JPH029795B2 - - Google Patents
Info
- Publication number
- JPH029795B2 JPH029795B2 JP22791382A JP22791382A JPH029795B2 JP H029795 B2 JPH029795 B2 JP H029795B2 JP 22791382 A JP22791382 A JP 22791382A JP 22791382 A JP22791382 A JP 22791382A JP H029795 B2 JPH029795 B2 JP H029795B2
- Authority
- JP
- Japan
- Prior art keywords
- tryptophan
- phosphonomethyl
- glycine
- producing
- bis
- 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
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 38
- 229960004799 tryptophan Drugs 0.000 claims description 26
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 8
- XDDAORKBJWWYJS-UHFFFAOYSA-N glyphosate Chemical compound OC(=O)CNCP(O)(O)=O XDDAORKBJWWYJS-UHFFFAOYSA-N 0.000 claims description 8
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 6
- 244000005700 microbiome Species 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 239000004471 Glycine Substances 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- IFGCUJZIWBUILZ-UHFFFAOYSA-N sodium 2-[[2-[[hydroxy-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyphosphoryl]amino]-4-methylpentanoyl]amino]-3-(1H-indol-3-yl)propanoic acid Chemical compound [Na+].C=1NC2=CC=CC=C2C=1CC(C(O)=O)NC(=O)C(CC(C)C)NP(O)(=O)OC1OC(C)C(O)C(O)C1O IFGCUJZIWBUILZ-UHFFFAOYSA-N 0.000 claims description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 18
- 238000000034 method Methods 0.000 description 8
- 239000002609 medium Substances 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 244000063299 Bacillus subtilis Species 0.000 description 6
- 235000014469 Bacillus subtilis Nutrition 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- 229910052799 carbon Inorganic materials 0.000 description 6
- OXHDYFKENBXUEM-UHFFFAOYSA-N glyphosine Chemical compound OC(=O)CN(CP(O)(O)=O)CP(O)(O)=O OXHDYFKENBXUEM-UHFFFAOYSA-N 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 150000001413 amino acids Chemical class 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
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- INPQIVHQSQUEAJ-UHFFFAOYSA-N 5-fluorotryptophan Chemical compound C1=C(F)C=C2C(CC(N)C(O)=O)=CNC2=C1 INPQIVHQSQUEAJ-UHFFFAOYSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- GNTVWGDQPXCYBV-UHFFFAOYSA-N Indolmycin Natural products O1C(NC)=NC(=O)C1C(C)C1=CNC2=CC=CC=C12 GNTVWGDQPXCYBV-UHFFFAOYSA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- RWZYAGGXGHYGMB-UHFFFAOYSA-N anthranilic acid Chemical compound NC1=CC=CC=C1C(O)=O RWZYAGGXGHYGMB-UHFFFAOYSA-N 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- GNTVWGDQPXCYBV-PELKAZGASA-N indolmycin Chemical compound O1C(NC)=NC(=O)[C@@H]1[C@H](C)C1=CNC2=CC=CC=C12 GNTVWGDQPXCYBV-PELKAZGASA-N 0.000 description 2
- 239000011785 micronutrient Substances 0.000 description 2
- 235000013369 micronutrients Nutrition 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- SNLOIIPRZGMRAB-QMMMGPOBSA-N (2s)-2-azaniumyl-3-(1h-pyrrolo[2,3-b]pyridin-3-yl)propanoate Chemical compound C1=CC=C2C(C[C@H]([NH3+])C([O-])=O)=CNC2=N1 SNLOIIPRZGMRAB-QMMMGPOBSA-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
- RSTKLPZEZYGQPY-UHFFFAOYSA-N 3-(indol-3-yl)pyruvic acid Chemical compound C1=CC=C2C(CC(=O)C(=O)O)=CNC2=C1 RSTKLPZEZYGQPY-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- 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 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 1
- 229930064664 L-arginine Natural products 0.000 description 1
- 235000014852 L-arginine Nutrition 0.000 description 1
