JPS6274293A - Production of l-isoleucine - Google Patents
Production of l-isoleucineInfo
- Publication number
- JPS6274293A JPS6274293A JP60215718A JP21571885A JPS6274293A JP S6274293 A JPS6274293 A JP S6274293A JP 60215718 A JP60215718 A JP 60215718A JP 21571885 A JP21571885 A JP 21571885A JP S6274293 A JPS6274293 A JP S6274293A
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- JP
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- Prior art keywords
- isoleucine
- culture
- medium
- microorganism
- produce
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/02—Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion
- C12N15/03—Bacteria
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- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
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- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
産業上の利用分野
本発明はプロトプラスト融合により得たし一イソロイ／
ン生産能を有するコリネバクテリウム属に属する微生物
を用いるし一イソロイシンの製造法に関する。[Detailed Description of the Invention] Industrial Field of Application The present invention relates to the isoloy/
This invention relates to a method for producing isoleucine using a microorganism belonging to the genus Corynebacterium that has the ability to produce isoleucine.
Ｌ−イソロイシンはアミノ酸製剤として医薬品として有
用である。L-isoleucine is useful as an amino acid preparation as a medicine.
従来の技術
従来、発酵法によるし一インロインンの製造法としては
ミクロコツカス、グルタミクムに嘱し、スレオニン、メ
チオニン、ホモセリン又はロインン要求性を存する微生
物を用いる方法（特公昭３８−７０９１号公報）、ブレ
ビバクテリウム・フラバムに属し、α−アミ７−β−ヒ
ドロキ／吉草酸耐性及びプリン又はリジン要求性を有す
る微生物を用いる方法（特開昭４９−８５２８８号公報
）、α−アミノ−β−ヒドロキ／吉草酸及び０−メチル
スレオニン相性を有するｊ敢生物を用いる方法（特開昭
４９−９３５８６号公報）、ブレビバクテリウム属又は
コリネバクテリウム、属に属し、α−アミノ−β−ヒド
ロキシ吉草酸耐性でかつエチオニン、２−チアゾールア
ラニン及びＳ−（２−アミノエチル）−Ｌ−システィン
のうち少なくとも１つに耐性を有する微生物を用いる方
法（特開昭５０−１０１５８２号公報）、コリネバクテ
リウム屈に属し、イソロイノンアナログ耐性でかつメチ
オニン又はリジン要求性を有する微生物を用いる方法（
特開昭４９−１１４５２号公報）、コリネバクテリウム
属又はブレビバクテリウム属に属し、ビタミン−Ｐ耐性
を有する微生物を用いる方法（特開昭５７１６３７号公
報）、コリネバクテリウム属に属し、アルギニン要求性
を有する微生物を用いる方法（特開昭５９−１０６２９
５号公報）、コリネバクテリウム属に属し、フルオロピ
ルビン酸感受性を有する微生物を用いる方法（特開昭５
９−１０６２９４号公報）等が知られている。Conventional technology Conventionally, fermentation methods have been used to produce loins, including Micrococcus and Glutamicum, and methods using microorganisms that have threonine, methionine, homoserine, or loin auxotrophy (Japanese Patent Publication No. 7091/1983), and Brevibacterium. A method using a microorganism belonging to U.um flavum and having α-amino-7-β-hydroxy/valeric acid resistance and purine or lysine auxotrophy (Japanese Unexamined Patent Publication No. 1985-85288), α-amino-β-hydroxy/valeric acid A method using biological organisms having compatibility with grass acid and 0-methylthreonine (Japanese Unexamined Patent Publication No. 49-93586), which belongs to the genus Brevibacterium or Corynebacterium and is resistant to α-amino-β-hydroxyvaleric acid. and a method using a microorganism resistant to at least one of ethionine, 2-thiazolealanine, and S-(2-aminoethyl)-L-cysteine (Japanese Patent Application Laid-open No. 101582/1982), which belongs to the Corynebacterium genus , a method using microorganisms that are resistant to isoloinone analogs and have methionine or lysine auxotrophy (
JP-A-49-11452), a method using microorganisms belonging to the genus Corynebacterium or Brevibacterium and having vitamin-P resistance (JP-A-571637); A method using microorganisms with
5), a method using microorganisms belonging to the genus Corynebacterium and sensitive to fluoropyruvate (Japanese Patent Laid-open No. 5
9-106294) and the like are known.
