JP4537862B2 - Highly efficient production method of organic acid by aerobic bacteria - Google Patents
Highly efficient production method of organic acid by aerobic bacteria Download PDFInfo
- Publication number
- JP4537862B2 JP4537862B2 JP2005010928A JP2005010928A JP4537862B2 JP 4537862 B2 JP4537862 B2 JP 4537862B2 JP 2005010928 A JP2005010928 A JP 2005010928A JP 2005010928 A JP2005010928 A JP 2005010928A JP 4537862 B2 JP4537862 B2 JP 4537862B2
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- Prior art keywords
- organic acid
- reaction
- aerobic
- succinic acid
- production
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 238000004519 manufacturing process Methods 0.000 title claims description 102
- 150000007524 organic acids Chemical class 0.000 title description 93
- 241001148470 aerobic bacillus Species 0.000 title description 9
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 claims description 51
- 239000012429 reaction media Substances 0.000 claims description 43
- 230000012010 growth Effects 0.000 claims description 36
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 24
- 150000007514 bases Chemical class 0.000 claims description 24
- 239000001384 succinic acid Substances 0.000 claims description 23
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 12
- 238000006386 neutralization reaction Methods 0.000 claims description 12
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 11
- 150000003112 potassium compounds Chemical class 0.000 claims description 10
- 239000002994 raw material Substances 0.000 claims description 10
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 claims description 9
- 150000001720 carbohydrates Chemical class 0.000 claims description 8
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 8
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- 238000012258 culturing Methods 0.000 claims description 5
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- 229940093915 gynecological organic acid Drugs 0.000 description 8
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- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 4
- CWERGRDVMFNCDR-UHFFFAOYSA-N thioglycolic acid Chemical compound OC(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-N 0.000 description 4
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- 239000008272 agar Substances 0.000 description 3
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
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Images
Description
本発明は、好気性細菌による乳酸やコハク酸等の有機酸の製造方法に関する。さらに詳しくは、好気性細菌を用いて特定の製造条件、方法による高効率な有機酸の製造方法に関するものである。 The present invention relates to a method for producing an organic acid such as lactic acid or succinic acid by an aerobic bacterium. More specifically, the present invention relates to a highly efficient method for producing an organic acid using aerobic bacteria under specific production conditions and methods.
乳酸やコハク酸、フマール酸等のジカルボン酸およびこれらの誘導体は、高分子合成原料、化粧品用途、医薬原料そして食品添加剤用途など広い分野で使用されている。例えば、コハク酸は生分解性プラスチック原料として、そしてそのエステル誘導体は環境汚染をもたらさないクリーンな洗浄溶剤としてその需要がさらに拡大すると期待されている。 Dicarboxylic acids such as lactic acid, succinic acid, and fumaric acid, and derivatives thereof are used in a wide range of fields such as polymer synthesis raw materials, cosmetics, pharmaceutical raw materials, and food additives. For example, the demand for succinic acid is expected to expand further as a biodegradable plastic raw material, and its ester derivative as a clean cleaning solvent that does not cause environmental pollution.
また、一方において、これら有機酸類は石油化学資源原料からも製造されるが、再生可能資源であるグルコース等糖類を原料とする生物的製造方法は、より環境調和型製造技術であるとの観点からも期待されているものである。 On the other hand, these organic acids are also produced from petrochemical resource raw materials, but the biological production method using saccharides such as glucose, which is a renewable resource, as a raw material is more environmentally friendly production technology. Is also expected.
生物的製造方法による有機酸の製造技術について、従来より、多くの方法が提案されている。例えば、大腸菌を好気的雰囲気下にインキュベートし、該菌体を急速増殖させた後、嫌気的雰囲気下の嫌気的代謝反応による炭素源からのカルボン酸の製造方法において、好気的増殖培地および嫌気的反応培地における炭素源濃度を規定することによるカルボン酸生産物収量を向上させる方法（特許文献１参照）、好気性細菌であるコリネ型細菌による好気的雰囲気下の有機酸製造方法において、好気的反応培地の炭素源（原料糖質）濃度や反応温度を規定する方法（特許文献２および３参照）等も知られている。 Conventionally, many methods have been proposed for organic acid production techniques using biological production methods. For example, in a method for producing carboxylic acid from a carbon source by anaerobic metabolic reaction in an anaerobic atmosphere after incubating E. coli in an aerobic atmosphere and rapidly growing the cells, an aerobic growth medium and In a method for improving the yield of a carboxylic acid product by defining a carbon source concentration in an anaerobic reaction medium (see Patent Document 1), an organic acid production method in an aerobic atmosphere by a coryneform bacterium that is an aerobic bacterium, A method of defining the carbon source (raw material carbohydrate) concentration and reaction temperature of the aerobic reaction medium (see Patent Documents 2 and 3) is also known.
また、生物的製造方法による有機酸の生成に際しては、生成した有機酸や微生物の各種代謝反応由来の酸性物質により反応培地のｐＨが低下するため、塩基性物質による中和処理が通常行なわれている。これは、ｐＨの低下による微生物の生育阻害もしくは死滅を防止し、目的とする有機酸の生産性の低下を防ぐための方法である。 In addition, when organic acids are produced by a biological production method, the pH of the reaction medium is lowered by the produced organic acids and acidic substances derived from various metabolic reactions of microorganisms. Yes. This is a method for preventing microbial growth inhibition or death due to a decrease in pH and preventing a decrease in the productivity of a target organic acid.
有機酸の製造工程において中和処理に使用される中和剤として、アンモニア（水酸化アンモニウム）、炭酸アンモニウム、尿素、アルカリ金属もしくはアルカリ土類金属の水酸化物、炭酸塩そして重炭酸塩やこれらの塩基性化合物の組成物が使用されることは良く知られている。 As neutralizing agents used in the neutralization process in the production process of organic acids, ammonia (ammonium hydroxide), ammonium carbonate, urea, alkali metal or alkaline earth metal hydroxide, carbonate and bicarbonate, and these It is well known that basic compound compositions are used.
中和処理に使用される塩基性化合物を選択する場合は、加えられた塩基性化合物は、一般に目的とする有機酸と反応して有機酸の塩を形成してしまうので、有機酸生成反応工程の下流にあたる有機酸塩を目的の遊離の有機酸に変換する工程（例えば、塩析法、電気透析法、イオン交換樹脂分離法、アンモニウム塩の反応晶析法やエステル化蒸留分離法など）との関連において選択されている（特許文献４参照）。 When selecting a basic compound to be used for the neutralization treatment, the added basic compound generally reacts with the target organic acid to form a salt of the organic acid. A step of converting an organic acid salt downstream of the target to a free organic acid (for example, salting out method, electrodialysis method, ion exchange resin separation method, ammonium salt reaction crystallization method, esterification distillation separation method, etc.) (Refer to Patent Document 4).
有機酸生成反応培地の中和処理の効果は、いずれも、前記した如くｐＨの低下を防止し目的とする有機酸の生産効率を高めようとするものであるが、より一層の生産効率の高い中和処理技術が求められている。
本発明は、好気性細菌を好気条件下で培養増殖し、増殖した菌体による還元条件下の反応培地での有機酸を製造する方法において、該細菌の培養増殖工程および有機酸製造工程で中和処理に用いられる塩基性化合物を特定することにより、有機酸生成反応が従来技術よりも高効率に実施できることを見出した結果、到達したものである。 The present invention relates to a method for producing an organic acid in a reaction medium under a reducing condition by an aerobic bacterium cultured under an aerobic condition, and in the culturing and growing step of the bacterium and the organic acid producing step. As a result of finding out that an organic acid generation reaction can be carried out with higher efficiency than in the prior art by specifying a basic compound used in the neutralization treatment, the present invention has been achieved.