- 239000004395 L-leucine Substances 0.000 description 1
- 235000019454 L-leucine Nutrition 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
- CZCIKBSVHDNIDH-NSHDSACASA-N N(alpha)-methyl-L-tryptophan Chemical compound C1=CC=C2C(C[C@H]([NH2+]C)C([O-])=O)=CNC2=C1 CZCIKBSVHDNIDH-NSHDSACASA-N 0.000 description 1
- CZCIKBSVHDNIDH-UHFFFAOYSA-N Nalpha-methyl-DL-tryptophan Natural products C1=CC=C2C(CC(NC)C(O)=O)=CNC2=C1 CZCIKBSVHDNIDH-UHFFFAOYSA-N 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-N Nitrous acid Chemical compound ON=O IOVCWXUNBOPUCH-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000297434 Stenotarsus subtilis Species 0.000 description 1
- 229930006000 Sucrose Natural products 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
- JETSKDPKURDVNI-UHFFFAOYSA-N [C].[Ca] Chemical compound [C].[Ca] JETSKDPKURDVNI-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000001580 bacterial effect Effects 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
- 159000000007 calcium salts Chemical class 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000007423 decrease Effects 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
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical class CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 159000000014 iron salts Chemical class 0.000 description 1
- 229960003136 leucine Drugs 0.000 description 1
- 159000000003 magnesium salts Chemical class 0.000 description 1
- 150000002696 manganese Chemical class 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 229940066779 peptones Drugs 0.000 description 1
- 229960005190 phenylalanine Drugs 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000013587 production medium Substances 0.000 description 1
- 235000019710 soybean protein Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- WPLOVIFNBMNBPD-ATHMIXSHSA-N subtilin Chemical compound CC1SCC(NC2=O)C(=O)NC(CC(N)=O)C(=O)NC(C(=O)NC(CCCCN)C(=O)NC(C(C)CC)C(=O)NC(=C)C(=O)NC(CCCCN)C(O)=O)CSC(C)C2NC(=O)C(CC(C)C)NC(=O)C1NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C1NC(=O)C(=C/C)/NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C2NC(=O)CNC(=O)C3CCCN3C(=O)C(NC(=O)C3NC(=O)C(CC(C)C)NC(=O)C(=C)NC(=O)C(CCC(O)=O)NC(=O)C(NC(=O)C(CCCCN)NC(=O)C(N)CC=4C5=CC=CC=C5NC=4)CSC3)C(C)SC2)C(C)C)C(C)SC1)CC1=CC=CC=C1 WPLOVIFNBMNBPD-ATHMIXSHSA-N 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
本発明は、発酵法によるＬ−トリプトフアン
（以下、トリプトフアンと記す）の製造法に関す
る。
従来トリプトフアンの製造法としては、トリプ
トフアンの前駆物質であるアントラニル酸、イン
ドール或は３−インドールピルビン酸よりトリプ
トフアンを製造する方法が知られている。
これら前駆物質を使用する方法に対し、前駆物
質を使用しないで糖類等を炭素源とし、バチルス
属に属しトリプトフアンアナログに耐性をを有す
る変異株を使用して直接発酵法によりトリプトフ
アンを生産する方法（特公昭48−18828、特公昭
53−39517）が開発されている。
そこで、本発明者らはバチルス属の微生物を用
いて更に糖類等の炭素源からトリプトフアンを直
接発酵法により安価に製造する方法を開発すべく
研究を行つた結果、バチルス属の上記のようなト
リプトフアンアナログ耐性の他にＮ−（ホスホノ
メチル）グリシン又はＮ，Ｎ−ビス−（ホスホノ
メチル）グリシンに耐性を付与した微生物の中に
従来知られているものより更に大量のＬ−トリプ
トフアンを生産する能力を有する菌株があること
を見い出した。本発明はこの知見に基づいて更に
研究の結果完成されたものである。
本発明の方法で使用される変異株はバチルス属
に属し、Ｎ−（ホスホノメチル）グリシンまたは
Ｎ，Ｎ−ビス−（ホスホノメチル）グリシンに耐
性を有し、かつＬ−トリプトフアンを生産する能
力を有する微生物であり、例えば次のような変異
株が使用される。
バチルス・ズブチリス（５−FTr、PMGr）
AJ11986 FERM−P6847
バチルス・ズブチリス（５−FTr、BPMGr）
AJ11987 FERM−P6848
バチルス・ズブチリス（５−FTr、IMr、
PMGr） AJ11988 FERM−P6849
バチルス・ズブチリス（５−FTr、IMr、
BPMGr） AJ11989 FERM−P6850
５−FTr：５−フルオロトリプトフアン耐性
IMr：インドールマイシン耐性
PMG：Ｎ−（ホスホノメチル）グリシン
BPMG：Ｎ，Ｎ−ビス−（ホスホノメチル）グリ
シン
これら本発明で使用される変異株は、バチルス
属のトリプトフアンアナログ耐性のＬ−トリプト
フアン生産菌を親株とし、これに通常の変異誘導
操作、例えば、紫外線照射或はＮ−メチル−
N′−ニトロ−Ｎ−ニトロソグアニジン、亜硝酸
等の化学薬剤処理を施した後、親株が生育できる
ような濃度のＮ−（ホスホノメチル）グリシンま
