JPS6359680B2 - - Google Patents

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Publication number
JPS6359680B2
JPS6359680B2 JP54069800A JP6980079A JPS6359680B2 JP S6359680 B2 JPS6359680 B2 JP S6359680B2 JP 54069800 A JP54069800 A JP 54069800A JP 6980079 A JP6980079 A JP 6980079A JP S6359680 B2 JPS6359680 B2 JP S6359680B2
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Prior art keywords
atp
medium
methanol
culture
adenosine
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JPS55162996A (en
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Priority to JP6980079A priority Critical patent/JPS55162996A/en
Priority to GB8017934A priority patent/GB2052504B/en
Priority to DE19803020851 priority patent/DE3020851C2/en
Priority to FR8012332A priority patent/FR2458588A1/en
Priority to SU802933104A priority patent/SU1144619A3/en
Publication of JPS55162996A publication Critical patent/JPS55162996A/en
Publication of JPS6359680B2 publication Critical patent/JPS6359680B2/ja
Granted legal-status Critical Current

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/26Preparation of nitrogen-containing carbohydrates
    • C12P19/28N-glycosides
    • C12P19/30Nucleotides
    • C12P19/32Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Description

【発明の詳細な説明】 本発明はアデノシン−5′−トリフオスフエート
(以下ATPと略称する)を発酵法により製造する
方法に関する。 ATPは生体内で高エネルギーリン酸化合物と
してエネルギー代謝に重要な役割を示しているも
ので、医薬、生化学試薬として重要視されてい
る。また、近年、ATPを使用するバイオリアク
ターに於てATPを効率よく使用するために、
ATP再生系にアデノシン−5′−ジフオスフエー
トをATPに再生する酵素ピルビン酸キナーゼを
使用することが検討されているなどフラビン・ア
デニンジヌクレオチド、ニコチンアミド・アデニ
ンジヌクレオチドなどの補酵素や生化学物質を生
産する上に於てATPの安価な供給が望まれてい
る。 従来、ATPの製造法としては直接動物の筋肉
から単離精製する方法、有機化学的合成法、5′−
アデニル酸を酵素的にリン酸化する方法および発
酵法などがある。これらのうち、発酵法として
は、例えばアデニンまたはその誘導体を含む培地
にブレビバクテリウム・アンモニアゲネスに属す
る細菌を培養してATPを生成蓄積する方法(特
公昭41−17634号)、ATP生産能を有する微生物
を界面活性剤を含む培地に培養し、培養物から
ATPを採取する方法(特公昭49−28996号)、培
地中におけるATPの生成が最大に達した時点で
フエノール性水酸基を有する化合物を添加する方
法(特開昭53−6490号)などが提案されている。 しかし、これらの発酵法による従来法ではいず
れも培地中にアデニンもしくはその誘導体、例え
ばアデノシンを存在させて発酵させるものであ
り、このアデニンやその誘導体を基質としてこれ
をリン酸化することによりATPを生成蓄積させ
るものである。したがつて、従来法は高価なアデ
ニン又はその誘導体を原料とするものであるから
工業上有利な方法とは言えない。 本発明者はATPを発酵法により工業的に安価
に製造し得る方法について検討した結果、アデニ
ンもしくはその誘導体に代えてメタノールもしく
はメタノールと同一代謝経路を示す化学物質、お
よび特定範囲量の無機リン酸を基質として含む培
地にメタノール資化性を有するATP生産菌を培
養することにより、ATPが高濃度で培地中に蓄
積することを見出し、本発明をなすに至つた。 したがつて、本発明はATPを工業的に有利に
製造し得る方法を供給することを目的とする。 以下本発明を詳しく説明する。 本発明は前述したごとく、メタノールもしくは
メタノールと同一代謝経路を示す化学物質を基質
として含む培地を使用するものであるから、本発
明で利用するATP生産菌はメタノール資化性を
有するものでなければならない。メタノール資化
性を有するATP生産菌としては次のものが例示
し得る。 メチロモナス属(Methylomonas)に属するメ
チロモナス・プロバスFERM−P3193、メチロモ
ナス・メチロフオーラATCC21369;シユードモ
ナス属(Pseudomonas)に属するシユードモナ
ス・メチロトロフアNCIB10508、シユードモナ
ス・ロセアNCIB10597、シユードモナス・イン
スエータATCC21276、シユードモナス・メタノ
リカATCC21704、シユードモナス・メタニカ
ATCC21439;プロタミノバクター属
(Protaminobacter)に属するプロタミノバクタ
ー・ルーバーATCC8457、プロタミノバクター・
カンジダスATCC21372;アクロモバクター属
(Achromobacter)に属するアクロモバクター・
メタノロフイラATCC21275;コリネバクテリウ
ム属(Corynebacterium)に属するコリネバクテ
リウム・エスピーATCC21232;およびバチルス
属(Bacillus)に属するバチルス・セレウス
ATCC14579、バチルス・ズブチリスvar.
NB1001FERM−P1373。 上掲した細菌を利用して有用物質を生産する技
術として、従来、メタノールを基質とする培地で
の培養による蛋白源としての菌体の増殖(特開昭
52−28988号)、フラビン・アデニンジヌクレオチ
ドの生産〔J.Ferment.Technol.55、630(1977)〕、
ビタミンB12の生産〔Appl.Microbiol.30、477
(1975)〕、脂質、多糖類の生産昭和53年度日本農
芸化学会大会講演要旨集、シンポジウムC1、微
生物の利用551〜556)、ならびにアミノ酸の生産
(前記講演要旨集)等が報告されているが、ATP
生産を目的とした上記細菌の利用についての報告
は未だみられない。 本発明は上掲したメタノール資化性を有する
ATP生産菌を、メタノール又はメタノールと同
一代謝経路を示す化学物質を基質とし、かつ無機
リン酸塩をリン酸根(PO4)として4〜35g/
を含む培地で培養することを特徴とする。ここで
“メタノールと同一代謝経路を示す化学物質”と
はメタン、メチルアミン、ホルムアルデヒド、ギ
酸のごとくメタノールと同一代謝経路で利用し得
る物質を意味する。メタノールならびに上記物質
は培地の炭素源として使用されるものであつて、
それらの培地中における濃度は高すぎると細菌の
生育を阻害するので留意すべきである。一般にそ
