JP2009284861A - Method for producing (s)-2-methylbutyric acid - Google Patents
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本発明は、香料、あるいは香料、医薬品などの製造原料として有用な（Ｓ）−２−メチル酪酸を高純度且つ高収率で製造する方法に関する。 The present invention relates to a method for producing (S) -2-methylbutyric acid, which is useful as a raw material for producing fragrances, fragrances, pharmaceuticals, and the like with high purity and high yield.
２−メチル酪酸は、天然には、アンゲリカ根油、ストロベリーなどの果実、アルコール飲料などに広く存在が知られる化合物である。２−メチル酪酸には（Ｓ）体および（Ｒ）体の２つの光学異性体があり、（Ｓ）体は甘いフルーツ香気を有し、そして（Ｒ）体は汗、チーズ様の香気を有している（非特許文献１）。市販品の多くはラセミ体の合成品として販売されており、果実、ナッツ、ココア、発酵乳などの調合香料の原料として用いられている（特許文献１〜５）。 Naturally, 2-methylbutyric acid is a compound widely known to exist in fruits such as angelica root oil and strawberry, alcoholic beverages and the like. 2-Methylbutyric acid has two optical isomers, (S) and (R), (S) has a sweet fruit aroma, and (R) has a sweaty, cheese-like aroma. (Non-Patent Document 1). Most of the commercially available products are sold as racemic synthetic products, and are used as raw materials for blended fragrances such as fruits, nuts, cocoa and fermented milk (Patent Documents 1 to 5).
一方、納豆菌であるバシラス サチリス（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）が納豆中の納豆臭の原因となる短鎖分岐脂肪酸である２−メチル酪酸、イソ酪酸、イソ吉草酸を生産することは知られている（非特許文献１）が、２−メチル酪酸、イソ酪酸およびイソ吉草酸の生合成経路はいまだに解明されていない。 On the other hand, it is known that Bacillus subtilis, which is a natto bacterium, produces 2-methylbutyric acid, isobutyric acid, and isovaleric acid, which are short-chain branched fatty acids that cause natto odor in natto. In Patent Document 1), the biosynthetic pathways of 2-methylbutyric acid, isobutyric acid and isovaleric acid have not yet been elucidated.
これまでこれらの化合物の光学異性体の製造に関する報告は意外に少なく、例えば、特許文献６にラセミ体の２−メチル酪酸または２−メチルペンタン酸に、（Ｓ）−２−メチル酪酸および（Ｓ）−２−メチルペンタン酸資化能を有する微生物、特にシュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）属に属する微生物を作用させて（Ｒ）−２−メチル酪酸または（Ｒ）−２−メチルペンタン酸を製造する方法が開示され、また、特許文献７に、チグリン酸をメタノール、エタノールのような溶媒に溶解し、ルテニウム−光学活性ホスフィンを加え、水素圧４〜１２５ｋｇ／ｃｍ２および反応温度５〜５０℃で１〜１００時間反応させることにより光学純度７０％ｅ．ｅ．以上の（Ｓ）−２−メチル酪酸を大量に得る製造法が開示されている程度である。 To date, there are surprisingly few reports on the production of optical isomers of these compounds. For example, Patent Document 6 discloses racemic 2-methylbutyric acid or 2-methylpentanoic acid to (S) -2-methylbutyric acid and (S And (R) -2-methylbutyric acid or (R) -2-methylpentanoic acid by producing a microorganism having the ability to assimilate 2-methylpentanoic acid, particularly a microorganism belonging to the genus Pseudomonas. Patent Document 7 discloses that tiglic acid is dissolved in a solvent such as methanol and ethanol, ruthenium-optically active phosphine is added, hydrogen pressure is 4 to 125 kg / cm2, and reaction temperature is 5 to 50 ° C. Optical reaction of 70% e. e. The production method for obtaining a large amount of the above (S) -2-methylbutyric acid is disclosed.
このように、従来の提案はいずれも合成品または半合成品の原料を用いた（Ｓ）−２−メチル酪酸または（Ｒ）−２−メチル酪酸の製造に関するものであり、天然の原料を用い、天然の方法、すなわち、植物体、微生物または酵素の作用により得られるナチュラルの（Ｓ）−２−メチル酪酸を高純度且つ高収率で製造する方法についての報告は見あたらない。
本発明の目的は、天然の原料を用い、ナチュラルな（Ｓ）−２−メチル酪酸を高純度且つ高収率で製造する方法を提供することである。 An object of the present invention is to provide a method for producing natural (S) -2-methylbutyric acid with high purity and high yield using natural raw materials.