たは／およびＮ，Ｎ−ビス−（ホスホノメチル）
グリシンを含有する寒天平板培地で培養し、該平
板培地上に生育するコロニーを分離することによ
つて得られる。
上記の親株としては、トリプトフアンアナログ
耐性の他にトリプトフアン生産に有用な性質を有
するトリプトフアン生産菌、例えば、Ｌ−アルギ
ニン、Ｌ−リジン、Ｌ−ロイシンもしくはＬ−フ
エニルアラニン要求性のトリプトフアン生産菌
（特公昭53−39517号公報）、更にはトリプトフア
ンアナログ耐性でかつインドールマイシン耐性の
トリプトフアン生産菌（特開昭56−92796号公報）
等が使用される。具体的としては次のようなトリ
プトフアンアナログ耐性のトリプトフアン生産菌
が使用される。
バチルス・ズブチリス（５−FTr） FT−145
FERM−P1783
バチルス・ズブチリス（５−FTr、IMr）
AJ11483 FERM−P5286
その他、本発明の変異株はバチルス属の野生株
を親株とし、これにＮ−（ホスホノメチル）グリ
シンまたは／およびＮ，Ｎ−ビス−（ホスホノメ
チル）グリシンの耐性を付与した後、トリプトフ
アンアナログ耐性を付与することによつても誘導
できる。
なお、上記のトリプトフアンアナログとはバチ
ルス属に属する微生物の生育を阻害し、この生育
阻害がＬ−トリプトフアンの存在によつて部分的
又は完全に解除されるような薬剤をいい、例えば
５−メチルトリプトフアン等の５，６又は７−低
級アルキルトリプトフアン、５−フルオロトリプ
トフアン等の５又は６−ハロゲノトリプトフア
ン、トリプトフアンハイドロキサメート、又は７
−アザトリプトフアン等がある。
以下、実験例にて本発明のＮ−（ホスホノメチ
ル）グリシン耐性あるいはＮ，Ｎ−ビス−（ホス
ホノメチル）グリシン耐性のトリプトフアン生産
菌のＮ−（ホスホノメチル）グリシンあるいはＮ，
Ｎ−ビス−（ホスホノメチル）グリシンに対する
耐性度を示す。
実験例
第１表に示す組成の最少培地にＮ−（ホスホノ
メチル）グリシンまたはＮ，Ｎ−ビス−（ホスホ
ノメチル）グリシンを第２表に示す濃度となるよ
う加え、験験管に４ml宛分注し加熱滅菌して液体
培地を作成した。上記液体培地に薬剤無添加培地
で24時間振盪培養した第２表に示したバチルス・
ズブチリスの変異株の培養液を0.1ml宛接種し、
30℃で48時間振盪培養を行い570nｍの吸光度を
測定することにより生育度を測定した。その結果
を第２表に示す。
The present invention relates to a method for producing L-tryptophan (hereinafter referred to as tryptophan) by a fermentation method. As a conventional method for producing tryptophan, a method for producing tryptophan from anthranilic acid, indole, or 3-indolepyruvic acid, which is a precursor of tryptophan, is known. In contrast to methods using these precursors, tryptophan is produced by direct fermentation using a mutant strain belonging to the genus Bacillus that is resistant to tryptophan analogs and using sugars as a carbon source without using precursors. Method (Tokuko Sho 48-18828, Tokko Sho
53-39517) has been developed. Therefore, the present inventors conducted research to develop a method to inexpensively produce tryptophan from carbon sources such as sugars by a direct fermentation method using microorganisms of the genus Bacillus. Ability to produce larger amounts of L-tryptophan than previously known in microorganisms that have been conferred resistance to N-(phosphonomethyl)glycine or N,N-bis-(phosphonomethyl)glycine in addition to resistance to tophan analogues. We discovered that there are strains that have the following. The present invention was completed as a result of further research based on this knowledge. The mutant strain used in the method of the present invention belongs to the genus Bacillus, is resistant to N-(phosphonomethyl)glycine or N,N-bis-(phosphonomethyl)glycine, and has the ability to produce L-tryptophan. For example, the following mutant strains are used. Bacillus subtilis (5-FT r , PMG r )
AJ11986 FERM-P6847 Bacillus subtilis (5-FT r , BPMG r )
AJ11987 FERM-P6848 Bacillus subtilis (5-FT r , IM r ,
PMG r ) AJ11988 FERM-P6849 Bacillus subtilis (5-FT r , IM r ,
BPMG r ) AJ11989 FERM-P6850 5-FT r : 5-fluorotryptophan resistance IM r : Indolmycin resistance PMG: N-(phosphonomethyl)glycine BPMG: N,N-bis-(phosphonomethyl)glycine These are used in the present invention The mutant strain to be developed uses an L-tryptophan-producing bacterium of the genus Bacillus that is resistant to tryptophan analogs as a parent strain, and is subjected to conventional mutagenesis procedures such as ultraviolet irradiation or N-methyl-
After treatment with chemical agents such as N'-nitro-N-nitrosoguanidine and nitrous acid, N-(phosphonomethyl)glycine or/and N,N-bis-(phosphonomethyl) at a concentration that allows the parent strain to grow.
