JP2926354B2 - Biological production of α-hydroxyisobutyric acid - Google Patents
Biological production of α-hydroxyisobutyric acidInfo
- Publication number
- JP2926354B2 JP2926354B2 JP14872590A JP14872590A JP2926354B2 JP 2926354 B2 JP2926354 B2 JP 2926354B2 JP 14872590 A JP14872590 A JP 14872590A JP 14872590 A JP14872590 A JP 14872590A JP 2926354 B2 JP2926354 B2 JP 2926354B2
- Authority
- JP
- Japan
- Prior art keywords
- hydroxyisobutyric acid
- hydroxyisobutyronitrile
- microorganism
- acid
- reaction
- 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.)
- Expired - Fee Related
Links
Description
【発明の詳細な説明】 〔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、これを脱水してメチル
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アクリル酸メチルに変換することが
できる。The present invention relates to a method for producing α-hydroxyisobutyric acid in a biological manner. α-Hydroxyisobutyric acid is not only a useful compound as a raw material for organic synthesis, but it can be converted to methyl methacrylate, which is important as a raw material monomer for methacrylic resin, by dehydrating and methylesterifying it.
ニトリル化合物を微生物学的に加水分解し対応する酸
を製造する方法としていくつかの提案があり、α−ヒド
ロキシニトリルの加水分解に関しては、例えば、バチル
ス層、バクテリジウム層、ミクロコッカス層、ブレビバ
クテリウム層の微生物によるラクトニトリルまたはヒド
ロキシアセトニトリルからの対応する酸の生産〔特公昭
58-15120号公報参照〕、トルロプシス属酵母による対応
するα−ヒドロキシニトリルからの光学活性なＬ−α−
ヒドロキシバレリアン酸およびＬ−α−ヒドロキシイソ
カプロン酸の生産〔Fukuda Y.,et al.J.Ferment.Techno
l.51 393（1973）参照〕、コリネバクテリウム層の微生
物を用いたグリコロニトリル、ラクトニトリルおよびア
セトンシアンヒドリンの加水分解による対応するα−ヒ
ドロキシ酸の生産〔特開昭61-56086号公報参照〕および
アルカリゲネス属、シュードモナス属、ロドシュードモ
ナス属、コリネバクテリウム属、アシネトバクター属、
バチルス層、マイコバクテリウム属、ロドコッカス属ま
たはキャンディダ属に属する微生物によるα−ヒドロキ
シニトリルからの光学活性なα−ヒドロキシ酸の生産
〔特開平2-84198号公報参照〕などが知られている。There are several proposals as a method for producing a corresponding acid by hydrolyzing a nitrile compound microbiologically.For hydrolysis of α-hydroxynitrile, for example, a Bacillus layer, a bacterium layer, a Micrococcus layer, a Brevibacterium, etc. Production of the corresponding acid from lactonitrile or hydroxyacetonitrile by microorganisms in the aluminum layer
58-15120], optically active L-α- from the corresponding α-hydroxynitrile by a Torulopsis yeast.
Production of hydroxyvaleric acid and L-α-hydroxyisocaproic acid [Fukuda Y., et al. J. Ferment.
l. 51 393 (1973)], production of the corresponding α-hydroxy acids by hydrolysis of glycolonitrile, lactonitrile and acetone cyanohydrin using microorganisms in the corynebacterium layer [JP-A-61-56086] And Alcaligenes, Pseudomonas, Rhodopseudomonas, Corynebacterium, Acinetobacter,
Production of an optically active α-hydroxy acid from α-hydroxynitrile by a microorganism belonging to the genus Bacillus, Mycobacterium, Rhodococcus or Candida (see Japanese Patent Application Laid-Open No. 2-84198) is known.
しかしながら、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生産に関しては唯一コリ
ネバクテリウム属の微生物を用いた方法〔前記、特開昭
61-56086号〕があるのみであり、同公報にみる限り該微
生物はグリコロニトリル，ラクトニトリルに対しては高
い加水分解活性を有するがアセトンシアンヒドリン（α
−ヒドロキシイソブチロニトリル）に対しては僅かな活
性しか有しない。However, regarding the production of α-hydroxyisobutyric acid from α-hydroxyisobutyronitrile, only a method using a microorganism belonging to the genus Corynebacterium (see the above-mentioned JP
No. 61-56086]. According to the publication, the microorganism has a high hydrolysis activity against glycolonitrile and lactonitrile, but acetone cyanohydrin (α
-Hydroxyisobutyronitrile) with little activity.
