JPS63192395A - Production of l-isoleucine - Google Patents
Production of l-isoleucineInfo
- Publication number
- JPS63192395A JPS63192395A JP2181087A JP2181087A JPS63192395A JP S63192395 A JPS63192395 A JP S63192395A JP 2181087 A JP2181087 A JP 2181087A JP 2181087 A JP2181087 A JP 2181087A JP S63192395 A JPS63192395 A JP S63192395A
- Authority
- JP
- Japan
- Prior art keywords
- aminobutyric acid
- isoleucine
- brevibacterium
- ethanol
- medium
- 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.)
- Pending
Links
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 title claims description 25
- 238000004519 manufacturing process Methods 0.000 title claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 22
- QWCKQJZIFLGMSD-UHFFFAOYSA-N alpha-aminobutyric acid Chemical compound CCC(N)C(O)=O QWCKQJZIFLGMSD-UHFFFAOYSA-N 0.000 claims abstract description 21
- 241000319304 [Brevibacterium] flavum Species 0.000 claims abstract description 13
- 241000186146 Brevibacterium Species 0.000 claims abstract description 12
- 244000005700 microbiome Species 0.000 claims abstract description 10
- QWCKQJZIFLGMSD-VKHMYHEASA-N L-alpha-aminobutyric acid Chemical compound CC[C@H](N)C(O)=O QWCKQJZIFLGMSD-VKHMYHEASA-N 0.000 claims abstract 3
- 229930182844 L-isoleucine Natural products 0.000 claims description 24
- 229960000310 isoleucine Drugs 0.000 claims description 24
- 238000012258 culturing Methods 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 abstract description 8
- 235000013305 food Nutrition 0.000 abstract description 2
- 150000001875 compounds Chemical class 0.000 abstract 2
- 235000015872 dietary supplement Nutrition 0.000 abstract 1
- 239000003814 drug Substances 0.000 abstract 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 18
- 238000000034 method Methods 0.000 description 14
- 239000002609 medium Substances 0.000 description 11
- 229960004295 valine Drugs 0.000 description 9
- 239000006227 byproduct Substances 0.000 description 8
- 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 4
- 241000894006 Bacteria Species 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- QWCKQJZIFLGMSD-GSVOUGTGSA-N D-alpha-aminobutyric acid Chemical compound CC[C@@H](N)C(O)=O QWCKQJZIFLGMSD-GSVOUGTGSA-N 0.000 description 3
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 150000001413 amino acids Chemical class 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
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 2
- 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
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 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
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 235000019157 thiamine Nutrition 0.000 description 2
- 239000011721 thiamine Substances 0.000 description 2
- QIVUCLWGARAQIO-OLIXTKCUSA-N (3s)-n-[(3s,5s,6r)-6-methyl-2-oxo-1-(2,2,2-trifluoroethyl)-5-(2,3,6-trifluorophenyl)piperidin-3-yl]-2-oxospiro[1h-pyrrolo[2,3-b]pyridine-3,6'-5,7-dihydrocyclopenta[b]pyridine]-3'-carboxamide Chemical compound C1([C@H]2[C@H](N(C(=O)[C@@H](NC(=O)C=3C=C4C[C@]5(CC4=NC=3)C3=CC=CN=C3NC5=O)C2)CC(F)(F)F)C)=C(F)C=CC(F)=C1F QIVUCLWGARAQIO-OLIXTKCUSA-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
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 229920002261 Corn starch Polymers 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
- 241001465754 Metazoa Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 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
- 102000003929 Transaminases Human genes 0.000 description 1
- 108090000340 Transaminases Proteins 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229940124277 aminobutyric acid Drugs 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 229940099112 cornstarch Drugs 0.000 description 1
- 239000013078 crystal Substances 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
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000004816 paper chromatography Methods 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
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 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
- 229960003495 thiamine Drugs 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、醗酵法によるＬ−イソロイシンの製造法に関
する。更に詳しくは、ブレビバクテリウム（Ｂｒｅｖｉ
ｂａｃｔｅｒｉｕｍ）属に屈し、エタノール資化性能を
有する微生物を、■、−又はＤＬ−α−アミノ酪酸を含
有する培地に好気的に培養するに際し、培地にＬ−又は
ＤＬ−α−アミノ酪酸を逐次添加することを逐次添加す
るＬ−イソロイシンの製造法に関する。DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for producing L-isoleucine by a fermentation method. For more information, see Brevibacterium (Brevi
When microorganisms belonging to the genus Bacterium and having ethanol assimilation ability are aerobically cultured in a medium containing - or DL-α-aminobutyric acid, L- or DL-α-aminobutyric acid is added to the medium. The present invention relates to a method for producing L-isoleucine in which sequential addition is performed.
