JPS61282092A - Production of l-lysine by fermentation method - Google Patents
Production of l-lysine by fermentation methodInfo
- Publication number
- JPS61282092A JPS61282092A JP60125499A JP12549985A JPS61282092A JP S61282092 A JPS61282092 A JP S61282092A JP 60125499 A JP60125499 A JP 60125499A JP 12549985 A JP12549985 A JP 12549985A JP S61282092 A JPS61282092 A JP S61282092A
- Authority
- JP
- Japan
- Prior art keywords
- lysine
- superoxide
- strain
- superoxide dismutase
- corynebacterium
- 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
Classifications
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (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） 本発明は発酵法によるＬ −リジンの製造法に関する。[Detailed description of the invention] (Industrial application field) The present invention relates to a method for producing L-lysine using a fermentation method.
さらに詳しくは、強化されたスーツや一オキシドジスム
ターゼ活性を有する！レピパクテリウム属又はコリネバ
クテリウム属のＬ−リジン生産菌を用いる発酵法による
Ｉ、−ＩＪジンを製造する方法に関する。More specifically, it has an enhanced suit and monooxide dismutase activity! The present invention relates to a method for producing I, -IJ gin by a fermentation method using L-lysine producing bacteria of the genus Lepipacterium or Corynebacterium.
（従来の技術）
従来、発酵法によるＬ−リジンの製造法として１ｓ−（
２−７ミノエチル）−Ｌ−システィン（以下ＡＥＣと記
す）耐性菌を使用する方法（％公昭４２−５５２１３号
）、ＡＥＣ耐性でかつＬ−ロイシン、Ｌ−ホモセリン、
Ｌ−プロリン、Ｌ−アルギ＝７、あ、いゆＬ−ア、＝□
よ、δｒＬａ−ｈＹ。(Prior art) Conventionally, as a method for producing L-lysine by a fermentation method, 1s-(
2-7minoethyl)-L-cysteine (hereinafter referred to as AEC) resistant bacteria (% Publication No. 42-55213), AEC-resistant and L-leucine, L-homoserine,
L-Proline, L-Argi = 7, A, IYL-A, =□
Yo, δrLa-hY.
変異株を使用する方法（％開昭４９−３６８８８号、特
開昭４９−８０２８９号、特公昭５１−２１０７８号）
、ＡＥＣ耐性でかつβ−ヒドロキシルロイシン（以下Ｈ
Ｌ！：記ｔ）等のロイシンアナログ耐性変異株を用いる
方法（特公昭５３−１８３３）、α−クロロカグロラク
タム（以下ＣＣＬと記す）耐性変異株を用いる方法（特
公昭５３−４３５９１）、γ−メチルリジン（以下ＭＬ
と記す）耐性変異株を用いる方法（特公昭５６−１９２
３５） 、フルオロピルビン酸（以下ＦＰと記す）感受
性変異株を使用する方法（特開昭５５−９７８３号）、
ピルビン酸キナーゼ活性が低下した変異株を使用する方
法（特開昭５８−１７０４８７号）などが知られている
。Method using mutant strains (% 1987-36888, JP 49-80289, JP 51-21078)
, AEC resistant and β-hydroxyleucine (hereinafter H
L! : A method using a leucine analog resistant mutant strain such as t) (Japanese Patent Publication No. 53-1833), a method using a mutant strain resistant to α-chlorocaglolactam (hereinafter referred to as CCL) (Japanese Patent Publication No. 53-43591), γ- Methylysine (hereinafter referred to as ML)
) method using resistant mutant strains (Special Publication No. 56-192)
35), a method using a fluoropyruvate (hereinafter referred to as FP) sensitive mutant strain (Japanese Unexamined Patent Publication No. 55-9783),
A method using a mutant strain with reduced pyruvate kinase activity (Japanese Unexamined Patent Publication No. 170487/1987) is known.
（本発明が解決しようとする問題点）
本発明が解決しようとする問題点は、更に安価に発酵法
によｆｉ Ｌ −リジンを製造する方法を確立すること
にある。(Problems to be Solved by the Present Invention) The problems to be solved by the present invention are to establish a method for producing fi L-lysine by a fermentation method at a lower cost.