本発明者らは上記の目的を達成すべく鋭意検討した結果、今回、バシラス（Ｂａｃｉｌｌｕｓ）属に属する（Ｓ）−２−メチル酪酸生産菌をＬ−イソロイシンを含む培地で培養することにより高純度且つ高収率で（Ｓ）−２−メチル酪酸が得られることを見出し、本発明の完成に至った。 As a result of intensive studies to achieve the above object, the present inventors have now obtained a high purity by culturing a (S) -2-methylbutyric acid-producing bacterium belonging to the genus Bacillus in a medium containing L-isoleucine. And it discovered that (S) -2-methylbutyric acid was obtained with a high yield, and came to completion of this invention.
かくして、本発明は、バシラス（Ｂａｃｉｌｌｕｓ）属に属する（Ｓ）−２−メチル酪酸生産菌をＬ−イソロイシンを含む培地で培養することを特徴とする（Ｓ）−２−メチル酪酸の製造方法を提供するものである。 Thus, the present invention provides a method for producing (S) -2-methylbutyric acid, characterized in that (S) -2-methylbutyric acid-producing bacteria belonging to the genus Bacillus are cultured in a medium containing L-isoleucine. It is to provide.
本発明によれば、バシラス（Ｂａｃｉｌｌｕｓ）属に属する（Ｓ）−２−メチル酪酸生産菌をＬ−イソロイシンを含む培地で培養することにより、ナチュラルの（Ｓ）−２−メチル酪酸を高純度且つ高収率で得ることができる。また、得られる（Ｓ）−２−メチル酪酸はラセミ体または（Ｒ）体の２−メチル酪酸に比べて、甘いフルーツ香気を有し、調合香料の原料として使用することができるのみならず、香料、医薬品などの原料として広い分野での利用が見込まれる。 According to the present invention, (S) -2-methylbutyric acid-producing bacteria belonging to the genus Bacillus are cultured in a medium containing L-isoleucine, whereby natural (S) -2-methylbutyric acid is highly purified and It can be obtained in high yield. In addition, (S) -2-methylbutyric acid obtained has a sweet fruit aroma as compared to racemic or (R) 2-methylbutyric acid, and can be used as a raw material for blended fragrances, It is expected to be used in a wide range of fields as a raw material for fragrances and pharmaceuticals.
本発明で使用することができるバシラス（Ｂａｃｉｌｌｕｓ）属に属する（Ｓ）−２−メチル酪酸生産菌としては、例えば、バシラス パミラス（Ｂａｃｉｌｌｕｓ ｐｕｍｉｌｕｓ）、バシラス メガテリウム（Ｂａｃｉｌｌｕｓ ｍｅｇａｔｅｒｉｕｍ）、バシラス サチリス（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）、バシラス サーキュランス（Ｂａｃｉｌｌｕｓ ｃｉｒｃｕｌａｎｓ）、バシラス シンプレックス（Ｂａｃｉｌｌｕｓ ｓｉｍｐｌｅｘ）、バシラス アゾトフォルマンス（Ｂａｃｉｌｌｕｓ ａｚｏｔｏｆｏｒｍａｎｓ）、バシラス サイクロサッカロリティカス（Ｂａｃｉｌｌｕｓ ｐｓｙｃｈｒｏｓａｃｃｈａｒｏｌｙｔｉｃｕｓ）などを挙げることができ、市場で入手できる菌株としては、例えば、Ｂａｃｉｌｌｕｓ ｐｕｍｉｌｕｓ ＮＢＲＣ１４３６７（独立行政法人 製品評価技術基盤機構による分譲菌株）、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ＪＣＭ２４９９、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ＪＣＭ２０１１８、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ＩＡＭ１２３１、Ｂａｃｉｌｌｕｓ ｃｉｒｃｕｌａｎｓ ＪＣＭ２５０４、Ｂａｃｉｌｌｕｓ ｍｅｇａｔｅｒｉｕｍ ＪＣＭ２５０６、Ｂａｃｉｌｌｕｓ ｓｉｍｐｌｅｘ ＪＣＭ１２３０７、Ｂａｃｉｌｌｕｓ ａｚｏｔｏｆｏｒｍａｎｓ ＪＣＭ１２２１０（以上、独立行政法人 理化学研究所バイオリソースセンター微生物材料開発室による分譲菌株）、Ｂａｃｉｌｌｕｓ ｐｓｙｃｈｒｏｓａｃｃｈａｒｏｌｙｔｉｃｕｓ ＡＴＣＣ２３２９６（アメリカン・タイプカルチャー・コレクション）などを挙げることができるが、これらに限定されるものではない。 Examples of (S) -2-methylbutyric acid-producing bacteria belonging to the genus Bacillus that can be used in the present invention include Bacillus pumilus, Bacillus megaterium, and Bacillus subtilis. , Bacillus circulans, Bacillus simplex, Bacillus azoformans, Bacillus cyclosaccharus, and the like. For example, Bacillus pumilus NBRC14367 (sale strain by the National Institute of Technology and Evaluation), Bacillus subtilis JCM2499, Bacillus subtilis JCM20118, Bacillus subtilis IAM1231, Bacillus circulans JCM2504, Bacillus megaterium JCM2506, Bacillus simplex JCM12307, Bacillus azotoformans JCM12210 (or more, National Institute of Physics and Chemistry Bacillius psychrosaccharolyticus ATCC 23296 (American Type Culture Collection) Emissions) and the like, but not limited thereto.
