JPH0362396B2 - - Google Patents
Info
- Publication number
- JPH0362396B2 JPH0362396B2 JP24137984A JP24137984A JPH0362396B2 JP H0362396 B2 JPH0362396 B2 JP H0362396B2 JP 24137984 A JP24137984 A JP 24137984A JP 24137984 A JP24137984 A JP 24137984A JP H0362396 B2 JPH0362396 B2 JP H0362396B2
- Authority
- JP
- Japan
- Prior art keywords
- ornithine
- medium
- cultured
- culture
- producing
- 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
Links
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 claims description 35
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 claims description 18
- 229960003104 ornithine Drugs 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 14
- DMSVGDIWEWBOGO-ZCFIWIBFSA-N (2s)-2-amino-2-(1,3-thiazol-2-yl)propanoic acid Chemical compound OC(=O)[C@@](N)(C)C1=NC=CS1 DMSVGDIWEWBOGO-ZCFIWIBFSA-N 0.000 claims description 6
- 241000186216 Corynebacterium Species 0.000 claims description 6
- BRBKOPJOKNSWSG-UHFFFAOYSA-N sulfaguanidine Chemical compound NC(=N)NS(=O)(=O)C1=CC=C(N)C=C1 BRBKOPJOKNSWSG-UHFFFAOYSA-N 0.000 claims description 6
- 229960004257 sulfaguanidine Drugs 0.000 claims description 6
- 244000005700 microbiome Species 0.000 claims description 4
- 238000000855 fermentation Methods 0.000 claims description 3
- 230000004151 fermentation Effects 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 claims description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- RHGKLRLOHDJJDR-BYPYZUCNSA-N L-citrulline Chemical compound NC(=O)NCCC[C@H]([NH3+])C([O-])=O RHGKLRLOHDJJDR-BYPYZUCNSA-N 0.000 description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000004202 carbamide Substances 0.000 description 4
- 229960002173 citrulline Drugs 0.000 description 4
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 3
- 239000004475 Arginine Substances 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 240000008042 Zea mays Species 0.000 description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 3
- 235000002017 Zea mays subsp mays 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
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 3
- 235000009697 arginine Nutrition 0.000 description 3
- 235000005822 corn Nutrition 0.000 description 3
- 239000002054 inoculum 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
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 3
- 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 2
- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 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 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 2
- 229930064664 L-arginine Natural products 0.000 description 2
- 235000014852 L-arginine Nutrition 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- RHGKLRLOHDJJDR-UHFFFAOYSA-N Ndelta-carbamoyl-DL-ornithine Natural products OC(=O)C(N)CCCNC(N)=O RHGKLRLOHDJJDR-UHFFFAOYSA-N 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- ZFOZVQLOBQUTQQ-UHFFFAOYSA-N Tributyl citrate Chemical compound CCCCOC(=O)CC(O)(C(=O)OCCCC)CC(=O)OCCCC ZFOZVQLOBQUTQQ-UHFFFAOYSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 235000013477 citrulline Nutrition 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 235000013379 molasses Nutrition 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- GGTYBZJRPHEQDG-WCCKRBBISA-N (2s)-2,5-diaminopentanoic acid hydrochloride Chemical compound Cl.NCCC[C@H](N)C(O)=O GGTYBZJRPHEQDG-WCCKRBBISA-N 0.000 description 1
- 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 1
- FSRFQOAAESLDSG-UHFFFAOYSA-N 1-nitro-2-nitrosoguanidine Chemical compound [O-][N+](=O)NC(=N)NN=O FSRFQOAAESLDSG-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
