JPH05219969A - Biological production of alpha-hydroxylisobutyric acid - Google Patents
Biological production of alpha-hydroxylisobutyric acidInfo
- Publication number
- JPH05219969A JPH05219969A JP4771792A JP4771792A JPH05219969A JP H05219969 A JPH05219969 A JP H05219969A JP 4771792 A JP4771792 A JP 4771792A JP 4771792 A JP4771792 A JP 4771792A JP H05219969 A JPH05219969 A JP H05219969A
- Authority
- JP
- Japan
- Prior art keywords
- alpha
- hydroxyisobutyronitrile
- acetone
- acid
- hydroxyisobutyric acid
- 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.)
- Pending
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明はα−ヒドロキシイソ酪酸
の生物学的製造法に関する。α−ヒドロキシイソ酪酸
は、有機合成原料として有用な化合物であるのみなら
ず、脱水してメチルエステル化することによりメタクリ
ル樹脂の原料モノマーとして重要なメタクリル酸メチル
に変換することができる。FIELD OF THE INVENTION The present invention relates to a biological process for producing α-hydroxyisobutyric acid. α-Hydroxyisobutyric acid is not only a compound useful as a raw material for organic synthesis, but can also be converted into methyl methacrylate, which is important as a raw material monomer for methacrylic resin, by dehydration and methyl esterification.
【０００２】[0002]
【従来の技術と問題点】α−ヒドロキシイソイソブチロ
ニトリルを微生物的に加水分解して対応する酸を製造す
る方法としては、コリネバクテリウム属の微生物を用い
たグリコロニトリル、ラクトニトリルおよびα−ヒドロ
キシイソブチロニトリルの加水分解による対応するα−
ヒドロキシ酸の製造法（特開昭61-56086号公報参照）が
知られているが、α−ヒドロキシイソブチロニトリルに
対する加水分解活性は僅かなものである。2. Description of the Related Art As a method for microbially hydrolyzing .alpha.-hydroxyisoisobutyronitrile to produce a corresponding acid, glycolonitrile, lactonitrile and .alpha. -Corresponding α- by hydrolysis of hydroxyisobutyronitrile
Although a method for producing a hydroxy acid (see Japanese Patent Laid-Open No. 61-56086) is known, its hydrolysis activity with respect to α-hydroxyisobutyronitrile is small.
【０００３】[0003]
【問題点を解決するための手段】本発明者らは、先に、
ロドコッカス属、シュードモナス属、アースロバクター
属またはブレビバクテリウム属に属する微生物を用いた
α−ヒドロキシイソブチロニトリルからのα−ヒドロキ
シイソ酪酸の製造法を見出し特許出願（特願平2-14872
5) したが、触媒活性が未だ低く、反応速度、α−ヒド
ロキシイソ酪酸蓄積濃度などの面で工業的に十分満足し
得るものではなかった。本発明者らは、これらの点を改
善すべくα−ヒドロキシイソブチロニトリルからα−ヒ
ドロキシイソ酪酸を微生物的に生産する際の反応条件に
ついて鋭意検討を行ったところ、反応系にアセトンを添
加することにより、反応速度とα−ヒドロキシイソ酪酸
蓄積濃度の両面が大きく改善されることを見い出し本発
明を完成した。[Means for Solving the Problems]
A method for producing α-hydroxyisobutyric acid from α-hydroxyisobutyronitrile using a microorganism belonging to the genus Rhodococcus, Pseudomonas, Arthrobacter or Brevibacterium was found and applied for a patent (Japanese Patent Application No. 2-14872).
5) However, the catalytic activity was still low, and it was not industrially sufficiently satisfactory in terms of reaction rate, α-hydroxyisobutyric acid accumulated concentration and the like. The present inventors have conducted extensive studies on reaction conditions when microbially producing α-hydroxyisobutyric acid from α-hydroxyisobutyronitrile in order to improve these points, and as a result, acetone was added to the reaction system. As a result, it was found that both the reaction rate and the concentration of α-hydroxyisobutyric acid accumulated were greatly improved, and the present invention was completed.
