JPH05317066A - Production of 3-hydroxynitrile compound by microorganism transformed by recombinant plasmid having malohydrin epoxydase gene - Google Patents
Production of 3-hydroxynitrile compound by microorganism transformed by recombinant plasmid having malohydrin epoxydase geneInfo
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- JPH05317066A JPH05317066A JP3062597A JP6259791A JPH05317066A JP H05317066 A JPH05317066 A JP H05317066A JP 3062597 A JP3062597 A JP 3062597A JP 6259791 A JP6259791 A JP 6259791A JP H05317066 A JPH05317066 A JP H05317066A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、１，３−ジハロ−２−
プロパノールをエピハロヒドリンに変換する活性および
その逆反応を触媒する活性を有する酵素（以下ハロヒド
リンエポキシダーゼと略す）遺伝子ＤＮＡをベクタープ
ラスミドに連結した組換え体プラスミドを宿主微生物に
導入した形質転換微生物による、３−ヒドロキシニトリ
ル化合物の製造法に関する。３−ヒドロキシニトリル化
合物は、種々の医薬品や生理活性物質の合成原料として
有用な物質として知られている。This invention relates to 1,3-dihalo-2-
An enzyme having an activity of converting propanol to epihalohydrin and an activity of catalyzing its reverse reaction (hereinafter abbreviated as halohydrin epoxidase) by a transformed microorganism in which a recombinant plasmid in which a gene DNA is ligated to a vector plasmid is introduced into a host microorganism. , A method for producing a 3-hydroxynitrile compound. The 3-hydroxynitrile compound is known as a substance useful as a raw material for synthesizing various drugs and physiologically active substances.
【０００２】[0002]
【発明の背景】脱ハロゲン化酵素の作用によりエピハロ
ヒドリンから４−ハロ−３−ヒドロキシブチロニトリル
を製造する方法は、先に、本発明者らの一部により見出
されている（特願平1-185992号明細書参照）。しかし、
これら脱ハロゲン化酵素を有する微生物の触媒能力は高
くなく、工業的に用いる場合には満足できるものではな
い。BACKGROUND OF THE INVENTION A method for producing 4-halo-3-hydroxybutyronitrile from epihalohydrin by the action of a dehalogenase has been previously found by a part of the present inventors (Japanese Patent Application No. Hei 10 (1999) -135242). 1-185992 specification). But,
The microorganisms having these dehalogenating enzymes have low catalytic ability and are not satisfactory for industrial use.
【０００３】[0003]
【発明が解決しようとする課題】遺伝子組換えの方法で
クローン化された脱ハロゲン化酵素遺伝子によるエピハ
ロヒドリンから４−ハロ−３−ヒドロキシブチロニトリ
ルへの変換反応では、菌体内に多数の遺伝子を存在させ
ることができるため微生物の触媒能力を従来の方法に比
して飛躍的に増大させることが期待できる。このような
状況下、本発明者らの一部は、エピハロヒドリンを４−
ハロ−３−ヒドロキシブチロニトリルに変換する酵素が
ハロヒドリンエポキシダーゼであることを見い出し、さ
らに微生物由来のハロヒドリンエポキシダーゼ酵素遺伝
子ＤＮＡをベクタ−プラスミドに挿入して組換え体プラ
スミドを得、この組換え体プラスミドを導入した形質転
換体によりハロヒドリンエポキシダーゼを発現させるこ
とに成功した。[Problems to be Solved by the Invention] In the conversion reaction of epihalohydrin to 4-halo-3-hydroxybutyronitrile by the dehalogenase gene cloned by the method of gene recombination, a large number of genes are Since it can be present, it can be expected to dramatically increase the catalytic ability of microorganisms as compared with conventional methods. Under these circumstances, some of the present inventors have reported that epihalohydrin has a 4-
It was found that the enzyme converting to halo-3-hydroxybutyronitrile is halohydrin epoxidase, and further, halohydrin epoxidase enzyme gene DNA derived from a microorganism was inserted into a vector-plasmid to obtain a recombinant plasmid. We succeeded in expressing halohydrin epoxidase by the transformant introduced with this recombinant plasmid.
【０００４】[0004]
【問題を解決するための手段】本発明は、この形質転換
体によるハロヒドリンエポキシダーゼ酵素反応が、エピ
ハロヒドリン以外の１，２−エポキシ化合物にも適用で
き、３−ヒドロキシニトリル化合物の製造に有用できる
ことを見い出し、本発明を完成させた。すなわち、本発
明は、微生物由来のハロヒドリンエポキシダーゼ酵素遺
伝子ＤＮＡをベクタープラスミドに連結した組換え体プ
ラスミドにより形質転換された形質転換微生物の培養
液、菌体または菌体処理物を、シアン化アルカリの存在
下で、下記一般式〔Ｉ〕で示される１，２−エポキシ化
合物に作用させ、これを下記一般式〔II〕で示される３
−ヒドロキシニトリル化合物に変換せしめることを特徴
とする３−ヒドロキシニトリル化合物の製造法、であ
る。INDUSTRIAL APPLICABILITY According to the present invention, the halohydrin epoxidase enzymatic reaction by this transformant can be applied to 1,2-epoxy compounds other than epihalohydrin, and is useful for the production of 3-hydroxynitrile compounds. They found what they could do and completed the present invention. That is, the present invention is for cyanating a culture solution, cells or treated cells of a transformed microorganism transformed with a recombinant plasmid in which a halohydrin epoxidase enzyme gene DNA derived from a microorganism is ligated to a vector plasmid. In the presence of an alkali, a 1,2-epoxy compound represented by the following general formula [I] is allowed to act on the 1,2-epoxy compound represented by the following general formula [II].