It is obtained by culturing on an agar plate medium containing glycine and separating colonies growing on the plate medium. The above-mentioned parent strain may include tryptophan-producing bacteria that have properties useful for tryptophan production in addition to tryptophan analog resistance, such as tryptophan-producing bacteria that require L-arginine, L-lysine, L-leucine, or L-phenylalanine. Bacteria (Japanese Patent Publication No. 53-39517), and tryptophan-producing bacteria resistant to tryptophan analogs and indolmycin (Japanese Patent Publication No. 56-92796)
etc. are used. Specifically, the following tryptophan analog-resistant tryptophan-producing bacteria are used. Bacillus subtilis (5-FT r ) FT-145
FERM-P1783 Bacillus subtilis (5-FT r , IM r )
AJ11483 FERM-P5286 In addition, the mutant strain of the present invention uses a wild strain of the genus Bacillus as a parent strain, and after imparting resistance to N-(phosphonomethyl)glycine or/and N,N-bis-(phosphonomethyl)glycine, It can also be induced by conferring resistance to tofuan analogs. The above-mentioned tryptophan analog refers to a drug that inhibits the growth of microorganisms belonging to the genus Bacillus, and this growth inhibition is partially or completely canceled by the presence of L-tryptophan, such as 5-tryptophan. 5,6 or 7-lower alkyltryptophans such as methyltryptophan, 5- or 6-halogenotryptophans such as 5-fluorotryptophan, tryptophan hydroxamates, or 7
-Azatryptophan etc. In the following experimental examples, N-(phosphonomethyl)glycine or N,
The degree of resistance to N-bis-(phosphonomethyl)glycine is shown. Experimental example Add N-(phosphonomethyl)glycine or N,N-bis-(phosphonomethyl)glycine to the concentration shown in Table 2 to a minimal medium with the composition shown in Table 1, and dispense 4 ml into test tubes. A liquid medium was prepared by heat sterilization. The Bacillus strains listed in Table 2 were cultured with shaking for 24 hours in the above liquid medium without any drug added.
Inoculate 0.1ml of the culture solution of the mutant strain of S. subtilis,
Growth was measured by culturing with shaking at 30° C. for 48 hours and measuring absorbance at 570 nm. The results are shown in Table 2.
【表】【table】
【表】
本発明で使用する培地は炭素源、窒素源、無機
塩類、その他必要に応じてアミノ酸、ビタミン等
の有機微量栄養素を含有する通常の栄養培地が使
用される。炭素源としてはグルコース、シユーク
ロース、マルトース、澱粉水解物、糖蜜等が使用
され、その他エタノール、酢酸、クエン酸等も単
独或は上記他の炭素源と併用して用いられる。窒
素源としては硫酸アンモニウム、塩化アンモニウ
ム、リン酸アンモニウム等のアンモニウム塩、硝
酸塩、尿素、ペプトン等有機或は無機の窒素源が
使用される。有機微量栄養素としてはアミノ酸、
ビタミン、脂肪酸、核酸、更にこれらのものを含
有するペプトン、カザミノ酸、酵母エキス、大豆
蛋白分解物等が使用され、生育にアミノ酸等を要
求する栄養要求性変異株を使用する場合には要求
される栄養素を補添することが必要である。無機
塩類としてはリン酸塩、マグネシウム塩、カルシ
ウム塩、鉄塩、マンガン塩等が使用される。
培養は通常の培養条件下で行えば良く、PHを５
ないし９、温度を20ないし４０℃に制御しつつ１
〜４日間振盪培養又は通気撹拌培養することによ
り培養液中に著量のトリプトフアンが蓄積され
る。培養中にPHが下がる場合には、炭素カルシウ
ムを別殺菌して加えるか又はアンモニア水、アン
モニアガス等のアルカリで中和する。又、有機酸
を炭素源とする場合はPHの上昇を鉱酸又は有機酸
で中和する。
培養液からトリプトフアンを採取する方法は、
公知のトリプトフアン回収方法に従つて行えば良
く、培養液から菌体を除去した後濃縮晶析する方
法或はイオン交換クロマトグラフイー等によつて
採取される。
以下、実施例にて説明する。
実施例 １
下記第３表に示した組成のトリプトフアン生産
用培地20mlを500ml容フラスコに分注し、110℃で
10分間加熱した後、第４表に示す微生物をそれぞ
れ1/3スラント量植えつけ30℃で96時間振盪培養
した。それぞれの培養液中のトリプトフアン生成
量は第４表の如くであつた。[Table] The medium used in the present invention is a conventional nutrient medium containing a carbon source, a nitrogen source, inorganic salts, and other organic micronutrients such as amino acids and vitamins as necessary. As the carbon source, glucose, sucrose, maltose, starch hydrolyzate, molasses, etc. are used, and ethanol, acetic acid, citric acid, etc. are also used alone or in combination with the other carbon sources mentioned above. As the nitrogen source, organic or inorganic nitrogen sources such as ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, nitrates, urea, and peptone are used. Organic micronutrients include amino acids,