本発明において培地としては、炭素源、窒素源、無機塩類などを含む通常の培地に、Ｌ−イソロイシンを加えた培地またはＬ−イソロイシンを比較的多く含むアミノ酸混合物、ペプチド、蛋白質もしくはこれらを組み合わせた培地を用いることができる。Ｌ−イソロイシンとしては、例えば、市販品であるＬ−イソロイシン（協和醗酵社製）、Ｌ−イソロイシン（味の素社製）、Ｌ−イソロイシン Ｗ５２７６０２（アルドリッチ社製）、Ｌ−イソロイシン（和光純薬社製）などを挙げることができ、また、Ｌ−イソロイシンを比較的多く含むアミノ酸混合物、ペプチドまたは蛋白質としては、例えば、チーズホエー、牛乳、鶏肉、鮭、大豆、小麦などを挙げることができる。 In the present invention, as a medium, a medium obtained by adding L-isoleucine to an ordinary medium containing a carbon source, a nitrogen source, inorganic salts, or the like, an amino acid mixture containing a relatively large amount of L-isoleucine, a peptide, a protein, or a combination thereof. A culture medium can be used. Examples of L-isoleucine include commercially available L-isoleucine (manufactured by Kyowa Hakko), L-isoleucine (manufactured by Ajinomoto Co., Inc.), L-isoleucine W527602 (manufactured by Aldrich), and L-isoleucine (manufactured by Wako Pure Chemical Industries, Ltd.). In addition, examples of the amino acid mixture, peptide, or protein containing a relatively large amount of L-isoleucine include cheese whey, milk, chicken, rice bran, soybean, and wheat.
培地に配合しうる炭素源としては、例えば、グルコース、フラクトースなどの糖類；クエン酸、リンゴ酸などの有機酸類；エタノール、グリセロールなどのアルコール類等が挙げられ、また、窒素源としては、例えば、硫酸アンモニウム、硝酸アンモニウムなどの無機窒素化合物；ペプトンなどの有機窒素源などを利用することができ、さらに、無機塩類としては、例えば、各種のリン酸塩、硫酸マグネシウムなどを使用することができ、そして培地には、さらに必要に応じて、微量の金属塩（鉄塩、カルシウム塩など）などを添加してもよい。 Examples of the carbon source that can be blended in the culture medium include saccharides such as glucose and fructose; organic acids such as citric acid and malic acid; alcohols such as ethanol and glycerol; and nitrogen sources such as Inorganic nitrogen compounds such as ammonium sulfate and ammonium nitrate; organic nitrogen sources such as peptone can be used, and as inorganic salts, for example, various phosphates, magnesium sulfate and the like can be used, and culture medium Further, if necessary, a trace amount of metal salt (iron salt, calcium salt, etc.) may be added.
前記の如き菌株を使用して、（Ｓ）−２−メチル酪酸を製造する方法としては、例えば、以上に述べた如き炭素源、窒素源、無機塩類などを含む培地にＬ−イソロイシンを添加して該菌株を培養する方法；炭素源、窒素源、無機塩類などを含む培地で該菌株を１日〜３日程度前培養した後、Ｌ−イソロイシンを添加してさらに培養する方法；該菌株を炭素源、窒素源、無機塩類などを含む培地で培養した後、遠心分離等の手段により菌体を集め、該菌体を緩衝液等に分散させ、Ｌ−イソロイシンを添加して反応させる方法；該菌株をそれ自体既知の方法で固定化し、Ｌ−イソロイシンを添加して反応させる方法等を挙げることができる。 As a method for producing (S) -2-methylbutyric acid using the above strain, for example, L-isoleucine is added to a medium containing a carbon source, a nitrogen source, inorganic salts and the like as described above. A method of culturing the strain; a method of pre-culturing the strain for about 1 to 3 days in a medium containing a carbon source, a nitrogen source, inorganic salts, etc., and further culturing by adding L-isoleucine; A method in which after culturing in a medium containing a carbon source, a nitrogen source, inorganic salts, etc., the cells are collected by means of centrifugation or the like, the cells are dispersed in a buffer or the like, and L-isoleucine is added and reacted; Examples thereof include a method in which the strain is immobilized by a method known per se, and L-isoleucine is added and reacted.