の濃度はメタノール、メチルアミンでは0.2〜4
容量%、ギ酸、ホルムアルデヒドでは0.01〜0.1
容量%が適当であり、メタンについてはその溶解
度に相当する濃度で使用するとよい。 また、これらの炭素源は培養当初に培地に全量
添加してもよいが、最初は培地における炭素源の
濃度を低くしておき、培養の進行とともに炭素源
の消費に応じて追加的に補給すると、ATPの生
産上さらに好結果が得られる。 本発明は上述したごとき炭素源に加えて無機リ
ン酸塩をPO4として4〜35g/培地に存在させ
ることを必要とするものであるが、このような培
地における無機リン酸塩の濃度は通常の細菌培地
の数倍乃至数十倍に相当するものである。 なお、培地中の無機リン酸(PO4として)の濃
度が4g/より低いとATPの生産の増加がみ
られず、一方35g/より高くなるとATP生産
菌の増殖が抑制されてATPの生産量を低減する
ことになる。因みに、無機リン酸は培養により生
産されたATPが菌体外に分泌されてフオスフア
ターゼにより分解されるのを防止する作用をす
る。 ここで用いる無機リン酸塩は通常の発酵培地に
使用されるものであつて、例えば
(NH42HPO4、KH2PO4、K2HPO4のごときも
のが例示し得る。本発明では培地組成分として上
述した炭素源および無機リン酸塩に加えて、カリ
ウム、マグネシウム、鉄、マンガンなどの無機
塩、場合によつては亜鉛、コバルト、モリブデン
のごとき金属の無機塩;アンモニア、アンモニウ
ム塩、尿素、硝酸塩のごとき蓄素源を使用し得
る。また、界面活性剤や消泡剤を必要に応じて培
地へ添加することもできる。 本発明で使用する倍地組成を例示すると下記の
とおりである。 炭素源 0.05〜2容量% (NH42HPO4 5〜45g/ (PO4として3.5〜32g/) KH2PO4 0.5〜2.5g/ (PO4として0.35〜1、75g/) K2HPO4 0.5〜2.5g/ (PO4として0.275〜1.37g/) (NH42SO4 0〜1.0g/ MgSO4・7H2O 0.5〜5.0g/ FeSO4・7H2O 0.05〜0.5g/ CaCl2・2H2O 0〜0.2g/ MnSO4・4H2O 0〜0.2g/ 上述したごとき培地中における前掲のATP生
産菌の培養は、一般に、PH5.8〜9.0、好ましくは
6.0〜8.0、温度15〜50℃、好ましくは30〜40℃、
通気量0.5〜4.0V.V.M(空気量/培養液/
min)の条件下に40〜600rpmで撹拌しながら2
日乃至9日間行うとよい。なお、培地のPHの調整
はアンモニアのごときアルカリ性物質又は硫酸の
ごとき酸性物質、さらにはリン酸バツフアーのご
とき緩衝剤、尿素、炭酸カルシウムなどを使用し
得るが、アルカリ性物質を使用するときはアンモ
ニアは窒素源にもなるので特に好適である。 上述のごとき培養条件下で発酵させることによ
りATPを培地中に2〜12g/の高濃度で蓄積
させることができる。発酵終了後菌体を除去し、
生成したATPを公知の手法により分離、採取す
る。例えば、発酵済培地から菌体を除去したのち
の上澄液について活性炭ならびにアニオン交換樹
脂を利用した吸着、溶出による分画処理および濃
縮沈殿処理の組合せによりATPは採取し得る。 本発明によると実施例に示すごとく、培地にア
デニンやその誘導体を添加しなくてもATPを2
g/以上の高濃度で培地中に生成、蓄積し得
る。 以下に実施例を例示して本発明の効果を具体的
に説明する。 なお、各実施例中の培地中におけるATPの生
成量は下記のように表わされるATPとルシフエ
リン−ルシフエラーゼとの反応原理を利用し次の
ごとくして測定した。 (1) ルシフエリン+ルシフエラーゼ+ATPMg2 ―――→ アデニル−ルシフエリン+ピロリン酸塩 (2) アデニル−ルシフエリンO2 ―――→ アデニル−
オキシルシフエリン 上記(2)の反応で発生するケイ光の560〜580nm
の波長域の光の強さをCHEM−
GLOWPHOTOMETERJ4−7441(American
Instrument Company製)を用いて、一定時間測
定してその積分量を求め、これを予め作成してあ
る既知のATP標準液検量線と対比して、培養液
中のATPの生成量を測定する。 実施例 1 (NH42HPO47.0g、KH2PO41.0g、
K2HPO41.0g、MgSO4、7H2O1.0gおよび
FeSO4・7H2O0.1gをイオン交換水1に溶解し
た培地を300ml容の三角フラスコに各100ml分注
し、殺菌処理後メタノール1.6g宛を夫々に添加
してなる各培地に、上記組成の寒天斜面培地で予
め培養したメチロモナス・プロバスFERM−P
No.3193をそれぞれ接種し、30℃で振盪培養した。
培養開始後4日目に培養液中にATP2.8g/が
生成蓄積する。 この培養液1を80℃に5分間加温処理し、冷
却後遠心分離して菌体を除き、上澄液を3N塩酸
でPH3.5に調整したものを活性炭処理してATPを
活性炭に吸着させた後、1.4%アンモニア含有50
%アルコール溶液で活性炭に吸着したATPを溶
出する。この溶出液を低温下で減圧濃縮して過剰
のアンモニアを除いてPH8.0附近にする。 次いて、この濃縮液を予めCl型調整した強塩基
性陰イオン交換樹脂ダウエツクス1−X2(Cl)カ
ラムにATPを吸着させる。この吸着物をイオン
交換水で洗浄したのち、塩酸と塩化ナトリウムの
混合液(0.02M−HCl溶液(PH1.7)にNaClを溶
解して0.2M−Cl溶液としたもの)で溶出して
ATP画分を分取する。 この溶出液を苛性ソーダで中和後、これを活性
炭の充てんされているカラムに通し、15%アンモ
ニア水で溶出し、得られる溶出液を濃縮してアン
モニアを飛散させる。該濃縮液にメタノールを添
加することによりATPのナトリウム塩の結晶2.2
gを得た。 実施例 2 (NH42HPO47.0g、KH2PO41.0g、
K2HPO41.0g、MgSO4・7H2O2g、FeSO4
7H2O0.2gおよび消泡剤KS−66 0.2mlをイオン交
換水1に溶解した培地20を30容ジヤー・フ
アーメンターに仕込み、1.2Kg/cm2の加圧下120℃
で30分間殺菌処理する。冷却後、上記培地にメタ
ノール200mlを添加し、あらかじめ上記と同一の
培地で培養した母菌(メチロモナス・プロバス
FERM−PNo.3193)を2%接種し、35℃で500r.
p.mで撹拌しながら、毎分10/20液量/min
(0.5V.V.M)の通気をおこない、菌の増殖につれ
て毎分20/20/min(1.0V.V.M)と通気量を
増加させ、96時間培養をおこなう。 培養中のPHは14%アンモニア水で6.0〜7.2に自
動制御し、消費されるメタノールはガスクロマト
グラフイーで測定して0.3〜2.0%になるように自
動添加する。培養開始後96時間で培養液中に
ATP5.3g/が生成蓄積する。培養液中のATP
は実施例1に記載と同じ方法により回収する。 実施例 3 実施例2で使用した培地成分のうち
(NH42HPO4の量を20g/とする以外は実施
例2と同じ条件で培養を行なう。培養開始後96時
間で培養液中にATP12g/が生成・蓄積する。
ATPは実施例1と同じ方法により回収する。 実施例 4 実施例2で使用した培地成分のうち、
(NH42HPO4の量を30g/とする以外は実施
例2と同じ条件で培養を行なう。培養開始後96時
間で培養液中にATP7g/が生成・蓄積する。
ATPは実施例1と同じ方法により回収する。 比較例 1 実施例2で使用の培地成分のうち、
(NH42HPO4の量を3g/とする以外は実施
例2と同じ条件で培養を行なう。培養開始後96時
間で培養液中にATP0.2g/が生成するにとど
まる。 比較例 2 実施例2で使用の培地成分のうち、
(NH42HPO4の量を50g/とする以外は実施
例2と同じ条件で培養を行なう。培養開始後96時
間で培養液中にATP1.0g/が生成するにすぎ
ない。 実施例 5 下記表に示す各菌株を用い、実施例3に記載と
同じ培養条件で培養したときのATP生産量を下