- CXABZTLXNODUTD-UHFFFAOYSA-N 3-fluoropyruvic acid Chemical compound OC(=O)C(=O)CF CXABZTLXNODUTD-UHFFFAOYSA-N 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- 241000186146 Brevibacterium Species 0.000 description 1
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 1
- 241000186226 Corynebacterium glutamicum Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 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
- 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
- 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
- 238000005273 aeration Methods 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 235000012501 ammonium carbonate Nutrition 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 239000000908 ammonium hydroxide Substances 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- 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 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
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 238000003163 cell fusion method Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229920001429 chelating resin Polymers 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- -1 etc. Substances 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 229960001781 ferrous sulfate Drugs 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 235000015203 fruit juice Nutrition 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- GVALZJMUIHGIMD-UHFFFAOYSA-H magnesium phosphate Chemical compound [Mg+2].[Mg+2].[Mg+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O GVALZJMUIHGIMD-UHFFFAOYSA-H 0.000 description 1
- 239000004137 magnesium phosphate Substances 0.000 description 1
- 229960002261 magnesium phosphate Drugs 0.000 description 1
- 229910000157 magnesium phosphate Inorganic materials 0.000 description 1
- 235000010994 magnesium phosphates Nutrition 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 229940055726 pantothenic acid Drugs 0.000 description 1
- 235000019161 pantothenic acid Nutrition 0.000 description 1
- 239000011713 pantothenic acid Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
産業上の利用分野
本発明は発酵法によるL−オルニチンの製造法
に関する。
従来の技術
従来、L−オルニチンの製造法としては、コリ
ネバクテリウム属のアルギニン又はシトルリン栄
養要求株を用いる方法(特公昭35−18439号公
報)、ブレビバクテリウム属のアルギニン又はシ
トルリン栄養要求株を用いる方法(特公昭43−
8712号公報)が知られている。
発明が解決しようとする問題点
従来の方法においては、L−オルニチンの収量
はまだ満足すべきものではない。常に優れたL−
オルニチンの製造法が求められている。
問題点を解決するための手段
本発明によると、コリネバクテリウム属に属
し、2−チアゾリールアラニン、サルフアグアニ
ジン又は2−フルオロピルビン酸に耐性を有する
微生物を用いることにより著量のL−オルニチン
を製造することができる。
本発明に使用する微生物としては、コリネバク
テリウム属に属し、2−チアゾリールアラニン、
サルフアグアジン又は2−フルオロピルビン酸に
耐性を有し、かつL−オルニチン生産能を有する
微生物であれば、紫外線照射もしくは化学薬剤処
理で誘導される変異株又は細胞融合法、遺伝子走
査法で誘導される組換え株等、いずれも用いられ
る。
具体的には、コリネバクテリウム・グルタミク
ム(Coryhnebacterium glutamicum)H−3795
(2−チアゾリールアラニン耐性)(微工研条寄第
656号)(以下、H−3795と称す)、コリネバクテ
リウム・ダルタミクムH−3796(サルフアグアニ
ジン耐性)(微工研条寄第657号)(以下、H−
3796と称す)、コリネバクテリウム・ダクタミク
ムH−3797(サルフアグアニジンおよび2−チア
ゾリールアラニン耐性)(微工研条寄第658号)
(以下、H−3797と称す)、コリネバクテリウム・
ダルタミクムH−3798(サルフアグアニジンおよ
び2−フルオロピルビン酸耐性)(微工研条寄第
659号)(以下、H−3798と称す)等があげられ
る。
次に、本発明の変異株の採取法と各種薬剤添加
培地での菌株の生育を説明する。
(1) 変異株の採取法
イーストブイヨンスラント(肉エキス0.5
g/dl、酵母エキス0.5g/dl、ペプトン1
g/dl、食塩0.25g/dl、寒天2g/dl、PH
7.2)で30℃、一夜培養したコリネバクテリウ
ム・グルタミクムATCC13232(以下、
ATCC13232と称す)をトリスマレート緩衡液
(PH6.0)に菌体濃度108〜109/mlとなるように
懸濁した。ついで該懸濁液にN−メチル−
N′−ニトロ−N−ニトロソグアニジンを最終
濃度250μg/mlとなるよう添加後、30℃で20
分間放置した。その後、遠心分離して菌体を集
め緩衡液でトリスマレート(PH6.0)で洗浄し
た後、該菌体をサルフアグアニジン100μg/
mlを含む第1表に示す平板培地で30℃、5〜7
日間培養した。