（発明の背景）
Ｌ−イソロイシンは必須アミノ酸として、人間及び動物
の栄養上重要な役割をするアミノ酸であり、医療、食品
、飼料強化剤としてその需要が近年急激に増加しつつあ
る。Ｌ−イソロイシンの工業的製造法としては、他のア
ミノ酸の場合と同様に立体異性体が存在する為、化学合
成法ではＬ体のみの製造は困難であり、主に醗酵法によ
り生産が行われている０ｍｍ演法してはＤＬ−α−アミ
ノ酪酸、スレオニン等のＬ−イソロイシンの前駆物質を
使用する方法（特公昭４３−８７０９゜特公昭４０−２
８８０等）、前駆物質を特に加えない所謂直接醗酵法（
特公昭３８−７０９１．特開昭４９−９３５８６等）が
ある。(Background of the Invention) L-isoleucine is an amino acid that plays an important role in the nutrition of humans and animals as an essential amino acid, and its demand as a medical, food, and feed enrichment agent has been increasing rapidly in recent years. As for the industrial production method of L-isoleucine, it is difficult to produce only the L-isomer by chemical synthesis method because stereoisomers exist like other amino acids, so it is mainly produced by fermentation method. The 0 mm method using L-isoleucine precursors such as DL-α-aminobutyric acid and threonine (Japanese Patent Publication No. 43-8709゜Special Publication No. 40-2)
880, etc.), the so-called direct fermentation method in which no precursors are added (
Tokuko Showa 38-7091. JP-A-49-93586, etc.).
（従来の技術と課題）
本発明者らは、すでにＤＬ−α−アミノ酪酸を原料とす
るＬ−イソロイシンの製造法を堤案している（特公昭５
７−２６７５５号、特開昭５３−７５３８６号）が、該
方法によれば、副生アミノ酸は比較的少ないものの、Ｌ
−バリンの副生が認められる。(Prior art and problems) The present inventors have already proposed a method for producing L-isoleucine using DL-α-aminobutyric acid as a raw material (Japanese Patent Publication No. 5
7-26755, JP-A No. 53-75386), according to this method, although by-product amino acids are relatively small, L
- Valine by-product is observed.
Ｌ−バリンとＬ−イソロイシンの分離は比較的複雑な工
程を要することや、対原料収率を向上させる等の観点か
ら従来よりＬ−バリン副生の抑制方法の開発が望まれて
いた。The separation of L-valine and L-isoleucine requires a relatively complicated process, and from the viewpoint of improving the yield based on raw materials, it has been desired to develop a method for suppressing L-valine by-products.
本発明者らは、Ｌ−バリン副生と培養方法との相関につ
いて鋭意検討した結果、培養中培地のＬ−又はＤＬ−ア
ミノ酪酸の濃度を低濃度に保持させることにより、Ｌ−
バリンの副生量を大幅に低減下し、好収量でＬ−イソロ
イシンを生成出来ることを見出し本発明を完成するに到
った。As a result of intensive studies on the correlation between L-valine by-products and culture methods, the present inventors found that by maintaining the concentration of L- or DL-aminobutyric acid in the culture medium at a low concentration, L-valine
The present invention was completed by discovering that L-isoleucine can be produced in good yields by significantly reducing the amount of valine by-product.