記表に示す。 【表】
DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing adenosine-5'-triphosphate (hereinafter abbreviated as ATP) by a fermentation method. ATP plays an important role in energy metabolism as a high-energy phosphoric acid compound in living organisms, and is considered important as a pharmaceutical and biochemical reagent. In addition, in recent years, in order to use ATP efficiently in bioreactors that use ATP,
The use of pyruvate kinase, an enzyme that regenerates adenosine-5'-diphosphate into ATP, is being considered in the ATP regeneration system. A cheap supply of ATP is desired for production. Conventional methods for producing ATP include direct isolation and purification from animal muscle, organic chemical synthesis, and 5'-
Examples include a method of enzymatically phosphorylating adenylic acid and a fermentation method. Among these, fermentation methods include, for example, a method in which bacteria belonging to Brevibacterium ammoniagenes are cultivated in a medium containing adenine or its derivatives to produce and accumulate ATP (Special Publication No. 17634-1971), and a method in which ATP production capacity is Culture microorganisms with a surfactant in a medium containing surfactants, and
A method of collecting ATP (Japanese Patent Publication No. 49-28996) and a method of adding a compound having a phenolic hydroxyl group when the production of ATP in the medium reaches the maximum (Japanese Patent Publication No. 53-6490) have been proposed. ing. However, in all of these conventional fermentation methods, fermentation is carried out in the presence of adenine or its derivatives, such as adenosine, in the medium, and ATP is produced by phosphorylating this adenine or its derivatives as a substrate. It is something that accumulates. Therefore, since the conventional method uses expensive adenine or its derivatives as a raw material, it cannot be said to be an industrially advantageous method. As a result of studying a method for industrially producing ATP at low cost by fermentation, the present inventor found that instead of adenine or its derivatives, methanol or a chemical substance that shows the same metabolic pathway as methanol, and a specific range of inorganic phosphoric acid. The present inventors have discovered that ATP accumulates in a high concentration in a medium by culturing ATP-producing bacteria capable of assimilating methanol in a medium containing ATP as a substrate, leading to the present invention. Therefore, an object of the present invention is to provide a method that can industrially advantageously produce ATP. The present invention will be explained in detail below. As described above, the present invention uses a medium containing methanol or a chemical substance that exhibits the same metabolic pathway as methanol as a substrate, so the ATP-producing bacteria used in the present invention must be capable of assimilating methanol. No. Examples of ATP-producing bacteria capable of assimilating methanol include the following. Methylomonas probus FERM-P3193, Methylomonas methylophora ATCC21369, belonging to the genus Methylomonas; Pseudomonas methylotropha NCIB10508, Pseudomonas rosea NCIB10597, Pseudomonas insueta ATCC21276, Seudomonas belonging to the genus Pseudomonas Monas methanolica ATCC21704, Pseudomonas metanica
ATCC21439; Protaminobacter ruber belonging to the genus Protaminobacter ATCC8457;
Candida ATCC21372; Achromobacter belonging to the genus Achromobacter