発明が解決しようとする問題点
プロトプラスト融合により得たし一イソロイシン生産能
を有するコリネバクテリウム属に属する微生物を用いる
ことにより、親株より大量にＬ−インロインンを製造す
る方法は開発されていない。Problems to be Solved by the Invention No method has been developed for producing L-inroin in larger amounts than the parent strain by using a microorganism belonging to the genus Corynebacterium and having the ability to produce isoleucine obtained by protoplast fusion.
問題点を解決するための手段
本発明方法によると、プロトプラスト融合により得たし
一イソロイシン生産能を有するコリネバクテリウム属に
属する微生物を培地に培養し、培養物中にＬ−イソロイ
シンを生成蓄積させ、該培養物からＬ−イソロイシンを
収率よく採取することができる。Means for Solving the Problems According to the method of the present invention, a microorganism belonging to the genus Corynebacterium and having the ability to produce L-isoleucine obtained by protoplast fusion is cultured in a medium, and L-isoleucine is produced and accumulated in the culture. , L-isoleucine can be collected from the culture with good yield.
本発明に用いる微生物としてはＬ−イソロイシン生産性
を有する微生物と主発酵生産物がＬ−イソロイシン以外
のアミノ酸を生産する能力を有する微生物との間のプロ
トプラスト融合株であればいずれも用いられる。As the microorganism used in the present invention, any protoplast fusion strain between a microorganism capable of producing L-isoleucine and a microorganism capable of producing an amino acid other than L-isoleucine as a main fermentation product can be used.
具体的には、コリネバクテリウム・グルタミクム （Ｃ
ｏｒｙｎｅｂａｃｔｅｒｉｕｍ ｇｌｕｔａｍｉｃｕ
ｍ） Ｈ−３８５９（以下、Ｉ（−３８５’ｌと称
す）及びＨ−３８６０（以下、Ｈ−３８６０と称す）が
挙げられ、いずれも茨城県筑波郡谷田部町東１丁目１番
３の】ｍ産省工業技術院微生物工業技術研究所に微工研
条寄第８４９号及び第８５０号として昭和６０年７月１
６日に寄託されている。Specifically, Corynebacterium glutamicum (C
orynebacterium glutamicu
m) H-3859 (hereinafter referred to as I (-385'l)) and H-3860 (hereinafter referred to as H-3860), both located at 1-1-3 Higashi, Yatabe-cho, Tsukuba-gun, Ibaraki Prefecture]m On July 1, 1985, it was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of Industry, as Microtechnology Research Institute No. 849 and No. 850.
It was deposited on the 6th.
次にプロトプラスト融合株の取得例を示す。Next, an example of obtaining a protoplast fusion strain will be shown.
親株として、イソロイシン生産菌であるコリネバクテリ
ウム・グルタミクムＨ−３５０１（ＦＥＲＭ ＢＰ−
８４８）（以下、Ｈ−３５０１と称す）及びリジン生産
菌であるコリネバクテリウム・グルタミクムＨ−３１１
９（ＦＥＲＭ ＢＰ−１４９）（以下、Ｈ−３１１９
と称す）を用いる。As a parent strain, Corynebacterium glutamicum H-3501 (FERM BP-
848) (hereinafter referred to as H-3501) and Corynebacterium glutamicum H-311, a lysine-producing bacterium.
9 (FERM BP-149) (hereinafter referred to as H-3119
) is used.
