JPS5832594B2 - Method for producing L-valine by fermentation method - Google Patents
Method for producing L-valine by fermentation methodInfo
- Publication number
- JPS5832594B2 JPS5832594B2 JP11368279A JP11368279A JPS5832594B2 JP S5832594 B2 JPS5832594 B2 JP S5832594B2 JP 11368279 A JP11368279 A JP 11368279A JP 11368279 A JP11368279 A JP 11368279A JP S5832594 B2 JPS5832594 B2 JP S5832594B2
- Authority
- JP
- Japan
- Prior art keywords
- valine
- producing
- ribonucleoside
- resistance
- brevibacterium
- 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
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 title claims description 25
- 229960004295 valine Drugs 0.000 title claims description 13
- 238000004519 manufacturing process Methods 0.000 title claims description 5
- 238000000034 method Methods 0.000 title description 9
- 238000000855 fermentation Methods 0.000 title description 4
- 230000004151 fermentation Effects 0.000 title description 4
- 239000002342 ribonucleoside Substances 0.000 claims description 12
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 claims description 11
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 claims description 10
- FFEARJCKVFRZRR-UHFFFAOYSA-N L-Methionine Natural products CSCCC(N)C(O)=O FFEARJCKVFRZRR-UHFFFAOYSA-N 0.000 claims description 9
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 9
- 229930195722 L-methionine Natural products 0.000 claims description 9
- 229960004452 methionine Drugs 0.000 claims description 9
- PYMYPHUHKUWMLA-LMVFSUKVSA-N aldehydo-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims description 8
- 241000186146 Brevibacterium Species 0.000 claims description 7
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 4
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 4
- MRWXACSTFXYYMV-FDDDBJFASA-N nebularine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC=C2N=C1 MRWXACSTFXYYMV-FDDDBJFASA-N 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims 1
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 241000186226 Corynebacterium glutamicum Species 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 4
- 229930182844 L-isoleucine Natural products 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 229960000310 isoleucine Drugs 0.000 description 4
- YYAYLSOQGBAUHK-UHFFFAOYSA-N 2-(1,3-thiazol-2-ylamino)propanoic acid Chemical compound OC(=O)C(C)NC1=NC=CS1 YYAYLSOQGBAUHK-UHFFFAOYSA-N 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000001580 bacterial effect Effects 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
- 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 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 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
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 2
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-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
- UBORTCNDUKBEOP-UHFFFAOYSA-N L-xanthosine Natural products OC1C(O)C(CO)OC1N1C(NC(=O)NC2=O)=C2N=C1 UBORTCNDUKBEOP-UHFFFAOYSA-N 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- UBORTCNDUKBEOP-HAVMAKPUSA-N Xanthosine Natural products O[C@@H]1[C@H](O)[C@H](CO)O[C@H]1N1C(NC(=O)NC2=O)=C2N=C1 UBORTCNDUKBEOP-HAVMAKPUSA-N 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 229940029575 guanosine Drugs 0.000 description 2
- 229910001410 inorganic ion Inorganic materials 0.000 description 2
- 239000003456 ion exchange resin Substances 0.000 description 2
- 229920003303 ion-exchange polymer Polymers 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
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 2
- 229940045145 uridine Drugs 0.000 description 2
- UBORTCNDUKBEOP-UUOKFMHZSA-N xanthosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(NC(=O)NC2=O)=C2N=C1 UBORTCNDUKBEOP-UUOKFMHZSA-N 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 241000186216 Corynebacterium Species 0.000 description 1
- 241000807905 Corynebacterium glutamicum ATCC 14067 Species 0.000 description 1
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 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
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 241000319304 [Brevibacterium] flavum Species 0.000 description 1
- 239000000061 acid fraction Substances 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000003863 ammonium salts Chemical class 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
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229920001429 chelating resin Polymers 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- -1 ethanol Chemical class 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 150000008223 ribosides Chemical class 0.000 description 1
- 235000011121 sodium hydroxide Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 229960000344 thiamine hydrochloride Drugs 0.000 description 1
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 1
- 239000011747 thiamine hydrochloride Substances 0.000 description 1
- 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 1
- 239000004474 valine Substances 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
【発明の詳細な説明】 この発明は発酵法によるＬ−バリンの製造法に関する。[Detailed description of the invention] This invention relates to a method for producing L-valine by fermentation.
