JPH053793A - Simultaneous fermentation of basic amino acid and acidic amino acid - Google Patents
Simultaneous fermentation of basic amino acid and acidic amino acidInfo
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- JPH053793A JPH053793A JP3106959A JP10695991A JPH053793A JP H053793 A JPH053793 A JP H053793A JP 3106959 A JP3106959 A JP 3106959A JP 10695991 A JP10695991 A JP 10695991A JP H053793 A JPH053793 A JP H053793A
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- Prior art keywords
- amino acid
- acidic
- basic
- fermentation
- producing
- Prior art date
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Abstract
Description
【産業上の利用分野】本発明は、塩基性Ｌ−アミノ酸生
産菌を酸性Ｌ−アミノ酸産生条件下に培養するか或いは
塩基性Ｌ−アミノ酸生産菌と酸性Ｌ−アミノ酸生産菌を
混合培養することを特徴とする塩基性Ｌ−アミノ酸と酸
性Ｌ−アミノ酸の同時発酵法に関する。TECHNICAL FIELD The present invention relates to culturing a basic L-amino acid-producing bacterium under acidic L-amino acid-producing conditions or a mixed culture of a basic L-amino acid-producing bacterium and an acidic L-amino acid-producing bacterium. And a method for co-fermenting a basic L-amino acid and an acidic L-amino acid.
【従来の技術】従来発酵法による塩基性Ｌ−アミノ酸と
酸性Ｌ−アミノ酸の同時生産は知られていない。知られ
ていたのは塩基性Ｌ−アミノ酸及び酸性Ｌ−アミノ酸の
いずれか一方の１種のアミノ酸を培地中に蓄積させるも
のであった。この場合、２種以上のアミノ酸が培地中に
認められても、目的以外のアミノ酸は副生物程度の微量
（数１０ｍｇ／ｄｌ）存在するのみであった。換言すれ
ば、塩基性アミノ酸と酸性アミノ酸を同時に商業的蓄積
量で発酵生産する方法は種々のメリットが期待されるに
も拘わらず未だ知られていない。2. Description of the Related Art Conventionally, simultaneous production of basic L-amino acids and acidic L-amino acids by fermentation has not been known. It was known that one of the basic L-amino acids and the acidic L-amino acids was accumulated in the medium. In this case, even if two or more kinds of amino acids were found in the medium, the amino acids other than the target were present in a trace amount (several 10 mg / dl) as a by-product. In other words, a method of fermentatively producing a basic amino acid and an acidic amino acid at the same time in a commercially accumulated amount has not been known although various merits are expected.
【発明が解決しようとする課題】本発明は、塩基性アミ
ノ酸と酸性アミノ酸を同時に商業的蓄積量で発酵生産す
る方法を提供することを目的とする。SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for fermentatively producing a basic amino acid and an acidic amino acid simultaneously in a commercially accumulated amount.
【課題を解決するための手段】本発明者は、上記目的の
ために種々検討し、塩基性アミノ酸と酸性アミノ酸を同
時に生産する方法を確立し、本発明を完成した。本発明
は、前記のように、塩基性Ｌ−アミノ酸生産菌を酸性Ｌ
−アミノ酸産生条件下に培養するか或いは塩基性Ｌ−ア
ミノ酸生産菌と酸性Ｌ−アミノ酸性産菌を混合培養する
ことを特徴とする塩基性Ｌ−アミノ酸と酸性Ｌ−アミノ
酸の同時発酵法に関する。以下、本発明を詳細に説明す
る。本発明に云う塩基性Ｌ−アミノ酸にはＬ−リジン
（Ｌｙｓ）、Ｌ−アルギニン（Ａｒｇ）、Ｌ−ヒスチジ
ン（Ｈｉｓ）及びＬ−オルニチン（Ｏｒｎ）が含まれ、
酸性Ｌ−アミノ酸にはＬ−グルタミン酸（Ｇｌｕ）及び
Ｌ−アスパラギン酸（Ａｓｐ）が含まれる。先ず、塩基
性Ｌ−アミノ酸生産菌を使用し、これを酸性Ｌ−アミノ
酸産生条件下に培養して塩基性Ｌ−アミノ酸と酸性Ｌ−
アミノ酸を同時に発酵生産する本発明に係わる第１の方
法について説明する。発酵培地としては、炭素源、窒素
源、無機塩類、生育因子などを含有する栄養培地または
合成培地が用いられる。炭素源としては、グルコース，
フラクトース，シュークロース，糖蜜，デンプン，デン
プン加水分解物，果汁などの炭水化物、エタノール，メ
タノール，プロパノールなどのアルコール類、酢酸など
の有機酸類が使用できる。窒素源としては、硫酸アンモ
ニウム，硝酸アンモニウム，塩化アンモニウム，リン酸
アンモニウム，酢酸アンモニウム，アンモニア，アミン
類，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，各種発酵菌体および
その消化物が使用できる。栄養要求性を示す変異株を使
用する場合には、それらの要求物質を標品もしくはそれ
を含有する天然物として添加する。発酵は、通気攪拌，
振盪培養などの好気的条件下で行う。培養温度は２４〜
４０℃、培養日数は２〜７日間である。培養液のｐＨは
５〜９の範囲に維持する。ｐＨの調節には尿素，炭酸カ
ルシウム，アンモニアガス，アンモニア水などを用い
る。 これらの発酵培地及び発酵条件は従来公知のアミ
ノ酸発酵に採用されているものであるが、本方法では発
酵条件として更に塩基性Ｌ−アミノ酸と酸性Ｌ−アミノ
酸を同時に発酵する条件をも採用しなければならない。
これについて以下詳述する。従来知られている塩基性Ｌ
−アミノ酸生産菌は、通常、酸性Ｌ−アミノ酸生産菌で
あるブレビバクテリウム属細菌、コリネバクテリウム属
細菌などのコリネ型細菌を各種の変異処理に付して得ら
れている。例えば、ブレビバクテリウム・ラクトファー
メンタムＡＴＣＣ １３８６９にニトロソグアニジンを
変異剤として変異処理をほどこし、ＡＥＣ（Ｓ−２−ア
ミノエチル−Ｌ−システイン）の耐性を付けたリジン生
産菌ブレビバクテリウム・ラクトファーメンタムＡＴＣ
Ｃ ２１７９８がある。一方、コリネ型細菌に属する酸
性Ｌ−アミノ酸生産菌は、通常ビオチン要求性である
が、ビオチンが充分量存在する培地（１０μｇ／ｌ以
上）では菌体が増殖するのみで酸性Ｌ−アミノ酸の蓄積
は殆んど見られない。酸性Ｌ−アミノ酸を蓄積させるた
めには菌体の増殖を抑制することが必要で、そのために
は低ビオチン濃度の培地を使用するかビオチンが充分量
存在する培地の場合はポリオキシエチレンソルビタンモ
ノパルミテート（ＰＥＳＰ）、ポリオキシエチレンソル
ビタンモノステアレート（ＰＥＳＳ）、ポリオキシエチ
レンソルビタンモノラウレート（ＰＥＳＬ）などの界面
活性剤又はペニシリン、セファロジンなどのラクタム系
抗生物質を培養の初発又は途上で培地に添加する必要が
ある。因みにこのようなコリネ型細菌に属する酸性Ｌ−
アミノ酸生産菌から変異誘導された塩基性Ｌ−アミノ酸
生産菌はやはりビオチン要求性であるが、これを培養し
て塩基性Ｌ−アミノ酸を発酵生産する場合は、元の酸性
Ｌ−アミノ酸生産菌を培養して酸性Ｌ−アミノ酸を発酵
生産する場合とは異なり、培地に充分量のビオチンが存
在していてもよく、ビオチンを低濃度に制限する必要は
ない。本発明の方法に使用できるＬ−アミノ酸生産菌の
例を表１に示す。Means for Solving the Problems The present inventor has conducted various studies for the above purpose, established a method for simultaneously producing a basic amino acid and an acidic amino acid, and completed the present invention. In the present invention, as described above, a basic L-amino acid-producing bacterium is treated with an acidic L-amino acid.
-A method for simultaneous fermentation of a basic L-amino acid and an acidic L-amino acid, which comprises culturing under a condition for producing an amino acid or culturing a basic L-amino acid-producing bacterium and an acidic L-amino acid-producing bacterium in a mixed culture. Hereinafter, the present invention will be described in detail. The basic L-amino acids referred to in the present invention include L-lysine (Lys), L-arginine (Arg), L-histidine (His) and L-ornithine (Orn),
Acidic L-amino acids include L-glutamic acid (Glu) and L-aspartic acid (Asp). First, a basic L-amino acid-producing bacterium is used, which is cultured under acidic L-amino acid producing conditions to produce a basic L-amino acid and an acidic L-amino acid.
The first method according to the present invention for simultaneously fermenting and producing amino acids will be described. As the fermentation medium, a nutrient medium or synthetic medium containing a carbon source, a nitrogen source, inorganic salts, growth factors and the like is used. As a carbon source, glucose,
Fructose, sucrose, molasses, starch, starch hydrolyzate, carbohydrates such as fruit juice, alcohols such as ethanol, methanol and propanol, and organic acids such as acetic acid can be used. As a nitrogen source, ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, ammonium acetate, ammonia, amines, peptone, meat extract, yeast extract, corn steve liquor, casein hydrolyzate, various fermented cells and their digestion Things can be used. When using a mutant strain showing an auxotrophy, those required substances are added as a standard product or a natural product containing the same. Fermentation is aeration stirring,
Perform under aerobic conditions such as shaking culture. Culture temperature is 24 ~
40 ° C., the number of days of culture is 2 to 7 days. The pH of the culture solution is maintained within the range of 5-9. Urea, calcium carbonate, ammonia gas, aqueous ammonia, etc. are used to adjust the pH. These fermentation media and fermentation conditions have been adopted for conventionally known amino acid fermentation, but in the present method, the conditions for simultaneously fermenting basic L-amino acids and acidic L-amino acids must also be adopted as fermentation conditions. I have to.
This will be described in detail below. Conventionally known basic L
-The amino acid-producing bacterium is usually obtained by subjecting coryneform bacteria such as Brevibacterium and Corynebacterium, which are acidic L-amino acid-producing bacteria, to various mutation treatments. For example, Brevibacterium lactofermentum ATCC 13869 was subjected to a mutation treatment using nitrosoguanidine as a mutagen to give AEC (S-2-aminoethyl-L-cysteine) resistance to a lysine-producing bacterium, Brevibacterium lactopher. Mental ATC
There is C 21798. On the other hand, an acid L-amino acid-producing bacterium belonging to a coryneform bacterium usually requires biotin, but in a medium in which a sufficient amount of biotin is present (10 μg / l or more), the cells only grow and the acid L-amino acid accumulates. Can hardly be seen. In order to accumulate acidic L-amino acids, it is necessary to suppress the growth of bacterial cells. For that purpose, use a medium with a low biotin concentration or, in the case of a medium in which a sufficient amount of biotin is present, polyoxyethylene sorbitan monopalmi. Tate (PESP), polyoxyethylene sorbitan monostearate (PESS), polyoxyethylene sorbitan monolaurate (PESL), or other surfactants or lactam antibiotics such as penicillin and cephalodin are added to the medium at the beginning or on the way of culture. Need to be added. By the way, acidic L- that belongs to such coryneform bacteria
The basic L-amino acid-producing bacterium mutated from the amino acid-producing bacterium is also biotin-auxotrophic, but when the basic L-amino acid is fermentatively produced by culturing this, the original acidic L-amino acid-producing bacterium is used. Unlike the case where the acidic L-amino acid is fermentatively produced by culturing, biotin may be present in the medium in a sufficient amount, and it is not necessary to limit biotin to a low concentration. Table 1 shows examples of L-amino acid-producing bacteria that can be used in the method of the present invention.
【表１】
しかして、本発明者は、上のような塩基性Ｌ−アミノ酸
生産菌を元の酸性Ｌ−アミノ酸生産菌を使用して酸性Ｌ
−アミノ酸を発酵生産する培養条件下に培養したとこ
ろ、極めて予期せざることに、塩基性Ｌ−アミノ酸のみ
ならす酸性Ｌ−アミノ酸をも同時に蓄積することを見出
したのである。尤も、この場合塩基性Ｌ−アミノ酸の対
炭素源収率は低下し、酸性Ｌ−アミノ酸が低下分に相当
する量で蓄積する。かくして、塩基性Ｌ−アミノ酸生産
菌が上のようなビオチン栄養要求性細菌である場合、そ
の酸性Ｌ−アミノ酸産生条件は低ビオチン濃度の培地を
使用するかビオチンが充分量存在する培地の場合はＰＥ
ＳＰなどの界面活性剤又はペニシリンなどのラクタム系
抗生物質を培養の初発又は途上で培地に添加することで
ある。 低ビオチン濃度とは濃度０．５〜１０μｇ／ｌ
のビオチン濃度である。こうすることによって例えばＬ
ｙｓと共にＧｌｕを生産することができる。培養の初発
又は途上で培地に添加するＰＥＳＰなどの界面活性剤及
びペニシリンなどのラクタム系抗生物質の添加量は、界
面活性剤については０．０１〜０．５ｇ／ｄｌ程度、ペ
ニシリンについては０．１〜１０Ｕ／ｍｌ程度である。
こうすることによって、例えばＬｙｓと共にＧｌｕを生
産することができる。次に、本発明に係わる第２の方
法、すなわち、塩基性Ｌ−アミノ酸生産菌と酸性Ｌ−ア
ミノ酸生産菌を併用して、混合培養する塩基性Ｌ−アミ
ノ酸と酸性Ｌ−アミノ酸の同時発酵法について説明す
る。塩基性Ｌ−アミノ酸生産菌及び酸性Ｌ−アミノ酸生
産菌がブレビバクテリウム属細菌、コリネバクテリウム
属細菌などのコリネ型細菌、枯草菌、などのバチルス属
細菌、大腸菌などのエシエリヒア属細菌等に属すること
は周知であり、これらのアミノ酸生産菌は広く使用され
る。又、発酵培地及び発酵条件は、第１の方法に関して
前記した従来公知のアミノ酸発酵に採用されているもの
でよい。第２の方法における塩基性Ｌ−アミノ酸と酸性
Ｌ−アミノ酸の同時発酵条件は塩基性Ｌ−アミノ酸生産
菌を使用して塩基性Ｌ−アミノ酸を発酵蓄積させる条件
と酸性Ｌ−アミノ酸生産菌を使用して酸性Ｌ−アミノ酸
を発酵蓄積させる条件とを単に相加的に併用するだけで
よい。しかして、２種の発酵条件はいずれも前記した従
来公知のアミノ酸発酵条件として一括されるものであっ
て、両者間に実質的な相違はない。相違のあるのは、例
えば、混合培養する２種の細菌の一方のみが栄養要求性
で他方が栄養要求性のない場合であるが、この場合栄養
要求性のある細菌の培養条件として培地に要求栄養物質
を添加することが必要なのに対して栄養要求性のない細
菌の培養条件として培地に要求栄養物質を特に加える必
要がない。栄養要求性細菌と非栄養要求性細菌の混合培
養の場合、両者の発酵条件の相加的併用とは培地に該栄
養物質を添加することである。非栄養要求性細菌と栄養
要求性細菌を混合培養して一方の細菌に塩基性Ｌ−アミ
ノ酸を産生せしめ、他方の細菌に酸性Ｌ−アミノ酸を産
生せしめる場合、当該要求栄養物質を培地に添加しても
非栄養要求性細菌のアミノ酸産生には何らの悪影響も及
ぼさないのである。前記のような同時発酵の条件を採用
することにより、本発明の方法（第１及び第２）によれ
ば、塩基性Ｌ−アミノ酸と酸性Ｌ−アミノ酸を同時に各
々商業的蓄積量、すなわち、約５００ｍｇ／ｄｌ以上の
蓄積量で培地中に蓄積させることができる。発酵終了液
から生成蓄積したアミノ酸を分離取得するには常法でよ
く、例えばイオン交換樹脂法（例えば、発酵終了液から
先ず陽イオン交換樹脂により塩基性アミノ酸を吸着分離
し、ついで陰イオン交換樹脂により酸性アミノ酸を吸着
分離する。）によることができる。塩基性Ｌ−アミノ酸
と酸性Ｌ−アミノ酸の混合物の用途に対してはもちろん
両種のアミノ酸を相互に分離することは不要である。前
記のように本発明の塩基性Ｌ−アミノ酸と酸性Ｌ−アミ
ノ酸の同時発酵法は種々のメリットを有するが、以下こ
れらについて説明する。培地中のイオン量の低減に関し
て云えば、例えば、Ｌｙｓ発酵の場合、従来のＬｙｓ単
独発酵法では生成するＬｙｓを中和するために酸性イオ
ンが０．２〜０．４Ｍ必要であったが、本発明の方法を
用いてＬｙｓ及びＧｌｕの同時発酵を行うことにより
０．１〜０．２Ｍと必要イオン量が半減できる。発酵生
産性の向上（アミノ酸生成速度の向上）に関し云えば、
例えば、従来のＬｙｓ単独発酵の場合、菌体・時間当り
のＬｙｓ生成速度は０．０３〜０．０４ｈｒ−１である
が、本発明の方法を用いてＬｙｓ及びＧｌｕの同時発酵
を行うことによりＬｙｓ及びＧｌｕの合計生成速度は
０．０６〜０．０８ｈｒ−１と約２倍に向上する。培地
成分の節減省略に関して云えば、例えば、従来のＬｙｓ
発酵の場合、培地成分として硫酸アンモニウム及びアン
モニアが２〜６ｇ／ｄｌ必要なのに対し、本発明の方法
を用いてＬｙｓ及びＧｌｕの同時発酵を行うことにより
硫酸アンモニウムは必要なくアンモニアも１ｇ／ｄｌ以
下でよい。発酵液処理（アミノ酸の単離操作）での簡略
化に関して云えば、例えば、従来のＬｙｓ発酵の場合、
処理工程で脱塩工程が必要となるが、本発明の方法によ
りＬｙｓ及びＧｌｕの同時発酵を行うことにより処理工
程での脱塩工程が不要になる。廃液の環境保全負荷の低
減に関して云えば、例えば、従来のＬｙｓ発酵の場合、
その製造工程より排出される廃液には活性汚泥処理やそ
の後に続く脱窒処理により除去すべき窒素化合物が含有
されているが、本発明の方法によりＬｙｓ及びＧｌｕの
同時発酵を行えば、この廃液中の窒素化合物をほとんど
ゼロにすることができる。[Table 1] Therefore, the present inventor uses the original acidic L-amino acid-producing bacterium to convert the acidic L-amino acid-producing bacterium into the acidic L-amino acid-producing bacterium.