培養細胞からプロトプラストを形成させるためには例え
ば栄養培地ＮＢ（粉末ブイヨン２０ｇ１酵母エキス５ｇ
を純水１１に含みｐ Ｈ７，２に調整した培地）に菌を
接種し、振盪培養する。比色計によって６６０ｎｍにお
ける吸光度（００）を測定し、対数増殖期の初期（ＯＤ
＝０．１〜０．２）に培養液中０．１〜０．８ ｕ ／
ｍｌの濃度になるようにペニシリンＧを添加する。培養
を続けて、さらにＯＤが０．３〜０．５に増加したとこ
ろで細胞を集菌し、５５Ｍ培地〔グルコース１０ｇ５Ｎ
Ｈ，Ｃ１４ｇ、尿素２ｇ、酵母エキス１ｇ、Ｉ＜）４２
Ｆ’０４１ ｇ 、 Ｋ ２ ＨＰ Ｏ４３ｇ 、 Ｍ
ｇ Ｃｌ ２・６６Ｈ２Ｏ１４，Ｆｅ５０＊７Ｈ２０
１０ｍｇ、ＭｎＳＯ４・４〜６日２０ Ｑ、２ｍｇ１Ｚ
ｎＳＯ４・７Ｈ２００，９ｍｇ、Ｃｕ５Ｏａ・５Ｈ２０
０，４ｍｇ、ＮａｚＢｍ○７＝１０Ｈｚ０ ０．０９ｍ
ｇ、（ＮＨ＜）ｓＭｏｅＯｚ＊−４８２００，０４のｇ
、ビオチン３０１１！Ｚ、サイアミン塩酸塩１ｍｇを純
水１１に含み、ｐ Ｈ７，２に調整した培地、アミノ酸
要求性の菌の培養には、要求アミノ酸５０■／ｍｌを追
加〕で洗浄する。次に菌体をＰＦＭ培地（３３Ｍ２倍希
釈液中にシュクロース０．４ Ｍ 、 Ｍ ｇ Ｃｌ
２ ・６Ｈ２００，０１Ｍを含み、ｐ ）ｉ ７．
Ｏ〜８．５に調整した培地）に再懸濁する。この細胞懸
濁液にリゾチーム（０，２〜２■／ｍｌ）を加え、３０
〜３７℃で１６時間処理する。In order to form protoplasts from cultured cells, for example, nutrient medium NB (20 g of powdered broth 1 5 g of yeast extract)
The bacteria are inoculated into a medium (containing 11% pure water and adjusted to pH 7.2) and cultured with shaking. The absorbance (00) at 660 nm was measured by a colorimeter, and the absorbance (00) at 660 nm was measured at
= 0.1-0.2) in culture medium at 0.1-0.8 u/
Add penicillin G to a concentration of ml. Continuing the culture, when the OD further increased to 0.3 to 0.5, the cells were harvested and added to 55M medium [glucose 10g 5N
H, C14g, urea 2g, yeast extract 1g, I<)42
F'041g, K2HP O43g, M
g Cl2・66H2O14,Fe50*7H20
10mg, MnSO4・4-6 days 20Q, 2mg1Z
nSO4・7H200, 9mg, Cu5Oa・5H20
0.4mg, NazBm○7=10Hz0 0.09m
g, (NH<)sMoeOz*-48200,04g
, Biotin 3011! Z, a medium containing 1 mg of thiamine hydrochloride in pure water 11 and adjusted to pH 7.2; for culturing amino acid-requiring bacteria, add 50 μl/ml of requisite amino acids]. Next, the bacterial cells were cultured in PFM medium (33M 2-fold diluted solution containing 0.4 M sucrose, M g Cl
2 ・6H200,01M, p)i 7.
Resuspend in medium adjusted to 0~8.5. Add lysozyme (0.2 to 2 μ/ml) to this cell suspension and
Treat at ~37°C for 16 hours.
プロトプラストの形成を光学顕微鏡で確認する。Confirm protoplast formation using an optical microscope.