Methanolophylla ATCC21275; Corynebacterium sp. ATCC21232, which belongs to the genus Corynebacterium; and Bacillus cereus, which belongs to the genus Bacillus.
ATCC14579, Bacillus subtilis var.
NB1001FERM−P1373. As a technology for producing useful substances using the above-mentioned bacteria, conventional methods have been to grow bacterial cells as a protein source by culturing them in a medium with methanol as a substrate (Japanese Unexamined Patent Publication No.
52-28988), production of flavin adenine dinucleotide [J. Ferment. Technol. 55, 630 (1977)],
Production of vitamin B 12 [Appl.Microbiol.30, 477
(1975)], production of lipids and polysaccharides, collection of lecture abstracts from the 1975 Japanese Society of Agricultural Chemistry Conference, Symposium C 1 , Utilization of Microorganisms 551-556), and production of amino acids (collection of lecture abstracts mentioned above). There is, but ATP
There are no reports yet on the use of the above bacteria for production purposes. The present invention has the above-mentioned methanol assimilation ability.
ATP-producing bacteria are used as substrates for methanol or a chemical substance that shows the same metabolic pathway as methanol, and inorganic phosphate as a phosphate radical (PO 4 ) at 4 to 35 g/
It is characterized by being cultured in a medium containing. Here, "chemical substances exhibiting the same metabolic pathway as methanol" means substances that can be utilized in the same metabolic pathway as methanol, such as methane, methylamine, formaldehyde, and formic acid. Methanol and the above substances are used as carbon sources for the culture medium,
It should be noted that if their concentration in the medium is too high, bacterial growth will be inhibited. Generally, the concentration is 0.2 to 4 for methanol and methylamine.
Volume %, 0.01 to 0.1 for formic acid and formaldehyde
Volume % is appropriate, and methane is preferably used at a concentration corresponding to its solubility. In addition, these carbon sources may be added in their entirety to the medium at the beginning of culture, but it is recommended to keep the concentration of carbon sources in the medium low at the beginning and supplement them as the carbon source is consumed as the culture progresses. , more favorable results in ATP production can be obtained. In addition to the carbon sources mentioned above, the present invention requires the presence of inorganic phosphate as PO 4 in the medium of 4 to 35 g/medium, but the concentration of inorganic phosphate in such a medium is usually This corresponds to several times to several tens of times that of the bacterial culture medium. Note that if the concentration of inorganic phosphoric acid (as PO 4 ) in the medium is lower than 4g/, no increase in ATP production will be observed, while if it is higher than 35g/, the growth of ATP-producing bacteria will be suppressed and the amount of ATP produced will decrease. This will reduce the Incidentally, inorganic phosphoric acid acts to prevent ATP produced during culture from being secreted outside the bacterial cell and being degraded by phosphatase. The inorganic phosphate used here is one used in ordinary fermentation media, and examples thereof include (NH 4 ) 2 HPO 4 , KH 2 PO 4 , and K 2 HPO 4 . In the present invention, in addition to the above-mentioned carbon source and inorganic phosphate as medium components, inorganic salts such as potassium, magnesium, iron, and manganese, and in some cases, inorganic salts of metals such as