（発明の構成及び効果）
本発明者らは、ブレビバクテリウム（Ｂｒｅｖｉｂａｃ
Ｌｅｒｉｕｓ）属に属し、エタノール資化性を有する微
生物をＬ−又はＤＬ−α−アミノ酪酸を含有する培地に
好気的に培養して、培地中にＬ−イソロイシンを製造す
るに際し、前述の如く培地中のし−又はＤＬ−α−アミ
ノ酪酸の濃度を低濃度に保持しつつこれらのアミノ酪酸
を培地に逐次添加して培養することによりＬ−バリンの
副生が抑制され、Ｌ−インロイシンが、培地中に高収量
で蓄積することを見出し、本発明に到達した。培地中の
し−又はＤＬ−α−アミノ酪酸の濃度とＬ−バリン副生
量の相関は本発明による新規な知見であり、本発明は新
規な方法である。(Structure and Effects of the Invention) The present inventors have discovered that Brevibacterium
When producing L-isoleucine in the medium by aerobically cultivating a microorganism belonging to the genus Lerius and capable of assimilating ethanol in a medium containing L- or DL-α-aminobutyric acid, as described above, By sequentially adding these aminobutyric acids to the medium and culturing while maintaining the concentration of L- or DL-α-aminobutyric acid in the medium at a low concentration, the by-production of L-valine is suppressed, and L-inleucine was found to accumulate in the culture medium in high yield, leading to the present invention. The correlation between the concentration of L- or DL-α-aminobutyric acid in the medium and the amount of L-valine byproduct is a novel finding by the present invention, and the present invention is a novel method.
（発明の具体的方法）
本発明に使用される微生物はブレビバクテリウム（Ｂｒ
ｅｖｉｂａｃｔｅｒｉｕｍ） ｍに属しエタノール資化
性のものであれば好い、このなかにはＬ−イソロイシン
生産菌が含まれる。該生産菌は例えば、ブレビバクテリ
ウム・フラバム（ＢｒｅｖｉｂａｃｔｅｒｉｕＩ 目ａ
ｖｕｍ） Ｍ Ｊ −２３３（微工研菌寄 第３０６８
号）、ブレビバクテリウム・フラバム（Ｂｒｅｖｉｂａ
ｃＬｅｒｉｕｍ Ｎａｖｕｍ） ＭＪ−２３３−ＡＢ
−４１（微工研菌寄 第３８１２号）、ブレビバクテリ
ウム・フラバム（Ｂｒｅｖｉｂａｃｔｅｒｉｕｍ ｆ
ｌａｖｕｗ） Ｍ Ｊ −２３３−ＡＢＴ−１１（微工
研菌寄 第８４２３号）及びブレビバクテリウム・フラ
バム（Ｂｒｅｖｉｂａｃｔｅｒｉｕｍ ｆｌａｖｕ
ｍ） ＭＪ−２３３−ＡＢＤ−２１（微工研菌寄 第
８０５５号）等であり、本発明に好適に用いられる。(Specific method of the invention) The microorganism used in the present invention is Brevibacterium (Br.
evibacterium) that can assimilate ethanol, including L-isoleucine-producing bacteria. The producing bacterium is, for example, Brevibacterium flavum (Brevibacterium order a).
vum) M J-233 (Feikoken Bacillus No. 3068
), Brevibacterium flavum (Breviba
cLerium Navum) MJ-233-AB
-41 (Feikoken Bacteria No. 3812), Brevibacterium flavum (Brevibacterium f.
lavuw) M J -233-ABT-11 (Feikoken Bibori No. 8423) and Brevibacterium flavu
m) MJ-233-ABD-21 (Feikoken Bibori No. 8055) and the like, which are suitably used in the present invention.
なお、上記の（微工研菌寄 第３８１２号）は（微工研
菌寄 第３０６８号）を親株としてＤＬ−α−アミノ酪
酸耐性を積極的に付与されたエタノール資化性微生物で
ある（特公昭５９−２８３９８号公報３〜４欄参照）、
（微工研菌寄 第８４２３号）は、（微工研菌寄 第３
０６８号）を親株としたし一α−アミノ酪酸トランスア
ミナーゼ高活性変異株である（特願昭６０−１９０６０
９号明細書３〜５頁参照）、また、（微工研菌寄第８０
５５号）は（微工研菌寄 第３０６８号）を親株とした
Ｄ−α−アミノ酪酸デアミナーゼ高活性変異株である（
特願昭６０−０１７５０１号明細書５〜７頁参照）。In addition, the above-mentioned (Feikoken Bokuyori No. 3812) is an ethanol-assimilating microorganism that has been actively imparted with DL-α-aminobutyric acid resistance using (Feikoken Bikyouri No. 3068) as its parent strain ( (See columns 3 and 4 of Japanese Patent Publication No. 59-28398),
(Feikoken Bikori No. 8423) is (Feikoku Kenbiyori No. 3)
068) as the parent strain and is a mutant strain with high activity of α-aminobutyric acid transaminase (Patent application No. 19060-1980).