INDUSTRIAL APPLICATION FIELD The present invention relates to a method for producing L-ornithine by fermentation. Conventional technology Conventionally, methods for producing L-ornithine include a method using an arginine or citrulline auxotroph of the genus Corynebacterium (Japanese Patent Publication No. 18439/1983), a method using an arginine or citrulline auxotroph of the genus Brevibacterium, Method to use (Special Public Interest Publication 43-
8712) is known. Problems to be Solved by the Invention In the conventional methods, the yield of L-ornithine is still not satisfactory. Always excellent L-
There is a need for a method for producing ornithine. Means for Solving the Problems According to the present invention, a significant amount of L- Ornithine can be produced. The microorganisms used in the present invention belong to the genus Corynebacterium, and include 2-thiazolylalanine,
Microorganisms that are resistant to sulfaguazine or 2-fluoropyruvate and have the ability to produce L-ornithine can be induced by mutant strains induced by ultraviolet irradiation or chemical treatment, cell fusion methods, or gene scanning methods. Any recombinant strain etc. can be used. Specifically, Coryhnebacterium glutamicum H-3795
(2-Thiazolylalanine resistance)
656) (hereinafter referred to as H-3795), Corynebacterium daltamicum H-3796 (sulfaguanidine resistance) (Feikoken Joyori No. 657) (hereinafter referred to as H-
3796), Corynebacterium dactamicum H-3797 (resistant to sulfaguanidine and 2-thiazolylalanine) (Feikoken Article No. 658)
(hereinafter referred to as H-3797), Corynebacterium
Daltamicum H-3798 (resistant to sulfaguanidine and 2-fluoropyruvate)
No. 659) (hereinafter referred to as H-3798). Next, the method for collecting the mutant strain of the present invention and the growth of the strain in various drug-added media will be explained. (1) Method for collecting mutant strains Yeast bouillon slant (meat extract 0.5
g/dl, yeast extract 0.5g/dl, peptone 1
g/dl, salt 0.25g/dl, agar 2g/dl, PH
Corynebacterium glutamicum ATCC13232 (hereinafter referred to as
ATCC13232) was suspended in trismalate buffer (PH6.0) to a bacterial cell concentration of 10 8 to 10 9 /ml. Then, N-methyl-
After adding N'-nitro-N-nitrosoguanidine to a final concentration of 250 μg/ml,
Leave it for a minute. Thereafter, the bacterial cells were collected by centrifugation, washed with trismalate (PH6.0) in a buffer solution, and then the bacterial cells were collected with 100 μg of sulfaguanidine/
30°C, 5-7 ml of plate culture shown in Table 1.
Cultured for 1 day.
【表】
出現した平板寒天上のコロニーからL−オル
ニチン生産性の向上した変異株、H−3796を得
た。
さらに、第2表に示す条件以外は上記の方法
と同様にしてH−3795、H−3797およびH−
3798を得た。[Table] A mutant strain H-3796 with improved L-ornithine productivity was obtained from the colonies that appeared on the flat agar plate. Furthermore, H-3795, H-3797 and H-3795, H-3797 and H-
Got 3798.
【表】
(2) 薬剤耐性株の薬剤含有培地での生育
上記(1)の方法で取得したH−3795、H−
3796、H−3797、H−3798及びATCC13232を
イーストブイヨンスラントに接種し、30℃で24
時間培養した。ついで、該培養液を生理食塩水
に懸濁した。
該懸濁液を第3表に示した濃度のサルフアグ
アニジン、2−チアゾリールアラニン又は2−
フルオロピルビン酸を含んだ第1表に示す平板
培地に接種し(108個/plate)、30℃で48時間
培養した。その結果を第3表に示す。[Table] (2) Growth of drug-resistant strains in drug-containing medium H-3795 and H- obtained by method (1) above
3796, H-3797, H-3798 and ATCC13232 were inoculated into yeast bouillon slant and incubated at 30℃ for 24 hours.
Cultured for hours. Then, the culture solution was suspended in physiological saline. The suspension was treated with sulfaguanidine, 2-thiazolylalanine or 2-thiazolylalanine at the concentrations shown in Table 3.
The cells were inoculated onto a plate medium containing fluoropyruvate shown in Table 1 (10 8 cells/plate) and cultured at 30°C for 48 hours. The results are shown in Table 3.