本発明者らは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生成する微生物について高い
加水分解活性を有し、しかも生成したα−ヒドロキシイ
ソ酪酸を分解する性質のない微生物の探索を鋭意行った
結果、ロドコッカス属に属する微生物に目的とする活性
を見い出し本発明を完成した。The present inventors have enthusiastically searched for a microorganism which has a high hydrolysis activity for a microorganism which produces α-hydroxyisobutyric acid from α-hydroxyisobutyronitrile and which has no property of decomposing the produced α-hydroxyisobutyric acid. As a result, the present inventors found the desired activity in microorganisms belonging to the genus Rhodococcus and completed the present invention.
すなわち、本発明は、α−ヒドロキシイソブチロニト
リルを微生物の作用による加水分解反応によりα−ヒド
ロキシイソ酪酸に変換するα−ヒドロキシイソ酪酸の製
造法において、使用する微生物が、ロドコッカス ロド
クロウス（Rhodococcus rhodochrous）ATCC12674、同AT
CC19140および同ATCC33258、並びにロドコッカス エリ
スロポリス（Rhodococcus erythropolis）IFM155、同IF
O12320および同IFO12538であ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 relates to a method for producing α-hydroxyisobutyric acid, which converts α-hydroxyisobutyronitrile into α-hydroxyisobutyric acid by a hydrolysis reaction by the action of a microorganism, wherein the microorganism used is Rhodococcus rhodochrous. ATCC12674, AT
CC19140 and ATCC33258, and Rhodococcus erythropolis IFM155 and IF
A biological production method of α-hydroxyisobutyric acid, characterized by being O12320 and IFO12538.
本発明で使用する微生物は、上記のとおりであり、ま
たこれらの変異株を用いることもできる。The microorganism used in the present invention is as described above, and these mutants can also be used.
これらの微生物は公知であり、各々アメリカン タイ
プカルチュア コレクション（ATCC）、財団法人酪酸研
究所（IFO）および千葉大学真核微生物研究センター（I
FM）から容易に入手することができる。These microorganisms are known, and are known to the American Type Culture Collection (ATCC), the Butyric Acid Research Institute (IFO), and the Research Center for Eukaryotic Microorganisms (I
FM).
次に本発明の一般的実施態様について説明する。本発
明に使用される微生物の培地には酸素誘導物質としてプ
ロピオニトリルやイソブチロニトリルなどのニトリル化
合物、またはアセトアミド、プロピオアミドなどのアミ
ド化合物を添加し、炭素源としては通常資化し得るグル
コース、グリセロールなどを、窒素源としては硫酸アン
モニウム、硝酸アンモニウムなどを、また無機栄養素と
しては塩化マグネシウム、塩化第二鉄などを使用するこ
とができる。またこれらの培地に酵母エキス、肉エキス
などの天然培地を添加したものを用いることができる。Next, general embodiments of the present invention will be described. The medium of the microorganism used in the present invention is added with a nitrile compound such as propionitrile or isobutyronitrile as an oxygen inducer, or an amide compound such as acetamide or propioamide. Glycerol and the like, ammonium sulfate and ammonium nitrate as a nitrogen source, and magnesium chloride, ferric chloride and the like as inorganic nutrients can be used. In addition, those obtained by adding a natural medium such as yeast extract and meat extract to these mediums can be used.
培養条件は好気条件下でpH4〜10、温度20〜50℃の範
囲で選べばよく、培地日数は１〜10日の範囲で活性が最
大となるまで培養すればよい。Culture conditions may be selected under aerobic conditions at a pH of 4 to 10 and a temperature of 20 to 50 ° C. The culture may be performed for 1 to 10 days until the activity is maximized.
加水分解反応は液体培地、または平板培地上にて培養
した菌体を採取し、必要に応じ固定化菌体、粗酵素、固
定化酵素などの菌体処理物を調製し、水、緩衝液中にて
α−ヒドロキシイソブチロニトリルと接触させればよ
い。菌体使用量は0.01〜10重量％、α−ヒドロキシイソ
ブチロニトリル濃度は0.01〜50重量％、反応温度は５〜
60℃、好ましくは10〜40℃、反応pHは４〜11、好ましく
は６〜10で、0.5〜100時間反応させればよい。In the hydrolysis reaction, cells cultured on a liquid medium or a plate medium are collected, and immobilized cells, crude enzymes, and treated cells such as immobilized enzymes are prepared as necessary. May be contacted with α-hydroxyisobutyronitrile. The amount of cells used is 0.01 to 10% by weight, the concentration of α-hydroxyisobutyronitrile is 0.01 to 50% by weight, and the reaction temperature is 5 to 5.