(Refer to pages 3 to 5 of the specification of No. 9)
No. 55) is a D-α-aminobutyric acid deaminase high activity mutant (
(See pages 5-7 of the specification of Japanese Patent Application No. 60-017501).
以下、更に本発明のＬ−イソロイシンの製造法を具体的
に説明する。Hereinafter, the method for producing L-isoleucine of the present invention will be further explained in detail.
本発明の菌体調製に使用する培地組成は、好ましくはエ
タノールを主炭素源とするが、特に主炭素源の限定は無
く、一般の微生物に使用されるもので良い、窒素源とし
てはアンモニア、硫酸アンモニウム、塩化アンモニウム
、硝酸アンモニウム、尿素等を単独若しくは混合して用
いることが出来る。The medium composition used for the preparation of microbial cells of the present invention preferably uses ethanol as the main carbon source, but there is no particular limitation on the main carbon source, and any material used for general microorganisms may be used.As the nitrogen source, ammonia, Ammonium sulfate, ammonium chloride, ammonium nitrate, urea, etc. can be used alone or in combination.
無機塩としては、リン酸−水素カリウム、リン酸二水素
カリウム、硫酸マグネシウム等が用いられる。この他に
菌の生育及びＬ−イソロイシン生成に必要であれば、ペ
プトン、肉エキス、酵母エキス、コーンステイープリカ
ー、カザミノ酸、各種ビタミン等の栄養素を培地に添加
し用いる。As the inorganic salt, potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, etc. are used. In addition, nutrients such as peptone, meat extract, yeast extract, cornstarch liquor, casamino acid, and various vitamins may be added to the medium if necessary for bacterial growth and L-isoleucine production.
Ｌ−イソロイシンの前駆体となるＬ−又はＤＬ−α−ア
ミノ酪酸の初発濃度は、好ましくはそれぞれ、５ｍＭ
〜３０ｍＭ、１０ｍＭ 〜６０ｍＭである。培養中の培
地のＬ−又はＤＬ−α−アミノ酪酸の濃度は、液体クロ
マトグラフィーにより経時的に測定し、好ましくはＬ−
α−アミノ酪酸は３０ｍＭ、またＤＬ−ａ−アミノ酪酸
では６０ｍＭを越えないようにする。培養は通気攪拌、
振盪等の好気的的条件下で行い、培養温度は２０〜４０
℃、好ましくは２５〜３５℃で行う、培養途中のｐｉ−
１は５〜１０．好ましくは７〜８付近にて行い、培養中
のｐＨの調整には酸、アルカリを添加して行う。The initial concentration of L- or DL-α-aminobutyric acid, which is a precursor of L-isoleucine, is preferably 5 mM each.
~30mM, 10mM ~60mM. The concentration of L- or DL-α-aminobutyric acid in the culture medium is measured over time by liquid chromatography, preferably L- or DL-α-aminobutyric acid.
α-aminobutyric acid should not exceed 30mM, and DL-a-aminobutyric acid should not exceed 60mM. Culture with aeration and agitation.
It is carried out under aerobic conditions such as shaking, and the culture temperature is 20-40℃.
℃, preferably 25 to 35℃, pi-
1 is 5-10. Preferably, the pH is around 7 to 8, and the pH during cultivation is adjusted by adding acid or alkali.
培養開始時のエタノール濃度は好ましくは１〜５容量％
、更に好ましくは２〜３容量％が適する。The ethanol concentration at the start of culture is preferably 1 to 5% by volume.
, more preferably 2 to 3% by volume.
培養期間は１〜９日間、最適期間は４〜７日間である。The culture period is 1-9 days, with an optimal period of 4-7 days.
