JPS6115695A - Preparation of l-isoleucine by fermentation method - Google Patents
Preparation of l-isoleucine by fermentation methodInfo
- Publication number
- JPS6115695A JPS6115695A JP13446084A JP13446084A JPS6115695A JP S6115695 A JPS6115695 A JP S6115695A JP 13446084 A JP13446084 A JP 13446084A JP 13446084 A JP13446084 A JP 13446084A JP S6115695 A JPS6115695 A JP S6115695A
- Authority
- JP
- Japan
- Prior art keywords
- isoleucine
- corynebacterium
- brevibacterium
- strain
- resistance
- 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.)
- Granted
Links
Landscapes
- 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〕
L−4ソロイシンはアミノ酸輸液及び総合アミノ酸製剤
の重要な成分である。本発明はこのL−イソロイシンを
発酵法で製造する方法を改良するものである。DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] L-4 Soleucine is an important component of amino acid infusions and comprehensive amino acid preparations. The present invention improves the method for producing L-isoleucine by fermentation.
ブレビバクテリウム属及びコリネバクテリウム属の微生
物がL−イソロイシン生産能を有するためには、α−ア
ミノ−β−ヒドロキシ吉草m(以下AHVと略す)等へ
の耐性を付右せしめれば良いことがわかっている。更に
、前記の薬剤耐性に加えてO−メチルスレオニン耐性、
β−ヒドロキシロイシン耐性又fd )リクロロアラニ
ン耐性を付与すること、及びプリン系物質又はリジン等
の要求性を付与することによりL−イソロイシンの生産
能が向上することは知られている。In order for microorganisms of the genus Brevibacterium and Corynebacterium to have the ability to produce L-isoleucine, it is sufficient to impart resistance to α-amino-β-hydroxyvalerin (hereinafter abbreviated as AHV), etc. I know that. Furthermore, in addition to the drug resistance mentioned above, O-methylthreonine resistance,
It is known that L-isoleucine production ability can be improved by imparting resistance to β-hydroxyleucine or fd)lichloroalanine, and by imparting requirements for purine substances or lysine.
L−イソロイシン発酵収率及び蓄積を向上させることは
工業生産上に於て、重要な問題である。Improving L-isoleucine fermentation yield and accumulation is an important issue in industrial production.
本発明は上記問題点を解決するためになされたものであ
り従来より知られているブレビバクテリウム属及びコリ
ネバクテリウム属に属するL−イソロイシン生産能を有
する微生物を【゛くC良して1Wに発酵収率の向上した
菌株を見いだすべく研究した結果、α−ケトマロン酸(
以下α−KMと略す。)に耐性を付与した菌株の中に、
従来のし一イソロイシン生産菌よシも晶収率でL−イソ
ロイシンを生産する菌株が存在することを発見した。The present invention has been made in order to solve the above-mentioned problems. As a result of research to find strains with improved fermentation yield, we found that α-ketomalonic acid (
Hereinafter, it will be abbreviated as α-KM. ) among the strains that have conferred resistance to
In addition to conventional isoleucine-producing bacteria, it has been discovered that there are strains that produce L-isoleucine at a high crystal yield.
即ち、本発明は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−双耐性を有し、且つL−イソロイ
シン生産能を有する微生物を液体培地中で培養し、培地
中に生成蓄積したL−イソ本発明において用いられる微
生物は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−店耐性を有し、かつL−イソロイシン生産
能を有する変異株である。That is, the present invention involves culturing a microorganism belonging to the genus Brevibacterium or Corynebacterium and having α-bitolerance and the ability to produce L-isoleucine in a liquid medium, and producing and accumulating L-isoleucine in the medium. Iso The microorganism used in the present invention belongs to the genus Brevibacterium or Corynebacterium, and is a mutant strain having α-store resistance and the ability to produce L-isoleucine.