培養は好気条件下に行うことができ、例えば、空気中での振盪培養、通気撹拌培養などの方法を採用することができる。培養温度は約２０℃〜約４０℃の範囲内、そして培地のｐＨは６〜８の範囲内が好ましい。また、培養日数は特に限定されないが、通常、１日〜１４日程度が実用上好ましい。 The culture can be performed under aerobic conditions. For example, methods such as shaking culture in air and aeration and agitation culture can be employed. The culture temperature is preferably in the range of about 20 ° C to about 40 ° C, and the pH of the medium is preferably in the range of 6-8. The number of days for cultivation is not particularly limited, but usually about 1 to 14 days is practically preferable.
Ｌ−イソロイシンの培地に対する添加濃度は、用いる培養方法等により異なり、厳密には限定されないが、一般には１ｇ／Ｌ〜１００ｇ／Ｌ、好ましくは１０ｇ／Ｌ〜３０ｇ／Ｌの範囲内を例示することができる。Ｌ−イソロイシンの添加濃度が１００ｇ／Ｌを超えると、微生物の生育阻害が起こり、Ｌ−イソロイシンから（Ｓ）−２−メチル酪酸への変換率が悪くなり、１ｇ／Ｌ未満では、（Ｓ）−２−メチル酪酸の生成量が少なすぎ、生産コストが上がるので好ましくない。 The concentration of L-isoleucine added to the medium varies depending on the culture method used and is not strictly limited, but is generally in the range of 1 g / L to 100 g / L, preferably 10 g / L to 30 g / L. Can do. When the added concentration of L-isoleucine exceeds 100 g / L, the growth of microorganisms is inhibited, and the conversion rate from L-isoleucine to (S) -2-methylbutyric acid becomes worse, and when it is less than 1 g / L, (S) Since the amount of 2-methylbutyric acid produced is too small and production costs increase, it is not preferable.
培地または培養後の菌体に上記濃度範囲でＬ−イソロイシンを加えることにより、培地中に０．１％〜８％程度の（Ｓ）−２−メチル酪酸を生成させることができる。 By adding L-isoleucine in the above-mentioned concentration range to the culture medium or cultured cells, about 0.1% to 8% (S) -2-methylbutyric acid can be produced in the culture medium.
Ｌ−イソロイシンから（Ｓ）−２−メチル酪酸への変換率は、添加するＬ−イソロイシンの量などにもよるが、通常、５０％〜１０５％、好ましくは８０％〜１０５％の範囲内である。 Although the conversion rate from L-isoleucine to (S) -2-methylbutyric acid depends on the amount of L-isoleucine added, etc., it is usually in the range of 50% to 105%, preferably 80% to 105%. is there.
以上に述べた培養により生産される（Ｓ）−２−メチル酪酸を含有する培地または反応液からの（Ｓ）−２−メチル酪酸の分離、精製は、それ自体既知の方法で行うことができ、例えば、培地または反応液に塩酸を加えた後、塩化ナトリウム、エーテルを加え、攪拌抽出し、濾過助剤を加えて濾過した濾液のエーテル層を分離し、これに無水硫酸マグネシウムを加え、蒸留を行いエーテルを除去することにより、粗製の（Ｓ）−２−メチル酪酸を得ることができる。さらに、常法に従い、例えば、減圧蒸留により分画することにより、通常、７５％ｅ．ｅ．以上、好ましくは９５〜１００％ｅ．ｅ．の範囲内の光学純度、より好ましくはの９９．５〜１００％ｅ．ｅ．の範囲内の光学純度で精製（Ｓ）−２−メチル酪酸を得ることができる。 Separation and purification of (S) -2-methylbutyric acid from a medium or reaction solution containing (S) -2-methylbutyric acid produced by the culture described above can be performed by a method known per se. For example, after adding hydrochloric acid to the culture medium or the reaction solution, sodium chloride and ether are added, extracted by stirring, the filter aid is added, the ether layer of the filtrate is separated, and anhydrous magnesium sulfate is added thereto and distilled. By removing the ether, crude (S) -2-methylbutyric acid can be obtained. Furthermore, in accordance with a conventional method, for example, by fractionation by vacuum distillation, usually 75% e.e. e. Or more, preferably 95-100% e.e. e. Of optical purity within the range of 99.5 to 100% e.e. e. (S) -2-methylbutyric acid can be obtained with an optical purity within the range of.
本発明の微生物菌株を利用する方法により得ることのできる（Ｓ）−２−メチル酪酸は、甘いフルーツ様の香気・香味を有しており、これを添加した嗜好性の高い各種の香料組成物は調合素材として利用することができる。 (S) -2-methylbutyric acid that can be obtained by the method using the microbial strain of the present invention has a sweet fruit-like aroma and flavor, and various flavor compositions with high palatability to which this is added Can be used as a blending material.