【０００４】すなわち、本発明は、α−ヒドロキシイソ
ブチロニトリルを微生物的に加水分解してα−ヒドロキ
シイソ酪酸に変換する方法において、反応系にアセトン
を共存させることを特徴とするα−ヒドロキシイソ酪酸
の生物学的製造法、である。That is, the present invention is a method of microbially hydrolyzing α-hydroxyisobutyronitrile to convert it to α-hydroxyisobutyric acid, wherein acetone is allowed to coexist in the reaction system. Is a biological process for the production of isobutyric acid.
【０００５】本発明の効果がアセトンのどのような作用
機構によるものかは明らかではないが、水性媒体中にお
けるα−ヒドロキシイソブチロニトリル←→アセトン＋
青酸の解離平衡が、アセトンの添加によりα−ヒドロキ
シイソブチロニトリルの側に傾き反応系内の青酸濃度が
減少すること、あるいは微量の遊離した青酸が過剰量存
在するアセトンにより速やかに補足されること等が関与
し、これにより青酸によって引き起こされていた加水分
解酵素の活性低下が軽減されたためと考えられる。しか
しながら、本発明はこのような作用機構により何ら限定
されるものではない。It is not clear what kind of mechanism of action of acetone the effect of the present invention is, but α-hydroxyisobutyronitrile ← → acetone + in an aqueous medium.
The dissociation equilibrium of hydrocyanic acid leans toward α-hydroxyisobutyronitrile due to the addition of acetone and the concentration of hydrocyanic acid in the reaction system decreases, or a trace amount of liberated hydrocyanic acid is rapidly supplemented by acetone in excess. It is considered that this is because the reduction of hydrolase activity caused by hydrocyanic acid was alleviated. However, the present invention is not limited to such an action mechanism.
【０００６】[0006]
【発明の具体的説明】本発明に用いられる微生物は、α
−ヒドロキシイソブチロニトリルを加水分解してα−ヒ
ドロキシイソ酪酸に変換する能力を有し、アセトン添加
の効果が顕著に現れる、例えば、ロドコッカス属の細菌
であり、具体的には、ロドコッカスロドクロス(Rhodoco
ccus rhodochrous) ATCC 19140株が挙げられる。この細
菌は公知であり、アメリカン タイプカルチャー コレ
クション(ATCC)から容易に入手することができる。DETAILED DESCRIPTION OF THE INVENTION The microorganism used in the present invention is α
-Hydroxyisobutyronitrile has the ability to hydrolyze and convert it into α-hydroxyisobutyric acid, and the effect of adding acetone is prominent, for example, a bacterium of the genus Rhodococcus, specifically Rhodococcus rhodocross. (Rhodoco
ccus rhodochrous) ATCC 19140 strain. This bacterium is known and can be easily obtained from the American Type Culture Collection (ATCC).
【０００７】本発明に使用される微生物の培養には、通
常資化し得る炭素源（グルコース、グリセロール、シュ
ークロース等）、窒素源（酵母エキス、ペプトン、硫酸
アンモニウム等）および微生物の生育に必要な無機栄養
源（りん酸水素二カリウム、塩化マグネシウム、塩化カ
ルシウム、塩化第二鉄、硫酸マンガン、硫酸亜鉛等）を
含有する培地が用いられる。また、培養の初期または中
期における、生育を大きく阻害しない濃度のニトリル類
（イソブチロニトリル、プロピオニトリル等）、該ニト
リルに対応するアミド類、ラクタム類（ε−カプロラク
タム、γ−ブチロラクタム等）などの添加は、より高い
酵素活性が得られるので好ましい。培養は好気的条件下
pH４〜10、温度20〜40℃の範囲で、１〜４日間程度行え
ばよい。In the culture of the microorganism used in the present invention, a carbon source (glucose, glycerol, sucrose, etc.) that can normally be assimilated, a nitrogen source (yeast extract, peptone, ammonium sulfate, etc.) and an inorganic substance necessary for the growth of the microorganism are used. A medium containing a nutrient source (dipotassium hydrogen phosphate, magnesium chloride, calcium chloride, ferric chloride, manganese sulfate, zinc sulfate, etc.) is used. Further, in the early or middle stage of the culture, nitriles (isobutyronitrile, propionitrile, etc.) at a concentration that does not significantly inhibit growth, amides corresponding to the nitrile, lactams (ε-caprolactam, γ-butyrolactam, etc.) The addition of such as is preferable because higher enzyme activity can be obtained. Culture under aerobic conditions
It may be carried out at a pH of 4 to 10 and a temperature of 20 to 40 ° C for about 1 to 4 days.