融合させようとする菌株の、上記の如くして得たプロト
プラストの数を計測し１：１になるように混合し、遠心
分離によりＰＦＭ培地で洗浄後０．１ｍｌに再懸濁する
。これに４０％ポリエチレングリコール（ＰＥＧ）４．
０００を含むＰＦＭ培地２．５ｍｌを加え静かに攪拌す
る。５分後、Ｑ、３ｍｌを例えばストレプトマイシン及
びリファンピシンをマーカーとした場合には、ストレプ
トマイシン１００■／ｍｌ及びリファンビンン１■／ｍ
ｌを含むＰＣＧ寒天平板培地（グルコース５ｇ、カザミ
ノ酸５ｇ、酵母エキス２．５ｇ、に２ＨＰＯ４３，５ｇ
、ＫＨ２ＰＯ４１，５ｇ、ＭｇＣｌ２・６Ｈ２００６４
１ｇ５ＦｅＳＯｎ・７Ｈ２０１０ｍｇ、ＭｎＳＯ４・４
〜６８２０ ２ｍｇ、ＺｎＳＯ４・７Ｈ２００，９ｍｇ
、 Ｃｕ ＳＯ＋ ・５ Ｈ２Ｏ０，４ｍｇ、 Ｎ ａ
ｚＢｓｏｔ・１０ＨｚＯＯ，０９ｍｇ、（Ｎ Ｈｔ ）
６ Ｍ ｏ ｓ ０２１・４ Ｈ２Ｏ０，０４ｍｇ−
ビオチン３０■、サイアミン塩酸塩２Ｉ＋Ｉｇ、コハク
酸ナトリウム１３５ｇ。The number of protoplasts of the strain to be fused obtained as described above is counted, mixed at a ratio of 1:1, washed with PFM medium by centrifugation, and resuspended in 0.1 ml. Add to this 40% polyethylene glycol (PEG).
Add 2.5 ml of PFM medium containing 000 and stir gently. After 5 minutes, 3 ml of Q, for example, if streptomycin and rifampicin are used as markers, streptomycin 100 / ml and rifampicin 1 / ml
PCG agar plate medium containing l (5g glucose, 5g casamino acids, 2.5g yeast extract, 43.5g 2HPO)
, KH2PO41.5g, MgCl2・6H20064
1g5FeSOn・7H2010mg, MnSO4・4
~6820 2mg, ZnSO4・7H200,9mg
, Cu SO+ ・5 H2O 0.4 mg, Na
zBsot・10HzOO, 09mg, (NHt)
6 Mos 021・4 H2O0.04mg-
Biotin 30■, thiamine hydrochloride 2I+Ig, sodium succinate 135g.
寒天１４ｇを１１中に含み、ｐ Ｈ７，４に調整した培
地に塗布する。11 contains 14 g of agar and is applied to a medium adjusted to pH 7.4.
３０℃で１２日間培養後に生じてきたストレプトマイシ
ン及びリファンピシン二重耐性株のコロニーを取得する
。Colonies of a streptomycin and rifampicin double resistant strain that have emerged after culturing at 30°C for 12 days are obtained.
Ｌ−イソロイシン生産菌とＬ−リジン生産菌の融合株２
００株を釣菌し、後述のイソロイシン生産試験にかけ、
親株よりインロイシン生産能が向上した菌株１（−３８
５９及びＨ−３８６０を取得する。Fusion strain 2 of L-isoleucine-producing bacteria and L-lysine-producing bacteria
00 strain was harvested and subjected to the isoleucine production test described below.
Strain 1 (-38
59 and H-3860.
細胞融合の親株であるＨ−３５０１はアルギニン要求性
のため最小培地〔グルコース０．５％、（ＮＨ，）、５
０．０．１５％、Ｋ）１２ＰＯ，０，１５％、Ｋ２ＨＰ
Ｏ４０，０５％、ＮａＣｊ２０．０１％、Ｍｇ５Ｏ，・
７Ｈ，ＯＯ，０５％、（： ａ Ｃｌ ２ ・２ ）（
２０１ｘ／ｍｌ、ＭｎＣ１−４８２０７μｇ／ｍｌ。H-3501, the parent strain for cell fusion, requires minimal medium [glucose 0.5%, (NH,), 5%] because it requires arginine.
0.0.15%, K)12PO, 0.15%, K2HP
O40.05%, NaCj20.01%, Mg5O,・
7H,OO,05%, (: a Cl 2 ・2) (
201x/ml, MnC1-48207 μg/ml.
Ｆ ｅ Ｓ Ｃ４・７ Ｈ２Ｏ１０ｘｒ／ｍｌ、チアミ
ン塩酸塩０．ｌμｇ／ｍｌ、ビオチア０．０３％ｇ／ｍ
ｌ、寒天１．５％：Ｉ）Ｈ７，２）では生育せずその生
育にはアルギニン（５０ｒ／ｍｌ）の添加を必要とする
。又、融合株Ｈ−３８６０は親株と同様にその生育にア
ルギニンを必要としたが、融合株Ｈ−３８５９は最小培
地でも充分な生育ができるのが特徴である。F e S C4.7 H2O10xr/ml, thiamine hydrochloride 0. lμg/ml, biothia 0.03%g/m
It does not grow on 1.5% agar: I) H7, 2) and requires the addition of arginine (50 r/ml) for growth. Furthermore, although the fusion strain H-3860 required arginine for its growth like the parent strain, the fusion strain H-3859 is characterized by its ability to grow sufficiently even in a minimal medium.