-When cultured under the culture conditions for producing amino acids by fermentation, it was found that, unexpectedly, acidic L-amino acids that level out only basic L-amino acids also accumulate at the same time. However, in this case, the yield of the basic L-amino acid with respect to the carbon source decreases, and the acidic L-amino acid accumulates in an amount corresponding to the decrease. Thus, when the basic L-amino acid-producing bacterium is a biotin auxotrophic bacterium as described above, the acidic L-amino acid producing condition is that a medium having a low biotin concentration is used or a medium in which a sufficient amount of biotin is present is used. PE
A surfactant such as SP or a lactam antibiotic such as penicillin is added to the medium at the beginning or during the culture. Low biotin concentration is 0.5-10 μg / l
Is the biotin concentration of. By doing this, for example, L
Glu can be produced with ys. The amount of the surfactant such as PESP and the lactam antibiotic such as penicillin added to the medium at the beginning of the culture or in the middle of the culture is about 0.01 to 0.5 g / dl for the surfactant and 0. It is about 1 to 10 U / ml.
By doing so, for example, Glu can be produced together with Lys. Next, a second method according to the present invention, that is, a method for co-fermenting a basic L-amino acid and an acidic L-amino acid, in which a basic L-amino acid-producing bacterium and an acidic L-amino acid-producing bacterium are used in combination and mixed and cultured. Will be described. Basic L-amino acid-producing bacterium and acidic L-amino acid-producing bacterium belong to coryneform bacteria such as Brevibacterium, Corynebacterium, Bacillus subtilis, Bacillus, Escherichia bacterium, etc. It is well known that these amino acid-producing bacteria are widely used. Further, the fermentation medium and the fermentation conditions may be those employed in the conventionally known amino acid fermentation described above in regard to the first method. The basic L-amino acid and the acidic L-amino acid are simultaneously fermented in the second method under the conditions of fermenting and accumulating the basic L-amino acid by using the basic L-amino acid-producing bacterium and the acidic L-amino acid-producing bacterium. It is only necessary to use additively in combination with the conditions for fermentative accumulation of acidic L-amino acids. However, both of the two types of fermentation conditions are collectively described as the conventionally known amino acid fermentation conditions described above, and there is no substantial difference between the two. The difference is, for example, when only one of the two kinds of bacteria to be mixed-cultured is auxotrophic and the other is not auxotrophic. In this case, the culture conditions of the auxotrophic bacteria are required in the medium. It is not necessary to add the required nutrient substance to the medium as a culture condition for bacteria that are not auxotrophic while it is necessary to add the nutrient substance. In the case of mixed culture of an auxotrophic bacterium and a non-auxotrophic bacterium, the additive combination of fermentation conditions of both is to add the nutrient substance to the medium. When non-auxotrophic bacteria and auxotrophic bacteria are mixed and cultured so that one bacterium produces a basic L-amino acid and the other bacterium produces an acidic L-amino acid, the auxotrophic substance is added to the medium. However, it has no adverse effect on the amino acid production of non-auxotrophic bacteria. By adopting the conditions of the co-fermentation as described above, according to the method of the present invention (first and second), the basic L-amino acid and the acidic L-amino acid are simultaneously stored in a commercially accumulated amount, that is, about It can be accumulated in the medium at an accumulated amount of 500 mg / dl or more. A conventional method may be used to separate and obtain the amino acids produced and accumulated from the fermentation broth. For example, an ion exchange resin method (for example, a basic amino acid is first adsorbed and separated from the fermentation broth by a cation exchange resin, and then an anion exchange resin is used). The acidic amino acid is adsorbed and separated by. For the use of a mixture of basic L-amino acid and acidic L-amino acid, it is of course not necessary to separate the two amino acids from each other. As described above, the simultaneous fermentation method of the basic L-amino acid and the acidic L-amino acid of the present invention has various advantages, which will be described below. Regarding the reduction of the amount of ions in the medium, for example, in the case of Lys fermentation, 0.2 to 0.4 M of acidic ions were required to neutralize Lys produced in the conventional Lys single fermentation method. By carrying out the simultaneous fermentation of Lys and Glu using the method of the present invention, the required ion amount can be halved to 0.1 to 0.2M. Speaking of improvement of fermentation productivity (improvement of amino acid production rate),
For example, in the case of the conventional Lys single fermentation, the Lys production rate per cell / hour is 0.03 to 0.04 hr −1 , but by performing the simultaneous fermentation of Lys and Glu using the method of the present invention, The total production rate of Lys and Glu is 0.06 to 0.08 hr −1, which is approximately doubled. Speaking of omitting saving of medium components, for example, conventional Lys
In the case of fermentation, ammonium sulfate and ammonia are required to be 2 to 6 g / dl as medium components, whereas ammonium sulfate is not necessary and ammonia may be 1 g / dl or less by performing the simultaneous fermentation of Lys and Glu using the method of the present invention. Speaking of simplification in fermentation liquid treatment (isolation of amino acids), for example, in the case of conventional Lys fermentation,
Although a desalting step is required in the treatment step, the desalting step in the treatment step becomes unnecessary by carrying out the simultaneous fermentation of Lys and Glu by the method of the present invention. Regarding the reduction of environmental conservation load of waste liquid, for example, in the case of conventional Lys fermentation,
The waste liquid discharged from the manufacturing process contains nitrogen compounds to be removed by the activated sludge treatment and the subsequent denitrification treatment, but if the simultaneous fermentation of Lys and Glu is performed by the method of the present invention, this waste liquid is The nitrogen compounds in it can be almost zero.
【実施例】以下、本発明を実施例により更に詳細に説明
する。
実施例１（Ｌｙｓ生産菌の培養によるＬｙｓとＧｌｕの
同時生産）
粗糖を糖として１４０ｍｇ／ｍｌ、ＫＨ２ＰＯ４１ｍｇ
／ｍｌ、ＭｇＳＯ４・７Ｈ２Ｏ０．４ｍｇ／ｍｌ、Ｆｅ
ＳＯ４・７Ｈ２Ｏ １０ｍｇ／ｍｌ、ＭｎＳＯ４・４Ｈ
２Ｏ ２０μｇ／ｍｌ、尿素５ｍｇ／ｍｌ、大豆蛋白酸
加水分解物「味液」（登録商標）５μｌ／ｍｌ、ビオチ
ン５０μｇ／ｌ及びサイアミン６０μｇ／ｌを含有する
水溶液培地を調製し、その２０ｍｌづつを５００ｍｌ容
の振盪フラスコに入れ１１５℃で１０分間加熱滅菌し
た。この培地にＬｙｓ生産菌ブレビバクテリウム・ラク
トファーメンタム（ＡＴＣＣ ２１７９８）を接種し往
復振盪機により３１．５℃で培養を行った。培養中、培
養液をｐＨ６．５ないし８．０に保つように４００ｍｇ
／ｍｌの濃度の尿素水溶液を少量づつ添加した。培養液
の２６倍希釈液の５６２μｍおける吸光度が０．３０に
到達した時にＰＥＳＰ（ポリオキシエチレンソルビタン
モノパルミテート）を４ｍｇ／ｍｌ添加し、３０時間で
発酵を終了し、発酵液中に蓄積したＬ−リジン及びＬ−
グルタミン酸を液体クロマトグラフィーにより定量した
ところ、Ｌ−リジン３０ｍｇ／ｍｌ及びＬ−グルタミン
酸２２ｍｇ／ｍｌが蓄積していた。同様の培地に硫酸ア
ンモニウム２５ｍｇ／ｍｌ加えた培地と同じ菌株を用い
て、培養途中でＰＥＳＰを添加しないこと以外は同様の
方法によりリジン単独発酵を行ったところ、５５時間で
発酵が終了し、発酵液中にＬ−リジンが５３ｍｇ／ｍｌ
蓄積していた。Ｌ−リジンとＬ−グルタミン酸の同時発
酵の場合の培地中のイオン量は０．１３Ｍと後者のＬ−
リジン単独発酵の場合の０．２８Ｍの半量であった。ア
ミノ酸生成速度は、Ｌ−リジンとＬ−グルタミン酸の同
時発酵の場合、菌体・時間当りのＬ−リジンとＬ−グル
タミン酸の合計生成速度は０．０６６ｈｒ−１、Ｌ−リ
ジン単独発酵ではＬ−リジン生成速度が０．０３５ｈｒ
−１であった。培地成分の比較では、後者のＬ−リジン
単独発酵の場合には初発培地の硫酸アンモニウム及び初
発と追加添加の尿素由来のアンモニアを併せて３５ｍｇ
／ｍｌ必要なのに対して、Ｌ−リジンとＬ−グルタミン
酸の同時発酵の場合には硫酸アンモニウムはゼロ、尿素
由来のアンモニアは初発と追加添加併せても６ｍｇ／ｍ
ｌであった。さらに、得られた発酵液についてイオン交
換樹脂を用いた常法により処理を行った。イオン交換樹
脂にＬ−リジンやＬ−グルタミン酸を吸着させた残りの
樹脂貫流液中の塩濃度は、Ｌ−リジン単独発酵の場合に
は５％以上と極めて高く、該貫流液を有効利用をするた
めに脱塩が必要であった。一方Ｌ−リジンとＬ−グルタ
ミン酸の同時発酵の場合には２％以下と塩濃度が薄く脱
塩は不要であった。また、樹脂洗浄液等の廃液を活性汚
泥法により処理したところ、Ｌ−リジン単独発酵の廃液
では含有されたＢＯＤは除去し得たが窒素化合物が多量
残存し、さらに別途脱窒処理の必要性が認められたが、
Ｌ−リジンとＬ−グルタミン酸の同時発酵の廃液では活
性汚泥処理によりＢＯＤを除去した処理水中には多量の
窒素化合物は特に認められなかった。
実施例２（Ｌｙｓ生産菌の培養によるＬｙｓとＧｌｕ同
時生産）
グルコース １５％、リン酸第一カリ ０．１％、硫酸
マグネシウム・７水塩０．０４％、硫酸アンモニウム２
％、ビチオン １００γ／ｌ、ビタミンＢ１塩酸塩 ２
００γ／ｌ、鉄イオン及びマンガンイオン各２ｐｐｍ、
及び「味液」１％を含有するｐＨ７．０の水溶液培地を
小型ガラス製ジヤー・フアーメンターに３００ｍｌ分注
し、殺菌後、あらかじめ３０℃で２４時間ブイヨンスラ