本発明の変異株を得るには、下記の野生株に先にL−イ
ソロイシン生産能を付与し、次いでα−個耐性を付与し
ても良いし、又先にα−KM 耐性を付与し、次いで4
ンロイシン生産能を付与しても良い。To obtain the mutant strain of the present invention, the following wild strain may first be endowed with L-isoleucine production ability, and then alpha-KM resistance may be imparted, or alpha-KM resistance may be first imparted, then 4
The ability to produce leucine may also be imparted.
本変異株の親株となる野生株は、ブレビバクテリウム属
又はコリネバクテリウム属等のコリネホA、 ムI、−
グルタミン酸生産菌として知られているものであシ、例
えば以下のものがある。The wild strain that is the parent strain of this mutant strain is Corinepho A, Mu I, - of the genus Brevibacterium or Corynebacterium.
These are known as glutamic acid producing bacteria, such as the following.
ブレビバクテリウム・ラクトフェルメンタムATCC1
3869ブレビバクテリウム・デイパリカタム
ATCC14020ブレビバクテリウム・す、カロリテ
ィカム ATCC14066ゾレビパクテリウム・フ
ラバム ATCC14067コリネバクテ
リウム・グルラミ1ム ATCC13032コ
リネバクテリウム アセトアミドフィラム ATCC1
3870これらの親株よう本発明の変異株を得る方法は
、N−メチル−N′−二トローN−ニトロソグアニジン
処理する等の通常の変異誘導方法が適用できる。Brevibacterium lactofermentum ATCC1
3869 Brevibacterium deipalicatum
ATCC14020 Brevibacterium calolyticum ATCC14066 Zorevibacterium flavum ATCC14067 Corynebacterium gullamium ATCC13032 Corynebacterium acetamidophyllum ATCC1
3870. To obtain the mutant strains of the present invention from these parent strains, conventional mutagenesis methods such as treatment with N-methyl-N'-nitro-N-nitrosoguanidine can be applied.
変異処理した菌液から本発明の変異株を分離する方法は
α−個を含む培地で生育するような菌株を採取すること
によって行われる。The method for isolating the mutant strain of the present invention from a bacterial solution subjected to mutation treatment is carried out by collecting a strain that grows in a medium containing α- cells.
本発明に示す変異株の具体的な変異誘導方法とα−KM
に対する菌株の生育度の関係を以下に示す。Specific mutation induction method of the mutant strain shown in the present invention and α-KM
The relationship between the growth rate of the strain and the strain is shown below.
ブイヨン寒天スラント上に30℃で24時間生育させた
ブレビバクテリウム・フラバムAJ 3686FERM
−P 2433(ATCC14067,1: !J誘導
したAI(v耐性株)及びコリネバクテリウム・グルタ
ミクムAJ12150 FERM−P r76r7
4(ATCC13032,lニジ誘導したAHV耐性株
)の菌体をM/ 30 ’)ン酸緩衝液に懇濁し菌体濃
度108〜109/mlの菌体懸濁液に500μg−/
rniのN−メチA −N’−ニドo −N −ニトロ
ソグアニジンを加え30℃に20分間保持した。ついで
遠心分離して菌体を集め、M/30 リン酸緩衝液で良
く洗滌した後、下記組成の培地に接種[2,315℃で
2〜10日間培善した7、グルコース 1.
0 f/−/di尿 素
0.2KH2PO40,1
MgSO4・7.H2O0,I P7dlF e
SO4・7H200−002”MnSO4・7H20
0,002#
ビオチン 100 μ9−/lサイアーン
塩酸塩 100 #
α−KM O,2’;!/di寒 天
2.0寒天培地に生育した菌株
の中からL−イソロイシン生産能の高い苗株としてブレ
ビバクテリウム・7 ラバA ki 12152 、
FERM−P ワ6r7g (AJ(v耐性α−個
耐性)及びコリネバクテリウム・グルタミクA AJ
12153 、 FERM−P r76ワ6(AIrV
耐性a、 α−個耐性)を得た。Brevibacterium flavum AJ 3686FERM grown on broth agar slant at 30°C for 24 hours
-P 2433 (ATCC 14067, 1: !J-induced AI (v-resistant strain) and Corynebacterium glutamicum AJ12150 FERM-P r76r7
4 (ATCC13032, an AHV-resistant strain induced by liniculture) was suspended in M/30') acid buffer, and 500 μg/ml was added to a cell suspension with a cell concentration of 108 to 109/ml.