zinc, cobalt, and molybdenum; , ammonium salts, urea, nitrates, etc. may be used. Furthermore, a surfactant or an antifoaming agent can be added to the medium as necessary. Examples of the composition of the medium used in the present invention are as follows. Carbon source 0.05-2% by volume (NH 4 ) 2 HPO 4 5-45 g/ (3.5-32 g/ as PO 4 ) KH 2 PO 4 0.5-2.5 g/ (0.35-1.75 g/ as PO 4 ) K 2 HPO 4 0.5-2.5g/ (0.275-1.37g/ as PO 4 ) (NH 4 ) 2 SO 4 0-1.0g/ MgSO 4・7H 2 O 0.5-5.0g/ FeSO 4・7H 2 O 0.05-0.5g/ CaCl2.2H2O 0-0.2g / MnSO4.4H2O 0-0.2g /The culture of the ATP-producing bacteria described above in the medium described above generally has a pH of 5.8-9.0, preferably
6.0~8.0, temperature 15~50℃, preferably 30~40℃,
Aeration volume 0.5 to 4.0VVM (air volume/culture solution/
min) with stirring at 40 to 600 rpm.
It is best to do this for 1 to 9 days. In addition, to adjust the pH of the culture medium, alkaline substances such as ammonia or acidic substances such as sulfuric acid, buffers such as phosphate buffer, urea, calcium carbonate, etc. can be used, but when using alkaline substances, ammonia This is particularly suitable since it also serves as a nitrogen source. By fermenting under the above-mentioned culture conditions, ATP can be accumulated in the medium at a high concentration of 2 to 12 g/1. After fermentation is complete, remove the bacterial cells,
The generated ATP is separated and collected using a known method. For example, ATP can be collected from the supernatant after removing bacterial cells from the fermented medium by a combination of adsorption using activated carbon and anion exchange resin, fractionation treatment by elution, and concentration precipitation treatment. According to the present invention, as shown in the examples, ATP can be reduced by 2 without adding adenine or its derivatives to the culture medium.
It can be produced and accumulated in the culture medium at a high concentration of more than g/g/g. EXAMPLES The effects of the present invention will be specifically explained below with reference to Examples. The amount of ATP produced in the medium in each Example was measured as follows using the principle of the reaction between ATP and luciferin-luciferase expressed as below. (1) Luciferin + Luciferase + ATPMg 2 ---→ Adenyl-Luciferin + Pyrophosphate (2) Adenyl-Luciferin O 2 ----→ Adenyl-
Oxyluciferin 560-580nm of fluorescence generated in the reaction of (2) above
CHEM− is the intensity of light in the wavelength range of
GLOWPHOTOMETERJ4−7441 (American
(manufactured by Instrument Company) for a certain period of time to determine the integrated amount, and compare this with a known ATP standard solution calibration curve prepared in advance to measure the amount of ATP produced in the culture solution. Example 1 (NH 4 ) 2 HPO 4 7.0 g, KH 2 PO 4 1.0 g,
K 2 HPO 4 1.0 g, MgSO 4 , 7H 2 O 1.0 g and
Dispense 100 ml of a medium prepared by dissolving 0.1 g of FeSO 4 7H 2 O in 1 part of ion-exchanged water into 300 ml Erlenmeyer flasks, add 1.6 g of methanol to each medium after sterilization, and add the above composition to each medium. Methylomonas probus FERM-P cultured in advance on agar slants of
No. 3193 was inoculated and cultured with shaking at 30°C.
On the fourth day after the start of culture, 2.8 g/ATP is produced and accumulated in the culture solution. This culture solution 1 was heated to 80℃ for 5 minutes, cooled and centrifuged to remove the bacterial cells, and the supernatant was adjusted to pH 3.5 with 3N hydrochloric acid and treated with activated carbon to adsorb ATP on the activated carbon. After 1.4% ammonia content 50