ここで云う「高効率」とは、有機酸生成反応工程における糖類からの有機酸への生成速度が速く、かつ、生成反応培地における到達有機酸濃度が高くなることを意味する。 Here, “high efficiency” means that the production rate of saccharides to organic acids in the organic acid production reaction step is high, and the ultimate organic acid concentration in the production reaction medium is high.
生成速度の向上は反応装置当たりの有機酸生産能力の増大をもたらし、到達有機酸濃度を高くできればその下流の有機酸の分離・生成工程の合理化を図ることが出来るので、経済的にも生産エネルギー消費的にも本発明による実用化実施上の効果は非常に大きい。 Improvement in the production rate leads to an increase in the production capacity of organic acid per reactor, and if the concentration of the reached organic acid can be increased, the downstream organic acid separation and production process can be streamlined. In terms of consumption, the practical effect of the present invention is very large.
有機酸の生物的製造方法においては、使用する微生物の培養増殖工程や有機酸の製造反応工程で培地のｐＨ制御自体は如何なる塩基性化合物による中和処理でも可能である。
本発明は今までその使用が知られている塩基性化合物であっても、培養増殖工程で使用される
塩基性化合物と製造反応工程で使用される塩基性化合物の特定の組み合わせ方法によってのみ、本発明の特有の効果が発現することを見出した（後記実施例および参考例を参照）。この本発明の効果は、培養増殖工程や有機酸製造工程における特定のｐＨ制御（中和）に起因するものであるが、その理由は不明である。
In the method for biological production of organic acids, the pH control of the culture medium itself can be neutralized with any basic compound in the culture growth and growth steps of the microorganisms used and the organic acid production reaction step.
The present invention can be applied only to a specific combination method of a basic compound used in the culture growth step and a basic compound used in the production reaction step, even if the basic compound has been known for use so far. It has been found that the specific effects of the invention are manifested (see Examples and Reference Examples below). The effect of the present invention is due to specific pH control (neutralization) in the culture growth process and the organic acid production process, but the reason is unknown.
有機酸製造に使用する微生物の増殖培養工程で用いる塩基性化合物の種類が、有機酸製造反応の生産性（有機酸生成速度および到達有機酸培地濃度）に影響をもたらすことは、これまで、明らかにされていなかった事実であり、本発明により見出された意外な現象である。 It has been clear that the types of basic compounds used in the growth culture process of microorganisms used for organic acid production have an effect on the productivity of organic acid production reactions (the rate of organic acid production and the concentration of the organic acid medium reached). This is an unexpected phenomenon found by the present invention.
増殖培養工程での中和反応の履歴が、培地の異なるその後の有機酸生成反応工程における微生物の代謝活動にも影響を及ぼすことになるのである。 The history of the neutralization reaction in the growth culture process also affects the metabolic activity of microorganisms in the subsequent organic acid production reaction process in which the medium is different.
すなわち、本発明は、
（１）好気性細菌を好気条件下で培養増殖し、該菌体を用いて還元条件下の反応培地中で有機酸を製造する方法において、培養増殖工程および有機酸の製造工程で培地中の中和反応の為に使用される塩基性化合物がナトリウム化合物および／またはカリウム化合物であることを特徴とする高効率な有機酸の製造方法、
（２）好気性細菌がコリネ型細菌であることを特徴とする（１）に記載の有機酸の製造方法、
（３）好気性コリネ型細菌がコリネバクテリウム属菌またはブレビバクテリウム属菌であることを特徴とする（２）に記載の有機酸の製造方法、
（４）好気性コリネ型細菌が、独立行政法人産業技術総合研究所特許生物寄託センターに受託番号ＦＥＲＭ Ｐ−１９４４６としてまたは受託番号 ＦＥＲＭ Ｐ−１９４４７として寄託されているものであることを特徴とする（２）に記載の有機酸の製造方法、
（５）ナトリウム化合物および／またはカリウム化合物が、ナトリウムまたはカリウムの水酸化物、炭酸化物および重炭酸化物より選ばれることを特徴とする（１）記載の有機酸の製造方法、
（６）還元条件下の反応培地が、炭酸イオン、重炭酸イオンまたは二酸化炭素ガスを含有し、生成する有機酸が、コハク酸、フマール酸およびリンゴ酸から選ばれるジカルボン酸であることを特徴とする（１）記載の有機酸の製造方法、
（７）還元条件下の反応培地の酸化還元電位が−２００ｍＶ〜−５００ｍＶであることを特徴とする（１）に記載の有機酸の製造方法、
（８）培養増殖した好気性細菌を、培養増殖に使用した培養液から分離し、還元条件下で２〜１０時間放置した後、有機酸の製造工程に付することを特徴とする（１）記載の有機酸の製造方法、
である。
That is, the present invention
(1) In a method for culturing and growing aerobic bacteria under aerobic conditions and producing an organic acid in a reaction medium under reducing conditions using the cells, in the medium during the cultivation and growth step and the organic acid production step A highly efficient method for producing an organic acid, characterized in that the basic compound used for the neutralization reaction is a sodium compound and / or a potassium compound,
(2) The method for producing an organic acid according to (1), wherein the aerobic bacterium is a coryneform bacterium,
(3) The method for producing an organic acid according to (2), wherein the aerobic coryneform bacterium is Corynebacterium or Brevibacterium
(4) The aerobic coryneform bacterium is deposited under the accession number FERM P-19446 or the accession number FERM P-19447 at the National Institute of Advanced Industrial Science and Technology (AIST). (2) The manufacturing method of the organic acid as described in
(5) The method for producing an organic acid according to (1), wherein the sodium compound and / or the potassium compound is selected from sodium, potassium hydroxide, carbonate and bicarbonate.
(6) The reaction medium under reducing conditions contains carbonate ion, bicarbonate ion or carbon dioxide gas, and the generated organic acid is a dicarboxylic acid selected from succinic acid, fumaric acid and malic acid. The method for producing an organic acid according to (1),
(7) The method for producing an organic acid according to (1), wherein the oxidation-reduction potential of the reaction medium under reducing conditions is -200 mV to -500 mV,
(8) The aerobic bacteria that have been cultured and proliferated are separated from the culture solution used for the culture and are allowed to stand for 2 to 10 hours under reducing conditions, and then subjected to a process for producing an organic acid (1) A method for producing the organic acid according to the description,
It is.
本発明により、高分子合成原料、化粧品用途、医薬原料そして食品添加剤用途など広い分野で使用され、今後の需要の拡大が期待されている乳酸やコハク酸、フマール酸、リンゴ酸等のジカルボン酸などの高効率な有機酸の生物的製造が可能となるので、経済的にも生産エネルギー消費的にも実用化実施上の効果は非常に大きい。 According to the present invention, dicarboxylic acids such as lactic acid, succinic acid, fumaric acid and malic acid, which are used in a wide range of fields such as polymer synthesis raw materials, cosmetic applications, pharmaceutical raw materials and food additives, and are expected to expand in the future. As a result, it is possible to biologically produce highly efficient organic acids such as those described above, and therefore, the effect of practical application is very great both economically and in terms of production energy consumption.