The reaction may be performed at 60 ° C., preferably 10 to 40 ° C., and at a reaction pH of 4 to 11, preferably 6 to 10, for 0.5 to 100 hours.
かくしてα−ヒドロキシイソブチロニトリルは直接、
もしくはα−ヒドロキシイソブチルアミドを経由してα
−ヒドロキシイソ酪酸に転換、蓄積される。Thus, α-hydroxyisobutyronitrile is directly
Alternatively, α via α-hydroxyisobutyramide
-Converted to and accumulated in hydroxyisobutyric acid.
生成物の単離は濃縮、イオン交換、電気透析、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。Product isolation includes concentration, ion exchange, electrodialysis, extraction,
A known method such as crystallization can be used.
本発明はα−ヒドロキシイソブチロニトリルの加水分
解活性を有する微生物を用いることにより常温、常圧と
いう温和な条件下で反応を進行できα−ヒドロキシイソ
酪酸の工業的に有利な製造法を提供するものである。The present invention provides an industrially advantageous method for producing α-hydroxyisobutyric acid by using a microorganism having an activity of hydrolyzing α-hydroxyisobutyronitrile so that the reaction can proceed under mild conditions of normal temperature and normal pressure. Is what you do.
次に、本発明を実施例によりさらに詳細に説明する
が、本発明はこれら実施例に限定されるものではない。Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
実施例１ （１）培養 表１に示す微生物を下記の条件で培養した。Example 1 (1) Culture The microorganisms shown in Table 1 were cultured under the following conditions.
ｉ）培地〔単位:W/V〕 グリセロール ２％ 酵母エキス 0.3％ りん酸一カリウム 0.68％ りん酸二ナトリウム 0.71％ 硫酸ナトリウム 0.28％ 塩化マグネシウム 0.04％ 塩化カルシウム 0.004％ 硫酸マンガン ４×10-4％ 塩化鉄 ６×10-5％ 硫酸亜鉛 ３×10-5％ 寒天 1.8％ pH 7.5 さらに、誘導剤としてイソブチロニトリル（IBN）0.2
％またはα−クロルプロピオニトリル（CPN）0.05％を
添加した。i) Medium [unit: W / V] Glycerol 2% Yeast extract 0.3% Monopotassium phosphate 0.68% Disodium phosphate 0.71% Sodium sulfate 0.28% Magnesium chloride 0.04% Calcium chloride 0.004% Manganese sulfate 4 × 10 -4 % Chloride Iron 6 × 10 -5 % Zinc sulfate 3 × 10 -5 % Agar 1.8% pH 7.5 In addition, isobutyronitrile (IBN) 0.2
% Or α-chloropropionitrile (CPN) 0.05% was added.
ii）培養条件 斜面培地から１白金耳の菌体を採り、上記平板培地上
に塗布し、30℃で48時間好気条件下に培養した。ii) Culture conditions One platinum loop of bacterial cells was collected from the slant medium, spread on the plate medium, and cultured at 30 ° C. for 48 hours under aerobic conditions.
（２）加水分解反応 平板培地から菌体を採取し遠心分離により各々の菌体
を0.05Mりん酸緩衝液（pH6.0）で３回洗浄した。沈殿菌
体を1.5mlの同様の緩衝液に再懸濁し終濃度25mMのα−
ヒドロキシイソブチロニトリルを添加し25℃で10時間振
盪しながら反応を行った。反応終了後、各々の反応液を
遠心分離し菌体を除去し遠心上清中のα−ヒドロキシイ
ソ酪酸の含量を液体クロマトグラフィー（カラム;SHODE
X ODS F511A、キャリア;0.2M H3PO4、30℃、モニター;2
08nm）により分析した。なお比較のため、特開昭61-560
86号公報記載のコリネバクテリウム ニトリロフィラス
ATCC21419株についても上記同様に培養および加水分解
反応を行った。(2) Hydrolysis reaction The cells were collected from the plate medium, and each cell was washed three times with 0.05 M phosphate buffer (pH 6.0) by centrifugation. The precipitated cells were resuspended in 1.5 ml of the same buffer and the final concentration of 25 mM α-
Hydroxyisobutyronitrile was added, and the reaction was carried out while shaking at 25 ° C. for 10 hours. After completion of the reaction, each reaction solution was centrifuged to remove cells, and the content of α-hydroxyisobutyric acid in the centrifuged supernatant was determined by liquid chromatography (column; SHODE
X ODS F511A, carrier; 0.2MH 3 PO 4 , 30 ° C, monitor; 2
08 nm). For comparison, see JP-A-61-560.