ント上で生育させたＬｙｓ生産菌ブレビバクテリウム・
フラバム（ＡＴＣＣ ２１１２７）を接種した。つい
で、これを３１℃で培養を開始し、約１０時間経過した
後、ペニシリンを６Ｕ／ｍｌの濃度になるように添加し
培養を続け、１８時間培養したところ、発酵終了時には
培地中に３．７５ｇ／ｄｌのＬ−リジンと２．０５ｇ／
ｄｌのＬ−グルタミン酸が生成蓄積していた。この発酵
終了液１ｌから、遠心分離によって菌体を除去し、上清
液からイオン交換樹脂をもちいる常法にしたがってＬ−
リジンおよびＬ−グルタミン酸を分離し、精製して、Ｌ
−リジン塩酸塩の結晶２８．１ｇとＬ−グルタミン酸ソ
ーダの結晶１８．５ｇを得た。
実施例３（Ｈｉｓ生産菌の培養によるＨｉｓとＧｌｕの
同時生産）
Ｈｉｓ生産菌ブレビバクテリウム・フラバム（ＡＴＣＣ
２１４０６）を下記種培養培地に培種し、２０時間３
１℃にて通気攪拌培養を行った。
種培養培地：グリコース ３ｇ／ｄｌ、尿素 ０．３ｇ
／ｄｌ、ＫＨ２ＰＯ４ ５０．１ｇ／ｄｌ、ＭｇＳＯ４
・７Ｈ２Ｏ ０．０４ｇ／ｄｌ、ＦｅおよびＭｎイオン
各２ｐｐｍ、ビオチン２００μｇ／ｌ、サイアミン塩酸
塩３００μｇ／ｌ、大豆タンパク塩酸加水分解液（総窒
素７％）１ｍｌ／ｄｌ、酵母エキス０．５ｇ／ｄｌ及び
肉エキス０．５ｇ／ｄｌを含有するｐＨ７．２の水溶液
培地。一方、１ｌ容小型ガラス製ジヤーフアーメンター
に下記組成の主発酵培地を３００ｍｌ分注し、殺菌し
た。これに上記種培養液を１５ｍｌ接種し、３１℃にて
通気毎分１／１容の攪拌培養を開始した。
主発酵培地：グルコース １０ｇ／ｄｌ、硫酸アンモニ
ウム ０．５ｇ／ｄｌ、ＫＨ２ＰＯ４０．１ｇ／ｄｌ、
ＭｇＳＯ４・７Ｈ２Ｏ ０．０４ｇ／ｄｌ、Ｆｅおよび
Ｍｎイオン各２ｐｐｍ、ビオチン１００μｇ／ｌ、サイ
アミン塩酸塩２００μｇ／ｌ及び大豆タンパク塩酸加水
分解液（総窒素７％）２ｍｌ／ｄｌを含有するｐＨ７．
２水溶液培地。３１℃で培養中ｐＨが低下したときに、
アンモニアガスを添加してｐＨを７．０〜７．５に維持
した。培養開始後１０時間経過後ポリオキシエチレンソ
ルビタンモノパルミテートを０．４ｇ／ｄｌの濃度とな
るように添加し、培養を続けた。１８時間培養後、発酵
終了液中にはＬ−ヒスチジン０．８ｇ／ｄｌおよびＬ−
グルタミン酸１・２ｇ／ｄｌの蓄積が認められた。発酵
終了液から遠心分離により菌体を除去し、その上清液よ
りイオン交換樹脂をもちいる常法にしたがってＬ−ヒス
チジン結晶１．９ｇおよびＬ−グルタミン酸結晶２．８
ｇを得た。
実施例４（Ｇｌｕ生産菌とＬｙｓ生産菌の混合培養によ
るＧｌｕとＬｙｓの同時生産）
Ｇｌｕ生産菌コリネバクテリウム・グルタミクム（ＮＲ
ＲＬ Ｂ−１２１３８）およびＬｙｓ生産菌ブレビバク
テリウム・ラクトファーメンタム（ＡＴＣＣ２１７９
９）を各スラント上より１白金耳かきとり、下記組成の
種培養水溶液培地５０ｍｌにそれぞれ接種し、１８時
間、３１℃にて通気攪拌培養をおこなって、おのおのの
種培養液を調製した。
種培養培地組成：
グルコース １．５％
酢酸アンモニウム ０．３％
尿 素 ０．１％
ＫＨ２ＰＯ４ ０．１％
ＭｇＳＯ４・７Ｈ２Ｏ ０．０４％
Ｆｅ＋＋ ２ｐｐｍ
Ｍｎ＋＋ ２ｐｐｍ
ビオチン ５０μｇ／ｌ
サイアミン塩酸塩 ２００μｇ／ｌ
大豆タンパク塩酸加水分解液
濃縮物（総窒素７％） ３％
ｐＨ７．５
一方、１ｌ容小型ガラス製ジヤー・フアーメンターに下
記組成の主発酵水溶液培地を３００ｍｌ分注し、常法に
より殺菌した。これらに上記の種培養液を同時にそれぞ
れ１５ｍｌ宛接種し、３１℃にて通気毎分１／１容の攪
拌培養を開始した。
主発酵培地：
グルコース ２％
酢酸アンモニウム ０．５％
尿 素 ０．２％
ＫＨ２ＰＯ４ ０．１％
ＭｇＳＯ４・７Ｈ２Ｏ ０．０４％
Ｆｅ＋＋ ２ｐｐｍ
Ｍｎ＋＋ ２ｐｐｍ
ビオチン ５０μｇ／ｌ
サイアミン塩酸塩 ６０μｇ／ｌ
大豆タンパク塩酸加水分解液
濃縮物（総窒素７％） ３％
ｐＨ７．５
培養液中に酢酸と酢酸アンモニウムとの混合液（酢酸：
酵酸アンモニウムの混合液のモル比は１：０．２５、混
合液の酢酸濃度は６０％）を培地のｐＨを７．２〜８．
０の間に保持するように連続的、あるいは間けつ的に添
加して、３０℃で７２時間培養をおこなった。結果はＬ
−グルタミン酸３７ｇ／ｌ及びＬ−リジン５４ｇ／ｌの
蓄積が認められた。
実施例５（Ａｓｐ生産菌とＡｒｇ生産菌の混合培養によ
るＡｓｐとＡｒｇの同時生産）
Ａｓｐ生産菌コリネバクテリウム・グルタミクム（ＦＥ
ＲＭ ＢＰ−２１７８及びＡｒｇ産菌ブレビバクテリウ
ム・フラバム（ＡＴＣＣ ２１４９３）を下記組成の種
培養水溶液培地にそれぞれ別々に接種し、１８時間３０
℃にて通気攪拌培養を行った。
種培養培地組成：グルコース ３％、ＫＨ２ＰＯ４
０．１％、ＭｇＳＯ４・７Ｈ２Ｏ ０．０４％、Ｆｅ
＋＋２ｐｐｍ、Ｍｎ＋＋ ２ｐｐｍ、「味液」３ｍｌ／
ｄｌ、ビオチン ５μｇ／ｌ、ビタミンＢ１．塩酸塩
３００γ／ｌ、尿素 ０．３％、ｐＨ７．２。一方、小
型ガラス製ジヤーフアーメンターに下記組成よりなる主
発酵水溶液培地を３００ｍｌ分注し殺菌した。これに上
記各種培養液を同時に各々１５ｖ／ｖ％接種し、３１℃
にて１５００ｒ．ｐ．ｍ．、通気毎分１／１容にて通気
攪拌培養を開始した。
主発酵培地組成：エチルアルコール １．５％、硫酸ア
ンモニウム ０．５％、ＫＨ２ＰＯ４０．１％、ＭｇＳ
Ｏ４・７Ｈ２Ｏ ０．０４％、Ｆｅ＋＋ ２ｐｐｍ、
「味液」（登録商標）２ｍｌ／ｄｌ、ビオチン ５μｇ
／１、ビタミン Ｂ１・塩酸塩 ３００γ／ｌ、ｐＨ
７．２。培養中ｐＨが低下したときにアンモニアガスを
添加してｐＨを７．０〜７．５に維持した。エチルアル
コールは、その消費をガスクロマトグラフで定量し、そ
の濃度が０．０１％前後に減少したときに追添加した。
４８時間培養後、培養終了液中はＬ−アスパラギン酸が
１．３０ｇ／ｄｌおよびＬ−アルギニンが１．０５ｇ／
ｄｌ生成していた。発酵終了液よりイオン交換樹脂を用
いる常法によりＬ−アスパラギン酸の結晶が２．１８ｇ
及びＬ−アルギニンの結晶が１．９５ｇ得られた。
実施例６（Ａｓｐ生産菌とＨｉｓ生産菌の混合培養によ
るＡｓｐとＨｉｓの同時生産）
シユークロース １３ｇ／ｄｌ、尿素 ０．５ｇ／ｄ
ｌ、ＫＨ２ＰＯ４０．１ｇ／ｄｌ、ＭｇＳＯ４・７Ｈ２
Ｏ ０．０４ｇ／ｄｌ、ＦｅおよびＭｎイオン各２ｐｐ
ｍ、ビオチン ５μｇ／ｌ、サイアミン塩酸塩 ２００
μｇ／ｌ及び大豆タンパク塩酸加水分解物（総窒素７
％）０．３ｍｌ／ｄｌを含みｐＨ７．２に調節した水溶
液培地を調製し、その２０ｍｌを５００ｍｌ容の振とう
フラスコに分注した。殺菌後あらかじめブイヨンスラン
ト上で生育させたＡｓｐ生産菌ブレビバクテリウム・グ
ルタミクム（ＦＥＲＭ ＢＰ−２１７８）及びＨｉｓ生
産菌ブレビバクテリウム・フラバム（ＡＴＣＣ ２１４
０６）を同じフラスコに接種し、３１℃にて７２時間振
とう培養した。培養中、ｐＨが６．５から８．０の間に
なるように４０ｇ／ｄｌの濃度の尿素水溶液を少量づつ
添加した。培養終了液中には、Ｌ−アスパラギン酸が
０．９５ｇ／ｄｌ及びＬ−ヒスチジンが０．８５ｇ／ｄ
ｌ蓄積していた。培養終了液から遠心分離によって菌体
を除いて得た上澄液をイオン交換樹脂を用いる常法によ
り精製し、Ｌ−アスパラギン酸の結晶１．７５ｇとＬ−
ヒスチジンの結晶１．６ｇを得た
実施例７（Ｇｌｕ生産菌とＬｙｓ生産菌の混合培養によ
るＧｌｕとＬｙｓの同時生産）
グルコース ５０ｍｇ／ｍｌ、尿素 ２ｍｇ／ｍｌ、Ｋ
Ｈ２ＰＯ４ １ｍｇ／ｍｌ、ＭｇＳＯ４．７Ｈ２Ｏ
０．４ｍｇ／ｍｌ、ＦｅＳＯ４・７Ｈ２Ｏ １０μｇ／
ｍｌ、ＭｎＳＯ４・４Ｈ２Ｏ ８μｇ／ｍｌ、大豆蛋白
酸加水分解物「味液」５μｌ／ｍｌ、サイアミン塩酸塩
１０．０μｇ／ｄｌ及びビオチン０．２５μｇ／ｄｌを
含有する水溶液培地を調製しその２０ｍｌを５００ｍｌ
容の振盪フラスコに入れ１１５℃で１０分間加熱殺菌し
た。この培地にＧｌｕ生産菌ブレビバクテリウム・ラク
トファーメンタム（ＡＴＣＣ １３８６９）及びＬｙｓ
生産菌ブレビバクテリウム・ラクトファーメンタム（Ａ
ＴＣＣ ２１８００）を接種し、振とうしつつ３１．５
℃にて培養した。培養中、培養液をｐＨ６．５ないし
８．０に保つように４５０ｍｇ／ｍｌの濃度の尿素溶液
を少量ずつ添加し、３０時間で培養を終了した。培養液
中に蓄積したＬ−グルタミン酸及びＬ−リジンを定量し
た。その結果、Ｌ−グルタミン酸が１０．５ｍｇ／ｍｌ
及びＬ−リジンが１５．３ｍｇ／ｍｌ蓄積していた。EXAMPLES The present invention will now be described in more detail with reference to examples. Example 1 (Simultaneous production of Lys and Glu by culturing Lys-producing bacteria) 140 mg / ml of crude sugar as sugar, 1 mg of KH 2 PO 4
/ Ml, MgSO 4 · 7H 2 O0.4mg / ml, Fe
SO 4 · 7H 2 O 10mg / ml, MnSO 4 · 4H
2 O 20 μg / ml, urea 5 mg / ml, soybean protein acid hydrolyzate “Taste liquid” (registered trademark) 5 μl / ml, biotin 50 μg / l, and thiamine 60 μg / l were prepared as an aqueous medium, and each 20 ml was prepared. Was placed in a 500 ml shake flask and sterilized by heating at 115 ° C for 10 minutes. This medium was inoculated with the Lys-producing bacterium Brevibacterium lactofermentum (ATCC 21798) and cultured at 31.5 ° C. with a reciprocating shaker. 400 mg to maintain the pH of the culture at 6.5 to 8.0 during culturing.
An aqueous urea solution having a concentration of / ml was added little by little. PESP (polyoxyethylene sorbitan monopalmitate) was added at 4 mg / ml when the absorbance at 562 μm of the 26-fold diluted culture solution reached 0.30, and the fermentation was completed in 30 hours and accumulated in the fermentation solution. L-lysine and L-
When glutamic acid was quantified by liquid chromatography, 30 mg / ml of L-lysine and 22 mg / ml of L-glutamic acid were accumulated. Using the same strain as the medium containing 25 mg / ml of ammonium sulfate in the same medium, lysine alone was fermented by the same method except that PESP was not added during the culture, and the fermentation was completed in 55 hours. L-lysine in 53 mg / ml
Had accumulated. In the case of co-fermentation of L-lysine and L-glutamic acid, the amount of ions in the medium was 0.13 M, and the latter L-
It was half of 0.28 M in the case of lysine fermentation alone. Regarding the amino acid production rate, in the case of simultaneous fermentation of L-lysine and L-glutamic acid, the total production rate of L-lysine and L-glutamic acid per cell / time was 0.066 hr −1 , and in L-lysine single fermentation L-lysine Lysine production rate is 0.035hr
It was -1 . In the case of the latter L-lysine single fermentation, 35 mg of ammonium sulfate of the initial medium and 35 mg of ammonia derived from urea and additional urea were added in comparison of medium components.
/ Ml is required, whereas in the case of simultaneous fermentation of L-lysine and L-glutamic acid, ammonium sulfate is zero, and urea-derived ammonia is 6 mg / m even if the initial and additional additions are combined.
It was l. Further, the obtained fermented liquor was treated by an ordinary method using an ion exchange resin. The salt concentration in the remaining resin flow-through liquid obtained by adsorbing L-lysine or L-glutamic acid on the ion exchange resin is extremely high at 5% or more in the case of L-lysine single fermentation, and the flow-through liquid is effectively used. Therefore, desalting was necessary. On the other hand, in the case of simultaneous fermentation of L-lysine and L-glutamic acid, the salt concentration was low at 2% or less and desalting was unnecessary. Further, when the waste liquid such as the resin washing liquid was treated by the activated sludge method, the BOD contained in the waste liquid of the L-lysine single fermentation could be removed, but a large amount of nitrogen compounds remained, and further denitrification treatment was required. Accepted,
In the waste liquid of the simultaneous fermentation of L-lysine and L-glutamic acid, a large amount of nitrogen compounds was not found in the treated water from which BOD was removed by the activated sludge treatment. Example 2 (Simultaneous production of Lys and Glu by culturing Lys-producing bacteria) Glucose 15%, primary potassium phosphate 0.1%, magnesium sulfate heptahydrate 0.04%, ammonium sulfate 2
%, Biotin 100γ / l, vitamin B 1 hydrochloride 2
00γ / 1, iron ion and manganese ion 2ppm each,
And 300 ml of a pH 7.0 aqueous solution medium containing 1% of "taste liquid" was poured into a small glass jar and fermenter, sterilized, and pre-grown on a broth slant at 30 ° C for 24 hours. Bacterium
Flavan (ATCC 21127) was inoculated. Then, the culture was started at 31 ° C., and after about 10 hours, penicillin was added at a concentration of 6 U / ml to continue the culture, and the culture was continued for 18 hours. 75 g / dl L-lysine and 2.05 g /
dl of L-glutamic acid was produced and accumulated. From the 1 l of the fermentation-finished liquid, cells were removed by centrifugation, and the supernatant was mixed with L- by an ordinary method using an ion exchange resin.