【０００８】加水分解反応は、上記方法により得た、例
えば ATCC 19140 株の培養液、培養液から分離した菌
体、あるいは該菌体処理物（菌体破砕物、抽出酵素、固
定化菌体・酵素等）を、水性媒体中でアセトンの共存下
α−ヒドロキシイソブチロニトリルと接触させればよ
い。The hydrolysis reaction is carried out by the above-mentioned method, for example, a culture solution of ATCC 19140 strain, cells separated from the culture solution, or a treated product of the cells (crushed cells, extracted enzyme, immobilized cells, etc.). The enzyme) may be contacted with α-hydroxyisobutyronitrile in the presence of acetone in an aqueous medium.
【０００９】アセトンの濃度は、使用する基質としての
α−ヒドロキシイソブチロニトリルの濃度、反応温度、
ｐＨ等により異なるが、通常 0.5〜50重量％の範囲で最
適な濃度を選択すればよい。基質であるα−ヒドロキシ
イソブチロニトリルの濃度は 0.1〜10重量％、ｐＨ４〜
10、好ましくは６〜８、微生物の使用量は、乾燥菌体量
として0.01〜５重量％、反応温度は氷点〜50℃、好まし
くは２〜30℃の範囲でそれぞれ選べばよい。また反応が
進行するに伴い減少するα−ヒドロキシイソブチロニト
リルを反応液に逐次添加しながら反応を行ってもよい。The concentration of acetone depends on the concentration of α-hydroxyisobutyronitrile used as a substrate, the reaction temperature,
Although it depends on pH and the like, it is usually necessary to select an optimum concentration within the range of 0.5 to 50% by weight. The concentration of the substrate α-hydroxyisobutyronitrile is 0.1 to 10% by weight, and the pH is 4 to 4.
10, preferably 6 to 8, the amount of the microorganism to be used is 0.01 to 5% by weight as the dry cell amount, and the reaction temperature is to be selected in the range of freezing point to 50 ° C, preferably 2 to 30 ° C. The reaction may be carried out while successively adding α-hydroxyisobutyronitrile, which decreases as the reaction proceeds, to the reaction solution.
【００１０】[0010]
【発明の効果】本発明によれば、α−ヒドロキシイソブ
チロニトリルは長時間にわたり効率よくα−ヒドロキシ
イソ酪酸に変換され、また反応液中への高濃度の蓄積も
可能となる。According to the present invention, α-hydroxyisobutyronitrile can be efficiently converted into α-hydroxyisobutyric acid over a long period of time, and a high concentration can be accumulated in the reaction solution.
【００１１】α−ヒドロキシイソ酪酸の単離は、遠心分
離などにより菌体を除去後、濃縮、電気透析、イオン交
換、抽出、晶析などの公知の方法を利用して行うことが
できる。Isolation of α-hydroxyisobutyric acid can be carried out by using known methods such as concentration, electrodialysis, ion exchange, extraction, crystallization, etc. after removing the cells by centrifugation or the like.
【００１２】[0012]
【実施例】以下、実施例により本発明を具体的に説明す
るが、本発明は該実施例により限定されるものではな
い。なお、特に説明がない限り実施例中の％は重量％を
示すEXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to the examples. Unless otherwise specified,% in the examples indicates% by weight.