本発明で使用される好気性細菌は、有機酸の生成能力を有していれば特に限定されないが、コリネバクテリウム（Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ）属、ブレビバクテリウム（Ｂｒｅｖｉｂａｃｔｅｒｉｕｍ）属、アースロバクター（Ａｒｔｈｒｏｂａｃｔｅｒ）属、マイクロコッカス（Ｍｉｃｒｏｃｏｃｃｕｓ）属、バチルス（Ｂａｃｉｌｌｕｓ）属に属する微生物等が挙げられる。 The aerobic bacterium used in the present invention is not particularly limited as long as it has an ability to produce an organic acid. And microorganisms belonging to the genus Micrococcus and the genus Bacillus.
これら好気性細菌のうちでも好ましいのは、コリネ型細菌であり、バージーズ・マニュアル・デターミネイティブ・バクテリオロジー〔（Ｂａｒｇｅｙｓ Ｍａｎｕａｌ ｏｆ Ｄｅｔｅｒｍｉｎａｔｉｖｅ Ｂａｃｔｅｒｉｏｌｏｇｙ， ８， ５９９、１９７４）〕に定義されている、好気性、グラム陽性、非抗酸性、胞子形成能を有さない桿菌微生物である。
具体的には、コリネバクテリウム グルタミカムＲ（Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ ｇｌｕｔａｍｉｃｕｍ Ｒ：独立行政法人産業技術総合研究所特許生物寄託センター 受託番号ＦＥＲＭ Ｐ−１８９７６），ＡＴＣＣ１３０３２、ＡＴＣＣ１３０５８、ＡＴＣＣ１３０５９、等が挙げられる。
Among these aerobic bacteria, coryneform bacteria are preferable, and are defined in the Barges Manual of Detergent Bacteriology [(Bergeys Manual of Determinative Bacteriology, 8, 599, 1974)]. It is a Neisseria gonorrhoeae microorganism that has no Gram-positive, non-acid-fast and sporulation ability.
Specific examples include Corynebacterium glutamicum R (Independent Administrative Institution, National Institute of Advanced Industrial Science and Technology, Patent Biodeposition Center Accession No. FERM P-18976), ATCC 13032, ATCC 13058, ATCC 13059, and the like.
また、これらコリネ型細菌は自然界に存在する野性株の変異株（例えば、受託番号ＦＥＲＭ Ｐ−１８９７７、受託番号 ＦＥＲＭ Ｐ−１８９７８ 特開２００４−８９０２９号公報記載）や人為的な遺伝子組換え体（例えば、受託番号ＦＥＲＭ Ｐ−１９４４６、受託番号 ＦＥＲＭ Ｐ−１９４４７；特開２００３−２８１９３３に記載）であってもよい。 In addition, these coryneform bacteria are wild-type mutant strains existing in nature (for example, accession number FERM P-18777, accession number FERM P-18978 described in JP 2004-89029 A) and artificial gene recombinants ( For example, accession number FERM P-19446, accession number FERM P-19447; described in JP2003-281933) may be used.
独立行政法人産業技術総合研究所特許生物寄託センターに受託番号ＦＥＲＭ Ｐ−１９４４６、受託番号 ＦＥＲＭ Ｐ−１９４４７として寄託されているコリネ型細菌の形質転換体を使用することが好ましい。特定の有機酸（例えば、乳酸、コハク酸、リンゴ酸、クエン酸など）を製造するための形質転換体の創製は、公知の技術に従ってよい。 It is preferable to use a transformant of coryneform bacteria deposited under the accession number FERM P-19446 and the accession number FERM P-19447 at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary. Creation of a transformant for producing a specific organic acid (for example, lactic acid, succinic acid, malic acid, citric acid, etc.) may be performed according to a known technique.
本発明に係わる有機酸の製造方法においては、まず、上記好気性細菌を好気条件下で増殖培養する。
この好気的増殖培養は、炭素源、窒素源、無機塩、ビタミン等の細菌の増殖に必要な栄養源を含む合成または天然培地で通気条件下培養することで好都合に実施できる。
In the method for producing an organic acid according to the present invention, first, the aerobic bacteria are grown and cultured under aerobic conditions.
This aerobic growth culture can be conveniently carried out by culturing under aerated conditions in a synthetic or natural medium containing nutrient sources necessary for the growth of bacteria such as carbon sources, nitrogen sources, inorganic salts, and vitamins.
炭素源として、例えばグルコース、ショ糖、フルクトース等の糖類やエタノール等のアルコール類が、窒素源としては、例えばアンモニア、硫酸アンモニウム、塩化アンモニウム、硝酸アンモニウムまたは尿素等をそれぞれ単独もしくは混合して用いることが出来る。また、無機塩として、例えばリン酸一水素カリウム、リン酸ニ水素カリウム、硫酸マグネシウム、硫酸マンガン等が一般に使用することが出来る。この他にも必要に応じて、ペプトン、肉エキス、酵母エキス、コーンスティープリカー、カザミノ酸またはビオチンもしくはチアミン等の各種ビタミン等の栄養素を培地に適宜添加することも出来る。 Examples of the carbon source include sugars such as glucose, sucrose, and fructose, and alcohols such as ethanol, and examples of the nitrogen source include ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, and urea. . As inorganic salts, for example, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate and the like can be generally used. In addition to these, nutrients such as peptone, meat extract, yeast extract, corn steep liquor, casamino acid or various vitamins such as biotin or thiamine can be appropriately added to the medium as necessary.
培養は、通常、通気攪拌または振盪等の好気的条件下、約２０℃〜約４０℃、好ましくは約２５℃〜約３５℃の温度で行うことが出来る。培養開始時の炭素源濃度は、約１〜２０％（Ｗ／Ｖ）、好ましくは約２〜５％（Ｗ／Ｖ）である。また、培養期間は通常１〜７日間程度である。 Culturing can usually be performed at a temperature of about 20 ° C. to about 40 ° C., preferably about 25 ° C. to about 35 ° C. under aerobic conditions such as aeration stirring or shaking. The carbon source concentration at the start of the culture is about 1 to 20% (W / V), preferably about 2 to 5% (W / V). The culture period is usually about 1 to 7 days.
本培養増殖工程において重要なことは、本増殖培養工程では、菌の増殖に伴い培地中に酢酸等の酸性物質が産生ないし分泌されるので、培地のｐＨを５〜９付近、好ましくは６．５〜８．５に調整するに際して、中和のために使用される塩基性化合物を選択することである。塩基性化合物がナトリウム化合物および／またはカリウム化合物から選ばれることが重要であって、アンモニアやアルカリ土類金属化合物の塩基性化合物を用いてもｐＨ調製自体は可能であるが、本発明の効果は得られない。 What is important in the main culture and growth step is that in the main growth and culture step, acidic substances such as acetic acid are produced or secreted in the medium as the bacteria grow, so the pH of the medium is preferably around 5 to 9, preferably 6. In adjusting to 5 to 8.5, the basic compound used for neutralization is selected. It is important that the basic compound is selected from a sodium compound and / or a potassium compound, and pH adjustment itself is possible even using a basic compound such as ammonia or an alkaline earth metal compound, but the effect of the present invention is I can't get it.
選択されるナトリウム化合物および／またはカリウム化合物は、ナトリウムまたはカリウムの水酸化物、炭酸化合物および重炭酸化合物等から選ばれるものであることが好ましい。これら選択される塩基性化合物の複数種を併用しても良い。また、それらの塩基性化合物の溶液濃度も培地のｐＨが上記範囲で調製される限り、適宜定め使用することが出来る。具体的には、水酸化ナトリウム、水酸化カリウム、炭酸ナトリウム、炭酸カリウム、重炭酸ナトリウムもしくは重炭酸カリウム、またはそれらの混合物が用いられる。 The selected sodium compound and / or potassium compound is preferably selected from sodium or potassium hydroxide, carbonate compound, bicarbonate compound and the like. A plurality of selected basic compounds may be used in combination. Further, the solution concentration of these basic compounds can be determined and used as long as the pH of the medium is adjusted within the above range. Specifically, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate or potassium bicarbonate, or a mixture thereof is used.