第１表に親株及び融合株の生育状況を示す。Table 1 shows the growth status of the parent strain and the fused strain.
本発明に使用する培地組成としては使用菌株の利用しう
る炭素源、窒素源、無機物その他の必要な栄養素を程良
く含有するものであれば合成培地及び天然培地のいずれ
も使用できる。As for the medium composition used in the present invention, both synthetic and natural media can be used as long as they contain appropriate amounts of carbon sources, nitrogen sources, inorganic substances, and other necessary nutrients that can be utilized by the bacterial strain used.
炭素源としては、グルコース、シュクロース、フラクト
ース、ＩＩＩ、デンプン、デンプン加水分解物などの炭
水化物、酢酸、プロピオン酸、ギ酸、フマール酸、リン
ゴ酸などの有機酸、メタノール、エタノール、プロパツ
ールなどのアルコールが用いられる。Carbon sources include carbohydrates such as glucose, sucrose, fructose, III, starch, and starch hydrolysates, organic acids such as acetic acid, propionic acid, formic acid, fumaric acid, and malic acid, and alcohols such as methanol, ethanol, and propatool. is used.
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、酢酸アンモニウム、リン酸アンモニウム
などの各種無機酸や有機酸のアンモニウム塩、尿素、ア
ミン類、その地合窒素化合物、ならびにペプトン、肉エ
キス、酵母エキス、コーン・スチーブ・リカー、カゼ・
イン加水分解物、大豆粕酸加水分解物、各種発酵菌体お
よびその消化物などが用いられる。Nitrogen sources include ammonium salts of various inorganic and organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, urea, amines, and their formed nitrogen compounds, as well as peptone, meat extract, yeast extract, corn stave liquor, cold
Ino hydrolyzate, soybean meal acid hydrolyzate, various fermented microbial cells and digested products thereof, etc. are used.
無機物としては、リン酸第−カリウム、リン酸第二カリ
ウム、リン酸マグネシウム、硫酸マグネシウム、塩化ナ
トリウム、硫酸第一鉄、硫酸マンガン、硫酸銅、炭酸カ
ルシウムなどが用いられる。As the inorganic substance, potassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc. are used.
勿論本発明に使用する微生物が生育のために特定の栄養
素を必要とする場合には、その栄養素を適当１培地中に
存在させなければならないが、これらの物質は窒素源と
して例示した天然物に含まれて添加される場合がある。Of course, if the microorganisms used in the present invention require specific nutrients for growth, those nutrients must be present in an appropriate medium, but these substances can be substituted with the natural products exemplified as nitrogen sources. May be included and added.
培養は振盪培養あるいは深部通気攪拌培養などの好気的
条件下で行なう。培養温度は通常２０〜１０℃、好まし
くは２５〜３５℃の範囲で、培地のｐＨは５〜９の範囲
で、好ましくは中性付近に保持することが望ましい。培
地のｐ）（調節は、炭酸カルシウム、無機又は有機の酸
、アルカリ溶液、尿素、アンモニア、ｐＨ緩衝剤などに
よって行う。Cultivation is performed under aerobic conditions such as shaking culture or deep aeration agitation culture. The culture temperature is usually in the range of 20 to 10°C, preferably 25 to 35°C, and the pH of the culture medium is in the range of 5 to 9, preferably around neutrality. p) (Adjustment of the medium is carried out with calcium carbonate, inorganic or organic acids, alkaline solutions, urea, ammonia, pH buffers, etc.).
培養期間は通常２〜７日間で培養液中にＬ−インロイン
ンが生成蓄積する。The culture period is usually 2 to 7 days, and L-inroin is produced and accumulated in the culture solution.