（問題点を解決するための手段）
本発明者らは上述の問題点を解決するためにＬ−リジン
生産菌の菌株改良によって発酵収率を高めるべく種々検
討し九結果、Ｌ−リジン生産菌のス−７４−オキシドシ
スムターゼ活性ｔｉめることによってこの菌のＬ −Ｉ
Ｊレシン産能を大巾に高めうろことを見出し、これに基
いて本発明を完成するに至っ九。(Means for Solving the Problems) In order to solve the above-mentioned problems, the present inventors conducted various studies to increase the fermentation yield by improving the strain of L-lysine-producing bacteria, and as a result, found that The L-I of this bacterium was reduced by decreasing the su-74-oxidocysmutase activity of
They discovered that the production capacity of J-resin could be greatly increased, and based on this, they completed the present invention.
すなわち本発明は、ブレビバクテリウム属を念はコリネ
バクテリウム属に属し、強化されたスーパーオキシドジ
スムターゼ活性を有シ、かつＬ−リジン生産能を有する
変異株を培養して培養液中にＬ−リジンを生成、蓄積せ
しめ、これを採取することを特徴とする発酵法によるＬ
−リジンの製造方法に関するものである。That is, the present invention involves culturing a mutant strain of the Brevibacterium genus, specifically the Corynebacterium genus, having enhanced superoxide dismutase activity and L-lysine producing ability, and injecting L-lysine into the culture solution. L produced by a fermentation method characterized by producing and accumulating lysine and collecting it.
- It relates to a method for producing lysine.
本発明に使用する微生物は、ブレビバクテリウム属また
はコリネバクテリウム属に属しＬ−リジン生産能を有す
るものを変異処理し、得られた変異株のうちからスーノ
ヤーオキシド増産剤、スーパ−オキシドジスムターゼ誘
導抑制剤、スーパーオキシドラジカル反応促進剤または
スーパーオキシ本発明で誘導する変異株の親株としては
ブレビパフチリウム属又はコリネバクテリウム属に属す
る微生物であれば種、菌株を問わずどのような菌株でも
良いが、以下に示すような、いわゆるコリネフォームの
Ｌ−グルタミン酸生産菌として知られるものが好適であ
る。The microorganisms used in the present invention belong to the genus Brevibacterium or Corynebacterium and have the ability to produce L-lysine. Dismutase induction inhibitors, superoxide radical reaction promoters, or superoxy As the parent strain of the mutant strain induced by the present invention, any microorganism belonging to the genus Brevipaftillium or Corynebacterium, regardless of its species or strain, can be used. Although any bacterial strain may be used, the so-called coryneform L-glutamic acid producing bacteria shown below are suitable.
グレピパクテリウム・デパリカタム ＡＴＣＣ
１４０２０ブレビバクテリウム・フラブム
ＡＴＣＣ１４０６７！レビバクテリウム・ラクト７ア
ーメンタム ＡＴＣＣ１３８６９グレピパクテリウム・
ロゼラム ＡＴＣＣ１３８２５コリネバク
テリウム・アセトアシドフィラム ＡＴＣＣ１３８７０
コリネバクテリウム・リリウム ＡＴＣＣ
１５９９０菌株としては、これら野生株の他に、リジン
生産に有利な性質、例えばＡＥＣ耐性、ＣＣＬ耐性、■
、耐性、ホモセリン要求性、アラニン要求性、フルオロ
ピルビン酸感受性、スレオニン感受性、メチオニン感受
性などの性質を有する変異株を使用することもできる。Grepipacterium deparicatum ATCC
14020 Brevibacterium flavum
ATCC14067! Levibacterium lacto 7 amentum ATCC13869 Grepipacterium
Roserum ATCC13825 Corynebacterium acetoacidophyllum ATCC13870
Corynebacterium Lilium ATCC
In addition to these wild strains, the 15990 strain has properties that are advantageous for lysine production, such as AEC resistance, CCL resistance,
Mutant strains having properties such as resistance, homoserine auxotrophy, alanine auxotrophy, fluoropyruvate sensitivity, threonine sensitivity, methionine sensitivity, etc. can also be used.