発酵法によるＬ−バリンの製造法としてはブレビバクテ
リウム属あるいはコリネバクテリウム属のＬ−インロイ
シン及びＬ−メチオニン等の栄養要求性を有する変異株
を用いる方法が知られている。As a method for producing L-valine by a fermentation method, a method using a mutant strain of the genus Brevibacterium or Corynebacterium that has auxotrophy for L-inleucine, L-methionine, etc. is known.
本発明者らは上記の変異株を改良して更に生産能の高い
変異株を得るべく研究した結果、ブレビバクテリウム属
のＬ−インロイシン及びＬ−メチオニン要求性変異株よ
りＤ−ＩＪボース、プリンリボヌクレオシド又はピリミ
ジンリボヌクレオシドのうちより高いＬ−バリン生産能
を有する変異株を多数分離することに成功した。The present inventors conducted research to improve the above-mentioned mutant strain to obtain a mutant strain with even higher productivity. As a result, D-IJbose, We succeeded in isolating a large number of mutant strains that have a higher ability to produce L-valine among purinibonucleosides and pyrimidine ribonucleosides.
即ち、この発明は、ブレビバクテリウム属に属し、Ｌ−
インロイシン及びＬ−メチオニン要求性並びにＤ−リボ
ース、プリンリボヌクレオシド又はピリミジンリボヌク
レオシド耐性を有し更にＬ−バリン生産能を有する変異
株を培養し、培地中にＬ−バリンを生成蓄積せしめ、こ
れを採取するＬ−バリンの製造法である。That is, this invention belongs to the genus Brevibacterium, and L-
A mutant strain that has inleucine and L-methionine auxotrophy and resistance to D-ribose, purine ribonucleoside, or pyrimidine ribonucleoside and also has the ability to produce L-valine is cultured, and L-valine is produced and accumulated in the medium. This is a method for producing L-valine.
本発明の変異株は具体的には、以下のものがある。Specifically, the mutant strains of the present invention include the following.
ブレビバクテリウム・ラクトファーメンタムＡＪ １
１４６０（ＦＥＲＭ−Ｐ ５１８１）（Ｌ−イソロイ
シン要求性、Ｌ−メチオニン要求性、２チアゾールアラ
ニン耐性、Ｄ−リボース耐性）ブレビバクテリウム・ラ
クトファーメンタムＡＪ １１４６１（ＦＥＲＭ−Ｐ
５１８２）（Ｌ−イソロイシン要求性、Ｌ−メチオ
ニン要求性、２−チアゾールアラニン耐性、グアノシン
耐性）ブレビバクテリウム・ラクトファーメンタムＡＪ
１１４６２（ＦＥＲＭ−Ｐ ５１８３）（Ｌ−イソロ
イシン要求性、Ｌ−メチオニン要求性、２−チアゾール
アラニン耐性、ウリジン耐性）
ブレビバクテリウム・フラブム ＡＪ １１４６３（
ＦＥＲＭ−Ｐ ５１８４）（Ｌ−インロイシン要求性
、Ｌ−メチオニン要求性、キサントシン耐性）本発明の
変異株は上記に例示したもののほかに、ブレビバクテリ
ウム・ラクトファーメンタムＡＴＣＣ１３８６９、ブレ
ビバクテリウム・フラブムＡＴＣＣ１４０６７、ブレビ
バクテリウム・デイバリカタムＡＴＣＣ１４０２０，ブ
レビバクテリウム・サラカロリティカムＡＴＣＣ１４０
６６等のブレビバクテリウム属のいわゆるグルタミン酸
生産菌を親株としてＬ−イソロイシン及びＬ−メチオニ
ン要求性並びにＤ−ＩＪボース、プリンリボヌクレオシ
ド又はピリミジンリボヌクレオシドに耐性を有する変異
株を誘導すれば著量のＬ−バリンを生成蓄積する能力を
有する菌株が高い頻度で得られる。Brevibacterium lactofermentum AJ 1
1460 (FERM-P 5181) (L-isoleucine requirement, L-methionine requirement, 2-thiazolealanine resistance, D-ribose resistance) Brevibacterium lactofermentum AJ 11461 (FERM-P
5182) (L-isoleucine requirement, L-methionine requirement, 2-thiazolealanine resistance, guanosine resistance) Brevibacterium lactofermentum AJ
11462 (FERM-P 5183) (L-isoleucine requirement, L-methionine requirement, 2-thiazolealanine resistance, uridine resistance) Brevibacterium flavum AJ 11463 (
FERM-P 5184) (L-inleucine auxotrophy, L-methionine auxotrophy, xanthosine resistance) In addition to those exemplified above, the mutant strains of the present invention include Brevibacterium lactofermentum ATCC13869, Brevibacterium flavum ATCC14067, Brevibacterium deivalicatum ATCC14020, Brevibacterium salacalolyticum ATCC140
If a mutant strain having auxotrophy for L-isoleucine and L-methionine and resistance to D-IJ bose, purinibonucleoside, or pyrimidine ribonucleoside is derived from a so-called glutamate-producing bacterium of the genus Brevibacterium such as Brevibacterium 66 as a parent strain, a significant amount of Bacterial strains capable of producing and accumulating L-valine are obtained with high frequency.