【表】
:非常に良好な生育 +:良好な生育
−:非生育
本発明で使用する培地としては、炭素源、窒素
源、無機物、その他生育促進物質を含むものであ
れば、天然培地又は人工培地のいずれでもよい。
炭素源としては、グルコース、フラクトース、シ
ユクロース、マルトース、デンプン加水分解液、
果汁、廃糖蜜、有機酸、アルコール等が使用され
る。
窒素源としては、アンモニア、塩化アンモニウ
ム、硫酸アンモニウム、リン酸アンモニウム、硝
酸アンモニウム、炭酸アンモニウム、水酸化アン
モニウム、尿素、酵母エキス、コーン・スチー
プ・リカー、ペプトン等が使用される。
無機物としては、リン酸−カリウム、リン酸二
カリウム、硫酸マグネシウム等、硫酸第一鉄、硫
酸マンガン、硫酸亜鉛等が使用される。
生育促進物質としては、アミノ酸やパントテン
酸、ニコチン酸、サイアミン塩酸塩等のビタミン
類が用いられる。
又、使用する微生物がL−シトルリン、又はL
−アルギニンの栄養要求性を有している場合には
L−アルギニンを培地中に100〜300mg/存在せ
しめれば良い。
培養は通気撹拌等の好気的条件下で行い、培養
温度は24〜40℃、PHは5.5〜8.5、培養日数は1〜
4日である。PHの調整には、尿素、炭酸カルシウ
ム、アンモニア水、アンモニアガス、リン酸マグ
ネシウム等が使用される。
発酵液からのL−オルニチンの採取は、常法、
例えばイオン交換樹脂法、濃縮法、有機溶媒添加
法等で行う。
実施例 1
種菌として、H−3796を用いた。
該菌株をブイヨンスラント上で30℃、24時間培
養した。該培養物の1白金耳をグルコース70g/
、硫酸アンモニウム30g/、KH2PO4、0.5
g/、MgSO40.125g/、FeSO4・7H2O0.02
g/、MnSO4・7H2O0.02g/、ZnSO4・
7H2O0.01g/、ビオチン100γ/、サイアミ
ン塩酸塩1mg/、コーン・スチープ・リカー10
g/、尿素3g/、L−アルキニン250mg/
、クエン酸ブチル3ml/、CaCO330g/
の組成からなる倍地20mlを含有する250ml三角フ
ラスコに接種し、30℃で48時間培養した。培養液
中に蓄積されたL−オルニチンは30.6g/(塩
酸塩換算)であつた。
次いで、培養液1から菌体を除去して、得ら
れた液をアンバーライト(H+型)(ロームアン
ドハウス社製)カラムに流し、水洗した。ついで
吸着物を5%アンモニア水で溶出した。L−ホル
ニチン区分を集めて減圧濃縮した。得られた残渣
にL−オルニチンと等モルの塩酸を加えた。生じ
た粗結晶を含水メタノールから再結晶することに
よりL−オルニチン塩酸塩27.5gを得た。
対象として、H−3796の代わりにATCC13232
を用いる以外は前記と同様にしてL−オルニチン
26.8g/(塩酸塩換算)を得た。
実施例 2
実施例1において、種菌としてH−3796の代わ
りに第4表に示す菌株を用いる以外は実施例1と
同様に行つた。
その結果を第4表に示す。[Table]: Very good growth +: Good growth -: No growth The medium used in the present invention may be a natural medium or an artificial medium as long as it contains carbon sources, nitrogen sources, inorganic substances, and other growth-promoting substances. Any medium may be used.
Carbon sources include glucose, fructose, sucrose, maltose, starch hydrolyzate,
Fruit juice, blackstrap molasses, organic acids, alcohol, etc. are used. As the nitrogen source, ammonia, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium nitrate, ammonium carbonate, ammonium hydroxide, urea, yeast extract, corn steep liquor, peptone, etc. are used. As the inorganic substance, potassium phosphate, dipotassium phosphate, magnesium sulfate, etc., ferrous sulfate, manganese sulfate, zinc sulfate, etc. are used. As growth promoting substances, amino acids and vitamins such as pantothenic acid, nicotinic acid, and thiamine hydrochloride are used. In addition, the microorganism used is L-citrulline or L-citrulline.