培養終了後、培養液から菌体などの沈殿物を除去し、公
知のイオン交換処理法、濃縮法、吸着法、塩析法などを
併用することにより、培養液からし一インロイシンを回
収することができる。After the cultivation is completed, precipitates such as bacterial bodies are removed from the culture solution, and mustard-inleucine is recovered from the culture solution by using a combination of known ion exchange treatment methods, concentration methods, adsorption methods, salting-out methods, etc. be able to.
以下に実施例を示す。Examples are shown below.
実施例１
種菌として融合株）（−３８５９及びＨ−３８６０並び
に親株Ｈ−３５０１及びＨ−３１１９を用いた。Example 1 Fusion strains (-3859 and H-3860) and parent strains H-3501 and H-3119 were used as inoculum.
これらの菌株をグルコース５０ｇ／ｌ、酵母エキス１０
ｇ／ｎ 、ペプトン１０ｇ／ｆ、尿素３ｇ／ｌ、食塩
２．５ ｇ ／ Ｉ！、コーンステイープリカー５ｇ／
ｌおよびビオチン５０■／ｌからなる種培地（ｐＨ７，
２）２０ｍｌを含む３０ Ｑｍｌｍｌ用フラスコに接種
し、２８℃で２４時間、ロータリーシェーカー（回転数
：２１０ｒｐｍ）上で振盪培養した。この種培養液２ｎ
ｌを２９ｍｌの下記組成の発酵培地を含む３０３ｍｌ容
三角フラスコに接種し、２８℃で７２時間＊盪培饗（回
転数：２１０ｒｐｍ）した。These strains were mixed with glucose 50g/l and yeast extract 10g/l.
g/n, peptone 10 g/f, urea 3 g/l, salt 2.5 g/I! , cornstarch liquor 5g/
Seed medium (pH 7,
2) It was inoculated into a 30 Qml flask containing 20ml, and cultured with shaking on a rotary shaker (rotation speed: 210 rpm) at 28°C for 24 hours. This seed culture solution 2n
1 was inoculated into a 303 ml Erlenmeyer flask containing 29 ml of a fermentation medium having the following composition, and cultured at 28° C. for 72 hours (rotation speed: 210 rpm).
培養液中に生成したし一イソロインン及びＬ−ＩＪジン
の債を第２表に示す。Table 2 shows the amounts of L-isolinine and L-IJine produced in the culture solution.
発酵培地の組成
廃Ｕ蜜（グルコース換算）７０ｇ／ｆｆ、コーンステイ
ープリカー５ｇ／Ｌ塩化アンモニウム２０ｇ／Ｌ尿素２
ｇ／１２、Ｋ ｌ（２Ｐ ０４ ２ ｇ ／ ｎ、Ｍ
ｇ５Ｏ，−７１（２００，５ｇ／ＡＳＦｅＳＯ＋・７Ｈ
２００，Ｏｉｇ＃、ＭｎＣｎｚ’４ｌ−（２００，０１
ｇ／ｊ”、Ｃ＋１ＳＯ４・５Ｈ２００，０１ｇ／ＬＣａ
ＣＬ・２Ｈ２０Ω、０１ｇ／ｌ、Ｚｎ５Ｏ＊・７）（２
０１，ｍｇ／ｊ！、 ＮｉＣ１！ｚ １ａ＋ｇ／ｊ！
、モリブデン酸７７％７−４水塩１ｍｇ／ｌ’、Ｃ０Ｃ
Ｌ・６ｔ（２０１ｍｇ／β、パントテン酸カルンウム１
０ｍｇ／ｌ、ニコチン酸１ｍｇ／ｆｆ、ビオチン５０γ
／Ａ）、Ｉ）Ｈ７，４
なおアルギニン要求株の場合は、アルギニン塩酸塩を０
．５ ｇ ’、ｌ’ｌ添加。Composition of fermentation medium Waste honey (glucose equivalent) 70g/ff, cornstarch liquor 5g/L ammonium chloride 20g/L urea 2
g/12, K l (2P 04 2 g/n, M
g5O, -71 (200,5g/ASFeSO+・7H
200, Oig#, MnCnz'4l-(200,01
g/j”, C+1SO4・5H200,01g/LCa
CL・2H20Ω, 01g/l, Zn5O*・7) (2
01,mg/j! , NiC1! z 1a+g/j!