Elute the ATP adsorbed on activated carbon with a % alcohol solution. This eluate is concentrated under reduced pressure at low temperature to remove excess ammonia and the pH is around 8.0. Next, ATP is adsorbed from this concentrated solution on a strongly basic anion exchange resin Dowex 1-X2 (Cl) column which has been previously adjusted to Cl type. After washing this adsorbate with ion-exchanged water, it was eluted with a mixture of hydrochloric acid and sodium chloride (NaCl was dissolved in 0.02M-HCl solution (PH1.7) to make a 0.2M-Cl solution).
Separate the ATP fraction. After neutralizing this eluate with caustic soda, it is passed through a column packed with activated carbon and eluted with 15% aqueous ammonia, and the resulting eluate is concentrated to scatter the ammonia. 2.2 Crystallization of the sodium salt of ATP by adding methanol to the concentrate
I got g. Example 2 (NH 4 ) 2 HPO 4 7.0 g, KH 2 PO 4 1.0 g,
K 2 HPO 4 1.0g, MgSO 4・7H 2 O2g, FeSO 4
Culture medium 20 in which 0.2 g of 7H 2 O and 0.2 ml of antifoam agent KS-66 were dissolved in 1 part of ion-exchanged water was placed in a 30-volume jar fermenter and heated at 120°C under a pressure of 1.2 kg/cm 2.
Sterilize for 30 minutes. After cooling, add 200 ml of methanol to the above medium, and add mother bacteria (Methylomonas probus) that was previously cultured in the same medium as above.
FERM-P No. 3193) was inoculated at 2% and incubated at 35°C for 500 r.
10/20 liquid volume/min while stirring at pm
(0.5VVM), and as the bacteria grow, increase the aeration rate to 20/20/min (1.0VVM) and culture for 96 hours. The pH during the culture is automatically controlled to 6.0-7.2 using 14% ammonia water, and methanol consumed is automatically added to 0.3-2.0% as measured by gas chromatography. into the culture medium 96 hours after the start of culture.
ATP5.3g/ is generated and accumulated. ATP in culture medium
is recovered by the same method as described in Example 1. Example 3 Culture is carried out under the same conditions as in Example 2, except that the amount of (NH 4 ) 2 HPO 4 among the medium components used in Example 2 is changed to 20 g/. 12 g/ATP is produced and accumulated in the culture solution 96 hours after the start of culture.
ATP is recovered using the same method as in Example 1. Example 4 Among the culture medium components used in Example 2,
Culture was carried out under the same conditions as in Example 2 except that the amount of (NH 4 ) 2 HPO 4 was 30 g/. 7g/ATP is produced and accumulated in the culture solution 96 hours after the start of culture.
ATP is recovered using the same method as in Example 1. Comparative Example 1 Among the medium components used in Example 2,
Culture was carried out under the same conditions as in Example 2 except that the amount of (NH 4 ) 2 HPO 4 was 3 g/. Only 0.2 g/ATP was produced in the culture solution 96 hours after the start of culture. Comparative Example 2 Among the medium components used in Example 2,
Culture was carried out under the same conditions as in Example 2 except that the amount of (NH 4 ) 2 HPO 4 was 50 g/. Only 1.0 g/ATP was produced in the culture solution 96 hours after the start of culture. Example 5 The ATP production amount when each strain shown in the table below was cultured under the same culture conditions as described in Example 3 is shown in the table below. 【table】