変異誘導方法としては、紫外線照射、Ｘ線照射、放射線
照射、変異誘発剤処理等の通常の方法が用いられば、例
えば２５０１ｎｇ／ＷｌｌのＮ−ニド０−Ｎ’−メチル
−Ｎ−ニトロソグアニジンにより３０’Ｃで２０分間処
理する方法等がある。As a mutation induction method, if a usual method such as ultraviolet irradiation, X-ray irradiation, radiation irradiation, or treatment with a mutagenic agent is used, for example, 2501 ng/Wll of N-nido0-N'-methyl-N-nitrosoguanidine can be used. There is a method of processing at 30'C for 20 minutes, etc.
プリンリボヌクレオシドとしては、イノシン、グアノシ
ン、キサントシン、アデノシン、２，６−ジアミツプリ
ンリボシド等が含まれ、ピリミジンリボヌクレオシドと
しては、ウリジン、シチジン等が含まれる。Examples of purine ribonucleosides include inosine, guanosine, xanthosine, adenosine, 2,6-diamitpurine riboside, and the like, and examples of pyrimidine ribonucleosides include uridine, cytidine, and the like.
ここでいうＤ−リボース、プリンリボヌクレオシドある
いはピリミジンリボヌクレオシド耐性菌とは親株が生育
できない量のＤ−ＩＪボース、プリンリボヌクレオシド
あるいはピリミジンリボヌクレオシドを含有する寒天培
地中で生育し得るような菌株をいう。The D-ribose, purine ribonucleoside, or pyrimidine ribonucleoside-resistant bacteria referred to here refers to a strain that can grow in an agar medium containing D-IJbose, purine ribonucleoside, or pyrimidine ribonucleoside in an amount that the parent strain cannot grow. say.
なお、Ｄ＝’Ｊボース及びプリンリボヌクレオシド及び
ピリミジンリボヌクレオシドに対する耐性度の測定は寒
天プレート濃度勾配によったが、本方法を具体的に以下
に示す。Note that the degree of resistance to D='J bose, purine ribonucleosides, and pyrimidine ribonucleosides was measured using an agar plate concentration gradient, and this method is specifically described below.
まず、例えば１０１０Ｘ５Ｘ３の上共蓋を有する方形ガ
ラス製容器を準備する本容器を蒸気殺菌し１奈た後、寒
天２ｇ／ｄｌ、を含む最少培地（グルコース３．６９／
ｄｌ 、 ＫＨ２ＰＯ４０，１９／ｄｉ、 Ｍｇ５０４
７Ｈ２００，１ｇ／ｄｉ 、 ＦｅＳＯ４−７Ｈ２００
，００１９／ｄｌ 、 Ｍｎ５０４−４ Ｈ２Ｏ０，０
０１１／ｄｌ、尿素１ｇ／ｄｌ、ビオチンｉ ｏ ｏ
ｐｇ／ｌ 、サイアミン塩酸塩２００ ｐｇ／１１．
Ｌ−イソロイシフ ３０ ｍ９７ｄｌ及びＤＬ−メチオ
ニ７３０ｍ９／ｄｌ（ｐ Ｈ８，０（苛性ソーダ水中和
））に、Ｄ−ＩＪボース、プリンリボヌクレオシドある
いはピリミジンリボヌクレオシドを５９７ｄｌになるよ
うに添加し蒸気殺菌して斜面プレートを作る。First, prepare a rectangular glass container, for example, 1010 x 5
dl, KH2PO40,19/di, Mg504
7H200, 1g/di, FeSO4-7H200
,0019/dl, Mn504-4 H2O0,0
011/dl, urea 1g/dl, biotin i o o
pg/l, thiamine hydrochloride 200 pg/11.