Vitamins, fatty acids, nucleic acids, and peptones containing these things, casamino acids, yeast extracts, soybean protein decomposition products, etc. are used, and when using auxotrophic mutant strains that require amino acids etc. for growth, It is necessary to supplement the nutrients. As the inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts, etc. are used. Cultivation can be carried out under normal culture conditions, with a pH of 5.
to 9, while controlling the temperature at 20 to 40℃ 1
A significant amount of tryptophan is accumulated in the culture solution by shaking culture or aerating and agitating culture for ~4 days. If the pH decreases during culture, add calcium carbon after sterilization or neutralize with alkali such as aqueous ammonia or ammonia gas. In addition, when an organic acid is used as a carbon source, the increase in pH is neutralized with a mineral acid or an organic acid. How to collect tryptophan from culture fluid:
This may be carried out according to a known method for recovering tryptophan, and the tryptophan can be collected by removing the bacterial cells from the culture solution and then concentrating and crystallizing it, or by ion exchange chromatography. Examples will be described below. Example 1 20 ml of a tryptophan production medium having the composition shown in Table 3 below was dispensed into a 500 ml flask and incubated at 110°C.
After heating for 10 minutes, each microorganism shown in Table 4 was inoculated in a 1/3 slant amount and cultured with shaking at 30°C for 96 hours. The amount of tryptophan produced in each culture solution was as shown in Table 4.
【表】【table】
【表】【table】
Claims (1)
グリシンまたはＮ，Ｎ−ビス−（ホスホノメチル）
グリシンに耐性を有するＬ−トリプトフアン生産
性の微生物を液体培地中に好気的に培養し、培養
液中にＬ−トリプトフアンを生成せしめ、これを
採取することを特徴とするＬ−トリプトフアンの
製造法。1 Belongs to the genus Bacillus, N-(phosphonomethyl)
Glycine or N,N-bis-(phosphonomethyl)
A method for producing L-tryptophan, which comprises culturing L-tryptophan-producing microorganisms that are resistant to glycine aerobically in a liquid medium, producing L-tryptophan in the culture solution, and collecting the L-tryptophan. .
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP22791382A JPS59120092A (en) | 1982-12-27 | 1982-12-27 | Preparation of l-tryptophan by fermentation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP22791382A JPS59120092A (en) | 1982-12-27 | 1982-12-27 | Preparation of l-tryptophan by fermentation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS59120092A JPS59120092A (en) | 1984-07-11 |
| JPH029795B2 true JPH029795B2 (en) | 1990-03-05 |
Family
ID=16868255
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP22791382A Granted JPS59120092A (en) | 1982-12-27 | 1982-12-27 | Preparation of l-tryptophan by fermentation |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS59120092A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5528013B2 (en) * | 2009-06-04 | 2014-06-25 | 花王株式会社 | Alkaline protease high-producing bacteria |
| CN109133704A (en) * | 2018-08-09 | 2019-01-04 | 姜香 | A kind of preparation method of microorganism remediation cement-based material |
-
1982
- 1982-12-27 JP JP22791382A patent/JPS59120092A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS59120092A (en) | 1984-07-11 |
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