N-methyA-N'-nido-N-nitrosoguanidine of rni was added and kept at 30°C for 20 minutes. The cells were then collected by centrifugation, washed well with M/30 phosphate buffer, and then inoculated into a medium with the following composition [cultured at 2,315°C for 2 to 10 days, 7, glucose 1.
0 f/-/diurea
0.2KH2PO40,1 MgSO4・7. H2O0,I P7dlF e
SO4・7H200-002”MnSO4・7H20
0,002# Biotin 100 μ9-/l Sian hydrochloride 100 # α-KM O,2';! /di agar 2.0 Among the strains grown on agar medium, Brevibacterium 7 Lava Aki 12152 was selected as a seedling with high L-isoleucine production ability.
FERM-P Wa 6r7g (AJ (v resistant α- resistant) and Corynebacterium glutamicum A AJ
12153, FERM-P r76 Wa 6 (AIrV
Resistance a, α-number resistance) was obtained.
このようにしてイ!tられた変異株のα1讃耐性度を親
株と比較した。Like this! The α1 resistance of the mutant strain was compared with that of the parent strain.
グルコース0.5 F//di 、尿素0.2fild
i、硫安0、15’ 9/di 、 K2HO4’0.
39/de 、 K2HPO40,19/dl 、
MgSO4・7H200,01g/de 、 C
aC42’2H200,1m9/de、ビオチy100
ttl/l 、fイ7ミy塩酸塩100 fig/11
、 FeSO4−7H200,0021/di。Glucose 0.5 F//di, Urea 0.2 field
i, ammonium sulfate 0,15'9/di, K2HO4'0.
39/de, K2HPO40,19/dl,
MgSO4・7H200,01g/de, C
aC42'2H200, 1m9/de, Biochi y100
ttl/l, fi7myyhydrochloride100fig/11
, FeSO4-7H200,0021/di.
MnSO4’7H20o、o O21!/dll 、お
よび表に示す量のα−藷を含み、PI(7,0に調節し
た培地に天然培地(−!!母
プトン1 g/de 、酵箇エキス11i /di 、
NaC60,5Ii/dl 、 pH7,0)スラン
トで24時間培養した菌体を殺菌水に懸濁して接種し、
24時間培養して生育度を一度で測定した。MnSO4'7H20o, o O21! /dll, and the amount shown in the table, and the medium adjusted to PI (7,0) was mixed with a natural medium (-!! Momopton 1 g/de, yeast extract 11i/di,
NaC60.5Ii/dl, pH 7.0) Cells cultured in a slant for 24 hours were suspended in sterilized water and inoculated.
After culturing for 24 hours, the degree of growth was measured at once.
第 1 表
上述の変異株にO−メチルスレオニン耐性、β−ヒドロ
キシロイシン耐性又はトリフロロアラニン耐性のように
すてにL−イソロイシンの生産性を向上せしめることが
知られている性質を更に付加することによシ、収率が向
上する場合が多い。Table 1 Addition of properties known to improve the productivity of L-isoleucine, such as O-methylthreonine resistance, β-hydroxyleucine resistance, or trifluoroalanine resistance, to the above-mentioned mutant strains. In particular, the yield is often improved.
このような変異株を培養する際に用いる培地は、炭素源
、窒素源、無機イオン、上記要求性を満足させるべき物
質及び必要に応じビタミン等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 used for culturing such mutant strains is a normal medium containing a carbon source, a nitrogen source, inorganic ions, substances that should satisfy the above-mentioned requirements, and other organic micronutrients such as vitamins as necessary. .