上記のような培養方法によって得られる培養液中に生成
したＬ−イソロイシンの分離・Ｉｉｓは、イオン交換樹
脂処理法あるいは、沈澱法等により行うことが出来る。Separation/Iis of L-isoleucine produced in the culture solution obtained by the above culture method can be performed by an ion exchange resin treatment method, a precipitation method, or the like.
（実施例）
以下に実施例を示す、なお、Ｌ−インロイシン及び副生
Ｌ−バリンの定性は、ペーパークロマトグラフのＲｆ値
、電気泳動法の易動度及び微生物定量法による生物活性
値により確認した。定量はロイコノストック・メセンテ
ロイデス（Ｌｅｕｃｏｎｏｓｔｏｅ ｍｅｓｅｎｔｅｒ
ｏｉｄｅｓ） Ａ Ｔ ＣＣ８０４２を用いるマイクロ
バイオアッセイ法と高速液体クロマトグラフィー（島津
ＬＣ−５Ａ）とを併用して行った。また、下記の実施例
において％と表したのは重量％を意味する。(Example) Examples are shown below. The qualitative characteristics of L-inleucine and by-product L-valine are determined based on the Rf value of paper chromatography, the mobility of electrophoresis, and the biological activity value of microorganism quantitative method. confirmed. The determination was made using Leuconostoe mesenteroides.
A microbioassay method using AT CC8042 and high performance liquid chromatography (Shimadzu LC-5A) were used in combination. Moreover, in the following examples, % means weight %.
実施例−１
培地（尿素０．４％、硫酸アンモニウム１．４％、Ｋ
Ｈ２Ｐ Ｏ４０、０５％、Ｋ２ＨＰＯ，Ｑ。Example-1 Medium (urea 0.4%, ammonium sulfate 1.4%, K
H2P O40, 05%, K2HPO,Q.
０５％、Ｍｇ５Ｏ，・７Ｈ，ＯＯ，０５％、ＣａＣ１，
・２Ｈｚ Ｏ２ｐｐｍｓ ＦｅＳＯ４Ｈ７Ｈ２０２ｐ
ｐｍ、 Ｍｎ５Ｏａ ＋ ４〜６ Ｈｚ Ｏ２ｐｐｍ
、Ｚｎ５Ｏａ ・７Ｈｚ ０ ２ｐｐｍ。05%, Mg5O, 7H, OO, 05%, CaCl,
・2Hz O2ppms FeSO4H7H202p
pm, Mn5Oa + 4-6 Hz O2ppm
, Zn5Oa ・7Hz 0 2ppm.
ＮａＣｊ！ ２ｐＩ）ｍ１ビオチン ２００．ｕｇ／
Ｊ、チアミン・Ｈ（１１００μｇ／ｌ、カザミノ酸 ０
．１％、酵母エキス ０．１％ ）１００ｍｌを５００
ｍｌ容三角フラスコに分注、滅菌（滅菌後ｐＨ７，０）
した後ブレビバクテリウム・フラバム（Ｂｒｅｖｉｂａ
ｃｔｅｒｉｕｍ ｆｌａｖｕｍ） Ｍ Ｊ −２３３
（微工研菌寄 第３０６８号）を植菌し、無菌的にエタ
ノールを２ｍｌ加え、３０℃にて２日間振盪培養を行っ
た（前培養）。NaCj! 2pI) m1 biotin 200. ug/
J, Thiamine H (1100μg/l, Casamino acids 0
．． 1%, yeast extract 0.1%) 100ml to 500ml
Dispense into ml Erlenmeyer flask and sterilize (pH 7.0 after sterilization)
After that, Brevibacterium flavum (Breviba
cterium flavum) M J -233
(Feikoken Bokuyori No. 3068) was inoculated, 2 ml of ethanol was added aseptically, and shaking culture was performed at 30°C for 2 days (preculture).