, molybdic acid 77% 7-tetrahydrate 1 mg/l', C0C
L・6t (201mg/β, carunium pantothenate 1
0mg/l, nicotinic acid 1mg/ff, biotin 50γ
/A), I) H7,4 In the case of arginine auxotrophic strains, arginine hydrochloride was added to 0.
．． 5 g', l'l added.
第２表
（Ａｒｇ”）：アルギニン非要求性
（Ａｒｇ−） ・アルギニン要求性
融合株！−（−３８６０を用いて得たＬ−インロインン
含有培養液２００ｍｌから遠心分離により、菌体その他
の不純物を除いた上澄液を強酸性陽イオン交換樹脂ダイ
ヤイオン５ＫＩ（Ｈ”型）（三菱化成社製）のカラムに
通し、Ｌ−イソロイシンを吸着させ、水洗後０．５規定
のアンモニア水で溶出して、Ｌ−イソロイシン画分を集
め、濃縮してｐ Ｈ６，０２の等電点で晶出させること
により純度９９％のし一インロイシン８５０ｍｇを得た
。Table 2 (Arg"): Non-arginine auxotrophic (Arg-) - Arginine auxotrophic fusion strain!- The removed supernatant liquid was passed through a column of strongly acidic cation exchange resin Diaion 5KI (H” type) (manufactured by Mitsubishi Kasei Corporation) to adsorb L-isoleucine, and after washing with water, it was eluted with 0.5N ammonia water. The L-isoleucine fractions were collected, concentrated, and crystallized at an isoelectric point of pH 6.02 to obtain 850 mg of inleucine with a purity of 99%.
発明の効果
本発明方法により収率よくＬ−イソロイシンをｉ尋るこ
とができる。Effects of the Invention By the method of the present invention, L-isoleucine can be obtained in good yield.
Claims (3)
生産能を有するコリネバクテリウム属に属する微生物を
培地に培養し、培養物中にＬ−イソロイシンを生成蓄積
させ、該培養物からＬ−イソロイシンを採取することを
特徴とするＬ一イソロイシンの製造法。(1) Cultivating a microorganism belonging to the genus Corynebacterium having the ability to produce L-isoleucine obtained by protoplast fusion in a medium, producing and accumulating L-isoleucine in the culture, and collecting L-isoleucine from the culture. A method for producing L-isoleucine, characterized by the following.
有する微生物と主発酵生産物がＬ−イソロイシン以外の
アミノ酸を生産する能力を有する微生物との間で行われ
ることを特徴とする特許請求の範囲第１項記載の製造法
。(2) Protoplast fusion is carried out between a microorganism capable of producing L-isoleucine and a microorganism capable of producing an amino acid other than L-isoleucine as a main fermentation product. Manufacturing method described in section.
特許請求の範囲第２項記載の製造法。(3) The production method according to claim 2, wherein the amino acid is L-lysine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60215718A JPS6274293A (en) | 1985-09-28 | 1985-09-28 | Production of l-isoleucine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60215718A JPS6274293A (en) | 1985-09-28 | 1985-09-28 | Production of l-isoleucine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS6274293A true JPS6274293A (en) | 1987-04-06 |
Family
ID=16677024
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP60215718A Pending JPS6274293A (en) | 1985-09-28 | 1985-09-28 | Production of l-isoleucine |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6274293A (en) |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008044409A1 (en) | 2006-10-10 | 2008-04-17 | Ajinomoto Co., Inc. | Method for production of l-amino acid |
| WO2008075483A1 (en) | 2006-12-19 | 2008-06-26 | Ajinomoto Co., Inc. | Process for production of l-amino acid |
| WO2008102572A1 (en) | 2007-02-20 | 2008-08-28 | Ajinomoto Co., Inc. | Method for production of l-amino acid or nucleic acid |
| WO2009088049A1 (en) | 2008-01-10 | 2009-07-16 | Ajinomoto Co., Inc. | Method for production of desired substance by fermentation process |
| WO2009093703A1 (en) | 2008-01-23 | 2009-07-30 | Ajinomoto Co., Inc. | Method of producing l-amino acid |