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
、リジン生産に有利な性質を有する菌株を用いて、本発
明の変異株を得るには、紫外線照射、Ｘ線照射、変異誘
起剤処理等の一般に微生物を変異誘導する通常の方法を
用いればよい。変異誘起剤処理の例としては、２５０μ
９７１１１のＮ−二トローＮ′−メチルーＮ−二トロソ
グアニジンによって３０℃で２０分間処理する方法があ
る。In order to obtain the mutant strain of the present invention using a strain belonging to the Brevibacterium genus or Corynebacterium genus and having properties advantageous for lysine production, microorganisms such as ultraviolet irradiation, X-ray irradiation, mutagenic agent treatment, etc. are generally used. Any conventional method for inducing mutations may be used. An example of mutagen treatment is 250μ
There is a method of treating with N-nitro N'-methyl-N-nitrosoguanidine of 97111 at 30° C. for 20 minutes.
本発明においてはこのようにして得られた変異株カラス
−ツク−オキシド増産剤、スー／４− ＃ ＊シトシス
ムメーゼ誘導抑制剤、スーパ−オキシドラジカル反応促
進剤、またはスー・平−オキシドを形成する酸素を供給
する醸化剤に対する耐性株を選別取得する。In the present invention, the thus obtained mutant Karasu-Tsuku-oxide production enhancer, Su/4-#*cytosis induction inhibitor, superoxide radical reaction accelerator, or oxygen forming Su/4- Select and obtain strains that are resistant to the brewing agent that supplies them.
これらの薬剤はいずれもスーツ４−オキシド（０２−）
と不飽和脂肪酸の反応による過酸化脂質の形成をしやす
くするものである。すなわち、これらＯ薬剤耐性株は細
胞活性が低下しやすい環境下で増殖する細胞活性の強い
菌株である筈であシ、本発明者らはこのような菌株がＬ
−Ｉ７ジン生産用の菌株として極めてすぐれていること
を見出したのである。Both of these drugs are suit 4-oxide (02-)
This facilitates the formation of lipid peroxides through the reaction of unsaturated fatty acids with unsaturated fatty acids. In other words, these O drug-resistant strains are supposed to be strains with strong cell activity that proliferate in an environment where cell activity is likely to decrease.
-I7 It was discovered that this strain is extremely excellent as a strain for producing gin.
スーツ！−オキシド増産剤とは生体内においてスーパー
オキシドを増す作用を有するものであシ、例えばメチル
ビオロゲン、ニトロ７ラントイン、ビタミンに１、モル
フイン、ストレプトニグリン、アドリアマイシン、マイ
トマイシンＣ，ダウノマイシン、ブレオマイシン、β−
ラノ母コン、ｅｉａ−グラチナム（［Ｉ）ジアミノジク
ロライドの如きものである。suit! - Oxide production enhancers are those that have the effect of increasing superoxide in the living body, such as methyl viologen, nitro7lantoin, vitamin 1, morphine, streptonigrin, adriamycin, mitomycin C, daunomycin, bleomycin, β-
It is a compound such as lanocontainer, eia-gratinum ([I) diaminodichloride.
スーパーオキシドジスムターゼ誘導抑制剤とはスー・ぐ
−オキシドを過酸化水素に変える酵素であるスーパーオ
キシドジスムターゼの生体内における誘導生成を抑制す
るもので、例としてはピーーロマイシンを挙げることが
できる。A superoxide dismutase induction inhibitor is an agent that inhibits the induction of superoxide dismutase, an enzyme that converts sour oxide into hydrogen peroxide, in vivo, and pelomycin can be mentioned as an example.
スーツや−オキシドラジカル反応促進剤とは、スー・母
−オキシドが不飽和脂肪酸と反応して過酸化脂質を生成
する反応を促進するものであシ、例としてフェニルヒド
ラノンを挙げることができる。The suit or oxide radical reaction accelerator is one that promotes the reaction in which a soot mother oxide reacts with an unsaturated fatty acid to produce lipid peroxide, and phenylhydranone can be mentioned as an example.
酸化剤は生体内でスーパーオキシドを形成する酸素を供
給するものであシ、例えば過酸化ベンゾイル、過硫酸ア
ンモニウムの如きものである。The oxidizing agent is one that supplies oxygen that forms superoxide in vivo, such as benzoyl peroxide and ammonium persulfate.