Lysine and L-glutamic acid were separated and purified to give L
-28.1 g of crystals of lysine hydrochloride and 18.5 g of crystals of L-sodium glutamate were obtained. Example 3 (Simultaneous production of His and Glu by culturing a His-producing bacterium) His-producing bacterium Brevibacterium flavum (ATCC
21406) in the following seed culture medium for 20 hours 3
Aeration-agitation culture was performed at 1 ° C. Seed culture medium: Glucose 3 g / dl, urea 0.3 g
/ Dl, KH 2 PO 4 50.1 g / dl, MgSO 4
7H 2 O 0.04 g / dl, Fe and Mn ions 2 ppm each, biotin 200 μg / l, thiamine hydrochloride 300 μg / l, soybean protein hydrochloric acid hydrolyzed solution (total nitrogen 7%) 1 ml / dl, yeast extract 0.5 g / Dl and meat extract 0.5 g / dl pH 7.2 aqueous medium. On the other hand, 300 ml of a main fermentation medium having the following composition was poured into a 1 l small glass glass jar mentor and sterilized. To this, 15 ml of the above seed culture was inoculated, and stirring culture was started at 31 ° C. with aeration of 1/1 volume per minute. Main fermentation medium: glucose 10 g / dl, ammonium sulfate 0.5 g / dl, KH 2 PO 4 0.1 g / dl,
MgSO 4 · 7H 2 O 0.04g / dl, pH7 containing Fe and Mn ions each 2 ppm, biotin 100 [mu] g / l, thiamine hydrochloride 200 [mu] g / l and soy protein hydrochloride hydrolyzate (total nitrogen 7%) 2ml / dl ．
2 Aqueous medium. When the pH drops during culture at 31 ° C,
Ammonia gas was added to maintain the pH at 7.0-7.5. After 10 hours from the start of the culture, polyoxyethylene sorbitan monopalmitate was added so that the concentration was 0.4 g / dl, and the culture was continued. After culturing for 18 hours, 0.8 g / dl of L-histidine and L-
Accumulation of 1.2 g / dl of glutamic acid was observed. Cells were removed from the fermentation broth by centrifugation and the supernatant was 1.9 g of L-histidine crystals and 2.8 L-glutamic acid crystals according to a conventional method using an ion exchange resin.
g was obtained. Example 4 (Simultaneous production of Glu and Lys by mixed culture of Glu-producing bacterium and Lys-producing bacterium) Glu-producing bacterium Corynebacterium glutamicum (NR
RL B-12138) and Lys producing strain Brevibacterium lactofermentum (ATCC 2179).
One platinum loop of 9) was scraped from each slant, inoculated into 50 ml of a seed culture aqueous solution medium having the following composition, and aerated and agitated at 18 ° C. for 18 hours to prepare each seed culture solution. Seed culture medium composition: 0.3% 1.5% glucose ammonium acetate 0.1% urea KH 2 PO 4 0.1% MgSO 4 · 7H 2 O 0.04% Fe ++ 2ppm Mn ++ 2ppm biotin 50 [mu] g / l Cyamine hydrochloride 200 μg / l Soybean protein hydrochloric acid hydrolyzed liquid concentrate (total nitrogen 7%) 3% pH 7.5 On the other hand, 300 ml of a main fermentation aqueous solution medium having the following composition was dispensed into a 1 l small glass glass jar fermenter, Sterilized by a conventional method. 15 ml of each of the above seed culture solutions was simultaneously inoculated to each of these, and agitated culture with aeration of 1/1 volume per minute was started at 31 ° C. The main fermentation medium: Glucose 2% ammonium acetate 0.5% 0.2% Urea KH 2 PO 4 0.1% MgSO 4 · 7H 2 O 0.04% Fe ++ 2ppm Mn ++ 2ppm biotin 50 [mu] g / l thiamine hydrochloride 60 μg / l Soybean protein hydrochloric acid hydrolyzate concentrate (total nitrogen 7%) 3% pH 7.5 A mixture of acetic acid and ammonium acetate (acetic acid:
The ammonium fermentate mixed solution has a molar ratio of 1: 0.25, and the mixed solution has an acetic acid concentration of 60%) and a medium pH of 7.2 to 8.
It was continuously or intermittently added so as to be maintained at 0, and cultured at 30 ° C. for 72 hours. The result is L
-Accumulation of glutamic acid 37 g / l and L-lysine 54 g / l was observed. Example 5 (Simultaneous production of Asp and Arg by mixed culture of Asp-producing bacterium and Arg-producing bacterium) Asp-producing bacterium Corynebacterium glutamicum (FE
RM BP-2178 and Arg-producing bacterium Brevibacterium flavum (ATCC 21493) were separately inoculated into a seed culture aqueous solution medium having the following composition, and each was inoculated for 18 hours 30 hours.
Aeration-agitation culture was carried out at ℃. Seed culture medium composition: glucose 3%, KH 2 PO 4
0.1%, MgSO 4 · 7H 2 O 0.04%, Fe
++ 2ppm, Mn ++ 2ppm, "taste liquid" 3ml /
dl, biotin 5 μg / l, vitamin B 1 . Hydrochloride
300γ / l, urea 0.3%, pH 7.2. On the other hand, 300 ml of a main fermentation aqueous solution medium having the following composition was dispensed into a small glass jar fermenter for sterilization. 15 v / v% of each of the above various culture solutions were simultaneously inoculated into this, and the temperature was 31 ° C.
At 1500 r. p. m. The aeration-agitation culture was started at 1/1 volume per minute of aeration. Main fermentation medium composition: ethyl alcohol 1.5%, ammonium sulfate 0.5%, KH 2 PO 4 0.1%, MgS
O 4 · 7H 2 O 0.04% , Fe ++ 2ppm,
"Taste liquid" (registered trademark) 2 ml / dl, biotin 5 μg
/ 1, Vitamin B 1 / Hydrochloride 300γ / l, pH
7.2. When the pH decreased during the culture, ammonia gas was added to maintain the pH at 7.0 to 7.5. The consumption of ethyl alcohol was quantitatively determined by gas chromatography, and was added when the concentration decreased to around 0.01%.
After 48 hours of culturing, L-aspartic acid was 1.30 g / dl and L-arginine was 1.05 g / dl in the culturing solution.
dl had been produced. 2.18 g of crystals of L-aspartic acid were obtained from the fermentation broth by a conventional method using an ion exchange resin.
And 1.95 g of L-arginine crystals were obtained. Example 6 (Simultaneous production of Asp and His by mixed culture of Asp-producing strain and His-producing strain) Sucrose 13 g / dl, urea 0.5 g / d
l, KH 2 PO 4 0.1g / dl, MgSO 4 · 7H 2
O 0.04 g / dl, Fe and Mn ions 2 pp each
m, biotin 5 μg / l, thiamin hydrochloride 200
μg / l and soybean protein hydrochloric acid hydrolyzate (total nitrogen 7
%) 0.3 ml / dl was added to prepare an aqueous medium adjusted to pH 7.2, and 20 ml thereof was dispensed into a 500 ml shake flask. After sterilization, the Asp-producing bacterium Brevibacterium glutamicum (FERM BP-2178) and the His-producing bacterium Brevibacterium flavum (ATCC 214, which were previously grown on broth slant.
06) was inoculated into the same flask and shake-cultured at 31 ° C. for 72 hours. During the culture, an aqueous urea solution having a concentration of 40 g / dl was added little by little so that the pH was between 6.5 and 8.0. In the culture broth, L-aspartic acid was 0.95 g / dl and L-histidine was 0.85 g / d.
I had accumulated. The supernatant obtained by removing the cells from the culture-completed liquid by centrifugation was purified by a conventional method using an ion exchange resin, and 1.75 g of L-aspartic acid crystals and L-aspartic acid were obtained.
Example 7 in which 1.6 g of histidine crystals were obtained (simultaneous production of Glu and Lys by mixed culture of Glu-producing bacterium and Lys-producing bacterium) Glucose 50 mg / ml, urea 2 mg / ml, K
H 2 PO 4 1 mg / ml, MgSO 4 . 7H 2 O
0.4mg / ml, FeSO 4 · 7H 2 O 10μg /
ml, MnSO 4 .4H 2 O 8 μg / ml, soybean protein acid hydrolyzate “taste solution” 5 μl / ml, thiamine hydrochloride 10.0 μg / dl and biotin 0.25 μg / dl 20 ml to 500 ml
The mixture was placed in a shake flask and heated at 115 ° C. for 10 minutes for sterilization. Glu-producing bacterium Brevibacterium lactofermentum (ATCC 13869) and Lys were added to this medium.
Brevibacterium lactofermentum (A
TCC 21800) and shaken for 31.5
Incubated at ° C. During the culturing, a urea solution having a concentration of 450 mg / ml was added little by little so as to keep the pH of the culturing solution at 6.5 to 8.0, and the culturing was completed in 30 hours. L-glutamic acid and L-lysine accumulated in the culture solution were quantified. As a result, L-glutamic acid was 10.5 mg / ml.
And L-lysine accumulated 15.3 mg / ml.
【発明の効果】塩基性アミノ酸と酸性アミノ酸を同時に
商業的蓄積量で発酵生産することに係わる本発明により
得られるメリットは、培地中のイオン量を低減すること
が可能となり、延いては培養中の浸透圧の上昇を抑える
ことによる発酵生産性の向上（アミノ酸生成速度の向
上）、培地成分の節減省略、発酵液処理（アミノ酸の単
離操作）での簡略化、発酵液からアミノ酸を分離取得し
た後の廃液の環境保全負荷の低減等が可能となることで
ある。特に廃液の環境保全負荷の低減は、環境保全が強
く叫ばれている現今、看過し得ざるメリットである。総
じて、本発明によればアミノ酸生産における大巾なコス
トダウンの達成が可能となり、安価にアミノ酸を供給で
きる。[Advantages of the Invention] The merit obtained by the present invention relating to the fermentative production of basic amino acids and acidic amino acids at the same time in a commercially accumulated amount makes it possible to reduce the amount of ions in the medium, and in turn, during culture. Improvement of fermentation productivity (improvement of amino acid production rate) by suppressing increase in osmotic pressure, omission of reduction of medium components, simplification of fermentation liquid treatment (amino acid isolation operation), separation and acquisition of amino acids from fermentation liquid After that, it is possible to reduce the environmental protection load of the waste liquid. Especially, the reduction of environmental conservation load of waste liquid is an advantage that cannot be overlooked nowadays when environmental conservation is strongly demanded. In general, according to the present invention, it is possible to achieve a large cost reduction in amino acid production, and amino acids can be supplied at low cost.
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【手続補正書】[Procedure amendment]
【提出日】平成３年４月１２日[Submission date] April 12, 1991
【手続補正１】[Procedure Amendment 1]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】発明の詳細な説明[Name of item to be amended] Detailed explanation of the invention
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【発明の詳細な説明】Detailed Description of the Invention
【産業上の利用分野】本発明は、塩基性Ｌ−アミノ駿生
産菌を酸性Ｌ−アミノ酸産生条件下に培養するか或いは
塩基性Ｌ−アミノ酸生産菌と酸性Ｌ−アミノ酸生産菌を
混合培養することを特徴とする塩基性Ｌ−アミノ酸と酸
性Ｌ−アミノ酸の同時発酵法に関する。INDUSTRIAL APPLICABILITY The present invention cultivates a basic L-amino-green-producing bacterium under acidic L-amino acid-producing conditions or a mixed culture of a basic L-amino acid-producing bacterium and an acidic L-amino acid-producing bacterium. The present invention relates to a method for simultaneously fermenting a basic L-amino acid and an acidic L-amino acid.
【従来の技術】従来発酵法による塩基性Ｌ−アミノ酸と
酸性Ｌ−アミノ酸の同時生産は知られていない。知られ
ていたのは塩基性Ｌ−アミノ酸及び酸性Ｌ−アミノ酸の
いずれか一方の１種のアミノ酸を培地中に蓄積させるも
のであった。この場合、２種以上のアミノ酸が培地中に
認められても、目的以外のアミノ酸は副生物程度の微量
（数１０ｍｇ／ｄｌ）存在するのみであった。換言すれ
ば、塩基性アミノ酸と酸性アミノ酸を同時に商業的蓄積
量で発酵生産する方法は種々のメリットが期待されるに
も拘わらず未だ知られていない。2. Description of the Related Art Conventionally, simultaneous production of basic L-amino acids and acidic L-amino acids by fermentation has not been known. It was known that one of the basic L-amino acids and the acidic L-amino acids was accumulated in the medium. In this case, even if two or more kinds of amino acids were found in the medium, the amino acids other than the target were present in a trace amount (several 10 mg / dl) as a by-product. In other words, a method of fermentatively producing a basic amino acid and an acidic amino acid at the same time in a commercially accumulated amount has not been known although various merits are expected.
【発明が解決しようとする課題】本発明は、塩基性アミ
ノ酸と酸性アミノ酸を同時に商業的蓄積量で発酵生産す
る方法を提供することを目的とする。SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for fermentatively producing a basic amino acid and an acidic amino acid simultaneously in a commercially accumulated amount.