本発明によれば、例えば、炭酸飲料、果汁飲料、果実酒飲料類、乳飲料などの飲料類；アイスクリーム類、シャーベット類、アイスキャンディー類などの冷菓類；和・洋菓子、チューインガム類、パン類、コーヒー、紅茶、お茶、タバコなどの嗜好品類；和風スープ類、洋風スープ類などのスープ類；ハム、ソーセージなどの畜肉加工品；風味調味料、各種インスタント飲料乃至食品類、各種のスナック類などに、（Ｓ）−２−メチル酪酸を有効成分として含有する香料組成物の適当量を添加することにより、そのユニークな香気香味が賦与された飲食品類を提供することができる。また、本発明によれば、例えば、シャンプー類、ヘアクリーム類、その他の毛髪化粧料基剤；オシロイ、口紅、その他の化粧用基剤や化粧用洗剤類基剤などに、（Ｓ）−２−メチル酪酸を有効成分として含有する香料組成物の適当量を添加することにより、そのユニークな香気が賦与された化粧品類を提供することができる。さらにまた、本発明によれば、（Ｓ）−２−メチル酪酸を有効成分として含有する香料組成物の適当量が配合された洗濯用洗剤類、消毒用洗剤類、防臭洗剤類、その他各種の保健・衛生用洗剤類；歯磨き、ティッシュペーパー、トイレットペーパーなどの各種保健・衛生材料類；医薬品類などを提供することができる。 According to the present invention, for example, beverages such as carbonated drinks, fruit juice drinks, fruit liquor drinks, milk drinks; frozen confections such as ice creams, sherbets, ice candy; Japanese / Western confectionery, chewing gums, breads Taste products such as coffee, tea, tea, tobacco; soups such as Japanese-style soups and Western-style soups; processed meat products such as ham and sausages; flavor seasonings, various instant beverages and foods, various snacks, etc. In addition, by adding an appropriate amount of a fragrance composition containing (S) -2-methylbutyric acid as an active ingredient, food and drink with a unique aroma flavor can be provided. In addition, according to the present invention, for example, (S) -2 can be applied to shampoos, hair creams, other hair cosmetic bases; osiroy, lipstick, other cosmetic bases and cosmetic detergent bases. -By adding an appropriate amount of a perfume composition containing methylbutyric acid as an active ingredient, it is possible to provide cosmetics imparted with the unique aroma. Furthermore, according to the present invention, laundry detergents, disinfecting detergents, deodorant detergents, and other various types of ingredients containing an appropriate amount of a fragrance composition containing (S) -2-methylbutyric acid as an active ingredient. Health and hygiene detergents; various types of health and hygiene materials such as toothpaste, tissue paper, and toilet paper; pharmaceuticals and the like can be provided.
さらに、本発明の方法によって製造される（Ｓ）−２−メチル酪酸から、合成あるいは生化学的手法を用いて、医薬中間体、医薬品、各種工業製品を製造することもできる。 Furthermore, pharmaceutical intermediates, pharmaceuticals, and various industrial products can also be produced from (S) -2-methylbutyric acid produced by the method of the present invention using synthetic or biochemical techniques.
以下、実施例および比較例を挙げて本発明をさらに具体的に説明する。 Hereinafter, the present invention will be described more specifically with reference to examples and comparative examples.
実施例１
培 地
イーストペプトン１．５ｇ、塩化ナトリウム０．５ｇ、リン酸ナトリウム・１２水０．８ｇ、リン酸１カリウム０．２ｇ、イオン交換水５０ｇおよびＬ−イソロイシン（協和発酵社製）０．５ｇを混合、溶解し、培地を調製した。
Example 1
Medium yeast peptone 1.5 g, sodium chloride 0.5 g, sodium phosphate 12 water 0.8 g, potassium phosphate 0.2 g, ion-exchanged water 50 g and L-isoleucine (manufactured by Kyowa Hakko) 0.5 g After mixing and dissolving, a medium was prepared.
培養方法
Ｂａｃｉｌｌｕｓ ｐｕｍｉｌｕｓ ＮＢＲＣ１４３６７を植菌したスラントから一白金耳を取り、生理食塩水１０ｍｌに懸濁し、懸濁液１ｍｌを培地に接種し、３０℃、１００ｒｐｍで約５０時間培養を行った。培養は二連で行い、ｐＨおよび６６０ｎｍにおける吸光度（ＯＤ６６０）を測定した。
Culturing Method One platinum loop was taken from a slant inoculated with Bacillus pumilus NBRC14367, suspended in 10 ml of physiological saline, 1 ml of the suspension was inoculated into the medium, and cultured at 30 ° C. and 100 rpm for about 50 hours. The culture was performed in duplicate, and pH and absorbance at 660 nm (OD660) were measured.