次に、本培養培地（硫酸アンモニウム２．３％、ＫＨ２
ＰＯ２０，０５％、Ｋ、ＨＰＯ４０，０５％、Ｍｇ５ｏ
４ ・７Ｈ２００，０５％、Ｆｅ３Ｏ４・ＩＨ，０２
０ｐｐｍ、Ｍｎ５Ｏ，−ｎＨ２Ｏ２０ｐｐｍ、 ビオチ
ン２００．ｕｇ／ｊ！、チアミン・Ｉ Ｃ１１００μｇ
／ｌ、カザミノ酸０．３％、酵母エキス０．３％、ＤＬ
−α−アミノ酪ｒ！１０．５％）の１０１００Ｏを２１
容通気攪拌槽に仕込み、滅菌（１２０℃、２０分間）後
、エタノールの２０ｍｊ！と前記前培養物の２０ｍ１を
添加して、回転数１１００Ｏｒｐ、通気量ｌｖ ｖ ｍ
、温度３３℃、ｐＨ７，６にて２４時間培養を行った
。Next, the main culture medium (ammonium sulfate 2.3%, KH2
PO20.05%, K, HPO40.05%, Mg5o
4 ・7H200,05%, Fe3O4・IH,02
0ppm, Mn5O, -nH2O20ppm, biotin 200. ug/j! , Thiamin I C1100μg
/l, casamino acid 0.3%, yeast extract 0.3%, DL
-α-aminobutyro! 10.5%) of 10100O to 21
Pour into a well-ventilated stirring tank, sterilize (120°C, 20 minutes), and add 20mj of ethanol! and 20 ml of the above preculture, the rotation speed was 1100 Orp, and the aeration volume was lv v m.
The cells were cultured for 24 hours at a temperature of 33° C. and a pH of 7.6.
尚、エタノールは、培養中培地中の濃度が２容量％を越
えないように、約１〜２時間ごと断続的に添加した。Incidentally, ethanol was added intermittently about every 1 to 2 hours so that the concentration in the medium did not exceed 2% by volume during the culture.
また、ＤＬ−α−アミノ酪酸は６０ｍＭを越えないよう
に約１〜２時間ごとに逐次添加し、最終の添加後に添加
量の総量が延べ８０ｍＭとなるようにした。Further, DL-α-aminobutyric acid was added sequentially every 1 to 2 hours so as not to exceed 60 mM, and after the final addition, the total amount added was 80 mM.
培養終了後、遠心分離（４０００ｒｐｍ、１５分間、室
温）にて除菌した上清液中のＬ−イソロイシンを定量し
た。また、培養液５００ｍＪを強酸性陽イオン交換樹脂
（Ｈ＋型）のカラムに通してＬ−イソロイシンを吸着さ
せ、水洗後、０．５Ｎアンモニア水で溶出させたのち、
Ｌ−イソロイシン画分を濃縮し、冷エタノールでＬ−イ
ソロイシンの結晶を析出させた。結果を、後に掲げる第
１表に示した。After completion of the culture, L-isoleucine in the supernatant liquid was sterilized by centrifugation (4000 rpm, 15 minutes, room temperature) and quantified. In addition, 500 mJ of the culture solution was passed through a column of strongly acidic cation exchange resin (H+ type) to adsorb L-isoleucine, washed with water, and eluted with 0.5N ammonia water.
The L-isoleucine fraction was concentrated, and L-isoleucine crystals were precipitated with cold ethanol. The results are shown in Table 1 below.
尚、ＤＬ−α−アミノ酪酸を培養開始時に培地に一括し
て８０ ｍ Ｍ゛ 添加した場合のＬ−イソロイシン
の生成量及びＬ−バリンの副生量を比較例とした。In addition, the amount of L-isoleucine produced and the amount of by-product L-valine when DL-α-aminobutyric acid was added at once to the medium at 80 mM at the start of culture were used as a comparative example.
実施例−２
実施例−１と同様の条件にてブレビバクテリウム・フラ
バム（Ｂｒｅｖｉｂａｃｔｅｒｉｕｍ ｆｌａｖｕｍ
） Ｍ Ｊ −２３３−ＡＢ−４１（微工研菌寄 第３
８１２号）を培養し、更にＬ−イソロイシンの精製を行
った。結果を後に掲げる第２表に示した。Example-2 Brevibacterium flavum was grown under the same conditions as Example-1.