Claims (1)

【特許請求の範囲】 1 メチロモナス属、シユードモナス属、プロタ
ミノバクター属、アクロモバクター属、コリネバ
クテリウム属およびバチルス属からなる群から選
択される属に属するメタノール資化性を有するア
デノシン−5′−トリフオスフエート生産菌を、メ
タノール又はメタノールと同一代謝経路を示す化
学物質および無機リン酸塩をリン酸根(PO4)と
して4〜35g/を基質として含む培地中で培養
してアデノシン−5′−トリフオスフエートを生成
蓄積せしめて採取することを特徴とするアデノシ
ン−5′−トリフオスフエートの製造方法。 2 メタノールと同一代謝経路を示す化学物質が
メタン、メチルアミン、ホルムアルデヒドおよび
ギ酸からなる群から選択されるものである特許請
求の範囲第1項に記載の製造方法。
[Scope of Claims] 1. Adenosine-5 having the ability to assimilate methanol and belonging to a genus selected from the group consisting of Methylomonas, Pseudomonas, Protaminobacter, Achromobacter, Corynebacterium, and Bacillus. Adenosine-triphosphate-producing bacteria were cultured in a medium containing methanol or a chemical substance showing the same metabolic pathway as methanol, and 4 to 35 g of inorganic phosphate as a phosphate group (PO 4 ) as a substrate. 1. A method for producing adenosine-5'-triphosphate, which comprises producing, accumulating and collecting 5'-triphosphate. 2. The manufacturing method according to claim 1, wherein the chemical substance exhibiting the same metabolic pathway as methanol is selected from the group consisting of methane, methylamine, formaldehyde, and formic acid.
JP6980079A 1979-06-04 1979-06-04 Preparation of adenosine-5'-triphosphate through fermentation process Granted JPS55162996A (en)