ついで、本発明の好気性細菌の培養菌体を回収する。培養物から培養菌体を回収分離する方法としては、特に限定されず、例えば遠心分離や膜分離等の公知の手段を用いることができる。 Subsequently, the cultured cells of the aerobic bacterium of the present invention are collected. The method for recovering and separating cultured cells from the culture is not particularly limited, and for example, known means such as centrifugation and membrane separation can be used.
回収された培養菌体をアクリルアミドまたはカラギーナン等で固定化する等の処理を加え、得られる菌体処理物を次工程に用いることも出来る。 A treatment such as immobilizing the collected cultured microbial cells with acrylamide or carrageenan can be added, and the resulting microbial cell treated product can be used in the next step.
上記の如くして培養増殖工程より得られた菌体は、次の還元条件下の反応培地での有機酸製造工程に供せられる。 The cells obtained from the culture growth step as described above are subjected to the organic acid production step in the reaction medium under the following reducing conditions.
本発明の還元条件下の生化学反応による有機酸製造工程に於いては、本発明の好気性細菌の増殖分裂が抑制され、増殖に伴う分泌副生物の実質的な完全抑制を実現することが出来る。この観点からは、培養増殖工程から回収された菌体またはその菌体処理物が反応培地に供せられるときには、好気性細菌細胞内外の好気培養増殖時の環境状態が反応培地にもたらされない方法や条件を用いることが推奨される。つまり、目的とする有機酸生成反応培地は、培養増殖工程で生成し、菌体内外に存在する物質を実質的に含有しないことが好ましい。より具体的には、培養増殖工程で生成し、菌体外に放出された分泌副生物、および培養菌体内の好気的代謝機能により生成し菌体内に残存する物質が、反応培地に実質的に存在しない状態であることが推奨される。このような状態は、例えば、培養増殖後の培養液を遠心分離、膜分離等により除去すること、および／または培養後培養液から分離された菌体を還元条件下で２時間ないし１０時間程度放置することで実現される。 In the organic acid production process based on the biochemical reaction under the reducing conditions of the present invention, the growth and division of the aerobic bacteria of the present invention is suppressed, and a substantial complete suppression of secreted byproducts accompanying the growth can be realized. I can do it. From this point of view, when the microbial cells recovered from the culture growth process or the processed microbial cells are used in the reaction medium, the environmental state during the aerobic culture growth inside and outside the aerobic bacterial cells is not brought to the reaction medium. It is recommended to use methods and conditions. That is, it is preferable that the target organic acid production reaction medium is substantially free of substances that are produced in the culture growth process and are present inside and outside the cells. More specifically, secreted by-products generated in the culture growth process and released outside the cells and substances generated by the aerobic metabolic function in the cells and remaining in the cells are substantially contained in the reaction medium. It is recommended that it does not exist in In such a state, for example, the culture solution after culture growth is removed by centrifugation, membrane separation or the like, and / or the cells separated from the culture solution after culture are reduced for about 2 to 10 hours under reducing conditions. Realized by leaving it alone.
本工程においては、還元条件下の反応培地を用いる。反応培地は、還元条件下にあれば、固体状、半固体状または液体状等いずれの状態であってもよい。 In this step, a reaction medium under reducing conditions is used. The reaction medium may be in a solid, semi-solid, or liquid state as long as it is under reducing conditions.
本工程における還元条件下とは、反応系の酸化還元電位で規定され、反応培地の酸化還元電位が、好ましくは約−２００ｍＶ〜−５００ｍＶ程度、より好ましくは約−２５０ｍＶ〜−５００ｍＶ程度であることである。 The reducing conditions in this step are defined by the oxidation-reduction potential of the reaction system, and the oxidation-reduction potential of the reaction medium is preferably about -200 mV to -500 mV, more preferably about -250 mV to -500 mV. It is.
本発明の有機酸の生成反応において、生成反応系の酸化還元電位の規定が目的とする有機酸の効率的な生産に関してなぜ有効であるかの理由は明らかではないが、下記にその推定理由を記する。ただし、本発明はその推定理由になんら限定されるものではない。 In the organic acid production reaction of the present invention, the reason why the regulation of the redox potential of the production reaction system is effective for the efficient production of the target organic acid is not clear, but the reason for the estimation is described below. I will write. However, the present invention is not limited to the reason for the estimation.
本発明の目的生産物である有機酸は細菌の代謝機能に基づく生化学反応により産生される化合物である。微生物細胞内の生化学反応には各種の酸化還元反応が関与しており、電子の授受移動が行われている。酸化還元電位は反応系での電子の受容性、供与性の難易度を示す尺度の一つであるが、この電位は微生物細胞内で起こっている代謝経路を構成する各種反応（酸化還元反応）の状態や細胞内外との電子授受の状態を反映している。電位差計により直接測定される酸化還元電位は反応溶液と電極との電位であるが反応溶液の電位は細胞膜を介してある電位勾配を持って細胞内で生じている反応と相関している。即ち、酸化還元電位は細胞内外を含む反応系全体の酸化還元反応の総和を反映（各種反応の内容やその頻度等も含めて）したものである。 The organic acid which is the target product of the present invention is a compound produced by a biochemical reaction based on the metabolic function of bacteria. Various redox reactions are involved in biochemical reactions in microbial cells, and electrons are transferred. The oxidation-reduction potential is one of the scales showing the difficulty of accepting and donating electrons in the reaction system, but this potential is a variety of reactions (oxidation-reduction reactions) that constitute metabolic pathways occurring in microbial cells. And the state of electronic transfer between the inside and outside of the cell. The redox potential directly measured by the potentiometer is the potential between the reaction solution and the electrode, but the potential of the reaction solution correlates with the reaction occurring in the cell with a certain potential gradient through the cell membrane. That is, the oxidation-reduction potential reflects the sum of oxidation-reduction reactions in the entire reaction system including inside and outside the cell (including the contents and frequency of various reactions).
反応系の酸化還元電位に影響する因子としては、反応系雰囲気ガスの種類と濃度、反応温度、反応溶液ｐＨ、反応液中に存在する目的有機化合物生成のために使用される無機および有機の各種化合物濃度と組成等が考えられる。本発明における反応培地の酸化還元電位とは上記各種影響因子が統合されて示されるものである。従って、目的とする有機化合物への代謝経路には各種化学反応が関与し、これら化学反応は上記因子群の影響下にあるが、単一の酸化還元電位なる反応状態を規定する尺度により、効率的に目的有機化合物が生成され得る。 Factors affecting the oxidation-reduction potential of the reaction system include various types and concentrations of the reaction system atmosphere gas, reaction temperature, reaction solution pH, and various inorganic and organic substances used to produce the target organic compounds present in the reaction solution. The compound concentration and composition can be considered. The oxidation-reduction potential of the reaction medium in the present invention is shown by integrating the above various influencing factors. Therefore, various chemical reactions are involved in the metabolic pathway to the target organic compound, and these chemical reactions are under the influence of the above factors, but the efficiency is determined by a scale that defines the reaction state of a single redox potential. In particular, the target organic compound can be produced.