（Ｓ）−２−メチル酪酸の生成量（％）、変換率（％）の算出
培養液にブタノールを添加し、４Ｎ 塩酸でｐＨを１．７ に調整し、１００００ｒｐｍで１０分間遠心分離を行い、上清０．２μｌをガスクロマトグラフィー分析（以後、ＧＣ分析と呼ぶ）に供した。また、以下に示すブタノールを内部標準とする内部標準法により生成量を算出した。
Calculation of (S) -2-methylbutyric acid production (%) and conversion rate (%) Butanol was added to the culture solution, pH was adjusted to 1.7 with 4N hydrochloric acid, and centrifugation was performed at 10,000 rpm for 10 minutes. Then, 0.2 μl of the supernatant was subjected to gas chromatography analysis (hereinafter referred to as GC analysis). Further, the production amount was calculated by an internal standard method using butanol as an internal standard shown below.
検量線の作成
ブタノールと２−メチル酪酸を任意の重量比で混合し、ＧＣ分析を行った。ＧＣ分析結果から得られたブタノールと２−メチル酪酸のＡＲＥＡ％の比を表１に示す。この結果から得られたＡＲＥＡ％の比をＸ軸、重量比をＹ軸にプロットし、ゼロ点を通過する検量線を作成し、その検量線から二次方程式 ｙ=１．３２６ｘ−０．０２１１（図１）を得た。
Preparation of calibration curve Butanol and 2-methylbutyric acid were mixed at an arbitrary weight ratio, and GC analysis was performed. Table 1 shows the ratio of AREA% between butanol and 2-methylbutyric acid obtained from the GC analysis results. The AREA% ratio obtained from this result is plotted on the X axis and the weight ratio is plotted on the Y axis, a calibration curve passing through the zero point is created, and a quadratic equation y = 1.326x−0.0211 is created from the calibration curve. (FIG. 1) was obtained.
（Ｓ）−２−メチル酪酸変換率％の算出
変換率％は、添加したＬ−イソロイシンから２−メチル酪酸へのモル変換率を表し、以下の式により算出した。その結果を後記表２にまとめて示す。
Calculation of (S) -2-methylbutyric acid conversion rate% The conversion rate% represents the molar conversion rate from added L-isoleucine to 2-methylbutyric acid, and was calculated by the following formula. The results are summarized in Table 2 below.
変換率％ ＝ {(２−メチル酪酸生成量×１．２９*)／Ｌ−イソロイシン添加量(％)｝ × １００
*Ｌ−イソロイシンの分子量／２−メチル酪酸の分子量＝１．２９
Conversion rate% = {(2-methylbutyric acid production amount × 1.29 * ) / L-isoleucine addition amount (%)} × 100
* Molecular weight of L-isoleucine / 2 Molecular weight of 2-methylbutyric acid = 1.29
実施例２
Ｂａｃｉｌｌｕｓ ｐｕｍｉｌｕｓ ＮＢＲＣ１４３６７に代え、Ｂ．ｍｅｇａｔｅｒｉｕｍ ＪＣＭ２５０６を用いる以外は実施例１と同様な方法で培養し、ｐＨ、ＯＤ６６０、（Ｓ）−２−メチル酪酸の生成量（％）および変換率（％）を測定または算出した。その結果を後記表２にまとめて示す。
Example 2
Instead of Bacillus pumilus NBRC14367, Culture was carried out in the same manner as in Example 1 except that megaterium JCM2506 was used, and pH, OD660, (S) -2-methylbutyric acid production (%) and conversion rate (%) were measured or calculated. The results are summarized in Table 2 below.
実施例３
Ｂａｃｉｌｌｕｓ ｐｕｍｉｌｕｓ ＮＢＲＣ１４３６７に代え、Ｂ．ｓｕｔｉｌｉｓ ＪＣＭ ２４９９を用いる以外は実施例１と同様な方法で培養し、ｐＨ、ＯＤ６６０、（Ｓ）−２−メチル酪酸の生成量（％）および変換率（％）を測定または算出した。その結果を後記表２にまとめて示す。
Example 3
Instead of Bacillus pumilus NBRC14367, The culture was carried out in the same manner as in Example 1 except that Sutilis JCM 2499 was used, and pH, OD660, (S) -2-methylbutyric acid production (%) and conversion rate (%) were measured or calculated. The results are summarized in Table 2 below.
実施例４
Ｂａｃｉｌｌｕｓ ｐｕｍｉｌｕｓ ＮＢＲＣ１４３６７に代え、Ｂ．ｓｉｍｐｌｅｘ ＪＣＭ１２３０７を用いる以外は実施例１と同様な方法で培養し、ｐＨ、ＯＤ６６０、（Ｓ）−２−メチル酪酸の生成量（％）および変換率（％）を測定または算出した。その結果を後記表２にまとめて示す。
Example 4
Instead of Bacillus pumilus NBRC14367, The culture was carried out in the same manner as in Example 1 except that simplex JCM12307 was used, and the pH, OD660, (S) -2-methylbutyric acid production (%) and conversion rate (%) were measured or calculated. The results are summarized in Table 2 below.