| WO2011013707A1 (en) | 2009-07-29 | 2011-02-03 | 味の素株式会社 | Method for producing l-amino acid |
| WO2014185430A1 (en) | 2013-05-13 | 2014-11-20 | 味の素株式会社 | Method for manufacturing l-amino acid |
| WO2015005406A1 (en) | 2013-07-09 | 2015-01-15 | 味の素株式会社 | Method for manufacturing useful substance |
| WO2015050234A1 (en) | 2013-10-02 | 2015-04-09 | 味の素株式会社 | Ammonia control apparatus and ammonia control method |
| WO2015060391A1 (en) | 2013-10-23 | 2015-04-30 | 味の素株式会社 | Method for producing target substance |
| EP3385389A1 (en) | 2017-04-03 | 2018-10-10 | Ajinomoto Co., Inc. | Method for producing l-amino acid from fructose |
| CN111925953A (en) * | 2019-12-30 | 2020-11-13 | 河南工业大学 | Glutamic acid producing strain and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS58158184A (en) * | 1982-03-15 | 1983-09-20 | Ajinomoto Co Inc | Fusion of protoplast of bacterium |
| JPS58158185A (en) * | 1982-03-15 | 1983-09-20 | Ajinomoto Co Inc | Breeding of amino acid-producing bacteria having improved rate of growth |
| JPS59113894A (en) * | 1982-12-17 | 1984-06-30 | Kyowa Hakko Kogyo Co Ltd | Preparation of l-glutamic acid by fermentation |
-
1985
- 1985-09-28 JP JP60215718A patent/JPS6274293A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS58158184A (en) * | 1982-03-15 | 1983-09-20 | Ajinomoto Co Inc | Fusion of protoplast of bacterium |
| JPS58158185A (en) * | 1982-03-15 | 1983-09-20 | Ajinomoto Co Inc | Breeding of amino acid-producing bacteria having improved rate of growth |
| JPS59113894A (en) * | 1982-12-17 | 1984-06-30 | Kyowa Hakko Kogyo Co Ltd | Preparation of l-glutamic acid by fermentation |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008044409A1 (en) | 2006-10-10 | 2008-04-17 | Ajinomoto Co., Inc. | Method for production of l-amino acid |
| WO2008075483A1 (en) | 2006-12-19 | 2008-06-26 | Ajinomoto Co., Inc. | Process for production of l-amino acid |
| WO2008102572A1 (en) | 2007-02-20 | 2008-08-28 | Ajinomoto Co., Inc. | Method for production of l-amino acid or nucleic acid |
| EP2749652A2 (en) | 2008-01-10 | 2014-07-02 | Ajinomoto Co., Inc. | A method for producing a target substance by fermentation |
| WO2009088049A1 (en) | 2008-01-10 | 2009-07-16 | Ajinomoto Co., Inc. | Method for production of desired substance by fermentation process |
| WO2009093703A1 (en) | 2008-01-23 | 2009-07-30 | Ajinomoto Co., Inc. | Method of producing l-amino acid |
| WO2011013707A1 (en) | 2009-07-29 | 2011-02-03 | 味の素株式会社 | Method for producing l-amino acid |
| WO2014185430A1 (en) | 2013-05-13 | 2014-11-20 | 味の素株式会社 | Method for manufacturing l-amino acid |
| WO2015005406A1 (en) | 2013-07-09 | 2015-01-15 | 味の素株式会社 | Method for manufacturing useful substance |
| EP3521433A1 (en) | 2013-07-09 | 2019-08-07 | Ajinomoto Co., Inc. | Process for producing l-glutamic acid |
| WO2015050234A1 (en) | 2013-10-02 | 2015-04-09 | 味の素株式会社 | Ammonia control apparatus and ammonia control method |
| WO2015060391A1 (en) | 2013-10-23 | 2015-04-30 | 味の素株式会社 | Method for producing target substance |
| EP3385389A1 (en) | 2017-04-03 | 2018-10-10 | Ajinomoto Co., Inc. | Method for producing l-amino acid from fructose |
| CN111925953A (en) * | 2019-12-30 | 2020-11-13 | 河南工业大学 | Glutamic acid producing strain and preparation method and application thereof |
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