反応培地の還元状態は簡便にはレサズリン指示薬（還元状態であれば、青色から無色への脱色）である程度推定できるが、正確には酸化還元電位差計（例えば、ＢＲＯＡＤＬＥＹ ＪＡＭＥＳ社製、ＯＲＰ Ｅｌｅｃｔｒｏｄｅｓ）を用いて測定する。本発明においては、反応培地に菌体またはその処理物を添加した直後から有機酸を採取するまでの間、還元条件を維持していることが好ましいが、反応時間の約５０％以上、より好ましくは約７０％以上、さらに好ましくは約９０％以上の時間、反応培地が還元条件下に保たれていることが望ましい。 The reduction state of the reaction medium can be easily estimated to some extent with a resazurin indicator (in the reduced state, decolorization from blue to colorless), but accurately, a redox potentiometer (for example, ORA Electrodes made by BROADELY JAMES) is used. Use to measure. In the present invention, it is preferable to maintain the reducing conditions immediately after adding the bacterial cells or the processed product to the reaction medium until collecting the organic acid, but more preferably about 50% or more of the reaction time. It is desirable that the reaction medium be kept under reducing conditions for about 70% or more, more preferably about 90% or more.
還元条件下にある反応培地の調整方法は、公知の方法を用いてよい。例えば、培養培地の液体媒体として、蒸留水などの代わりに反応培地用水溶液を使用してもよく、反応培地用水溶液の調整方法は、例えば硫酸還元微生物などの絶対嫌気性微生物用の培養液調整方法（Ｐｆｅｎｎｉｇ， Ｎ ｅｔ． ａｌ．（１９８１）：Ｔｈｅ ｄｉｓｓｉｍｉｌａｔｏｒｙ ｓｕｌｆａｔｅ−ｒｅｄｕｃｉｎｇ ｂａｃｔｅｒｉａ， Ｉｎ Ｔｈｅ Ｐｒｏｋａｒｙｏｔｅｓ，Ａ Ｈａｎｄｂｏｏｋ ｏｎ Ｈａｂｉｔａｔｓ， Ｉｓｏｌａｔｉｏｎ ａｎｄ Ｉｄｅｎｔｉｆｉｃａｔｉｏｎ ｏｆ Ｂａｃｔｅｒｉａ，Ｅｄ． ｂｙ Ｓｔａｒｒ， Ｍ． Ｐ． ｅｔ． ａｌ． ｐ．９２６−９４０， Ｂｅｒｌｉｎ， Ｓｐｒｉｎｇｅｒ Ｖｅｒｌａｇ．や「農芸化学実験書 第三巻、京都大学農学部 農芸化学教室編、１９９０年第２６刷、産業図書株式会社出版」）などが参考となり、所望する還元条件下の水溶液を得ることが出来る。 A known method may be used for adjusting the reaction medium under reducing conditions. For example, a reaction medium aqueous solution may be used as a liquid medium for the culture medium instead of distilled water. The method for preparing the reaction medium aqueous solution is, for example, preparation of a culture solution for absolute anaerobic microorganisms such as sulfate-reducing microorganisms. Method (Pfenig, N et. Al. (1981): The dissimilarity sulfate-reducing bacterium, In The Prokaryotes, A Handbook on Habitats. 926-940, Berlin, Springer Verlag., “Agricultural Chemistry Experiment Vol.3, Kyoto University Faculty of Agriculture, Department of Agricultural Chemistry, 1990” No. 26, published by Sangyo Tosho Co., Ltd.)) can be used as a reference to obtain an aqueous solution under the desired reducing conditions.
反応培地用水溶液の調整方法として、より具体的には反応培地用水溶液を加熱処理や減圧処理することにより溶解ガスを除去する方法等が挙げられる。より具体的には、約１０ｍｍＨｇ以下、好ましくは約５ｍｍＨｇ以下、より好ましくは約３ｍｍＨｇ以下の減圧下で、約１〜６０分程度、好ましくは５〜４０分程度、反応培地用水溶液を処理することにより、溶解ガス、特に溶解酸素を除去して還元条件下の反応培地用水溶液を作成することができる。 More specifically, the method for adjusting the aqueous solution for reaction medium includes a method for removing dissolved gas by subjecting the aqueous solution for reaction medium to heat treatment or reduced pressure treatment. More specifically, the reaction medium aqueous solution is treated for about 1 to 60 minutes, preferably about 5 to 40 minutes under reduced pressure of about 10 mmHg or less, preferably about 5 mmHg or less, more preferably about 3 mmHg or less. Thus, the dissolved gas, particularly dissolved oxygen can be removed to prepare an aqueous solution for reaction medium under reducing conditions.
また、適当な還元剤（例えば、チオグリコール酸、アスコルビン酸、システィン塩酸塩、メルカプト酢酸、チオール酢酸、グルタチオンそして硫化ソーダ等）を添加して還元条件下の反応培地用水溶液を調整することも出来る。また、場合により、これらの方法を適宜組み合わせることも有効な還元条件下の反応培地用水溶液を調整する方法となる。 In addition, an appropriate reducing agent (for example, thioglycolic acid, ascorbic acid, cysteine hydrochloride, mercaptoacetic acid, thiolacetic acid, glutathione, sodium sulfide, etc.) can be added to prepare an aqueous solution for reaction medium under reducing conditions. . In some cases, combining these methods as appropriate also becomes a method for preparing an aqueous solution for reaction medium under effective reducing conditions.
反応途中における還元条件の維持方法としては、反応系外からの酸素の混入を可能な限り防止することが望ましく、反応系を窒素ガス等の不活性ガスや炭酸ガス等で封入する方法が通常用いられる。酸素混入をより効果的に防止する方法としては、反応途中において本発明の好気性細菌の菌体内の代謝機能を効率よく機能させるために、反応系のｐＨ維持調整液の添加や各種栄養素溶解液を適宜添加する必要が生じる場合もあるが、このような場合には添加溶液から酸素を予め除去しておくことが有効である。 As a method for maintaining the reduction conditions during the reaction, it is desirable to prevent oxygen contamination from outside the reaction system as much as possible, and a method in which the reaction system is sealed with an inert gas such as nitrogen gas or carbon dioxide gas is usually used. It is done. As a method for more effectively preventing oxygen contamination, in order to efficiently function the metabolic function of the aerobic bacteria of the present invention during the reaction, addition of a pH maintenance adjustment solution of the reaction system and various nutrient solution In such a case, it is effective to remove oxygen from the added solution in advance.
反応系の酸化還元電位に影響する因子としては、反応系雰囲気ガスの種類と濃度、反応温度、反応溶液ｐＨ、目的とする有機酸生成のために使用される無機および有機の各種化合物濃度と組成等が考えられる。本発明における反応培地の酸化還元電位とは上記各種影響因子が統合されて示されるものである。 Factors affecting the oxidation-reduction potential of the reaction system include the type and concentration of the reaction system atmosphere gas, the reaction temperature, the reaction solution pH, and the concentration and composition of various inorganic and organic compounds used to produce the desired organic acid. Etc. are considered. The oxidation-reduction potential of the reaction medium in the present invention is shown by integrating the above various influencing factors.