炭素源としてはグルコース、シュクロース等の炭水化物
、酢酸等の有機酸等が、窒素源としてはアンモニア水、
アンモニアガス、アンモニウム塩等が好適である。無機
イオンとしてはカリイオン。Carbon sources include carbohydrates such as glucose and sucrose, organic acids such as acetic acid, etc., and nitrogen sources include aqueous ammonia and
Ammonia gas, ammonium salts, etc. are suitable. Potassium ion is an inorganic ion.
ナトリウムイオン、マグネシウムイオン、リン酸イオン
その他が必要に応じ適宜培地に添加される。Sodium ions, magnesium ions, phosphate ions, and others are added to the medium as needed.
培養は好気的条件が望ましく、培誉の間培地のPHを4
ないし8に温度を25℃ないし37℃に調節しつつ行え
ばよシ好ましい結果が得られる。かくして1ないし7日
間も培養すれば培地中に著量のL−イソロイシンが生成
蓄積される。培養液よpL−イノロイシンを採取する方
法はイオン交換樹脂による方法等通常の方法で採取でき
る。It is desirable to cultivate under aerobic conditions, and the pH of the medium should be kept at 4 during cultivation.
Preferable results can be obtained by controlling the temperature between 25°C and 37°C. Thus, if the culture is continued for 1 to 7 days, a significant amount of L-isoleucine will be produced and accumulated in the medium. pL-inoleucine can be collected from the culture solution by a conventional method such as using an ion exchange resin.
以下実施例にて説明する。This will be explained below using examples.
実施例1
グルコース10g/di、 (NH4)2S0471
1/di、KH2PO40,111/di、 MgSO
4・7H200,04g/di、 FeSO4・7H2
01m9/d1. MnSO4’4H201m9/dl
、サイアミン−HCL100μ!i/l、ビチオン10
0μI/l、大豆蛋白酸加水分解液60ダ/dj(全箪
素として)炭酸カルシウム5yZdec別殺菌)を含む
培地をPI(7,0に調節し、その20rnlを500
nll容肩伺フラスコに入れ加熱殺菌した。これに第
1表に示す菌株を一白金耳接種し、31.5℃に保ちつ
つ4日間振逼した。各菌株の培養液中には第2表に示す
量のL−イソロイシンが蓄積した。AJ12152
を上記の方法で培養して培養液11を得、これより遠心
分離にて菌体を除き、上滑を、強酸性イオン交換樹脂[
ダイヤイオンj 5K−IB (N1(4+型)に通過
させた。樹脂を水洗後、2N−アンモニア水にて溶出し
ついで溶出液を濃縮し、これよりL−イソロイシンの粗
結晶16.0.9を得た。Example 1 Glucose 10g/di, (NH4)2S0471
1/di, KH2PO40,111/di, MgSO
4.7H200,04g/di, FeSO4.7H2
01m9/d1. MnSO4'4H201m9/dl
, Thiamine-HCL100μ! i/l, bithion 10
A medium containing 0μI/l, soybean protein acid hydrolyzate 60 da/dj (as total silica) calcium carbonate 5yZdec separate sterilization) was adjusted to PI (7,0), and the 20rnl was sterilized to 500
The mixture was placed in a shoulder-length flask and sterilized by heating. A loopful of the bacterial strains shown in Table 1 was inoculated into this, and the mixture was shaken for 4 days while being kept at 31.5°C. The amount of L-isoleucine shown in Table 2 was accumulated in the culture solution of each strain. AJ12152
was cultured by the above method to obtain culture solution 11, from which the bacterial cells were removed by centrifugation, and the supernatant was treated with a strongly acidic ion exchange resin [
It was passed through Diaion J 5K-IB (N1 (4+ type). After washing the resin with water, it was eluted with 2N aqueous ammonia, and the eluate was concentrated. From this, crude crystals of L-isoleucine 16.0.9 I got it.