比較例１
実施例１の培地組成からＬ−イソロイシン０．５ｇを除いた培地を用いる以外は実施例１と同様な方法で培養し、ｐＨ、ＯＤ６６０および（Ｓ）−２−メチル酪酸の生成量（％）を測定または算出した。その結果を後記表２にまとめて示す。
Comparative Example 1
The culture was carried out in the same manner as in Example 1 except that a medium obtained by removing 0.5 g of L-isoleucine from the medium composition of Example 1 was used, and pH, OD660, and (S) -2-methylbutyric acid production amount (%) Was measured or calculated. The results are summarized in Table 2 below.
比較例２
実施例１の培地組成からＬ−イソロイシン０．５ｇを除いた培地を用いる以外は実施例２と同様な方法で培養し、ｐＨ、ＯＤ６６０および（Ｓ）−２−メチル酪酸の生成量（％）を測定または算出した。その結果を後記表２にまとめて示す。
Comparative Example 2
The culture was carried out in the same manner as in Example 2 except that a medium obtained by removing 0.5 g of L-isoleucine from the medium composition of Example 1 was used, and pH, OD660, and (S) -2-methylbutyric acid production amount (%) Was measured or calculated. The results are summarized in Table 2 below.
比較例３
実施例１の培地組成からＬ−イソロイシン０．５ｇを除いた培地を用いる以外は実施例３と同様な方法で培養し、ｐＨ、ＯＤ６６０および（Ｓ）−２−メチル酪酸の生成量（％）を測定または算出した。その結果を後記表２にまとめて示す。
Comparative Example 3
Culturing was carried out in the same manner as in Example 3 except that a medium obtained by removing 0.5 g of L-isoleucine from the medium composition of Example 1 was used, and pH, OD660, and (S) -2-methylbutyric acid production amount (%) Was measured or calculated. The results are summarized in Table 2 below.
〔（Ｓ）−２−メチル酪酸の生成量、変換率の比較〕 [Comparison of (S) -2-methylbutyric acid production and conversion]
表２に示すように、Ｌ−イソロイシンを添加した培地で培養した実施例１〜４の培養液は、いずれも、（Ｓ）−２−メチル酪酸を培地中に生産していることが確認された。特に、実施例１、２および３で用いたＢ．ｐｕｍｉｌｕｓ ＮＢＲＣ１４３６７、Ｂ．ｍｅｇａｔｅｒｉｕｍ ＪＣＭ２５０６およびＢ．ｓｕｂｔｉｌｉｓ ＪＣＭ２４９９による変換率は９１．１％、５３．１％および７６．０％と高かった。実施例４で用いたＢ．ｓｉｍｐｌｅｘ ＪＣＭ１２３０７はこれらに次ぐ３０．７％の変換率であった。また、培養物から分離した（Ｓ）−２−メチル酪酸を分析したところ、その光学純度は表２のように９９．５〜９９．９ｅ．ｅ．の範囲内の数値となり、非常に高い値であった。 As shown in Table 2, it was confirmed that all of the culture solutions of Examples 1 to 4 cultured in a medium supplemented with L-isoleucine produced (S) -2-methylbutyric acid in the medium. It was. In particular, the B.C. Pumilis NBRC14367, B.I. megaterium JCM2506 and B.I. The conversion rates by subtilis JCM2499 were as high as 91.1%, 53.1% and 76.0%. B. used in Example 4 simplex JCM12307 had the next conversion rate of 30.7%. Further, when (S) -2-methylbutyric acid separated from the culture was analyzed, its optical purity was 99.5 to 99.9 e. e. It was a numerical value within the range, and was a very high value.
一方、Ｌ−イソロイシンを添加しない培地で培養した比較例１〜３の培養液も（Ｓ）−２−メチル酪酸を培地中に生産していたが、その生産量（％）は０．０８〜０．０９と非常に低く、Ｌ−イソロイシンを添加した実施例１〜４の場合の約１／３〜１／９程度であった。 On the other hand, the culture solutions of Comparative Examples 1 to 3 cultured in a medium not added with L-isoleucine produced (S) -2-methylbutyric acid in the medium, but the production amount (%) was 0.08 to It was very low as 0.09, and was about 1/3 to 1/9 of Examples 1 to 4 to which L-isoleucine was added.