- If the medium has auxotrophy for arginine, 100 to 300 mg/L-arginine may be present in the medium. Culture is performed under aerobic conditions such as aeration and stirring, culture temperature is 24-40℃, pH is 5.5-8.5, and culture days are 1-40℃.
It's been 4 days. Urea, calcium carbonate, aqueous ammonia, ammonia gas, magnesium phosphate, etc. are used to adjust the pH. L-ornithine can be collected from the fermentation liquid using a conventional method.
For example, an ion exchange resin method, a concentration method, an organic solvent addition method, etc. are used. Example 1 H-3796 was used as an inoculum. The strain was cultured on bouillon slant at 30°C for 24 hours. One loopful of the culture was mixed with 70 g of glucose/
, ammonium sulfate 30g/, KH 2 PO 4 , 0.5
g/, MgSO 4 0.125g/, FeSO 4・7H 2 O0.02
g/, MnSO 4・7H 2 O0.02g/, ZnSO 4・
7H 2 O 0.01g/, Biotin 100γ/, Thiamine Hydrochloride 1mg/, Corn Steep Liquor 10
g/, urea 3g/, L-alkinine 250mg/
, butyl citrate 3ml/, CaCO 3 30g/
The cells were inoculated into a 250 ml Erlenmeyer flask containing 20 ml of a medium having the following composition, and cultured at 30°C for 48 hours. The amount of L-ornithine accumulated in the culture solution was 30.6 g/(in terms of hydrochloride). Next, the bacterial cells were removed from the culture solution 1, and the resulting solution was poured into an Amberlite (H + type) (manufactured by Rohm and Haus Co., Ltd.) column and washed with water. The adsorbed material was then eluted with 5% aqueous ammonia. The L-fornithine fraction was collected and concentrated under reduced pressure. Hydrochloric acid in an equimolar amount as L-ornithine was added to the obtained residue. The resulting crude crystals were recrystallized from aqueous methanol to obtain 27.5 g of L-ornithine hydrochloride. As a target, ATCC13232 instead of H-3796
L-ornithine was prepared in the same manner as above except for using
26.8 g/(in terms of hydrochloride) was obtained. Example 2 The same procedure as in Example 1 was carried out except that the strains shown in Table 4 were used instead of H-3796 as the inoculum. The results are shown in Table 4.
【表】
実施例 3
種菌として、H−3796を用いた。
該菌種をブイヨンスラント上で30℃、24時間培
養した。該培養物の1白金耳を廃糖蜜70g/
(gluose換算)、硫酸アンモニウム30g/、
KH2PO40.5/、MgSO40.125g/、尿素3
g/、コーン・スチープ・リカー10g/、サ
イアミン塩酸塩150γ/、クエン酸ブチル3
ml/、L−アルギニン250mg/、CaCO330
g/の組成からなる培地20mlを含有する250ml
三角フラスコに接種し、30℃で48時間培養した。
培養液中には27.5g/(塩酸塩換算)のL−オ
ルニチンが蓄積された。
対照として、H−3796の代わりにATCC13232
を用いる以外は前記と同様にしてL−オルニチン
22.1g/(塩酸塩換算)を得た。
発明の効果
本発明方法により著量のL−オルニチンを得る
ことができる。[Table] Example 3 H-3796 was used as the inoculum. The bacterial strain was cultured on a bouillon slant at 30°C for 24 hours. Add 1 platinum loop of the culture to 70g of blackstrap molasses/
(gluose equivalent), ammonium sulfate 30g/,
KH 2 PO 4 0.5/, MgSO 4 0.125g/, Urea 3
g/, corn steep liquor 10g/, thiamine hydrochloride 150γ/, butyl citrate 3
ml/, L-Arginine 250mg/, CaCO 3 30
250 ml containing 20 ml of medium with a composition of
It was inoculated into an Erlenmeyer flask and cultured at 30°C for 48 hours.