本有機酸製造工程においても重要なことは、本有機酸製造工程では生成する有機酸等の酸性物質により反応培地のｐＨが低下するので、反応培地のｐＨを５〜９付近、好ましくは６．５〜８．５に調整するが、その際に中和のために使用される塩基性化合物を選択することである。好気条件下の培養増殖工程の場合と同じく、塩基性化合物がナトリウム化合物および／またはカリウム化合物から選ばれることが重要であって、アンモニアやアルカリ土類金属化合物の塩基性化合物を用いてもｐＨ調製自体は可能であるが、本発明の効果は得られない。このことは、培養増殖工程時と有機酸製造工程時に同じ塩基性化合物を用いて、ｐＨ調製を行なえば本発明の効果が得られることを意味するものではない。 What is important in this organic acid production process is that the pH of the reaction medium is lowered by an acidic substance such as an organic acid produced in this organic acid production process, so the pH of the reaction medium is around 5 to 9, preferably 6. In this case, the basic compound used for neutralization is selected. It is important that the basic compound is selected from a sodium compound and / or a potassium compound as in the case of the culture growth step under an aerobic condition. Even if a basic compound such as ammonia or an alkaline earth metal compound is used, the pH is Although the preparation itself is possible, the effect of the present invention cannot be obtained. This does not mean that the effects of the present invention can be obtained by adjusting the pH using the same basic compound during the culture growth step and the organic acid production step.
後記の参考例でもって明らかにされている如く、培養増殖工程時と有機酸製造工程時のｐＨ調整剤として、例えば、アンモニアを用いても本発明の効果は得られない。そして、培養増殖工程時のｐＨ調整剤にアンモニアが用いられた場合には、有機酸製造工程時のｐＨ調整剤にナトリウム化合物および／またはカリウム化合物から選ばれる塩基性化合物、すなわち、水酸化ナトリウム、水酸化カリウム、炭酸ナトリウム、炭酸カリウム、重炭酸ナトリウムまたは重炭酸カリウムを用いても、やはり、本発明の効果は得られないのである。 As will be clarified in the reference examples described later, even if ammonia is used as a pH adjuster during the culture growth process and the organic acid production process, the effect of the present invention cannot be obtained. When ammonia is used as the pH adjuster during the culture growth step, the basic compound selected from sodium compounds and / or potassium compounds as the pH adjuster during the organic acid production step, that is, sodium hydroxide, Even if potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate or potassium bicarbonate is used, the effect of the present invention cannot be obtained.
選択されるナトリウム化合物および／またはカリウム化合物は、ナトリウムまたはカリウムの水酸化物、炭酸化合物および重炭酸化合物等から選ばれる。これら選択される塩基性化合物の複数種を併用しても良い。また、それらの塩基性化合物の溶液濃度も有機酸製造反応培地のｐＨが上記範囲で調製される限り、適宜定め使用することが出来る。 The selected sodium compound and / or potassium compound is selected from sodium or potassium hydroxide, carbonate compound and bicarbonate compound. A plurality of selected basic compounds may be used in combination. Further, the solution concentration of these basic compounds can be determined and used as long as the pH of the organic acid production reaction medium is adjusted within the above range.
反応培地には、有機酸生成の原料となる糖類および目的とする有機酸がＴＣＡ代謝回路に介在するコハク酸等ジカルボン酸の場合には、二酸化炭素ガスや炭酸イオン又は重炭酸イオンが含まれていることが好ましい。炭酸イオン又は重炭酸イオンは、炭酸ナトリウム、炭酸カリウム、重炭酸ナトリウム、重炭酸カリウムとして、反応系に添加されてよい。 The reaction medium contains carbon dioxide gas, carbonate ion or bicarbonate ion in the case of dicarboxylic acid such as succinic acid in which the saccharide used as a raw material for organic acid generation and the target organic acid intervene in the TCA metabolic circuit. Preferably it is. Carbonate ions or bicarbonate ions may be added to the reaction system as sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate.
糖類としては、グルコース、ガラクトース、フルクトースもしくはマンノースなどの単糖類、セロビオース、ショ糖もしくはラクトース、マルトースなどの二糖類、またはデキストリンもしくは可溶性澱粉などの多糖類などが挙げられる。なかでも、グルコースが好ましい。この場合、グルコースは０．５〜５００ｇ／Ｌ（リットル）の濃度範囲で使用される。 Examples of the saccharide include monosaccharides such as glucose, galactose, fructose or mannose, disaccharides such as cellobiose, sucrose or lactose and maltose, and polysaccharides such as dextrin and soluble starch. Of these, glucose is preferable. In this case, glucose is used in a concentration range of 0.5 to 500 g / L (liter).
炭酸イオン又は重炭酸イオンは、炭酸又は重炭酸もしくはこれらの塩あるいは炭酸ガスから供給されるものである。これら炭酸イオン又は重炭酸イオンは１〜５００ｍＭ、好ましくは２〜３００ｍＭの濃度範囲で使用される。炭酸ガスが供給される場合は、５０ｍｇ／Ｌ〜２５ｇ／Ｌ、好ましくは、１００ｍｇ／Ｌ〜１５ｇ／Ｌの濃度で溶液中に含有するように供給される。 Carbonate ion or bicarbonate ion is supplied from carbonic acid, bicarbonate, a salt thereof, or carbon dioxide gas. These carbonate ions or bicarbonate ions are used in a concentration range of 1 to 500 mM, preferably 2 to 300 mM. When carbon dioxide gas is supplied, it is supplied so as to be contained in the solution at a concentration of 50 mg / L to 25 g / L, preferably 100 mg / L to 15 g / L.
有機酸の生成反応に用いられる反応培地組成は、好気性細菌またはその処理物がその代謝機能を維持するために必要な成分、即ち、各種糖類等の炭素源と炭酸源の他にも、蛋白質合成に必要な窒素源、その他リン、カリウムまたはナトリウム等の無機塩類、さらに鉄、マンガンまたはカルシウム等の微量金属塩を含む。これらの添加量は所要反応時間、有機酸生成物の種類または用いられる好気性細菌の種類等により適宜定めることが出来る。用いる好気性細菌によっては特定のビタミン類の添加が好ましい場合もある。炭素源、窒素源、無機塩類、ビタミン、微量金属塩は、公知のもの、例えば増殖培養工程につき例示したものでよい。好気性細菌またはその菌体処理物と糖類との反応は、本発明の好気性細菌またはその菌体処理物が活動できる温度条件下で行われることが好ましく、好気性コリネ型細菌またはその菌体処理物の種類などにより適宜選択することができる。通常、約２５℃〜３５℃である。有機酸の生成反応は回分式、連続式いずれの生成方式も可能である。 The composition of the reaction medium used for the organic acid production reaction is a component necessary for the aerobic bacterium or its treated product to maintain its metabolic function, that is, in addition to carbon sources such as various sugars and carbonic acid sources, protein Nitrogen sources necessary for synthesis, other inorganic salts such as phosphorus, potassium or sodium, and trace metal salts such as iron, manganese or calcium. These addition amounts can be appropriately determined depending on the required reaction time, the type of organic acid product, the type of aerobic bacteria used, and the like. Depending on the aerobic bacterium used, the addition of specific vitamins may be preferred. Carbon sources, nitrogen sources, inorganic salts, vitamins, and trace metal salts may be known ones, for example, those exemplified for the growth culture step. The reaction between the aerobic bacterium or its cell-treated product and the saccharide is preferably performed under temperature conditions where the aerobic bacterium of the present invention or its cell-treated product can act, and the aerobic coryneform bacterium or its cell body. It can be appropriately selected depending on the type of processed material. Usually, it is about 25 degreeC-35 degreeC. The organic acid can be produced either batchwise or continuously.