これらの薬剤に対する耐性株の取得は通常の薬剤耐性株
取得方法に準じて行なえばよく、例えば前記薬剤のいず
れかを親株が生育しないような濃度で含有する寒天培地
に変異株を撒いて培養し、生育し九コロニーを採取すれ
ばよい。培地中の薬剤の濃度は薬剤の種類によって異な
）、例えばメチルピオロｒンの場合には１．５μシ’ａ
ｔ程Ｈ、ビューロマイシンの場合には０．５μ１１７ａ
ｔ 徨度、フェニルヒドラジンの場合には２０μシ旬程
度、過酸化ベンゾイルの場合には２０μ７９／ｒｄ程度
が適当である。その他の薬剤を用い゛る場合には予め培
養試験を行なフて適切な濃度を定めればよいことはいう
までもない。Obtaining strains resistant to these drugs can be carried out in accordance with the usual method for obtaining drug-resistant strains, for example, by spreading and culturing the mutant strain on an agar medium containing one of the above drugs at a concentration that does not allow the growth of the parent strain. , all you need to do is to grow and collect nine colonies. (The concentration of the drug in the medium varies depending on the type of drug), for example, in the case of methylpiolone, the concentration of drug is 1.5μ a
t H, in case of buromycin 0.5μ117a
In the case of phenylhydrazine, a suitable degree of t-tolerance is about 20μ, and in the case of benzoyl peroxide, it is about 20μ79/rd. Needless to say, when using other drugs, a culture test may be conducted in advance to determine the appropriate concentration.
また、グレピパクテリウム属またはコリネパクテリクム
属に属する野生株を変異処理し、得られた変異株の中か
らスーツ臂−オキシド増産剤、スー／４−オキクドソス
ムメーゼ鰐導抑制剤、スーツ４’−オキシドラジカル反
応促進剤ま九はスー・臂−オキシドを形成する酸素を供
給する酸化剤に耐性を有する株を選別し九後にこれらの
耐性株にリジン化してもリジン生産能を大巾に高めた菌
株を取得できる。In addition, wild strains belonging to the genus Grepipacterium or Corynepactericum were subjected to mutation treatment, and from among the obtained mutant strains, a suit arm oxide production enhancer, a crocodile induction inhibitor for suo/4-oxydososummase, etc. The 4'-oxide radical reaction accelerator is used to select strains that are resistant to oxidizing agents that supply oxygen to form soot-oxides. You can obtain strains with greatly increased
本発明に使用しうる微生物の例としては、スーツ々−オ
キシド増産剤については、メチルピオロｒ中シトジスム
ターゼ誘導抑制剤については、ピ。Examples of microorganisms that can be used in the present invention include: methyl pyrolytic acid, methylpyrolytic acid, methylpyrolytic acid, cytodismutase induction inhibitor, and cytodismutase induction inhibitor.
−ロマイシンに耐性を有するコリネバクテリウ・ム・ア
セトグルタミカムＡＪ １２，２′２３ （ＦＥＲＭ−
直こまた、スーツや−オキシドラジカル反応促進剤につ
いては、てスー・々−オキシドを形成する酸素を供給す
る酸化剤については、過酸化ベンゾイルに耐性を有すこ
のよ５な微生物を用いてＬ−リジンを生成蓄゛積させる
にはＬ −Ｕジン発酵に用いられる常法を用いて行なえ
ばよい。- Corynebacterium acetoglutamicum AJ 12,2'23 (FERM-
In addition, for suits and -oxide radical reaction accelerators, for oxidizing agents that supply oxygen to form tetanus-oxide, we can use these five microorganisms that are resistant to benzoyl peroxide. - Lysine can be produced and accumulated using a conventional method used for L-U gin fermentation.
すなわち、使用する培地としては、通常の炭素源、窒素
源、無機イオンその他の栄養素の含有する通常の培地を
用いる。炭素源としては、例えばサトウキビ、甜菜から
の糖汁あるいは廃糖蜜、澱粉加水分解物等の糖質原料等
ま九は酢酸等の有機酸等を用いればよい。That is, the medium to be used is a conventional medium containing a conventional carbon source, nitrogen source, inorganic ions, and other nutrients. As the carbon source, for example, sugar juice from sugar cane or sugar beet, or carbohydrate raw materials such as blackstrap molasses or starch hydrolyzate, or organic acids such as acetic acid may be used.