【課題を解決するための手段】本発明者は、上記目的の
ために種々検討し、塩基性アミノ酸と酸性アミノ酸を同
時に生産する方法を確立し、本発明を完成した。本発明
は、前記のように、塩基性Ｌ−アミノ酸生産菌を酸性Ｌ
−アミノ酸産生条件下に培養するか或いは塩基性Ｌ−ア
ミノ酸生産菌と酸性Ｌ−アミノ酸生産菌を混合培養する
ことを特徴とする塩基性Ｌ−アミノ酸と酸性Ｌ−アミノ
酸の同時発酵法に関する。以下、本発明を詳細に説明す
る。本発明に云う塩基性Ｌ−アミノ酸にはＬ−リジン
（Ｌｙｓ）、Ｌ−アルギニン（Ａｒｇ）、Ｌ−ヒスチジ
ン（Ｈｉｓ）及びＬ−オルニチン（Ｏｒｎ）が含まれ、
酸性Ｌ−アミノ酸にはＬ−グルタミン酸（Ｇｌｕ）及び
Ｌ−アスパラギン酸（Ａｓｐ）が含まれる。先ず、塩基
性Ｌ−アミノ酸生産菌を使用し、これを酸性Ｌ−アミノ
酸産生条件下に培養して塩基性Ｌ−アミノ酸と酸性Ｌ−
アミノ酸を同時に発酵生産する本発明に係わる第１の方
法について説明する。発酵培地としては、炭素源、窒素
源、無機塩類、生育因子などを含有する栄養培地または
合成培地が用いられる。炭素源としては、グルコース，
フラクトース，シュークロース，糖蜜，デンプン，デン
プン加水分解物，果汁などの炭水化物、エタノール，メ
タノール，プロパノールなどのアルコール類、酢酸など
の有機酸類が使用できる。窒素源としては、硫酸アンモ
ニウム，硝酸アンモニウム，塩化アンモニウム，リン酸
アンモニウム，酢酸アンモニウム，アンモニア，アミン
類，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，各種発酵菌体および
その消化物が使用できる。栄養要求性を示す変異株を使
用する場合には、それらの要求物質を標品もしくはそれ
を含有する天然物として添加する。発酵は、通気攪拌，
振盪培養などの好気的条件下で行う。培養温度は２４〜
４０℃、培養日数は２〜７日間である。培養液のｐＨは
５〜９の範囲に維持する。ｐＨの調節には尿素，炭酸カ
ルシウム，アンモニアガス，アンモニア水などを用い
る。 これらの発酵倍地及び発酵条件は従来公知のアミ
ノ酸発酵に採用されているものであるが、本方法では発
酵条件として更に塩基性Ｌ−アミノ酸と酸性Ｌ−アミノ
酸を同時に発酵する条件をも採用しなければならない。
これについて以下詳述する。従来知られている塩基性Ｌ
−アミノ酸生産菌は、通常、酸性Ｌ−アミノ酸生産菌で
あるブレビバクテリウム属細菌、コリネバクテリウム属
細菌などのコリネ型細菌を各種の変異処理に付して得ら
れている。例えば、ブレビバクテリウム・ラクトファー
メンタムＡＴＣＣ １３８６９にニトロソグアニジンを
変異剤として変異処理をほどこし、ＡＥＣ（Ｓ−２−ア
ミノエチル−Ｌ−システイン）の耐性を付けたリジン生
産菌ブレビバクテリウム・ラクトファーメンタムＡＴＣ
Ｃ ２１７９８がある。一方、コリネ型細菌に属する酸
性Ｌ−アミノ酸生産菌は、涌堂ビオチン要求性である
が、ビオチンが充分量存在する培地（１０μｇ／ｌ以
上）では菌体が増殖するのみで酸性Ｌ−アミノ酸の蓄積
は殆んど見られない。酸性Ｌ−アミノ酸を蓄積させるた
めには菌体の増殖を抑制することが必要で、そのために
は低ビオチン濃度の培地を使用するかビオチンが充分量
存在する培地の場合はポリオキシエチレンソルビタンモ
ノパルミテート（ＰＥＳＰ）、ポリオキシエチレンソル
ビタンモノステアレート（ＰＥＳＳ）、ポリオキシエチ
レンソルビタンモノラウレート（ＰＥＳＬ）などの界面
活性剤又はペニシリン、セファロリジンなどのラクタム
系抗生物質を培養の初発又は徐上で培地に添加する必要
がある。因みにこのようなコリネ型細菌に属する酸性Ｌ
−アミノ酸生産菌から変異誘導された塩基性Ｌ−アミノ
酸生産菌はやはりビオチン要求性であるが、これを培養
して塩基性Ｌ−アミノ酸を発酵生産する場合は、元の酸
性Ｌ−アミノ酸生産菌を培養して酸性Ｌ−アミノ酸を発
酵生産する場合とは異なり、培地に充分量のビオチンが
存在していてもよく、ビオチンを低濃度に制限する必要
はない。本発明の方法に使用できるＬ−アミノ酸生産菌
の例を表１に示す。Means for Solving the Problems The present inventor has conducted various studies for the above purpose, established a method for simultaneously producing a basic amino acid and an acidic amino acid, and completed the present invention. In the present invention, as described above, a basic L-amino acid-producing bacterium is treated with an acidic L-amino acid.
-A method for simultaneous fermentation of a basic L-amino acid and an acidic L-amino acid, which comprises culturing under a condition for producing an amino acid or culturing a basic L-amino acid-producing bacterium and an acidic L-amino acid-producing bacterium in a mixed culture. Hereinafter, the present invention will be described in detail. The basic L-amino acids referred to in the present invention include L-lysine (Lys), L-arginine (Arg), L-histidine (His) and L-ornithine (Orn),
Acidic L-amino acids include L-glutamic acid (Glu) and L-aspartic acid (Asp). First, a basic L-amino acid-producing bacterium is used, which is cultured under acidic L-amino acid producing conditions to produce a basic L-amino acid and an acidic L-amino acid.
The first method according to the present invention for simultaneously fermenting and producing amino acids will be described. As the fermentation medium, a nutrient medium or synthetic medium containing a carbon source, a nitrogen source, inorganic salts, growth factors and the like is used. As a carbon source, glucose,
Fructose, sucrose, molasses, starch, starch hydrolyzate, carbohydrates such as fruit juice, alcohols such as ethanol, methanol and propanol, and organic acids such as acetic acid can be used. As a nitrogen source, ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, ammonium acetate, ammonia, amines, peptone, meat extract, yeast extract, corn steve liquor, casein hydrolyzate, various fermented cells and their digestion Things can be used. When using a mutant strain showing an auxotrophy, those required substances are added as a standard product or a natural product containing the same. Fermentation is aeration stirring,
Perform under aerobic conditions such as shaking culture. Culture temperature is 24 ~
40 ° C., the number of days of culture is 2 to 7 days. The pH of the culture solution is maintained within the range of 5-9. Urea, calcium carbonate, ammonia gas, aqueous ammonia, etc. are used to adjust the pH. These fermentation mediums and fermentation conditions have been adopted in the conventionally known amino acid fermentation, but in the present method, the conditions for simultaneously fermenting basic L-amino acids and acidic L-amino acids are also adopted as fermentation conditions. There must be.
This will be described in detail below. Conventionally known basic L
-The amino acid-producing bacterium is usually obtained by subjecting coryneform bacteria such as Brevibacterium and Corynebacterium, which are acidic L-amino acid-producing bacteria, to various mutation treatments. For example, Brevibacterium lactofermentum ATCC 13869 was subjected to a mutation treatment using nitrosoguanidine as a mutagen to give AEC (S-2-aminoethyl-L-cysteine) resistance to a lysine-producing bacterium, Brevibacterium lactopher. Mental ATC
There is C 21798. On the other hand, an acid L-amino acid-producing bacterium belonging to a coryneform bacterium requires Kakudo biotin, but in a medium in which a sufficient amount of biotin is present (10 μg / l or more), only bacterial cells grow and acid L-amino acid Almost no accumulation is seen. In order to accumulate acidic L-amino acids, it is necessary to suppress the growth of bacterial cells. For that purpose, use a medium with a low biotin concentration or, in the case of a medium in which a sufficient amount of biotin is present, polyoxyethylene sorbitan monopalmi. Tate (PESP), polyoxyethylene sorbitan monostearate (PESS), polyoxyethylene sorbitan monolaurate (PESL) or other surfactants or penicillin, cephaloridine or other lactam antibiotics may be used at the beginning or after the initial culture. Must be added to the medium. By the way, acidic L belonging to such coryneform bacteria
-A basic L-amino acid-producing bacterium mutated from an amino acid-producing bacterium is also biotin-auxotrophic, but when the basic L-amino acid is fermentatively produced by culturing this, the original acidic L-amino acid-producing bacterium is used. Unlike the case where the acidic L-amino acid is fermentatively produced by culturing the bacterium, a sufficient amount of biotin may be present in the medium, and it is not necessary to limit the biotin to a low concentration. Table 1 shows examples of L-amino acid-producing bacteria that can be used in the method of the present invention.
【表１】
しかして、本発明者は、上のような塩基性Ｌ−アミノ酸
性産菌を元の酸性Ｌ−アミノ酸生産菌を使用して酸性Ｌ
−アミノ酸を発酵生産する培養条件下に培養したとこ
ろ、極めて予期せざることに、塩基性Ｌ−アミノ酸のみ
ならす酸性Ｌ−アミノ酸をも同時に蓄積することを見出
したのである。尤も、この場合塩基性Ｌ−アミノ酸の対
炭素源収率は低下し、酸性Ｌ−アミノ酸が低下分に相当
する量で蓄積する。かくして、塩基性Ｌ−アミノ酸生産
菌が上のようなビオチン栄養要求性細菌である場合、そ
の酸性Ｌ−アミノ酸産生条件は低ビオチン濃度の培地を
使用するかビオチンが充分量存在する培地の場合はＰＥ
ＳＰなどの界面活性剤又はペニシリンなどのラクタム系
抗生物質を培養の初発又は途上で培地に添加することで
ある。 低ビオチン濃度とは濃度０．５〜１０μｇ／ｌ
のビオチン濃度である。こうすることによって例えばＬ
ｙｓと共にＧｌｕを生産することができる。培養の初発
又は途上で培地に添加するＰＥＳＰなどの界面活性剤及
びペニシリンなどのラクタム系抗生物質の添加量は、界
面活性剤については０．０１〜０．５ｇ／ｄｌ程度、ペ
ニシリンについては０．１〜１０Ｕ／ｍｌ程度である。
こうすることによって、例えばＬｙｓと共にＧｌｕを生
産することができる。次に、本発明に係わる第２の方
法、すなわち、塩基性Ｌ−アミノ酸性産菌と酸性Ｌ−ア
ミノ酸生産菌を併用して、混合培養する塩基性Ｌ−アミ
ノ酸と酸性Ｌ−アミノ酸の同時発酵法について説明す
る。塩基性Ｌ−アミノ酸生産菌及び酸性Ｌ−アミノ酸生
産菌がブレビバクテリウム属細菌、コリネバクテリウム
属細菌などのコリネ型細菌、枯草菌、などのバチルス属
細菌、大腸菌などのエシエリヒア属細菌等に属すること
は周知であり、これらのアミノ酸生産菌は広く使用され
る。又、発酵培地及び発酵条件は、第１の方法に関して
前記した従来公知のアミノ酸発酵に採用されているもの
でよい。第２の方法における塩基性Ｌ−アミノ酸と酸性
Ｌ−アミノ酸の同時発酵条件は塩基性Ｌ−アミノ酸生産
菌を使用して塩基性Ｌ−アミノ酸を発酵蓄積させる条件
と酸性Ｌ−アミノ酸生産菌を使用して酸性Ｌ−アミノ酸
を発酵蓄積させる条件とを単に相加的に併用するだけで
よい。しかして、２種の発酵条件はいずれも前記した従
来公知のアミノ酸発酵条件として一括されるものであっ
て、両者間に実質的な相違はない。相違のあるのは、例
えば、混合培養する２種の細菌の一方のみが栄養要求性
で他方が栄養要求性のない場合であるが、この場合栄養
要求性のある細菌の培養条件として培地に要求栄養物質
を添加することが必要なのに対して栄養要求性のない細
菌の培養条件として培地に要求栄養物質を特に加える必
要がない。栄養要求性細菌と非栄養要求性細菌の混合培
養の場合、両者の発酵条件の相加的併用とは培地に該栄
養物質を添加することである。非栄養要求性細菌と栄養
要求性細菌を混合培養して一方の細菌に塩基性Ｌ−アミ
ノ酸を産生せしめ、他方の細菌に酸性Ｌ−アミノ酸を産
生せしめる場合、当該要求栄養物質を培地に添加しても
非栄養要求性細菌のアミノ酸産生には何らの悪影響も及
ぼさないのである。前記のような同時発酵の条件を採用
することにより、本発明の方法（第１及び第２）によれ
ば、塩基性Ｌ−アミノ酸と酸性Ｌ−アミノ酸を同時に各
々商業的蓄積量、すなわち、約５００ｍｇ／ｄｌ以上の
蓄積量で培地中に蓄積させることができる。発酵終了液
から生成蓄積したアミノ酸を分離取得するには常法でよ
く、例えばイオン交換樹脂法（例えば、発酵終了液から
先ず陽イオン交換樹脂により塩基性アミノ酸を吸着分離
し、ついで陰イオン交換樹脂により酸性アミノ酸を吸着
分離する。）によることができる。塩基性Ｌ−アミノ酸
と酸性Ｌ−アミノ酸の混合物の用途に対してはもちろん
両種のアミノ酸を相互に分離することは不要である。前
記のように本発明の塩基性Ｌ−アミノ酸と酸性Ｌ−アミ
ノ酸の同時発酵法は種々のメリットを有するが、以下こ
れらについて説明する。培地中のイオン量の低減に関し
て云えば、例えば、Ｌｙｓ発酵の場合、従来のＬｙｓ単
独発酵法では生成するＬｙｓを中和するために酸性イオ
ンが０．２〜０．４Ｍ必要であったが、本発明の方法を
用いてＬｙｓ及びＧｌｕの同時発酵を行うことにより
０．１〜．０．２Ｍと必要イオン量が半減できる。発酵
生産性の向上（アミノ酸生成速度の向上）に関し云え
ば、例えば、従来のＬｙｓ単独発酵の場合、菌体・時間
当りのＬｙｓ生成速度は０．０３〜０．０４ｈｒ−１で
あるが、本発明の方法を用いてＬｙｓ及びＧｌｕの同時
発酵を行うことによりＬｙｓ及びＧｌｕの合計生成速度
は０．０６〜０．０８ｈｒ−１と約２倍に向上する。培
地成分の節減省略に関して云えば、例えば、従来のＬｙ
ｓ発酵の場合、培地成分として硫酸アンモニウム及びア
ンモニアが２〜６ｇ／ｄｌ必要なのに対し、本発明の方
法を用いてＬｙｓ及びＧｌｕの同時発酵を行うことによ
り硫酸アンモニウムは必要なくアンモニアも１ｇ／ｄｌ
以下でよい。発酵液処理（アミノ酸の単離操作）での簡
略化に関して云えば、例えば、従来のＬｙｓ発酵の場
合、処理工程で脱塩工程が必要となるが、本発明の方法
によりＬｙｓ及びＧｌｕの同時発酵を行うことにより処
理工程での脱塩工程が不要になる。廃液の環境保全負荷
の低減に関して云えば、例えば、従来のＬｙｓ発酵の場
合、その製造工程より排出される廃液には活性汚泥処理
やその後に続く脱窒処理により除去すべき窒素化合物が
含有されているが、本発明の方法によりＬｙｓ及びＧｌ
ｕの同時発酵を行えば、この廃液中の窒素化合物をほと
んどゼロにすることができる。[Table 1] Therefore, the present inventor uses the original acidic L-amino acid-producing bacterium to transform the basic L-amino acid-producing bacterium into the acidic L-amino acid-producing bacterium.
-When cultured under the culture conditions for producing amino acids by fermentation, it was found that, unexpectedly, acidic L-amino acids that level out only basic L-amino acids also accumulate at the same time. However, in this case, the yield of the basic L-amino acid with respect to the carbon source decreases, and the acidic L-amino acid accumulates in an amount corresponding to the decrease. Thus, when the basic L-amino acid-producing bacterium is a biotin auxotrophic bacterium as described above, the acidic L-amino acid producing condition is that a medium having a low biotin concentration is used or a medium in which a sufficient amount of biotin is present is used. PE
A surfactant such as SP or a lactam antibiotic such as penicillin is added to the medium at the beginning or during the culture. Low biotin concentration is 0.5-10 μg / l
Is the biotin concentration of. By doing this, for example, L
Glu can be produced with ys. The amount of the surfactant such as PESP and the lactam antibiotic such as penicillin added to the medium at the beginning of the culture or in the middle of the culture is about 0.01 to 0.5 g / dl for the surfactant and 0. It is about 1 to 10 U / ml.