D-IJ bose, purinibonucleoside, or pyrimidine ribonucleoside was added to 30 m97 dl of L-isoleusif and 730 m9/dl of DL-methioni (pH 8.0 (hydrated in caustic soda)) to a total volume of 597 dl, and the mixture was sterilized by steam and slanted. Make a plate.
この上に寒天を水平に重層して濃度勾配プレートを作る
。Agar is layered horizontally on top of this to create a concentration gradient plate.
この濃度勾配プレート上に天然培地（ペプトン１ｇ／ｄ
ｉ、イーストエキス１ ｇ／ｃｌｌ ｔ Ｎａ Ｃｌ
ｏ、 ５ｇ／ｄｉ 、 （ｐＨ７，０）スラントで２４
時間培養した各種薬剤耐性株を無菌水で懸濁してその菌
液を線状に塗りつけ、３１．５℃、２４時間培養して生
育帯の長さを測定した。Natural medium (peptone 1 g/d
i, yeast extract 1 g/cll t Na Cl
o, 5g/di, (pH 7,0) 24 with slant
The various drug-resistant strains that had been cultured for several hours were suspended in sterile water, the bacterial solution was applied in a linear pattern, and the cells were cultured at 31.5° C. for 24 hours, and the length of the growth zone was measured.
以上の方法で測定した試験菌株のＤ−ＩＪボース、プリ
ンリボヌクレオシド又はピリミジンリボヌクレオシド耐
性に関する試験結果を第１表に示す。Table 1 shows the test results regarding D-IJ bose, purinribonucleoside, or pyrimidine ribonucleoside resistance of the test strains measured by the above method.
ＡＴＣＣ１３８６９はＡＪ１１４６０．ＡＪ１１４６１
、及びＡＪ１１４６２の親株（野性株うであり、ＡＴＣ
Ｃ１４０６７はＡＣ１１４６３の親株（野性株）である
。ATCC13869 is AJ11460. AJ11461
, and the parent strain of AJ11462 (wild strain, ATC
C14067 is the parent strain (wild strain) of AC11463.
これらの微生物を培養する培地は炭素源、窒素源、無機
イオン及び更に必要に応じその他の有機微量栄養素を含
有する通常の培地である。The medium in which these microorganisms are cultured is a conventional medium containing a carbon source, a nitrogen source, inorganic ions and, if necessary, other organic micronutrients.
炭素源トしては、グルコース、シュークロース及びこれ
らを含有する糖蜜、澱粉加水分解物などの炭水化物、エ
タノール等のアルコール、その他が使用できる。As the carbon source, carbohydrates such as glucose, sucrose and molasses containing these, starch hydrolysates, alcohols such as ethanol, and others can be used.
窒素源としては、アンモニアガス、アンモニア水、アン
モニウム塩、尿素等が好適である。Suitable nitrogen sources include ammonia gas, aqueous ammonia, ammonium salts, urea, and the like.
無機イオンとしては、カリイオン、燐酸イオンマグネシ
ウムイオン等が必要に応じ適宜添加される。As the inorganic ions, potassium ions, phosphate ions, magnesium ions, etc. are added as appropriate.
有機微量栄養素としてはＬ−インロイシン、Ｌ−メチオ
ニン等の被要求物質が培地に添加されるが、更に必要に
応じビタミンその他のアミノ酸適が適宜使用される。Required substances such as L-inleucine and L-methionine are added to the medium as organic micronutrients, and vitamins and other amino acids are also used as appropriate.
培地は好気的条件が好ましい。The medium is preferably under aerobic conditions.
培養の間、培地のｐＨは５ないし９に、温度は２４ない
し３７℃に調節すれば最も好ましい結果が得られる。The most favorable results can be obtained by adjusting the pH of the medium to 5 to 9 and the temperature to 24 to 37° C. during culturing.