優素源も通常のＬ−ＩＪジン発酵に用いられるアンモニ
ウム塩、アンモニア水、尿素等を用いればよく、その他
リン酸イオン、マグネシウムイオン等の有機イオン及び
サイアミン等のビタミンを必要に応じて適宜使用する。Ammonium salts, aqueous ammonia, urea, etc. used in normal L-IJ gin fermentation may be used as the element source, and organic ions such as phosphate ions, magnesium ions, and vitamins such as thiamine may be used as necessary. do.
栄養要求性株のような特定の物質を生育に必要とする菌
株を用いるときにはこれらの物質そのものを培地に加え
るか、これらを含む蛋白加水分解物、ペプトン、コーン
ステーｆ＋）カー、肉工Φス、酵母エキスなどを加えた
培地を用いればよい。When using bacterial strains that require specific substances for growth, such as auxotrophic strains, these substances themselves must be added to the culture medium, or protein hydrolysates containing these substances, peptone, cornstarch, meat processing A medium to which yeast extract or the like may be added may be used.
培養条件についても、温度３０〜４０℃、ＰＨ６〜８の
範囲内で好気的条件で実施する等常法によりて実施すれ
ばよい。また、発酵液からＬ −Ｉ７ゾンを取得する方
法も常法に従って行なえばよいことはいうまでもない。Regarding the culture conditions, the culture may be carried out by a conventional method, such as under aerobic conditions at a temperature of 30 to 40°C and a pH of 6 to 8. Furthermore, it goes without saying that L-I7zone can be obtained from the fermentation broth by any conventional method.
本発明はＬ−グルタミン酸生産菌にそのスーツ４’−オ
キシドジスムターゼ活性を強化することによってＬ−リ
ジン生産能を高めるという全く新規な知見に係るもので
あシ、本発明の方法によってＬ−リジンを簡便な手段で
安価に取得できる。The present invention relates to the completely new finding that L-lysine production ability is increased by enhancing the suit 4'-oxide dismutase activity of L-glutamic acid producing bacteria. It can be obtained easily and inexpensively.
次に、本発明の方法に使用する微生物の取得例を示す。Next, an example of obtaining microorganisms used in the method of the present invention will be shown.
親株として下記に示す微生物を使用した。The microorganism shown below was used as a parent strain.
！レビパクテリウム・ラクトフェルメンタムＡＪ ３９
９０ 、ＦＥＲＭ−Ｐ ３３８７ （ＡＥＣｒ、ＭＬｒ
、ｋｔ＊−）！レピパクテリウム・フラバムＦ’ＡＥＣ
１−３０。! Levipacterium lactofermentum AJ 39
90, FERM-P 3387 (AECr, MLr
,kt*-)! Lepipacterium flavum F'AEC
1-30.
ＦｇＲＭ−Ｐ ２８２ （ｋＥＣ’ ）コリネバクテリ
ウム・アセトグルタミカム ＡＪ３７９２ 、 ＦＥ
ＲＭ−Ｐ ２６５０ （ＡＥＣ’ 、 β−ＨＩ、ｒ）
これらの菌株をブイヨンスラントで２４時間培養し、得
られた各菌株をいずれも２５０μｌ／ｒｕｔのＮ−二ト
ローＮ／ −メチル−Ｎ−ニトロソクアニジンによって
３０℃で２０分間処理して変異株を得九〇
イーストエキス１ ｉｔ／ｍ ｓペグトンＩＦ／ｄｇ、
食塩０、５１１７ｄｌおよび寒天２１７ｄｌよシなる培
地に、メチルビオロゲン１．５μｇ〜、ピューロマイク
７０．５μｇ７ｍｔ、 フェニルヒドラノン２０μ７
７７ｒｄ、過酸化ベンゾイル１０μｂ包を添加し、いず
れもｐＨ７，０に調整後１２０Ｃで１５分間殺菌して寒
天培地を調製し念。FgRM-P 282 (kEC') Corynebacterium acetoglutamicum AJ3792, FE
RM-P 2650 (AEC', β-HI, r)
These strains were cultured in bouillon slant for 24 hours, and each strain was treated with 250 μl/rut of N-nitro-N/-methyl-N-nitrosoquanidine at 30°C for 20 minutes to isolate mutant strains. Toku90 Yeast Extract 1 it/m s Pegton IF/dg,
In a medium consisting of 0.5117 dl of sodium chloride and 217 dl of agar, 1.5 μg ~ of methyl viologen, 70.5 μg of Puromic, 7 mt, and 20 μ7 of phenylhydranone.