27.5 g/(hydrochloride equivalent) of L-ornithine was accumulated in the culture solution. As a control, ATCC13232 instead of H-3796
L-ornithine was prepared in the same manner as above except for using
22.1 g/(in terms of hydrochloride) was obtained. Effects of the Invention A significant amount of L-ornithine can be obtained by the method of the present invention.
Claims (1)
ールアラニン、サルフアグアニジン又は2−フル
オロピルビン酸に耐性を有し、かつL−オルニチ
ン生産能を有する微生物を培地に培養し、培養液
中にL−オルニチンを生成蓄積させ、生成蓄積し
たL−オルニチンを採取することを特徴とする発
酵法によるL−オルニチンの製造法。1. A microorganism belonging to the genus Corynebacterium that is resistant to 2-thiazolylalanine, sulfaguanidine, or 2-fluoropyruvate and capable of producing L-ornithine is cultured in a medium, and L-ornithine is added to the culture solution. - A method for producing L-ornithine by a fermentation method, which comprises producing and accumulating ornithine and collecting the produced and accumulated L-ornithine.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP24137984A JPS61119194A (en) | 1984-11-15 | 1984-11-15 | Production of l-ornithine through fermentation process |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP24137984A JPS61119194A (en) | 1984-11-15 | 1984-11-15 | Production of l-ornithine through fermentation process |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS61119194A JPS61119194A (en) | 1986-06-06 |
JPH0362396B2 true JPH0362396B2 (en) | 1991-09-25 |
Family
ID=17073406
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP24137984A Granted JPS61119194A (en) | 1984-11-15 | 1984-11-15 | Production of l-ornithine through fermentation process |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS61119194A (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5210636B2 (en) | 2005-10-04 | 2013-06-12 | 協和発酵バイオ株式会社 | Composition for improving subjective symptoms of fatigue |
CN101460159B (en) | 2006-06-07 | 2011-11-23 | 协和发酵生化株式会社 | Fatigue-reducing agent |
DE102010003419B4 (en) | 2010-03-30 | 2019-09-12 | Evonik Degussa Gmbh | Process for the fermentative production of L-ornithine |
RU2496867C2 (en) | 2011-04-25 | 2013-10-27 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО "АГРИ") | Method to produce l-amino acid of glutamate family using coryneformic bacterium |
US20150025147A1 (en) | 2012-03-02 | 2015-01-22 | Kyowa Hakko Bio Co., Ltd | Enhancer for eating activity and/or gastrointestinal activity |
-
1984
- 1984-11-15 JP JP24137984A patent/JPS61119194A/en active Granted
Also Published As
Publication number | Publication date |
---|---|
JPS61119194A (en) | 1986-06-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JPS6234397B2 (en) | ||
US4169763A (en) | Process for the production of L-lysine by fermentation | |
JPH0430275B2 (en) | ||
JPS6257315B2 (en) | ||
JPS62195293A (en) | Production of l-isoleucine by fermentation method | |
JPH0362395B2 (en) | ||
US5188947A (en) | Process and microorganism for producing l-ornithine by corynebacterium, brevibacterium, or athrobacter | |
JPH0362396B2 (en) | ||
US5098835A (en) | Process for producing l-threonine by fermentation | |
JPS6251112B2 (en) | ||
KR920007402B1 (en) | Process for producing l-lysine | |
JP2578474B2 (en) | Method for producing L-glutamic acid | |
JPH057493A (en) | Production of l-valine by fermentation | |
JP2578468B2 (en) | Method for producing L-arginine by fermentation | |
JP3006939B2 (en) | Method for producing L-lysine | |
JPH0644871B2 (en) | Fermentation method for producing L-leucine | |
US5019503A (en) | Method for production of l-threonine | |
JPH0347838B2 (en) | ||
JP3100763B2 (en) | Method for producing L-arginine by fermentation | |
JP2817228B2 (en) | Method for producing L-glutamic acid | |
JPH0411195B2 (en) | ||
JPH0314436B2 (en) | ||
JPH0657155B2 (en) | Method for producing L-valine | |
JPS6234398B2 (en) | ||
JPS5988094A (en) | Production of l-lysine by fermentation |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EXPY | Cancellation because of completion of term |