B2 (en) | Method for producing L-proline |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20060327 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20060427 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20060710 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20060727 |
|
| A911 | Transfer to examiner for re-examination before appeal (zenchi) |
Free format text: JAPANESE INTERMEDIATE CODE: A911 Effective date: 20060829 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20061010 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20061013 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 3869788 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| S531 | Written request for registration of change of domicile |
Free format text: JAPANESE INTERMEDIATE CODE: R313532 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20091020 Year of fee payment: 3 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20111020 Year of fee payment: 5 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20111020 Year of fee payment: 5 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20131020 Year of fee payment: 7 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20131020 Year of fee payment: 7 |
|
| S533 | Written request for registration of change of name |
Free format text: JAPANESE INTERMEDIATE CODE: R313533 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20131020 Year of fee payment: 7 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| LAPS | Cancellation because of no payment of annual fees |