77rd and a 10 μb packet of benzoyl peroxide were added, and after adjusting the pH to 7.0, sterilization was carried out at 120C for 15 minutes to prepare an agar medium.
前記変異株をこの寒天培地に撒いて３１．５℃で４８時
間培養し、生成したコロニーを採取して各薬剤耐性株を
取得し、表１に記載された各ＡＪ番号を付与し友。The mutant strain was spread on this agar medium and cultured at 31.5°C for 48 hours, and the resulting colonies were collected to obtain each drug-resistant strain, which was assigned each AJ number listed in Table 1.
このようにして得られた各薬剤耐性株および親株につい
て各薬剤の濃度を変えて生育試験を行なっ之結果を次に
示す。Growth tests were conducted on each of the drug-resistant strains and the parent strain thus obtained, with varying concentrations of each drug.The results are shown below.
試験方法としては、いずれの菌株もブイヨンスラントで
２４時間培養し、イーストエキスｌ １ｌｄｌ−ベグト
ン１１７ｍ １食塩０．５Ｉ殉および寒天２１１／ｄｉ
を含有するｐＨ７，０の培地を直径８ｔＭのシャーレに
入れて調製し几寒天プレート上に菌数がグレート当シ１
０５〜１０個になるように撒い友。そして、その上に第
１表に示す濃度の各薬剤を含有するペーノ量−ディスク
をおいて１６〜４８時間培養後、形成される生育阻止円
の存在の有無によりて生育度を求めた。各薬剤耐性株お
よびその親株について得られた結果を８１表に示す。な
お、表中井、＋は菌の生育度を表わし、−は生育が観察
されなかりたものを表わしている。As a test method, all strains were cultured for 24 hours in bouillon slant, yeast extract 1 1 ldl-Begton 117 ml 1 common salt 0.5 I and agar 211/di.
A medium containing pH 7.0 was prepared by placing it in a Petri dish with a diameter of 8 tM, and the number of bacteria was 1.
Sprinkle so that there are 05 to 10 pieces. Then, a peno disk containing each drug at the concentration shown in Table 1 was placed thereon and cultured for 16 to 48 hours, and the degree of growth was determined based on the presence or absence of a growth inhibition circle formed. Table 81 shows the results obtained for each drug-resistant strain and its parent strain. In addition, + indicates the degree of growth of the bacteria, and - indicates that no growth was observed.
第 １ 表
前述の各薬剤耐性株およびその親株のスー・量−オキシ
ドシスムターゼ活性を測定し九結果を第２表に示す。Table 1 The oxidocysmutase activity of each of the above-mentioned drug-resistant strains and their parent strains was measured and the results are shown in Table 2.
測定は次のようにして行なりた。まず、各菌株の培養液
２０ゴを１０．００ Ｏｒｐｍで１０分間遠心して沈殿
物を集め、ＰＨ７の０．１Ｍリン酸緩衝液で２回洗浄し
た。洗浄した沈殿物に前記リン酸緩衝液を２０１４加え
て懸濁し、超音波処理ｔ−５分間行なりてから１０．０
０ Ｏｒｐｍで１０分間遠心し、得られた上清液を試料
とした。The measurements were carried out as follows. First, 20 cultures of each strain were centrifuged at 10.00 Orpm for 10 minutes to collect precipitates, which were washed twice with 0.1M phosphate buffer, pH 7. The washed precipitate was suspended by adding 2014 ml of the phosphate buffer solution, and subjected to ultrasonication for 5 minutes, and then 10.0
The mixture was centrifuged at 0 Orpm for 10 minutes, and the resulting supernatant was used as a sample.