By doing so, for example, Glu can be produced together with Lys. Next, the second method according to the present invention, that is, the basic L-amino acid-producing bacterium and the acidic L-amino acid-producing bacterium are used in combination, and the mixed L-amino acid and the acidic L-amino acid are simultaneously fermented. The method will be explained. Basic L-amino acid-producing bacterium and acidic L-amino acid-producing bacterium belong to coryneform bacteria such as Brevibacterium, Corynebacterium, Bacillus subtilis, Bacillus, Escherichia bacterium, etc. It is well known that these amino acid-producing bacteria are widely used. Further, the fermentation medium and the fermentation conditions may be those employed in the conventionally known amino acid fermentation described above in regard to the first method. The basic L-amino acid and the acidic L-amino acid are simultaneously fermented in the second method under the conditions of fermenting and accumulating the basic L-amino acid by using the basic L-amino acid-producing bacterium and the acidic L-amino acid-producing bacterium. It is only necessary to use additively in combination with the conditions for fermentative accumulation of acidic L-amino acids. However, both of the two types of fermentation conditions are collectively described as the conventionally known amino acid fermentation conditions described above, and there is no substantial difference between the two. The difference is, for example, when only one of the two kinds of bacteria to be mixed-cultured is auxotrophic and the other is not auxotrophic. In this case, the culture conditions of the auxotrophic bacteria are required in the medium. It is not necessary to add the required nutrient substance to the medium as a culture condition for bacteria that are not auxotrophic while it is necessary to add the nutrient substance. In the case of mixed culture of an auxotrophic bacterium and a non-auxotrophic bacterium, the additive combination of fermentation conditions of both is to add the nutrient substance to the medium. When non-auxotrophic bacteria and auxotrophic bacteria are mixed and cultured so that one bacterium produces a basic L-amino acid and the other bacterium produces an acidic L-amino acid, the auxotrophic substance is added to the medium. However, it has no adverse effect on the amino acid production of non-auxotrophic bacteria. By adopting the conditions of the co-fermentation as described above, according to the method of the present invention (first and second), the basic L-amino acid and the acidic L-amino acid are simultaneously stored in a commercially accumulated amount, that is, about It can be accumulated in the medium at an accumulated amount of 500 mg / dl or more. A conventional method may be used to separate and obtain the amino acids produced and accumulated from the fermentation broth. For example, an ion exchange resin method (for example, a basic amino acid is first adsorbed and separated from the fermentation broth by a cation exchange resin, and then an anion exchange resin is used). The acidic amino acid is adsorbed and separated by. For the use of a mixture of basic L-amino acid and acidic L-amino acid, it is of course not necessary to separate the two amino acids from each other. As described above, the simultaneous fermentation method of the basic L-amino acid and the acidic L-amino acid of the present invention has various advantages, which will be described below. Regarding the reduction of the amount of ions in the medium, for example, in the case of Lys fermentation, 0.2 to 0.4 M of acidic ions were required to neutralize Lys produced in the conventional Lys single fermentation method. By performing the simultaneous fermentation of Lys and Glu using the method of the present invention, 0.1-. The required amount of ions can be halved to 0.2M. Speaking of improvement of fermentation productivity (improvement of amino acid production rate), for example, in the case of conventional Lys single fermentation, the Lys production rate per cell / hour is 0.03 to 0.04 hr −1. By carrying out the co-fermentation of Lys and Glu using the method of the invention, the total production rate of Lys and Glu is improved from 0.06 to 0.08 hr −1, which is about double. Speaking of omitting saving of medium components, for example, conventional Ly
In the case of s fermentation, ammonium sulfate and ammonia are required as a medium component in an amount of 2 to 6 g / dl, whereas by performing the simultaneous fermentation of Lys and Glu using the method of the present invention, ammonium sulfate is not required and ammonia is also 1 g / dl.
The following is sufficient. Regarding the simplification in the fermentation liquid treatment (amino acid isolation operation), for example, in the case of conventional Lys fermentation, a desalting step is required in the treatment step, but the method of the present invention simultaneously ferments Lys and Glu. By carrying out the step, the desalting step in the processing step becomes unnecessary. Regarding the reduction of environmental conservation load of waste liquid, for example, in the case of conventional Lys fermentation, the waste liquid discharged from the production process contains nitrogen compounds to be removed by activated sludge treatment and subsequent denitrification treatment. However, according to the method of the present invention, Lys and Gl
If the simultaneous fermentation of u is performed, the nitrogen compounds in this waste liquid can be reduced to almost zero.
【実施例】以下、本発明を実施例により更に詳細に説明
する。
実施例１（Ｌｙｓ生産菌の培養によるＬｙｓとＧｌｕの
同時生産）
粗糖を糖として１４０ｍｇ／ｍｌ、ＫＨ２ＰＯ４１ｍｇ
／ｍｌ、ＭｇＳＯ４・７Ｈ２Ｏ０．４ｍｇ／ｍｌ、Ｆｅ
ＳＯ４・７Ｈ２Ｏ １０ｍｇ／ｍｌ、ＭｎＳＯ４・４Ｈ
２Ｏ ２０μｇ／ｍｌ、尿素５ｍｇ／ｍｌ、大豆蛋白酸
加水分解物「味液」（登録商標）５μｌ／ｍｌ、ビオチ
ン５０μｇ／ｌ及びサイアミン６０μｇ／ｌを含有する
水溶液培地を調製し、その２０ｍｌづつを５００ｍｌ容
の振盪フラスコに入れ１１５℃で１０分間加熱滅菌し
た。この培地にＬｙｓ生産菌ブレビバクテリウム・ラク
トファーメンタム（ＡＴＣＣ ２１７９８）を接種し往
復振盪機により３１．５℃で培養を行った。培養中、培
養液をｐＨ６．５ないし８．０に保つように４００ｍｇ
／ｍｌの濃度の尿素水溶液を少量づつ添加した。培養液
の２６倍希釈液の５６２μｍおける吸光度が０．３０に
到達した時にＰＥＳＰ（ポリオキシエチレンソルビタン
モノパルミテート）を４ｍｇ／ｍｌ添加し、３０時間で
発酵を終了し、発酵液中に蓄積したＬ−リジン及びＬ−
グルタミン酸を液体クロマトグラフィーにより定量した
ところ、Ｌ−リジン３０ｍｇ／ｍｌ及びＬ−グルタミン
酸２２ｍｇ／ｍｌが蓄積していた。同様の培地に硫酸ア
ンモニウム２５ｍｇ／ｍｌ加えた培地と同じ菌株を用い
て、培養途中でＰＥＳＰを添加しないこと以外は同様の
方法によりリジン単独発酵を行ったところ、５５時間で
発酵が終了し、発酵液中にＬ−リジンが５３ｍｇ／ｍｌ
蓄積していた。Ｌ−リジンとＬ−グルタミン酸の同時発
酵の場合の培地中のイオン量は０．１３Ｍと後者のＬ−
リジン単独発酵の場合の０．２８Ｍの半量であった。ア
ミノ酸生成速度は、Ｌ−リジンとＬ−グルタミン酸の同
時発酵の場合、菌体・時間当りのＬ−リジンとＬ−グル
タミン酸の合計生成速度は０．０６６ｈｒ−１、Ｌ−リ
ジン単独発酵ではＬ−リジン生成速度が０．０３５ｈｒ
−１であった。培地成分の比較では、後者のＬ−リジン
単独発酵の場合には初発培地の硫酸アンモニウム及び初
発と追加添加の尿素由来のアンモニアを併せて３５ｍｇ
／ｍｌ必要なのに対して、Ｌ−リジンとＬ−グルタミン
酸の同時発酵の場合には硫酸アンモニウムはゼロ、尿素
由来のアンモニアは初発と追加添加併せても６ｍｇ／ｍ
ｌであった。さらに、得られた発酵液についてイオン交
換樹脂を用いた常法により処理を行った。イオン交換樹
脂にＬ−リジンやＬ−グルタミン酸を吸着させた残りの
樹脂貫流液中の塩濃度は、Ｌ−リジン単独発酵の場合に
は５％以上と極めて高く、該貫流液を有効利用をするた
めに脱塩が必要であった。一方Ｌ−リジンとＬ−グルタ
ミン酸の同時発酵の場合には２％以下と塩濃度が薄く脱
塩は不要であった。また、樹脂洗浄液等の廃液を活性汚
泥法により処理したところ、Ｌ−リジン単独発酵の廃液
では含有されたＢＯＤは除去し得たが窒素化合物が多量
残存し、さらに別途脱窒処理の必要性が認められたが、
Ｌ一リジンとＬ−グルタミン酸の同時発酵の廃液では活
性汚泥処理によりＢＯＤを除去した処理水中には多量の
窒素化合物は特に認められなかった。
実施例２（Ｌｙｓ生産菌の培養によるＬｙｓとＧｌｕ同
時生産）
グルコース １５％、リン酸第一カリ ０．１％、硫酸
マグネシウム・７水塩０．０４％、硫酸アンモニウム２
％、ビチオン １００γ／ｌ、ビタミンＢ１塩酸塩 ２
００γ／ｌ、鉄イオン及びマンガンイオン各２ｐｐｍ、
及び「味液」１％を含有するｐＨ７．０の水溶液培地を
小型ガラス製ジヤー・フアーメンターに３００ｍｌ分注
し、殺菌後、あらかじめ３０℃で２４時間ブイヨンスラ
ント上で生育させたＬｙｓ生産菌ブレビバクテリウム・
フラバム（ＡＴＣＣ ２１１２７）を接種した。つい
で、これを３１℃で培養を開始し、約１０時間経過した
後、ペニシリンを６Ｕ／ｍｌの濃度になるように添加し
培養を続け、１８時間培養したところ、発酵終了時には
培地中に３．７５ｇ／ｄｌのＬ−リジンと２．０５ｇ／
ｄｌのＬ−グルタミン酸が生成蓄積していた。この発酵
終了液１ｌから、遠心分離によって菌体を除去し、上清
液からイオン交換樹脂をもちいる常法にしたがってＬ−
リジンおよびＬ−グルタミン酸を分離し、精製して、Ｌ
−リジン塩酸塩の結晶２８．１ｇとＬ−グルタミン酸ソ
ーダの結晶１８．５ｇを得た。
実施例３（Ｈｉｓ生産菌の培養によるＨｉｓとＧｌｕの
同時生産）
Ｈｉｓ生産菌ブレビバクテリウム・フラバム（ＡＴＣＣ
２１４０６）を下記種培養培地に培種し、２０時間３
１℃にて通気攪拌培養を行った。
種培養培地：グリコース ３ｇ／ｄｌ、尿素 ０．３ｇ
／ｄｌ、ＫＨ２ＰＯ４ ５０．１ｇ／ｄｌ、ＭｇＳＯ４
・７Ｈ２Ｏ ０．０４ｇ／ｄｌ、ＦｅおよびＭｎイオン
各２ｐｐｍ、ビオチン２００μｇ／ｌ、サイアミン塩酸
塩３００μｇ／ｌ、大豆タンパク塩酸加水分解液（総窒
素７％）１ｍｌ／ｄｌ、酵母エキス０．５ｇ／ｄｌ及び
肉エキス０．５ｇ／ｄｌを含有するｐＨ７．２の水溶液
培地。一方、１ｌ容小型ガラス製ジヤーフアーメンター
に下記組成の主発酵培地を３００ｍｌ分注し、殺菌し
た。これに上記種培養液を１５ｍｌ接種し、３１℃にて
通気毎分１／１容の攪拌培養を開始した。
主発酵培地：グルコース １０ｇ／ｄｌ、硫酸アンモニ
ウム ０．５ｇ／ｄｌ、ＫＨ２ＰＯ４０．１ｇ／ｄｌ、
ＭｇＳＯ４・７Ｈ２Ｏ ０．０４ｇ／ｄｌ、Ｆｅおよび
Ｍｎイオン各２ｐｐｍ、ビオチン１００μｇ／ｌ、サイ
アミン塩酸塩２００μｇ／ｌ及び大豆タンパク塩酸加水
分解液（総窒素７％）２ｍｌ／ｄｌを含有するｐＨ７．
２水溶液培地。３１℃で培養中ｐＨが低下したときに、
アンモニアガスを添加してｐＨを７．０〜７．５に維持
した。培養開始後１０時間経過後ポリオキシエチレンソ
ルビタンモノパルミテートを０．４ｇ／ｄｌの濃度とな
るように添加し、培養を続けた。１８時間培養後、発酵
終了液中にはＬ−ヒスチジン０．８ｇ／ｄｌおよびＬ−
グルタミン酸１．２ｇ／ｄｌの蓄積が認められた。発酵
終了液から遠心分離により菌体を除去し、その上清液よ
りイオン交換樹脂をもちいる常法にしたがってＬ−ヒス
チジン結晶１．９ｇおよびＬ−グルタミン酸結晶２．８
ｇを得た。
実施例４（Ｇｌｕ生産菌とＬｙｓ生産菌の混合培養によ
るＧｌｕとＬｙｓの同時生産）
Ｇｌｕ生産菌コリネバクテリウム・グルタミクム（ＮＲ
ＲＬ Ｂ−１２１３８）およびＬｙｓ生産菌ブレビバク
テリウム・ラクトファーメンタム（ＡＴＣＣ２１７９
９）を各スラント上より１白金耳かきとり、下記組成の
種培養水溶液培地５０ｍｌにそれぞれ接種し、１８時
間、３１℃にて通気攪拌培養をおこなって、おのおのの
種培養液を調製した。
種培養培地組成：
グルコース １．５％
酢酸アンモニウム ０．３％
尿 素 ０．１％
ＫＨ２ＰＯ４ ０．１％
ＭｇＳＯ４・７Ｈ２Ｏ ０．０４％
Ｆｅ＋＋ ２ｐｐｍ
Ｍｎ＋＋ ２ｐｐｍ
ビオチン ５０μｇ／ｌ
サイアミン塩酸塩 ２００μｇ／ｌ
大豆タンパク塩酸加水分解液
濃縮物（総窒素７％） ３％
ｐＨ７．５
一方、１ｌ容小型ガラス製ジヤー・フアーメンターに下
記組成の主発酵水溶液培地を３００ｍｌ分注し、常法に
より殺菌した。これらに上記の種培養液を同時にそれぞ
れ１５ｍｌ宛接種し、３１℃にて通気毎分１／１容の攪
拌培養を開始した。主発酵培地：
グルコース ２％
酢酸アンモニウム ０．５％
尿 素 ０．２％
ＫＨ２ＰＯ４ ０．１％
ＭｇＳＯ４・７Ｈ２Ｏ ０．０４％
Ｆｅ＋＋ ２ｐｐｍ
Ｍｎ＋＋ ２ｐｐｍ
ビオチン ５０μｇ／ｌ
サイアミン塩酸塩 ６０μｇ／ｌ
大豆タンパク塩酸加水分解液
濃縮物（総窒素７％） ３％
ｐＨ７．５
培養液中に酢酸と酢酸酸アンモニウムとの混合液（酢
酸：酵酸アンモニウムの混合液のモル比は１：０．２
５、混合液の酢酸濃度は６０％）を培地のｐＨを７．２
〜８．０の間に保持するように連続的、あるいは間けつ
的に添加して、３０℃で７２時間培養をおこなった。結
果はＬ−グルタミン酸３７ｇ／ｌ及びＬ−リジン５４ｇ
／ｌの蓄積が認められた。
実施例５（Ａｓｐ生産菌とＡｒｇ生産菌の混合培養によ
るＡｓｐとＡｒｇの同時生産）
Ａｓｐ生産菌コリネバクテリウム・グルタミクム（ＦＥ
ＲＭ ＢＰ−２１７８）及びＡｒｇ産菌ブレビバクテリ
ウム・フラバム（ＡＴＣＣ ２１４９３）を下記組成の
種培養水溶液培地にそれぞれ別々に接種し、１８時間３
０℃にて通気攪拌培養を行った。
種培養培地組成：グリコース ３％、ＫＨ２ＰＯ４
０．１％、ＭｇＳＯ４・７Ｈ２Ｏ ０．０４％、Ｆｅ
＋＋ ２ｐｐｍ、Ｍｎ＋＋、２ｐｐｍ、「味液」３ｍｌ
／ｄｌ、ビオチン ５μｇ／ｌ、ビタミンＢ１・塩酸塩
３００ｒ／ｌ、尿素 ０．３％、ｐｈ７．２。一方、
小型ガラス製ジャーファーメンターに下記組成よりなる
主発酵水溶液倍地を３００ｍｌ分注し雑菌した。これに
上気各種培養液を同時に各々１５ｖ／ｖ％接種し、３１
℃にて１５００ｒ．ｐ．ｍ、通気攪拌培養を開始した。
主発酵灰地組織：エチルアルコール １．５％、硫酸ア
ンモニウム ０．５％、ＫＨ２ＰＯ４０．１％ＭｇＳＯ
４・７Ｈ２Ｏ ０．０４％、Ｆｅ＋＋２ｐｐｍ、「味
液」（登録商標）２ｍｌ／ｄｌ、ビオチン ５μｇ／
１、ビタミン Ｂ１・塩酸塩 ３００γ／ｌ、ｐＨ７．
２。培養中ｐＨが低下したときにアンモニアガスを添加
してｐＨを７．０〜７．５に維持した。エチルアルコー
ルは、その消費をガスクロマトグラフで定量し、その濃
度が０．０１％前後に減小したときに追添加した。４８
時間倍養後、倍養終了液中はＬ−アスパラギン酸が１．
３０ｇ／ｄｌおよびＬ−アルギニンが１．０５ｇ／ｄｌ
生成していた。発酵終了液よりイオン交換樹脂を用いる
常法によりＬ−アスパラギン酸の結晶が２．１８ｇ及び
Ｌ−アルギニンの結晶が１．９５ｇ得られた。
実施例６（Ａｓｐ生産菌とＨｉｓ生産菌の混合培養によ
るＡｓｐとＨｉｓの同時生産）
シュークロース １３ｇ／ｄｌ、尿素 ０．５ｇ／ｄ
ｌ、ＫＨ２ＰＯ４０．１ｇ／ｄｌ、ＭｇＳＯ４・７Ｈ２
Ｏ ０．０４ｇ／ｄｌ、ＦｅおよびＭｎイオン各２ｐｐ
ｍ、ビオチン ５μｇ／ｌ、サイアミン塩酸塩 ２００
μｇ／ｌ及び大豆タンパク塩酸加水分解物（総窒素７
％）０．３ｍｌ／ｄｌを含みｐＨ７．２に調節した水溶
液培地を調製し、その２０ｍｌを５００ｍｌ容の振とう
フラスコに分注した。殺菌後あらかじめブイヨンスラン
ト上で生育させたＡｓｐ生産菌コリネバクテリウム・グ
ルタミクム（ＦＥＲＭ ＢＰ−２１７８）及びＨｉｓ生
産菌ブレビバクテリウム・フラバム（ＡＴＣＣ ２１４
０６）を同じフラスコに接種し、３１℃にて７２時間振
とう培養した。培養中、ｐＨが６．５から８．０の間に