) M J -233-AB-41 (Microtechnology Research Institute 3rd
No. 812) was cultured, and L-isoleucine was further purified. The results are shown in Table 2 below.
実施Ｎ−３
実施例−１と同様の条件にてブレビバクテリウム・フラ
バム（Ｂｒｅｖｉｂａｃｔａｒｉｕｓ ｆｌａｖｕｓ）
Ｍ Ｊ −２３３−ＡＢＴ−１１（微工研菌寄 第８
４２３号）を培養し、あとＬ−イソロイシンの精製を行
った。結果を後に掲げる第３表に示した。Implementation N-3 Brevibacterium flavus under the same conditions as Example-1
M J -233-ABT-11 (Fiber Engineering Laboratory No. 8
No. 423) was cultured, and L-isoleucine was purified. The results are shown in Table 3 below.
実施例−４
実施例−１と同様の条件にてブレビバクテリウム・フラ
バム（Ｂｒｅｖｉｂａｃｔｅｒｉｕｍ＋ ｆｌａｖｕ
ｍ） Ｍ Ｊ −２３３−ＡＢＣ−２１（微工研菌寄
第８０５５号）を培養し、あとＬ−イソロイシンの精製
を行った。結果を後に掲げる第４表に示した。Example-4 Brevibacterium flavum (Brevibacterium + flavu) was grown under the same conditions as Example-1.
m) M J -233-ABC-21 (Microtechnical Research Institute
No. 8055) was cultured, and L-isoleucine was purified. The results are shown in Table 4 below.
第１表 第２表 第３表 第４表 （駆逢堀」Table 1 Table 2 Table 3 Table 4 (Kohohori)
Claims (3)
ｕｍ）属に属するエタノール資化性微生物をＬ−又はＤ
Ｌ−α−アミノ酪酸を含有する培地に好気的に培養して
培地中にＬ−イソロイシンを生成せしめるに際し、培養
中の培地にＬ−又はＤＬ−α−アミノ酪酸を逐次添加す
ることを特徴とするＬ−イソロイシンの製造法。(1) Brevibacterium
um) ethanol-assimilating microorganisms belonging to the genus L- or D
When culturing aerobically in a medium containing L-α-aminobutyric acid to produce L-isoleucine in the medium, L- or DL-α-aminobutyric acid is sequentially added to the medium during culturing. A method for producing L-isoleucine.
越えないようにＬ−又はＤＬ−α−アミノ酪酸を逐次添
加する特許請求の範囲第１項記載の製造法。(2) The production method according to claim 1, wherein L- or DL-α-aminobutyric acid is added sequentially so that the concentration of L-α-aminobutyric acid in the medium does not exceed 30 mM.
ｕｍ）属に属し、エタノール資化性を有する微生物がブ
レビバクテリウム・フラバム（Ｂｒｅｖｉｂａｃｔｅｒ
ｉｕｍ ｆｌａｖｕｍ）ＭＪ−２３３、ブレビバクテリ
ウム・フラバムＭＪ−２３３−ＡＢ−４１、ブレビバク
テリウム・フラバムＭＪ−２３３−ＡＢＴ−１１、ブレ
ビバクテリウム・フラバムＭＪ−２３３−ＡＢＤ−２１
である特許請求の範囲第１項又は同第２項記載の製造法
。(3) Brevibacterium
Brevibacterium flavum is a microorganism that belongs to the genus um and has the ability to assimilate ethanol.
Brevibacterium flavum) MJ-233, Brevibacterium flavum MJ-233-AB-41, Brevibacterium flavum MJ-233-ABT-11, Brevibacterium flavum MJ-233-ABD-21
The manufacturing method according to claim 1 or 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2181087A JPS63192395A (en) | 1987-02-03 | 1987-02-03 | Production of l-isoleucine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2181087A JPS63192395A (en) | 1987-02-03 | 1987-02-03 | Production of l-isoleucine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS63192395A true JPS63192395A (en) | 1988-08-09 |
Family
ID=12065411
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2181087A Pending JPS63192395A (en) | 1987-02-03 | 1987-02-03 | Production of l-isoleucine |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63192395A (en) |
-
1987
- 1987-02-03 JP JP2181087A patent/JPS63192395A/en active Pending
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