試験管にｐＨ１０，２の０．０５Ｍ炭酸ナトリウム緩衝
液２．４ ａｌ ｔ−とシ、３ｍＭキサンチン、３ｍＭ
ＥＤＴ人、０．１５％５％クシアルブミンよび０゜７５
２？ＩＭニトログルーテトラゾリウムを各々０．１ｄ添
加し次。これに前記試料０．１ ｍを加えて２５℃で１
０分間放置し、後述するキサンチンオキシダーゼ溶液０
．１ｄを加えすばやく攪拌して２５Ｃでインキ−ベート
した。２０分後に６ｍＭＣ託旬０．ＩＭｌを添加して反
応を停止させ、５６０ ｎｍにおける吸光度を測定した
。一方、試料のかわ９に蒸溜水を用いて同様の操作を行
ない、得られ九吸光度をブランクとした。In a test tube, add 0.05M sodium carbonate buffer at pH 10.2, 2.4 alt, 3mM xanthine, 3mM
EDT human, 0.15% 5% oxyalbumin and 0°75
2? Next, 0.1 d each of IM nitroglutetrazolium was added. Add 0.1 m of the above sample to this and heat at 25°C for 1
Leave to stand for 0 minutes, and add xanthine oxidase solution 0 to be described later.
．． 1d was added, stirred quickly, and incubated at 25C. After 20 minutes, 6mMC 0. The reaction was stopped by adding IMl and the absorbance was measured at 560 nm. On the other hand, the same operation was carried out using distilled water as the sample glue 9, and the obtained absorbance 9 was used as a blank.
活性単位は、このような測定条件でキサンチンオキシダ
ーゼ灰石を１／２阻害するスーツクーオキシドジスムタ
ーゼ活性を１単位とした。One unit of activity was defined as the activity of suction oxide dismutase that inhibits xanthine oxidase by half under these measurement conditions.
なお、前記キサンチンオキシダーゼ溶液は、ブランク試
験における吸光度が０．２３前後になるようにキサンチ
ンオキシダーゼを２Ｍ（ＮＨ４）２８０４で希釈し友も
のであシ、キサンチンオキシダーゼ濃度は約２．ｌＸ１
０ Ｍでありた。The xanthine oxidase solution was prepared by diluting xanthine oxidase with 2M (NH4) 2804 so that the absorbance in the blank test was around 0.23, and the xanthine oxidase concentration was about 2. lX1
It was 0M.
第 ２ 表 以下、実施例を示す。Table 2 Examples are shown below.
実施例１
グルコース３６１９／ｌＥｊ、塩化アンモニウム２０ｒ
ｒ９／ｋｌ、ＫＨ２ＰＯ４１ｗ／ｋｌ、Ｍｇ５Ｏ４・７
ａｑ Ｏ，４ｍ９／Ｍ 。Example 1 Glucose 3619/lEj, ammonium chloride 20r
r9/kl, KH2PO41w/kl, Mg5O4・7
aq O, 4m9/M.
Ｆ＠５Ｏ４４ａｑ １０ ｔｓｌ／ｍｌ、ＭｎＳＯ４４
ａｑ ８ ｆｉ１７７ｋｌ、大豆蛋白酸加水分解物（窒
素として）１Ｍｇ／ｒｔｔｌ 、サイアミン塩酸塩０．
１μシｎおよびピオチン０．３μ／１７ｆｎｌを含有す
る培地を調製し、その３０ｄづつを５００４容の振盪フ
ラスコに入れて１１５℃で１０分間を接種し、往復振盪
機によシ３１．５℃で培養を行っ次。４８時間で発酵を
終了し発酵液中に蓄積したＬ −ＩＪジンを測定し次。F@5O44aq 10 tsl/ml, MnSO44
aq 8 fi177kl, soybean protein acid hydrolyzate (as nitrogen) 1Mg/rttl, thiamine hydrochloride 0.
Prepare a medium containing 1μ sin and 0.3μ/17fnl piotin, put 30d each into a 5004 volume shaking flask, inoculate at 115°C for 10 minutes, and transfer to a reciprocating shaker at 31.5°C. Next, culture. Fermentation was completed in 48 hours, and L-IJ gin accumulated in the fermentation liquid was measured.
その結果、第３表に示すようにいずれの薬剤耐性株も良
好なＬ −ＩＪジン（塩酸塩換算）を蓄積していた。As a result, as shown in Table 3, all drug-resistant strains accumulated good L-IJ gin (in terms of hydrochloride).