Priority Applications (5)

Application Number Priority Date Filing Date Title
JP6980079A JPS55162996A (en) 1979-06-04 1979-06-04 Preparation of adenosine-5'-triphosphate through fermentation process
GB8017934A GB2052504B (en) 1979-06-04 1980-06-02 Process for the preparation of adenosine-5'-triphosphate by fermentation
DE19803020851 DE3020851C2 (en) 1979-06-04 1980-06-02 Adenosine 5'-triphosphate
FR8012332A FR2458588A1 (en) 1979-06-04 1980-06-03 PROCESS FOR THE PREPARATION OF ADENOSINE-5'-TRIPHOSPHATE BY FERMENTATION
SU802933104A SU1144619A3 (en) 1979-06-04 1980-06-03 Method of obtaining adenosine-5'-triphosphate

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP6980079A JPS55162996A (en) 1979-06-04 1979-06-04 Preparation of adenosine-5'-triphosphate through fermentation process

Publications (2)

Publication Number Publication Date
JPS55162996A JPS55162996A (en) 1980-12-18
JPS6359680B2 true JPS6359680B2 (en) 1988-11-21

Family

ID=13413170

Family Applications (1)

Application Number Title Priority Date Filing Date
JP6980079A Granted JPS55162996A (en) 1979-06-04 1979-06-04 Preparation of adenosine-5'-triphosphate through fermentation process

Country Status (5)

Country Link
JP (1) JPS55162996A (en)
DE (1) DE3020851C2 (en)
FR (1) FR2458588A1 (en)
GB (1) GB2052504B (en)
SU (1) SU1144619A3 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004261150A (en) 2003-03-04 2004-09-24 Ajinomoto Co Inc Method for producing objective material

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR1473629A (en) * 1966-03-31 1967-03-17 Kyowa Hakko Kogyo Kk Process for the production of 5'-purine nucleotides
GB1185123A (en) * 1967-05-15 1970-03-18 Kyowa Hakko Kogyo Kk Fermentation Processes Utilizing Gaseous Hydrocarbons
US3769165A (en) * 1968-06-14 1973-10-30 Kyowa Hakko Kogyo Kk Process for producing adenosine triphosphate and adenosine diphosphate
JPS5129537U (en) * 1974-08-27 1976-03-03
JPS51139677A (en) * 1975-05-24 1976-12-02 Kyowa Hakko Kogyo Co Ltd Process for cultivating methanol-assimilating microorganisms
JPS5530759Y2 (en) * 1975-06-12 1980-07-22

Also Published As

Publication number Publication date
DE3020851C2 (en) 1982-04-15
SU1144619A3 (en) 1985-03-07
FR2458588A1 (en) 1981-01-02
JPS55162996A (en) 1980-12-18
GB2052504B (en) 1983-08-10
GB2052504A (en) 1981-01-28
FR2458588B1 (en) 1983-07-29
DE3020851A1 (en) 1980-12-11

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