なるように４０ｇ／ｄｌの濃度の尿素水溶液を少量づつ
添加した。培養終了液中には、Ｌ−アスパラギン酸が
０．９５ｇ／ｄｌ及びＬ−ヒスチジンが０．８５ｇ／ｄ
ｌ蓄積していた。培養終了液から遠心分離によって菌体
を除いて得た上澄液をイオン交換樹脂を用いる常法によ
り精製し、Ｌ−アスパラギン酸の結晶１．７５ｇとＬ−
ヒスチジンの結晶１．６ｇを得た
実施例７（Ｇｌｕ生産菌とＬｙｓ生産菌の混合培養によ
るＧｌｕとＬｙｓの同時生産）
グルコース ５０ｍｇ／ｍｌ、尿素 ２ｍｇ／ｍｌ、Ｋ
Ｈ２ＰＯ４ １ｍｇ／ｍｌ、ＭｇｓＯ４・７Ｈ２Ｏ
０．４ｍｇ／ｍｌ、ＦｅＳＯ４・７Ｈ２Ｏ １０μｇ／
ｍｌ、ＭｎＳＯ４・４Ｈ２Ｏ ８μｇ／ｍｌ、大豆蛋白
酸加水分解物「味液」５μｌ／ｍｌ、サイアミン塩酸塩
１０．０μｇ／ｄｌ及びビオチン０．２５μｇ／ｄｌを
含有する水溶液培地を調製しその２０ｍｌを５００ｍｌ
容の振盪フラスコに入れ１１５℃で１０分間加熱殺菌し
た。この培地にＧｌｕ生産菌ブレビバクテリウム・ラク
トファーメンタム（ＡＴＣＣ １３８６９）及びＬｙｓ
生産菌ブレビバクテリウム・ラクトファーメンタム（Ａ
ＴＣＣ ２１８００）を接種し、振とうしつつ３１．５
℃にて培養した。培養中、培養液をｐＨ６．５ないし
８．０に保つように４５０ｍｇ／ｍｌの濃度の尿素溶液
を少量ずつ添加し、３０時間で培養を終了した。培養液
中に蓄積したＬ−グルタミン酸及びＬ−リジンを定量し
た。その結果、Ｌ−グルタミン酸が１０．５ｍｇ／ｍｌ
及びＬ−リジンが１５．３ｍｇ／ｍｌ蓄積していた。EXAMPLES The present invention will now be described in more detail with reference to examples. Example 1 (Simultaneous production of Lys and Glu by culturing Lys-producing bacteria) 140 mg / ml of crude sugar as sugar, 1 mg of KH 2 PO 4
/ Ml, MgSO 4 · 7H 2 O0.4mg / ml, Fe
SO 4 · 7H 2 O 10mg / ml, MnSO 4 · 4H
2 O 20 μg / ml, urea 5 mg / ml, soybean protein acid hydrolyzate “Taste liquid” (registered trademark) 5 μl / ml, biotin 50 μg / l, and thiamine 60 μg / l were prepared as an aqueous medium, and each 20 ml was prepared. Was placed in a 500 ml shake flask and sterilized by heating at 115 ° C for 10 minutes. This medium was inoculated with the Lys-producing bacterium Brevibacterium lactofermentum (ATCC 21798) and cultured at 31.5 ° C. with a reciprocating shaker. 400 mg to maintain the pH of the culture at 6.5 to 8.0 during culturing.
An aqueous urea solution having a concentration of / ml was added little by little. PESP (polyoxyethylene sorbitan monopalmitate) was added at 4 mg / ml when the absorbance at 562 μm of the 26-fold diluted culture solution reached 0.30, and the fermentation was completed in 30 hours and accumulated in the fermentation solution. L-lysine and L-
When glutamic acid was quantified by liquid chromatography, 30 mg / ml of L-lysine and 22 mg / ml of L-glutamic acid were accumulated. Using the same strain as the medium containing 25 mg / ml of ammonium sulfate in the same medium, lysine alone was fermented by the same method except that PESP was not added during the culture, and the fermentation was completed in 55 hours. L-lysine in 53 mg / ml
Had accumulated. In the case of co-fermentation of L-lysine and L-glutamic acid, the amount of ions in the medium was 0.13 M, and the latter L-
It was half of 0.28 M in the case of lysine fermentation alone. Regarding the amino acid production rate, in the case of simultaneous fermentation of L-lysine and L-glutamic acid, the total production rate of L-lysine and L-glutamic acid per cell / time was 0.066 hr −1 , and in L-lysine single fermentation L-lysine Lysine production rate is 0.035hr
It was -1 . In the case of the latter L-lysine single fermentation, 35 mg of ammonium sulfate of the initial medium and 35 mg of ammonia derived from urea and additional urea were added in comparison of medium components.
/ Ml is required, whereas in the case of simultaneous fermentation of L-lysine and L-glutamic acid, ammonium sulfate is zero, and urea-derived ammonia is 6 mg / m even if the initial and additional additions are combined.
It was l. Further, the obtained fermented liquor was treated by an ordinary method using an ion exchange resin. The salt concentration in the remaining resin flow-through liquid obtained by adsorbing L-lysine or L-glutamic acid on the ion exchange resin is extremely high at 5% or more in the case of L-lysine single fermentation, and the flow-through liquid is effectively used. Therefore, desalting was necessary. On the other hand, in the case of simultaneous fermentation of L-lysine and L-glutamic acid, the salt concentration was low at 2% or less and desalting was unnecessary. Further, when the waste liquid such as the resin washing liquid was treated by the activated sludge method, the BOD contained in the waste liquid of the L-lysine single fermentation could be removed, but a large amount of nitrogen compounds remained, and further denitrification treatment was required. Accepted,
In the waste liquid of the simultaneous fermentation of L-lysine and L-glutamic acid, a large amount of nitrogen compounds was not found in the treated water from which BOD was removed by the activated sludge treatment. Example 2 (Simultaneous production of Lys and Glu by culturing Lys-producing bacteria) Glucose 15%, primary potassium phosphate 0.1%, magnesium sulfate heptahydrate 0.04%, ammonium sulfate 2
%, Biotin 100γ / l, vitamin B 1 hydrochloride 2
00γ / 1, iron ion and manganese ion 2ppm each,
And 300 ml of a pH 7.0 aqueous solution medium containing 1% of "taste liquid" was poured into a small glass jar and fermenter, sterilized, and pre-grown on a broth slant at 30 ° C for 24 hours. Bacterium
Flavan (ATCC 21127) was inoculated. Then, the culture was started at 31 ° C., and after about 10 hours, penicillin was added at a concentration of 6 U / ml to continue the culture, and the culture was continued for 18 hours. 75 g / dl L-lysine and 2.05 g /
dl of L-glutamic acid was produced and accumulated. From the 1 l of the fermentation-finished liquid, cells were removed by centrifugation, and the supernatant was mixed with L- by an ordinary method using an ion exchange resin.
Lysine and L-glutamic acid were separated and purified to give L
-28.1 g of crystals of lysine hydrochloride and 18.5 g of crystals of L-sodium glutamate were obtained. Example 3 (Simultaneous production of His and Glu by culturing a His-producing bacterium) His-producing bacterium Brevibacterium flavum (ATCC
21406) in the following seed culture medium for 20 hours 3
Aeration-agitation culture was performed at 1 ° C. Seed culture medium: Glucose 3 g / dl, urea 0.3 g
/ Dl, KH 2 PO 4 50.1 g / dl, MgSO 4
7H 2 O 0.04 g / dl, Fe and Mn ions 2 ppm each, biotin 200 μg / l, thiamine hydrochloride 300 μg / l, soybean protein hydrochloric acid hydrolyzed solution (total nitrogen 7%) 1 ml / dl, yeast extract 0.5 g / Dl and meat extract 0.5 g / dl pH 7.2 aqueous medium. On the other hand, 300 ml of a main fermentation medium having the following composition was poured into a 1 l small glass glass jar mentor and sterilized. To this, 15 ml of the above seed culture was inoculated, and stirring culture was started at 31 ° C. with aeration of 1/1 volume per minute. Main fermentation medium: glucose 10 g / dl, ammonium sulfate 0.5 g / dl, KH 2 PO 4 0.1 g / dl,
MgSO 4 · 7H 2 O 0.04g / dl, pH7 containing Fe and Mn ions each 2 ppm, biotin 100 [mu] g / l, thiamine hydrochloride 200 [mu] g / l and soy protein hydrochloride hydrolyzate (total nitrogen 7%) 2ml / dl ．
2 Aqueous medium. When the pH drops during culture at 31 ° C,
Ammonia gas was added to maintain the pH at 7.0-7.5. After 10 hours from the start of the culture, polyoxyethylene sorbitan monopalmitate was added so that the concentration was 0.4 g / dl, and the culture was continued. After culturing for 18 hours, 0.8 g / dl of L-histidine and L-
Accumulation of 1.2 g / dl of glutamic acid was observed. Cells were removed from the fermentation broth by centrifugation and the supernatant was 1.9 g of L-histidine crystals and 2.8 L-glutamic acid crystals according to a conventional method using an ion exchange resin.
g was obtained. Example 4 (Simultaneous production of Glu and Lys by mixed culture of Glu-producing bacterium and Lys-producing bacterium) Glu-producing bacterium Corynebacterium glutamicum (NR
RL B-12138) and Lys producing strain Brevibacterium lactofermentum (ATCC 2179).
One platinum loop of 9) was scraped from each slant, inoculated into 50 ml of a seed culture aqueous solution medium having the following composition, and aerated and agitated at 18 ° C. for 18 hours to prepare each seed culture solution. Seed culture medium composition: 0.3% 1.5% glucose ammonium acetate 0.1% urea KH 2 PO 4 0.1% MgSO 4 · 7H 2 O 0.04% Fe ++ 2ppm Mn ++ 2ppm biotin 50 [mu] g / l Cyamine hydrochloride 200 μg / l Soybean protein hydrochloric acid hydrolyzed liquid concentrate (total nitrogen 7%) 3% pH 7.5 On the other hand, 300 ml of a main fermentation aqueous solution medium having the following composition was dispensed into a 1 l small glass glass jar fermenter, Sterilized by a conventional method. 15 ml of each of the above seed culture solutions was simultaneously inoculated to each of these, and agitated culture with aeration of 1/1 volume per minute was started at 31 ° C. The main fermentation medium: Glucose 2% ammonium acetate 0.5% 0.2% Urea KH 2 PO 4 0.1% MgSO 4 · 7H 2 O 0.04% Fe ++ 2ppm Mn ++ 2ppm biotin 50 [mu] g / l thiamine hydrochloride 60 μg / l Soybean protein hydrochloric acid hydrolyzate concentrate (total nitrogen 7%) 3% pH 7.5 A mixed solution of acetic acid and ammonium acetate in the culture solution (the molar ratio of the mixed solution of acetic acid: ammonium fermentate is 1: 0.2
5. The acetic acid concentration of the mixed solution is 60%) and the pH of the medium is 7.2.
It was continuously or intermittently added so as to maintain the temperature between ˜8.0, and cultured at 30 ° C. for 72 hours. The result was L-glutamic acid 37 g / l and L-lysine 54 g.
/ L accumulation was observed. Example 5 (Simultaneous production of Asp and Arg by mixed culture of Asp-producing bacterium and Arg-producing bacterium) Asp-producing bacterium Corynebacterium glutamicum (FE
RM BP-2178) and the Arg-producing bacterium Brevibacterium flavum (ATCC 21493) were separately inoculated into the seed culture aqueous solution medium having the following composition, and each of them was incubated for 3 hours for 18 hours.
Aeration-agitation culture was performed at 0 ° C. Seed culture medium composition: Glucose 3%, KH 2 PO 4
0.1%, MgSO 4 · 7H 2 O 0.04%, Fe
++ 2ppm, Mn ++ , 2ppm, "taste liquid" 3ml
/ Dl, biotin 5 μg / l, vitamin B 1 · hydrochloride 300 r / l, urea 0.3%, ph 7.2. on the other hand,
300 ml of a main fermentation aqueous solution medium having the following composition was dispensed into a small glass jar fermenter to inoculate bacteria. Each of them was inoculated with 15% v / v% of various cultures of Jikyu at the same time.