【００１３】実施例１ (1) 培 養 下記培地を100ml を含む500ml 三角フラスコに予め同培
地で培養したロドカッカス ロドクロス ATCC 19140株
を５ml接種し、30℃で３日間振とう培養を行った。Example 1 (1) Culture 5 ml of Rhodococcus rhodochros ATCC 19140 strain, which had been previously cultured in the same medium, was inoculated in a 500 ml Erlenmeyer flask containing 100 ml of the following medium, and shake culture was carried out at 30 ° C. for 3 days.
【００１４】 [0014]
【００１５】(2) 加水分解反応 反応液組成は、アセトンの濃度０〜5.80％、α−ヒドロ
キシイソブチロニトリル濃度２％、菌体濃度 OD630(630
nmの吸光度）＝９となるように調製し、30℃、pH７で17
時間反応を行った。(2) Hydrolysis reaction The reaction solution composition is such that the concentration of acetone is 0 to 5.80%, the concentration of α-hydroxyisobutyronitrile is 2%, and the cell concentration is OD 630 (630).
(absorbance at nm) = 9 and adjust at 30 ℃, pH 7 to 17
A time reaction was performed.
【００１６】(3) α−ヒドロキシイソ酪酸の定量 α−ヒドロキシイソ酪酸の生成量は、反応終了後、反応
液を遠心分離して菌体を除去した上清液を高速液体クロ
マトグラフィー〔Shodex ODS F-511A カラム〕により、
溶離液 0.2Ｍりん酸緩衝液(pH 2)を用い 208nm吸光度に
て検出し、α−ヒドロキシイソ酪酸の定量を行った。結
果を表−１に示す。(3) Quantification of α-hydroxyisobutyric acid The amount of α-hydroxyisobutyric acid produced was determined by high performance liquid chromatography [Shodex ODS] after the reaction was completed by centrifuging the reaction solution to remove the cells. F-511A column]
The eluent 0.2M phosphate buffer (pH 2) was used to detect the absorbance at 208 nm to quantify α-hydroxyisobutyric acid. The results are shown in Table-1.
【００１７】[0017]
【表１】 [Table 1]
Claims (3)
生物的に加水分解してα−ヒドロキシイソ酪酸に変換す
る方法において、反応系にアセトンを共存させることを
特徴とするα−ヒドロキシイソ酪酸の生物学的製造法。1. A method of converting α-hydroxyisobutyronitrile into α-hydroxyisobutyric acid by microbially hydrolyzing it, wherein acetone is allowed to coexist in the reaction system. Manufacturing method.
属である請求項１記載のα−ヒドロキシイソ酪酸の生物
学的製造法。2. The microorganism is Rhodococcus.
The method for producing α-hydroxyisobutyric acid according to claim 1, which is a genus.
0.5〜50重量％である請求項１記載α−ヒドロキシイソ
酪酸の生物学的製造法。3. The concentration of acetone coexisting in the reaction system is
The method for biologically producing α-hydroxyisobutyric acid according to claim 1, which is 0.5 to 50% by weight.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4771792A JPH05219969A (en) | 1992-02-05 | 1992-02-05 | Biological production of alpha-hydroxylisobutyric acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4771792A JPH05219969A (en) | 1992-02-05 | 1992-02-05 | Biological production of alpha-hydroxylisobutyric acid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH05219969A true JPH05219969A (en) | 1993-08-31 |
Family
ID=12783068
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP4771792A Pending JPH05219969A (en) | 1992-02-05 | 1992-02-05 | Biological production of alpha-hydroxylisobutyric acid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH05219969A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6582943B1 (en) | 2002-02-05 | 2003-06-24 | E. I. Du Pont De Nemours And Company | Method for producing 2-hydroxyisobutyric acid and methacrylic acid from acetone cyanohydrin |
-
1992
- 1992-02-05 JP JP4771792A patent/JPH05219969A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6582943B1 (en) | 2002-02-05 | 2003-06-24 | E. I. Du Pont De Nemours And Company | Method for producing 2-hydroxyisobutyric acid and methacrylic acid from acetone cyanohydrin |
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