上述のようにして反応培地で生成した有機酸塩類を分離・精製して有機酸を採取する。その方法はバイオプロセスで用いられる公知の方法を用いることが出来る。そのような公知の方法として、有機酸生成液の塩析法、再結晶法、有機溶媒抽出法、エステル化蒸留分離法、クロマトグラフィー分離法または電気透析法等があり、有機酸の特性に応じてその分離・精製採取法は適宜定めることが出来る。 An organic acid is collected by separating and purifying the organic acid salt produced in the reaction medium as described above. As the method, a known method used in a bioprocess can be used. Examples of such known methods include salting out organic acid production solutions, recrystallization methods, organic solvent extraction methods, esterification distillation separation methods, chromatographic separation methods or electrodialysis methods, depending on the characteristics of the organic acid. The separation / purification collection method can be determined as appropriate.
〔実施例〕
以下、実施例でもって本発明を説明するが、本発明はこれら実施例に限定されるものではない。
〔Example〕
EXAMPLES Hereinafter, although this invention is demonstrated with an Example, this invention is not limited to these Examples.
ａ）好気培養増殖
冷凍庫にて−８０℃で保存してあるコリネ型細菌株（独立行政法人産業技術総合研究所特許生物寄託センターに受託番号 ＦＥＲＭ Ｐ−１９４４７として寄託されているコリネ型細菌と同一株）を、プレート培養用培地であるＡ寒天培地（組成：尿素２ｇ、酵母エキス２ｇ、カザミノ酸７ｇ、硫安７ｇ、第一リン酸カリウム（ＫＨ２ＰＯ４）０．５ｇ、第二リン酸カリウム（Ｋ２ＨＰＯ４）５ｇ、硫酸マグネシウム・７水和物０．５ｇ、硫酸鉄・７水和物６ｍｇ、硫酸マンガン・１水和物４．２ｍｇ、ビオチン０．２ｍｇ、チアミン０．２ｍｇ、グルコース２０ｇ、寒天１．５％（Ｗ／Ｖ）そして蒸留水１Ｌ）に塗布し、３３℃、１２ｈｒ暗所に静置した。
a) Aerobic culture growth Coryneform bacterium strain preserved in a freezer at −80 ° C. (coryneform bacterium deposited under the accession number FERM P-19447 at the National Institute of Advanced Industrial Science and Technology (AIST)) Agar agar plate (composition: urea 2 g, yeast extract 2 g, casamino acid 7 g, ammonium sulfate 7 g, monobasic potassium phosphate (KH 2 PO 4 ) 0.5 g, diphosphate) 5 g of potassium (K 2 HPO 4 ), 0.5 g of magnesium sulfate heptahydrate, 6 mg of iron sulfate heptahydrate, 4.2 mg of manganese sulfate monohydrate, 0.2 mg of biotin, 0.2 mg of thiamine, Glucose (20 g), agar (1.5% (W / V), and distilled water (1 L)) were applied, and the mixture was allowed to stand in a dark place at 33 ° C. for 12 hours.
上記のプレート上で生育したコリネ型細菌株を、試験管培養用培地であるＡ培地（組成：寒天が含まれていないことを除けば上記Ａ寒天培地と同一成分組成）１０ｍｌに白金耳でもって植菌し、３３℃、１２ｈｒ、２００ｒｐｍで振盪培養した。 The coryneform bacterial strain grown on the above plate is placed in 10 ml of A medium (composition: the same composition as the above-mentioned A agar medium except that it does not contain agar) with platinum ears. Inoculated and cultured with shaking at 33 ° C., 12 hr, 200 rpm.
このようにして生育したコリネ型細菌株を、好気培養増殖用培地であるＡ−Ｕ培地（組成：尿素が含まれていないことを除けば上記Ａ培地と同一成分組成）の５００ｍｌが入っている容量１Ｌのジャーファーメンターに移し、３３℃、１０００ｒｐｍ、滅菌空気を１ｖｖｍで通気して、１３ｈｒ好気培養増殖を行なった。 The coryneform bacterial strain thus grown contains 500 ml of an AU medium (composition: the same composition as that of the A medium except that it does not contain urea), which is an aerobic culture growth medium. It was transferred to a 1 L jar fermenter, and aerobic culture growth was carried out for 13 hours by aeration at 33 ° C., 1000 rpm, and sterilized air at 1 vvm.
この間、５Ｎ（規定）濃度のＮａＯＨ（水酸化ナトリウム）水溶液を使用してジャーファーメンター槽内のｐＨを７．５に常時維持した。 During this time, the pH in the jar fermenter tank was constantly maintained at 7.5 using a 5N (normal) concentration NaOH (sodium hydroxide) aqueous solution.
このようにして培養増殖された菌体は、遠心分離（４℃、１０分、５０００ｘＧ）によって回収し、次の還元条件下の有機酸製造反応に供した。 The cells grown in this manner were collected by centrifugation (4 ° C., 10 minutes, 5000 × G) and subjected to the organic acid production reaction under the following reducing conditions.
ｂ）還元条件下の有機酸（コハク酸）製造反応
ａ）の好気培養増殖工程より回収された湿潤菌体１５０ｇ（Ｄｒｙ Ｃｅｌｌ換算約３０ｇ）を、コハク酸生成反応用溶液であるＢＴ−Ｕ培地（組成：硫安７ｇ、第一リン酸カリウム（ＫＨ２ＰＯ４）０．５ｇ、第二リン酸カリウム（Ｋ２ＨＰＯ４）０．５ｇ、硫酸マグネシウム・７水和物０．５ｇ、硫酸鉄・７水和物６ｍｇ、硫酸マンガン・１水和物４．２ｍｇ、ビオチン０．２ｍｇ、チアミン０．２ｍｇ、グルコース３６ｇ、重炭酸ソーダ（ＮａＨＣＯ３）１２．６ｇ、蒸留水１Ｌ）５００ｍｌの入っている容量１Ｌの反応槽に加え、３３℃にて緩やかに攪拌し、還元条件下のコハク酸生成反応を４８時間実施した。反応時の培地の酸化還元電位は、反応開始後直ちに急激に低下し、その後が約−４００ｍＶに維持してコハク酸生成反応が継続された。また、反応培地のグルコース濃度は、グルコースアナライザー（王子計測社製 ＢＦ−４）により測定し、グルコースが消費消失される都度、グルコースおよび炭酸イオン源としての重炭酸ソーダを追添加して反応を継続した。
b) Organic acid (succinic acid) production reaction under reducing conditions 150 g of wet cells recovered from the aerobic culture growth step of a) (about 30 g in terms of Dry Cell) were used as a solution for succinic acid production reaction. Medium (composition: ammonium sulfate 7 g, primary potassium phosphate (KH 2 PO 4 ) 0.5 g, dibasic potassium phosphate (K 2 HPO 4 ) 0.5 g, magnesium sulfate heptahydrate 0.5 g, iron sulfate・ Capacity containing 500 mg of heptahydrate 6 mg, manganese sulfate monohydrate 4.2 mg, biotin 0.2 mg, thiamine 0.2 mg, glucose 36 g, sodium bicarbonate (NaHCO 3 ) 12.6 g, distilled water 1 L) In addition to a 1 L reaction vessel, the mixture was gently stirred at 33 ° C., and a succinic acid production reaction under reducing conditions was performed for 48 hours. The oxidation-reduction potential of the medium during the reaction decreased rapidly immediately after the start of the reaction, and thereafter maintained at about −400 mV, and the succinic acid production reaction was continued. The glucose concentration in the reaction medium was measured with a glucose analyzer (BF-4 manufactured by Oji Scientific Co., Ltd.) and the reaction was continued by adding glucose and sodium bicarbonate as a carbonate ion source each time glucose was consumed and lost.