Corynebacterium nitriophilus described in JP 86
The ATCC21419 strain was also cultured and hydrolyzed in the same manner as described above.
結果を表１に示した。 The results are shown in Table 1.
実施例２ （１）培養 ロドコッカス エリスロポリスIFO12538を実施例１と
同様に培養した。 Example 2 (1) Culture Rhodococcus erythropolis IFO12538 was cultured in the same manner as in Example 1.
（２）加水分解反応 実施例１と同様して調製した菌体懸濁液に25mMのα−
ヒドロキシイソブチロニトリルを加え実施例１と同じ条
件で反応した。反応開始２時間と３時間後に各々25mMの
α−ヒドロキシイソブチロニトリルを再び加え合計75mM
添加した。４時間後、反応液を遠心分離することにより
除菌し、上清中のα−ヒドロキシイソ酪酸含量を実施例
１と同様に定量し、結果を表２に示した。(2) Hydrolysis reaction The cell suspension prepared in the same manner as in Example 1 was added with 25 mM α-
Hydroxyisobutyronitrile was added and reacted under the same conditions as in Example 1. Two hours and three hours after the start of the reaction, 25 mM each of α-hydroxyisobutyronitrile was added again to a total of 75 mM.
Was added. After 4 hours, the reaction solution was centrifuged to remove bacteria, and the content of α-hydroxyisobutyric acid in the supernatant was quantified in the same manner as in Example 1. The results are shown in Table 2.
───────────────────────────────────────────────────── フロントページの続き (58)調査した分野(Int.Cl.6，ＤＢ名) C12P 7/42 ＣＡ（ＳＴＮ) ＲＥＧＩＳＴＲＹ（ＳＴＮ)──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. 6 , DB name) C12P 7/42 CA (STN) REGISTRY (STN)
Claims (1)
物の作用による加水分解反応によりα−ヒドロキシイソ
酪酸に変換するα−ヒドロキシイソ酪酸の製造法におい
て、使用する微生物が、ロドコッカス ロドクロウス
（Rhodococcus rhodochrous）ATCC12674、同ATCC19140
および同ATCC33258、並びにロドコッカス エリスロポ
リス（Rhodococcus erythropolis）IFM155、同IFO12320
および同IFO12538であ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 for producing α-hydroxyisobutyric acid, which converts α-hydroxyisobutyronitrile into α-hydroxyisobutyric acid by a hydrolysis reaction under the action of a microorganism, wherein the microorganism used is Rhodococcus rhodochrous. ATCC12674, ATCC19140
And ATCC33258, and Rhodococcus erythropolis IFM155, IFO12320
And a method for biologically producing α-hydroxyisobutyric acid, which is IFO12538.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14872590A JP2926354B2 (en) | 1990-06-08 | 1990-06-08 | Biological production of α-hydroxyisobutyric acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14872590A JP2926354B2 (en) | 1990-06-08 | 1990-06-08 | Biological production of α-hydroxyisobutyric acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0440897A JPH0440897A (en) | 1992-02-12 |
| JP2926354B2 true JP2926354B2 (en) | 1999-07-28 |
Family
ID=15459217
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP14872590A Expired - Fee Related JP2926354B2 (en) | 1990-06-08 | 1990-06-08 | Biological production of α-hydroxyisobutyric acid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2926354B2 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3354757B2 (en) * | 1995-07-20 | 2002-12-09 | 三菱レイヨン株式会社 | Circular plasmid |
| DE69737696T2 (en) * | 1996-02-29 | 2008-01-10 | Nippon Soda Co. Ltd. | PROCESS FOR THE PREPARATION OF ALPHA HYDROXYLIC ACIDS WITH THE HELP OF MICROORGANISM AND NEW MICROORGANISM. |
| 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 |
| DE102006017760A1 (en) | 2006-03-24 | 2007-09-27 | Ufz-Umweltforschungszentrum Leipzig-Halle Gmbh | Enzymatic preparation of 2-hydroxy-2-methylcarboxylic acid comprises producing 3-hydroxycarboxylic acid in aqueous reaction solution, incubating the obtained solution and converting to 2-hydroxy-2-methylcarboxylic acid |
-
1990
- 1990-06-08 JP JP14872590A patent/JP2926354B2/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| 化学大辞典編集委員会編「化学大辞典９（縮刷版第７刷）」（昭４４−３−１５）、第１０１頁「メタクリル酸」の項 |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0440897A (en) | 1992-02-12 |
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