第 ３ 表
実施例２
廃蔗糖蜜を糖として８０〃旬、正２ＰＯ４１４旬、Ｍｇ
ＳＯ４・７ａｑ １ ｍ９／ｍｌ、大豆加水分解物を窒
素換算で１４旬および硫酸アンモニウム５００μｇ７ｒ
ｔｔｔの組成を有する培地を調製しくｐ）（７，０）、
その２０ゴづつを５００ｄ振盪フラスコに分注して加熱
殺菌し、あらかじめ別殺菌した炭酸カルシウムを１．９
加えた。この培地に下記の菌株を接種し、往復振盪機に
よ、９３１．５℃で培養を行つ次。Table 3 Example 2 Using waste cane molasses as sugar, 80 yen, regular 2PO 414 yen, Mg
SO4.7aq 1 m9/ml, soybean hydrolyzate 14 times in terms of nitrogen and ammonium sulfate 500μg7r
Prepare a medium having the composition of ttt p) (7,0),
Dispense 20 grams of each into 500 d shake flasks and heat sterilize them.
added. The following bacterial strains were inoculated into this medium and cultured at 931.5°C using a reciprocating shaker.
７２時間で発酵を終了し、発酵液中に蓄積し九Ｌ−リジ
ンを測定し比。その結果、第４表に示すようにいずれの
薬剤耐性株もＬ −ＩＪレシン塩酸塩換算）を良好に蓄
積し念。Fermentation was completed in 72 hours, and the nine L-lysine accumulated in the fermentation liquid was measured and compared. As a result, as shown in Table 4, all drug-resistant strains successfully accumulated L-IJ (resin hydrochloride equivalent).
第 ４ 表Table 4
Claims (1)
に属し、強化されたスーパーオキシドジスムターゼ活性
を有し、かつＬ−リジン生産能を有する変異株を培養し
て培養液中にＬ−リジンを生成、蓄積せしめ、これを採
取することを特徴とする発酵法によるＬ−リジンの製造
方法。 ２、変異株がスーパーオキシド増産剤、スーパーオキシ
ドジスムターゼ誘導抑制剤、スーパーオキシドラジカル
反応促進剤、またはスーパーオキシドを形成する酸素を
供給する酸化剤に耐性を有するものである特許請求の範
囲第１項記載の製造方法。[Scope of Claims] 1. A mutant strain belonging to the genus Brevibacterium or Corynebacterium that has enhanced superoxide dismutase activity and L-lysine production ability is cultured, and L-lysine is added to the culture solution. - A method for producing L-lysine by a fermentation method, which comprises producing, accumulating, and collecting lysine. 2. Claim 1, wherein the mutant strain is resistant to superoxide production agents, superoxide dismutase induction inhibitors, superoxide radical reaction promoters, or oxidizing agents that supply oxygen to form superoxide. Manufacturing method described.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60125499A JPS61282092A (en) | 1985-06-10 | 1985-06-10 | Production of l-lysine by fermentation method |
| FR8608398A FR2583061B1 (en) | 1985-06-10 | 1986-06-10 | PROCESS FOR PRODUCING L-LYSINE BY FERMENTATION |
| US07/469,687 US5179010A (en) | 1985-06-10 | 1990-01-26 | Fermentation process for producing L-lysine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60125499A JPS61282092A (en) | 1985-06-10 | 1985-06-10 | Production of l-lysine by fermentation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS61282092A true JPS61282092A (en) | 1986-12-12 |
| JPH0411196B2 JPH0411196B2 (en) | 1992-02-27 |
Family
ID=14911619
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP60125499A Granted JPS61282092A (en) | 1985-06-10 | 1985-06-10 | Production of l-lysine by fermentation method |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JPS61282092A (en) |
| FR (1) | FR2583061B1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002089253A (en) * | 2000-09-11 | 2002-03-27 | Ibiden Co Ltd | Holding seal material of catalytic converter for exhaust emission control |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2103617B (en) * | 1981-08-10 | 1986-05-21 | Kyowa Hakko Kogyo Kk | Production of l-lysine by fermentation and new micro-organisms obtained by protoplast fusion |
-
1985
- 1985-06-10 JP JP60125499A patent/JPS61282092A/en active Granted
-
1986
- 1986-06-10 FR FR8608398A patent/FR2583061B1/en not_active Expired
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002089253A (en) * | 2000-09-11 | 2002-03-27 | Ibiden Co Ltd | Holding seal material of catalytic converter for exhaust emission control |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0411196B2 (en) | 1992-02-27 |
| FR2583061B1 (en) | 1989-07-28 |
| FR2583061A1 (en) | 1986-12-12 |
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