第2表
菌 株 性 質
し−イソロイシン(9Ahブレビバクテリウム・
フラバム
AJ 3686 (FERM−P 2433) AHt
” ’ 0.6プレビバクテリウム・フラ
バムTable 2 Bacterial Strain Characteristics
- Isoleucine (9Ah Brevibacterium
Flavum AJ 3686 (FERM-P 2433) AHt
” ' 0.6 Previbacterium flavum
Claims (1)
α−ケトマロン耐性を有し、且つL−イソロイシン生産
能を有する微生物を液体培地中で培養し、培地中に生成
蓄積したL−イソロイシンを採取することを特徴とする
L−イソロイシンの製造方法。A microorganism that belongs to the genus Brevibacterium or Corynebacterium and has α-ketomalon resistance and is capable of producing L-isoleucine is cultivated in a liquid medium, and L-isoleucine produced and accumulated in the medium is collected. Characteristic method for producing L-isoleucine.
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP13446084A JPS6115695A (en) | 1984-06-29 | 1984-06-29 | Preparation of l-isoleucine by fermentation method |
EP85108049A EP0167132B1 (en) | 1984-06-29 | 1985-06-28 | Process for producing l-isoleucine by fermentation |
DE8585108049T DE3585052D1 (en) | 1984-06-29 | 1985-06-28 | METHOD FOR PRODUCING L-ISOLEUCIN BY FERMENTATION. |
US06/750,289 US4656135A (en) | 1984-06-29 | 1985-07-01 | Process for producing L-isoleucine by fermentation |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP13446084A JPS6115695A (en) | 1984-06-29 | 1984-06-29 | Preparation of l-isoleucine by fermentation method |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS6115695A true JPS6115695A (en) | 1986-01-23 |
JPH0362394B2 JPH0362394B2 (en) | 1991-09-25 |
Family
ID=15128848
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP13446084A Granted JPS6115695A (en) | 1984-06-29 | 1984-06-29 | Preparation of l-isoleucine by fermentation method |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS6115695A (en) |
Cited By (14)
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---|---|---|---|---|
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 |
JP2016514474A (en) * | 2013-06-11 | 2016-05-23 | シージェイ チェイルジェダング コーポレイション | Microorganism producing L-isoleucine and method for producing L-isoleucine using the same |
EP3385389A1 (en) | 2017-04-03 | 2018-10-10 | Ajinomoto Co., Inc. | Method for producing l-amino acid from fructose |
CN110541013A (en) * | 2019-10-06 | 2019-12-06 | 冯世红 | method for producing L-leucine by fermentation |
CN110607331A (en) * | 2019-10-22 | 2019-12-24 | 冯世红 | Process for preparing and extracting L-leucine |
-
1984
- 1984-06-29 JP JP13446084A patent/JPS6115695A/en active Granted
Cited By (19)
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---|---|---|---|---|
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 |
EP2749652A2 (en) | 2008-01-10 | 2014-07-02 | Ajinomoto Co., Inc. | A method for producing a target substance by fermentation |
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 |
US9885093B2 (en) | 2013-06-11 | 2018-02-06 | Cj Cheiljedang Corporation | L-isoleucine-producing microorganism and method of producing L-isoleucine using the same |
JP2016514474A (en) * | 2013-06-11 | 2016-05-23 | シージェイ チェイルジェダング コーポレイション | Microorganism producing L-isoleucine and method for producing L-isoleucine using the same |
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 |
CN110541013A (en) * | 2019-10-06 | 2019-12-06 | 冯世红 | method for producing L-leucine by fermentation |
CN110541013B (en) * | 2019-10-06 | 2023-03-10 | 新疆阜丰生物科技有限公司 | Method for producing L-leucine by fermentation |
CN110607331A (en) * | 2019-10-22 | 2019-12-24 | 冯世红 | Process for preparing and extracting L-leucine |
CN110607331B (en) * | 2019-10-22 | 2023-03-10 | 新疆阜丰生物科技有限公司 | Process for preparing and extracting L-leucine |
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Publication number | Publication date |
---|---|
JPH0362394B2 (en) | 1991-09-25 |
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