1500 r.p.C. p. m, aeration stirring culture was started. Main fermented ash texture: ethyl alcohol 1.5%, ammonium sulfate 0.5%, KH 2 PO 4 0.1% MgSO 4.
4 · 7H 2 O 0.04%, Fe ++ 2ppm, "Taste liquid" (registered trademark) 2 ml / dl, biotin 5 [mu] g /
1, vitamin B 1 · hydrochloride 300γ / l, pH 7.
2. When the pH decreased during the culture, ammonia gas was added to maintain the pH at 7.0 to 7.5. The consumption of ethyl alcohol was quantitatively determined by gas chromatography, and was added when the concentration decreased to around 0.01%. 48
After time doubling, L-aspartic acid was 1.
30 g / dl and L-arginine 1.05 g / dl
Was being generated. 2.18 g of L-aspartic acid crystals and 1.95 g of L-arginine crystals were obtained from the fermentation broth by a conventional method using an ion exchange resin. Example 6 (Simultaneous production of Asp and His by mixed culture of Asp-producing strain and His-producing strain) Sucrose 13 g / dl, urea 0.5 g / d
l, KH 2 PO 4 0.1g / dl, MgSO 4 · 7H 2
O 0.04 g / dl, Fe and Mn ions 2 pp each
m, biotin 5 μg / l, thiamin hydrochloride 200
μg / l and soybean protein hydrochloric acid hydrolyzate (total nitrogen 7
%) 0.3 ml / dl was added to prepare an aqueous medium adjusted to pH 7.2, and 20 ml thereof was dispensed into a 500 ml shake flask. After sterilization, the Asp-producing bacterium Corynebacterium glutamicum (FERM BP-2178) and the His-producing bacterium Brevibacterium flavum (ATCC 214) that had been previously grown on broth slant.
06) was inoculated into the same flask and shake-cultured at 31 ° C. for 72 hours. During the culture, an aqueous urea solution having a concentration of 40 g / dl was added little by little so that the pH was between 6.5 and 8.0. In the culture broth, L-aspartic acid was 0.95 g / dl and L-histidine was 0.85 g / d.
I had accumulated. The supernatant obtained by removing the cells from the culture-completed liquid by centrifugation was purified by a conventional method using an ion exchange resin, and 1.75 g of L-aspartic acid crystals and L-aspartic acid were obtained.
Example 7 in which 1.6 g of histidine crystals were obtained (simultaneous production of Glu and Lys by mixed culture of Glu-producing bacterium and Lys-producing bacterium) Glucose 50 mg / ml, urea 2 mg / ml, K
H 2 PO 4 1mg / ml, MgsO 4 · 7H 2 O
0.4mg / ml, FeSO 4 · 7H 2 O 10μg /
ml, MnSO 4 .4H 2 O 8 μg / ml, soybean protein acid hydrolyzate “taste” 5 μl / ml, thiamine hydrochloride 10.0 μg / dl and biotin 0.25 μg / dl 20 ml to 500 ml
The mixture was placed in a shake flask and heated at 115 ° C. for 10 minutes for sterilization. Glu-producing bacterium Brevibacterium lactofermentum (ATCC 13869) and Lys were added to this medium.
Brevibacterium lactofermentum (A
TCC 21800) and shaken for 31.5
Incubated at ° C. During the culturing, a urea solution having a concentration of 450 mg / ml was added little by little so as to keep the pH of the culturing solution at 6.5 to 8.0, and the culturing was completed in 30 hours. L-glutamic acid and L-lysine accumulated in the culture solution were quantified. As a result, L-glutamic acid was 10.5 mg / ml.
And L-lysine accumulated 15.3 mg / ml.
【発明の効果】塩基性アミノ酸と酸性アミノ酸を同時に
商業的蓄積量で発酵生産することに係わる本発明により
得られるメリットは、培地中のイオン量を低減すること
が可能となり、延いては培養中の浸透圧の上昇を抑える
ことによる発酵生産性の向上（アミノ酵中成速度の向
上）、培地成分の節減省略、発酵液処理（アミノ酸の単
離操作）での簡略化、発酵液からアミノ酸を分離取得し
た後の廃液の環境保全負荷の低減等が可能となることで
ある。特に廃液の環境保全負荷の低減は、環境保全が強
く叫ばれている現今、看過し得ざるメリットである。総
じて、本発明によればアミノ酸生産における大巾なコス
トダウンの達成が可能となり、安価にアミノ酸を供給で
きる。[Advantages of the Invention] The merit obtained by the present invention relating to the fermentative production of basic amino acids and acidic amino acids at the same time in a commercially accumulated amount makes it possible to reduce the amount of ions in the medium, and in turn, during culture. Of fermentative productivity by improving the osmotic pressure of Amino acid (improvement of amino fermentation rate), omission of saving medium components, simplification of fermentation liquor treatment (isolation of amino acid), amino acid removal from fermentation broth It is possible to reduce the environmental protection load of the waste liquid after separation and acquisition. Especially, the reduction of environmental conservation load of waste liquid is an advantage that cannot be overlooked nowadays when environmental conservation is strongly demanded. In general, according to the present invention, it is possible to achieve a large cost reduction in amino acid production, and amino acids can be supplied at low cost.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.5 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｃ１２Ｐ 13/20 6977−4Ｂ 13/24 Ｃ 6977−4Ｂ //(Ｃ１２Ｐ 13/08 Ｃ１２Ｒ 1:13） (Ｃ１２Ｐ 13/08 Ｃ１２Ｒ 1:15） (Ｃ１２Ｐ 13/10 Ｃ１２Ｒ 1:13） (Ｃ１２Ｐ 13/10 Ｃ１２Ｒ 1:15） (Ｃ１２Ｐ 13/14 Ｃ１２Ｒ 1:13） (Ｃ１２Ｐ 13/14 Ｃ１２Ｒ 1:15） (Ｃ１２Ｐ 13/20 Ｃ１２Ｒ 1:15） (Ｃ１２Ｐ 13/20 Ｃ１２Ｒ 1:13） (Ｃ１２Ｐ 13/24 Ｃ１２Ｒ 1:13） (Ｃ１２Ｐ 13/24 Ｃ１２Ｒ 1:15） (72)発明者 石井 俊昌 三重県四日市市大字日永1730 味の素株式 会社東海工場内 (72)発明者 吉原 康彦 佐賀県佐賀郡諸富町大字諸富津450 味の 素株式会社九州工場内─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 5 Identification code Office reference number FI technical display location C12P 13/20 6977-4B 13/24 C 6977-4B // (C12P 13/08 C12R 1:13 ) (C12P 13/08 C12R 1:15) (C12P 13/10 C12R 1:13) (C12P 13/10 C12R 1:15) (C12P 13/14 C12R 1:13) (C12P 13/14 C12R 1:15) ) (C12P 13/20 C12R 1:15) (C12P 13/20 C12R 1:13) (C12P 13/24 C12R 1:13) (C12P 13/24 C12R 1:15) (72) Inventor Toshimasa Ishii Mie Prefecture 1730 Hinode Nagaoka, Yokkaichi-shi Ajinomoto Co., Inc. Tokai Plant (72) Inventor Yasuhiko Yoshihara 450 Morojimi, Morotomi-cho, Saga-gun, Saga Prefecture Kyushu Plant, Ajinomoto Co., Inc.
Claims (6)
ミノ酸産生条件下に培養することを特徴とする塩基性Ｌ
−アミノ酸と酸性Ｌ−アミノ酸の同時発酵法。1. A basic L-amino acid-producing bacterium is cultured under an acidic L-amino acid-producing condition.
-A simultaneous fermentation method of an amino acid and an acidic L-amino acid.
テリウム属又はコリネバクテリウム属に属する細菌であ
ることを特徴とする請求項１記載の発酵法。2. The fermentation method according to claim 1, wherein the basic L-amino acid-producing bacterium is a bacterium belonging to the genus Brevibacterium or the genus Corynebacterium.
養要求性細菌でありかつ酸性Ｌ−アミノ酸産生条件が低
ビオチン培地であることを特徴とする請求項１又は２記
載の発酵法。3. The fermentation method according to claim 1 or 2, wherein the basic L-amino acid-producing bacterium is a biotin auxotrophic bacterium and the acidic L-amino acid producing condition is a low biotin medium.
養要求性細菌でありかつ酸性Ｌ−アミノ酸産生条件がビ
オチンを充分量含有する培地であって培養の初発又は途
上で界面活性剤又はラクタム系抗生物質を培地に含有せ
しめることであることを特徴とする請求項１又は２記載
の発酵法。4. A medium in which the basic L-amino acid-producing bacterium is a biotin auxotrophic bacterium and the acidic L-amino acid producing condition is a medium containing a sufficient amount of biotin, and a surfactant or a lactam system is used at the beginning or on the way of culturing. The fermentation method according to claim 1 or 2, wherein an antibiotic is contained in the medium.
ミノ酸生産菌を混合培養することを特徴とする塩基性Ｌ
−アミノ酸と酸性Ｌ−アミノ酸の同時発酵法。5. The basic L-amino acid-producing bacterium and the acidic L-amino acid-producing bacterium are mixed and cultured, and the basic L-amino acid is produced.
-A simultaneous fermentation method of an amino acid and an acidic L-amino acid.
性Ｌ−アミノ酸生産菌がブレビバクテリウム属及びコリ
ネバクテリウム属のいずれかに属する細菌であることを
特徴とする請求項５記載の発酵法。6. The fermentation according to claim 5, wherein the basic L-amino acid-producing bacterium and / or the acidic L-amino acid-producing bacterium is a bacterium belonging to either the genus Brevibacterium or the genus Corynebacterium. Law.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10695991A JP3245881B2 (en) | 1990-02-15 | 1991-02-15 | Simultaneous fermentation of basic and acidic amino acids |
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2-34066 | 1990-02-15 | ||
| JP3406690 | 1990-02-15 | ||
| JP3-100574 | 1991-02-04 | ||
| JP10057491 | 1991-02-04 | ||
| JP10695991A JP3245881B2 (en) | 1990-02-15 | 1991-02-15 | Simultaneous fermentation of basic and acidic amino acids |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH053793A true JPH053793A (en) | 1993-01-14 |
| JP3245881B2 JP3245881B2 (en) | 2002-01-15 |
Family
ID=27288315
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP10695991A Expired - Fee Related JP3245881B2 (en) | 1990-02-15 | 1991-02-15 | Simultaneous fermentation of basic and acidic amino acids |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3245881B2 (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995023224A1 (en) * | 1994-02-24 | 1995-08-31 | Ajinomoto Co., Inc. | Novel gene originating in corynebacterium and use thereof |
| EP0780477A4 (en) * | 1994-08-19 | 1999-06-30 | Ajinomoto Kk | Process for producing l-lysine and l-glutamic acid by fermentation |
| JP2002253273A (en) * | 2000-08-08 | 2002-09-10 | Roche Vitamins Ag | Method for producing target fermentation product |
| WO2005007848A1 (en) * | 2003-07-16 | 2005-01-27 | Ajinomoto Co., Inc. | Process for producing l-histidine with the use of bacterium belonging to enterobacteriaceae |
| CN102673891A (en) * | 2012-05-04 | 2012-09-19 | 深圳市华星光电技术有限公司 | Buffer stop block structure and corresponding package box body |
| WO2012147989A1 (en) | 2011-04-25 | 2012-11-01 | Ajinomoto Co.,Inc. | A method for producing an l-amino acid belonging to the glutamate family, using a coryneform bacterium |
| JP2013520196A (en) * | 2010-02-25 | 2013-06-06 | コルゲート・パーモリブ・カンパニー | Process for producing arginine using Corynebacterium glutamicum ATCC 21831 or Corynebacterium glutamicum ATCC 21493 in fermentation medium containing Cassava bagasse or jackfruit seed as a carbon source |
| JP2015198662A (en) * | 2015-06-01 | 2015-11-12 | コルゲート・パーモリブ・カンパニーＣｏｌｇａｔｅ−Ｐａｌｍｏｌｉｖｅ Ｃｏｍｐａｎｙ | Process for producing arginine using corynebacterium glutamicum atcc21831 or corynebacterium glutamicum atcc21493 in fermentation medium containing cassava bagasse, or jackfruit seeds as carbon source |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023136404A1 (en) * | 2022-01-11 | 2023-07-20 | 대상 주식회사 | Method for preparing seasoning material by mixed fermentation of heterogeneous microbes |
-
1991
- 1991-02-15 JP JP10695991A patent/JP3245881B2/en not_active Expired - Fee Related
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995023224A1 (en) * | 1994-02-24 | 1995-08-31 | Ajinomoto Co., Inc. | Novel gene originating in corynebacterium and use thereof |
| EP0780477A4 (en) * | 1994-08-19 | 1999-06-30 | Ajinomoto Kk | Process for producing l-lysine and l-glutamic acid by fermentation |
| EP1293560A3 (en) * | 1994-08-19 | 2004-12-01 | Ajinomoto Co., Inc. | Methods for producing L-lysine and L-glutamic acid by fermentation |
| JP2002253273A (en) * | 2000-08-08 | 2002-09-10 | Roche Vitamins Ag | Method for producing target fermentation product |
| WO2005007848A1 (en) * | 2003-07-16 | 2005-01-27 | Ajinomoto Co., Inc. | Process for producing l-histidine with the use of bacterium belonging to enterobacteriaceae |
| JPWO2005007848A1 (en) * | 2003-07-16 | 2006-11-16 | 味の素株式会社 | Process for producing L-histidine using bacteria of the family Enterobacteriaceae |
| JP4821321B2 (en) * | 2003-07-16 | 2011-11-24 | 味の素株式会社 | Process for producing L-histidine using bacteria of the family Enterobacteriaceae |
| JP2013520196A (en) * | 2010-02-25 | 2013-06-06 | コルゲート・パーモリブ・カンパニー | Process for producing arginine using Corynebacterium glutamicum ATCC 21831 or Corynebacterium glutamicum ATCC 21493 in fermentation medium containing Cassava bagasse or jackfruit seed as a carbon source |
| US9150892B2 (en) | 2010-02-25 | 2015-10-06 | Colgate-Palmolive Company | Process of producing arginine employing Corynebacterium glutamicum ATCC 21831 or Corynebacterium glutamicum ATCC 21493 in an afermantation medium comprising cassava bagasse or jackfruit seed as a carbon source |
| WO2012147989A1 (en) | 2011-04-25 | 2012-11-01 | Ajinomoto Co.,Inc. | A method for producing an l-amino acid belonging to the glutamate family, using a coryneform bacterium |
| CN102673891A (en) * | 2012-05-04 | 2012-09-19 | 深圳市华星光电技术有限公司 | Buffer stop block structure and corresponding package box body |
| JP2015198662A (en) * | 2015-06-01 | 2015-11-12 | コルゲート・パーモリブ・カンパニーＣｏｌｇａｔｅ−Ｐａｌｍｏｌｉｖｅ Ｃｏｍｐａｎｙ | Process for producing arginine using corynebacterium glutamicum atcc21831 or corynebacterium glutamicum atcc21493 in fermentation medium containing cassava bagasse, or jackfruit seeds as carbon source |
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