この間、５Ｎ（規定）濃度のＮａＯＨ（水酸化ナトリウム）水溶液を使用して反応槽内のｐＨを７．５に常時維持した。 During this time, the pH in the reaction vessel was constantly maintained at 7.5 using a 5N (normal) concentration NaOH (sodium hydroxide) aqueous solution.
反応槽内の生成したコハク酸量は逐次少量をサンプリングして液体クロマトグラフィーによる分析を行い、経時的な生成量の変動を調べた。 The amount of succinic acid produced in the reaction vessel was successively sampled and analyzed by liquid chromatography, and the change in production amount over time was examined.
その結果を表１に記す。（表１における生成速度とは反応開始後１時間から２時間程度の範囲における初期最大速度であり、到達最終濃度とは反応開始後４８時間後の反応槽内におけるコハク酸濃度である。） The results are shown in Table 1. (The production rate in Table 1 is the initial maximum rate in the range of about 1 to 2 hours after the start of the reaction, and the final concentration reached is the succinic acid concentration in the reaction vessel 48 hours after the start of the reaction.)
実施例１におけるａ）好気培養増殖工程およびｂ）還元条件下の有機酸製造反応工程で使用する中和剤として、それぞれＮＨ４ＯＨ（５Ｎアンモニア水溶液）を用いて培地のｐＨを７．５に維持する以外は、実施例１と同様の条件にて好気培養増殖および有機酸製造反応を行なった。表１に有機酸製造反応における生成速度と到達最終濃度を記す。 As a neutralizing agent used in the a) aerobic culture growth step and b) the organic acid production reaction step under reducing conditions in Example 1, NH 4 OH (5N ammonia aqueous solution) was used to adjust the pH of the medium to 7.5. The aerobic culture growth and the organic acid production reaction were carried out under the same conditions as in Example 1 except that the above conditions were maintained. Table 1 shows the production rate and the final concentration reached in the organic acid production reaction.
実施例１におけるａ）好気培養増殖工程における中和剤としてＮＨ４ＯＨ（５Ｎアンモニア水溶液）を用い、ｂ）有機酸製造反応工程における中和剤として５Ｎ（規定）濃度のＮａＯＨ（水酸化ナトリウム）溶液を用いて、それぞれの培地のｐＨを７．５に維持する以外は、実施例１と同様の条件にて好気培養増殖および有機酸製造反応を行なった。表１に有機酸製造反応における生成速度と到達最終濃度を記す。 In Example 1, a) NH 4 OH (5N ammonia aqueous solution) is used as a neutralizing agent in the aerobic culture growth step, and b) NaOH (sodium hydroxide) having a 5N (normal) concentration as a neutralizing agent in the organic acid production reaction step. ) Aerobic culture growth and organic acid production reaction were performed under the same conditions as in Example 1 except that the pH of each medium was maintained at 7.5 using the solution. Table 1 shows the production rate and the final concentration reached in the organic acid production reaction.
実施例１におけるａ）好気培養増殖工程における中和剤として、５Ｎ（規定）濃度のＮａＯＨ（水酸化ナトリウム）溶液を用い、ｂ）有機酸製造反応工程における中和剤としてＮＨ４ＯＨ（５Ｎアンモニア水溶液）を用いて、それぞれの培地のｐＨを７．５に維持する以外は、実施例１と同様の条件にて好気培養増殖および有機酸製造反応を行なった。表１に有機酸製造反応における生成速度と到達最終濃度を記す。 In Example 1 a) NaOH (sodium hydroxide) solution of 5N (normal) concentration is used as a neutralizing agent in the aerobic culture growth step, and b) NH 4 OH (5N) is used as the neutralizing agent in the organic acid production reaction step. The aerobic culture growth and the organic acid production reaction were performed under the same conditions as in Example 1 except that the pH of each medium was maintained at 7.5 using an aqueous ammonia solution. Table 1 shows the production rate and the final concentration reached in the organic acid production reaction.
実施例１におけるコハク酸製造初期反応培地のグルコース濃度を１．８ｗｔ％とする以外は全て実施例１と同様の条件、方法にて好気培養増殖およびコハク酸製造反応を行なった。表２にコハク酸製造反応における生成速度と到達最終濃度を記す。 The aerobic culture growth and succinic acid production reaction were carried out under the same conditions and method as in Example 1 except that the glucose concentration of the succinic acid production initial reaction medium in Example 1 was 1.8 wt%. Table 2 shows the production rate and the final concentration reached in the succinic acid production reaction.
実施例１におけるａ）好気培養増殖工程およびｂ）還元条件下のコハク酸製造反応工程で使用する中和剤として、それぞれ５Ｎ（規定）濃度のＫＯＨ（水酸化カリウム）溶液を用いて培地のｐＨを７．５に維持すること、およびコハク酸製造初期反応培地のグルコース濃度を７．２ｗｔ％とする以外は、実施例１と同様の条件にて好気培養増殖およびコハク酸製造反応を行なった。 表２にコハク酸製造反応における生成速度と到達最終濃度を記す。 As a neutralizing agent used in the a) aerobic culture growth step and b) the succinic acid production reaction step under reducing conditions in Example 1, a 5N (normal) concentration KOH (potassium hydroxide) solution was used, respectively. The aerobic culture growth and succinic acid production reaction were carried out under the same conditions as in Example 1 except that the pH was maintained at 7.5 and the glucose concentration of the succinic acid production initial reaction medium was 7.2 wt%. It was. Table 2 shows the production rate and the final concentration reached in the succinic acid production reaction.
本発明は、有機酸の生物的製造方法に関して、製造工程における反応条件および製造工程で中和処理に用いられる塩基性化合物を特定することにより、有機酸生成反応が従来技術よりも高効率に実施できる。ここで云う「高効率」とは、有機酸生成反応工程における糖類から有機酸への生成速度が速く、かつ、生成反応培地における到達有機酸濃度が高くなることを意味する。生成速度の向上は反応装置当たりの生産能力の増大をもたらし、到達有機酸濃度を高め、
その下流の有機酸の分離・生成工程の合理化を図ることが出来るので、経済的にも生産エネルギー消費的にも実用化実施上の効果は非常に大きい。
The present invention relates to a biological production method of an organic acid, and the reaction conditions in the production process and the basic compound used for the neutralization treatment in the production process are specified, so that the organic acid generation reaction is performed more efficiently than in the prior art. it can. The term “high efficiency” as used herein means that the production rate from saccharides to organic acids in the organic acid production reaction step is high, and the ultimate organic acid concentration in the production reaction medium is high. Increased production rate results in increased production capacity per reactor, increasing the organic acid concentration reached,
Since the downstream organic acid separation / generation process can be rationalized, the effect of practical application is very great both economically and in terms of production energy consumption.
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| JP2001514900A (en) * | 1997-08-18 | 2001-09-18 | アプライド カーボケミカルズ | Method for producing and purifying succinic acid |
| JP2003235593A (en) * | 2002-02-13 | 2003-08-26 | Mitsubishi Chemicals Corp | Method for producing organic acid |
| JP2003235592A (en) * | 2002-02-13 | 2003-08-26 | Mitsubishi Chemicals Corp | Method for producing organic acid |
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| JPH07115982A (en) * | 1993-10-22 | 1995-05-09 | Japan Energy Corp | Aerobic fermentation of hardly water-soluble substrate and production of fermentation product of hardly water-soluble substrate |
| JPH1033192A (en) * | 1996-07-24 | 1998-02-10 | Mitsubishi Chem Corp | Production of fumaric acid |
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