以上の結果より、バシラス属に属する菌株はいずれも（Ｓ）−２−メチル酪酸を生産する能力を有するが、Ｌ−イソロイシンを添加した培地で培養することにより、その生産性は大きく向上することが確認された。また、菌株の中ではＢａｃｉｌｌｕｓ ｐｕｍｉｌｕｓ ＮＢＲＣ１４３６７、Ｂ．ｍｅｇａｔｅｒｉｕｍ ＪＣＭ２５０６およびＢ．ｓｕｂｔｉｌｉｓ ＪＣＭ２４９９の生産性が良好であった。 From the above results, all strains belonging to the genus Bacillus have the ability to produce (S) -2-methylbutyric acid, but the productivity is greatly improved by culturing in a medium supplemented with L-isoleucine. Was confirmed. Among the strains, Bacillus pumilus NBRC14367, B. megaterium JCM2506 and B.I. The productivity of subtilis JCM2499 was good.
実施例５
前培養
ひだ付き三角フラスコに、イーストペプトン３．０ｇ、塩化ナトリウム１．０ｇ、リン酸ナトリウム・１２水１．５ｇ、リン酸１カリウム０．３ｇ、イオン交換水１００ｇおよびＬ−イソロイシン（協和発酵社製）１．０ｇを入れ、混合、溶解、殺菌し、培地とした。Ｂａｃｉｌｌｕｓ ｐｕｍｉｌｕｓ ＮＢＲＣ１４３６７を植菌したスラントから一白金耳取り、上記培地に接種し、３０℃、１００ｒｐｍで２９．５時間前培養を行った。
Example 5
In an Erlenmeyer flask with preculture folds, 3.0 g yeast peptone, 1.0 g sodium chloride, 1.5 g sodium phosphate / 12 water, 0.3 g potassium monophosphate, 100 g ion-exchanged water, and L-isoleucine (Kyowa Hakko) (Product made) 1.0 g was added, mixed, dissolved and sterilized to obtain a medium. One platinum ear was taken from the slant inoculated with Bacillus pumilus NBRC14367, inoculated into the above medium, and precultured at 30 ° C. and 100 rpm for 29.5 hours.
本培養
イーストペプトン３０ｇ、塩化ナトリウム１０ｇ、リン酸ナトリウム・１２水１５．３ｇ、リン酸１カリウム３．３ｇ、イオン交換水１０００ｇおよびＬ−イソロイシン（協和発酵社製）２２５ｇを混合溶解させた培地を用意し、２Ｌ容発酵槽を用いて本培養を行った。培地殺菌後、前培養液50ｍｌを本培養培地に接種し、３０℃、６００ｒｐｍ、通気量０．３〜０．４リットル／分で９０時間培養を行い、ｐＨおよびＯＤ６６０を測定した。また、実施例１と同様、ＧＣ分析により（Ｓ）−２−メチル酪酸の濃度を求め、生成量（％）および変換率（％）を算出した。
A culture medium in which 30 g of main culture yeast peptone, 10 g of sodium chloride, 15.3 g of sodium phosphate / 12 water, 3.3 g of monopotassium phosphate, 1000 g of ion-exchanged water and 225 g of L-isoleucine (manufactured by Kyowa Hakko) was mixed and dissolved. Prepared and main culture was performed using a 2 L fermenter. After the medium was sterilized, 50 ml of the preculture was inoculated into the main culture medium, cultured at 30 ° C., 600 rpm, aeration rate of 0.3 to 0.4 liter / min for 90 hours, and pH and OD660 were measured. Further, as in Example 1, the concentration of (S) -2-methylbutyric acid was determined by GC analysis, and the production amount (%) and the conversion rate (%) were calculated.
培養の結果
表３に２Ｌ容発酵槽を用いたＢａｃｉｌｌｕｓ ｐｕｍｉｌｕｓ ＮＢＲＣ１４３６７の培養におけるｐＨ、ＯＤ６６０、生成量（％）および変換率（％）の経時変化を示す。
Results of the culture Table 3 shows changes over time in pH, OD660, production (%) and conversion rate (%) in culture of Bacillus pumilus NBRC14367 using a 2 L fermenter.
表３に示すように、時間の経過とともに（Ｓ）−２−メチル酪酸の生成量（％）は増加し、９０時間後に２．０６％と最大になった。変換率も同様に上昇し、８６．５時間、９０時間では１０５．７、１０６．４％となった。これにより、添加したＬ−イソロイシンはすべて（Ｓ）−２−メチル酪酸に変換されていたことがわかる。また、９０時間後の（Ｓ）−２−メチル酪酸を分離し、分析したところ、その光学純度は９９．５ｅ．ｅ．であった。 As shown in Table 3, the production amount (%) of (S) -2-methylbutyric acid increased with time and reached a maximum of 2.06% after 90 hours. Similarly, the conversion rate increased to 105.7% and 106.4% at 86.5 hours and 90 hours, respectively. Thereby, it turns out that all the added L-isoleucine was converted into (S) -2-methylbutyric acid. Further, when (S) -2-methylbutyric acid after 90 hours was separated and analyzed, its optical purity was 99.5 e. e. Met.
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