発酵液からのＬ−バリンの採取はイオン交換樹脂法等に
より行われる。L-valine is collected from the fermentation liquid by an ion exchange resin method or the like.
実施例 １
下記の組成の培地を２０ｒＩＬｌ宛 ５００ｒｎｌ容振
盪フラスコに入れ１１０℃で１０分間蒸気殺菌した。Example 1 A medium having the following composition was placed in a 500rnl shaking flask containing 20ml and steam sterilized at 110°C for 10 minutes.
これにあらカルめペプトン１ ｇ／ｄｉ、酵母エキス１
ｇ／ｄｉ、グルコース０．５ ｇ／ｄｌ 、 Ｎａ
Ｃ１ｌ Ｏ，５ｇ／ｄｌ、寒天２ ｇ／ｄｌ（ｐＨ７
，０）を含むスラント上で生育せしめた第２表に示す菌
株を一白金耳づつ接種し、３１．５℃にて７２時間振盪
培養した。Add to this 1 g/di of peptone, 1 g/di of yeast extract.
g/di, glucose 0.5 g/dl, Na
C1l O, 5 g/dl, agar 2 g/dl (pH 7
The strains shown in Table 2 grown on slants containing .
ＡＪ１１４６０を上記と同様の方法で培養して培養液１
１を得、その培養液より遠心分離によって菌体および炭
酸カルシウムを除きその５００ｗＬｌを強力チオン交換
樹脂（ｒ Ａｍｂｅｒｌｉｔｅ Ｉ Ｒ−１２０ｊ〔Ｈ
型〕）のカラムに流した。AJ11460 was cultured in the same manner as above to obtain culture solution 1.
1 was obtained, bacterial cells and calcium carbonate were removed from the culture solution by centrifugation, and 500 wL of the culture solution was soaked in strong ion exchange resin (Amberlite I R-120j [H
type]) column.
３％アンモニア水で溶出し、アミノ酸画分を集め、脱塩
及び脱色を行い、減圧濃縮した。Elution was performed with 3% aqueous ammonia, and the amino acid fractions were collected, desalted and decolorized, and concentrated under reduced pressure.
アルコールを添加し、冷却下に保存後生成した結晶を集
めて乾燥した結果、純度９８％以上のＬバリン結晶１０
．１．９が得られた。After adding alcohol and storing under cooling, the resulting crystals were collected and dried, resulting in 10 L valine crystals with a purity of over 98%.
．． 1.9 was obtained.
Claims (1)
びＬ−メチオニン要求性並びにＤ−リボース、プリンリ
ボヌクレオシド又はピリミジンリボヌクレオシドに耐性
を有し更にＬ−バリン生産能を有する変異株を培養し、
培地中に生成蓄積したＬ−バリンを採取することを特徴
とするＬ−バリンの製造法。1. Cultivating a mutant strain belonging to the genus Brevibacterium that is auxotrophic for L-inleucine and L-methionine and resistant to D-ribose, purine ribonucleoside or pyrimidine ribonucleoside, and has the ability to produce L-valine,
A method for producing L-valine, which comprises collecting L-valine produced and accumulated in a culture medium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11368279A JPS5832594B2 (en) | 1979-09-05 | 1979-09-05 | Method for producing L-valine by fermentation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11368279A JPS5832594B2 (en) | 1979-09-05 | 1979-09-05 | Method for producing L-valine by fermentation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5639792A JPS5639792A (en) | 1981-04-15 |
| JPS5832594B2 true JPS5832594B2 (en) | 1983-07-14 |
Family
ID=14618496
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11368279A Expired JPS5832594B2 (en) | 1979-09-05 | 1979-09-05 | Method for producing L-valine by fermentation method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5832594B2 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU1675327A1 (en) * | 1986-12-24 | 1991-09-07 | Научно-Исследовательский Технологический Институт Аминокислот | Method for l-value preparation |
| JP3151073B2 (en) * | 1992-02-25 | 2001-04-03 | 協和醗酵工業株式会社 | Production of amino acids by fermentation |
| WO2013114991A1 (en) | 2012-02-01 | 2013-08-08 | 株式会社カネカ | Method for producing solid amino acid |
-
1979
- 1979-09-05 JP JP11368279A patent/JPS5832594B2/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5639792A (en) | 1981-04-15 |
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