A method for producing a 3-hydroxynitrile compound, which comprises converting the compound into a hydroxynitrile compound.
【０００５】[0005]
【化２】 〔R は炭素数１〜４のアルキル基を表す〕[Chemical 2] [R represents an alkyl group having 1 to 4 carbon atoms]
【０００６】本発明における形質転換微生物は、ハロヒ
ドリンエポキシダーゼ遺伝子ＤＮＡをベクタープラスミ
ドに連結した組換え体プラスミドにより形質転換された
微生物であり、この組換え体プラスミドとして、(1) 配
列番号：１で示されるアミノ酸配列またはその一部の配
列を有しハロヒドリンエポキシダ−ゼ活性を有するポリ
ペプチドをコードするＤＮＡ配列、(2) 配列番号：２で
示されるアミノ酸配列またはその一部の配列を有しハロ
ヒドリンエポキシダ−ゼ活性を有するポリペプチドをコ
ードするＤＮＡ配列、(3) 上記 (1)項のハロヒドリンエ
ポキシダ−ゼ活性を有するポリペプチドをコードするＤ
ＮＡ配列が、配列番号：３で示されるＤＮＡ配列または
その一部の配列からなるもの、(4) 上記 (2)項のハロヒ
ドリンエポキシダ−ゼ活性を有するポリペプチドをコー
ドするＤＮＡ配列が、配列番号：４で示されるＤＮＡ配
列またはその一部の配列からなるもの、の少なくとも一
つを含むものが挙げられる。The transformed microorganism of the present invention is a microorganism transformed with a recombinant plasmid in which halohydrin epoxidase gene DNA is ligated to a vector plasmid, and as the recombinant plasmid, (1) SEQ ID NO: A DNA sequence encoding a polypeptide having a halohydrin epoxidase activity, which has the amino acid sequence represented by 1 or a partial sequence thereof, (2) the amino acid sequence represented by SEQ ID NO: 2, or a partial sequence thereof A DNA sequence having a sequence and encoding a polypeptide having halohydrin epoxidase activity, (3) D encoding the polypeptide having halohydrin epoxidase activity according to the above item (1)
NA sequence consisting of the DNA sequence represented by SEQ ID NO: 3 or a part thereof, (4) the DNA sequence encoding the polypeptide having halohydrin epoxidase activity of (2) above. , Consisting of the DNA sequence represented by SEQ ID NO: 4 or a part of the sequence thereof, and the like.
【０００７】以下に本発明を具体的に説明する。本発明
で使用し得る形質転換微生物におけるＤＮＡ供与体微生
物としては、コリネバクテリウムsp. N-1074（微工研条
寄第2643号）、ミクロバクテリウム sp.N-4701（微工研
条寄第2644号）等が挙げられ、その菌学的性質はそれぞ
れ特開平2-291280号公報に記載されている。ベクターと
しては、プラスミドベクター（例えば pUC18 、pUC19
、pUC118、pUC119等）、ファージベクター（例えばλg
t11等）のいずれでもよい。また、形質転換に用いられ
る宿主微生物としては、エシェリシア コリ(E. coli)
JM105 株あるいは同 JM109株が挙げられるが、特に、こ
れらに限定されるものではなく、他の宿主生物を用いる
こともできる。一例として、コリネバクテリウム sp. N
-1074 （微工研条寄第2643号）のハロヒドリンエポキシ
ダーゼ遺伝子の E. coli JM109株 へのクローニング
を、以下に示す。The present invention will be specifically described below. Examples of the DNA donor microorganism in the transformant microorganism that can be used in the present invention include Corynebacterium sp. N-1074 (Microtechnical Laboratory Article No. 2643), Microbacterium sp. N-4701 (Microtechnical Laboratory Article). No. 2644) and the like, and their mycological properties are described in JP-A-2-291280. Vectors include plasmid vectors (eg pUC18, pUC19
, PUC118, pUC119, etc.), phage vectors (eg λg
t11 or the like). The host microorganism used for transformation is Escherichia coli (E. coli).
Examples thereof include the JM105 strain and the JM109 strain, but the present invention is not particularly limited thereto, and other host organisms can be used. As an example, Corynebacterium sp. N
Cloning of the halohydrin epoxidase gene of -1074 (Microtechnology Research Institute No. 2643) into E. coli JM109 strain is shown below.
【０００８】(1) コリネバクテリウム sp. N-1074 染色
体ＤＮＡの調製とＤＮＡライブラリーの作成：コリネバ
クテリウム sp. N-1074 から Saito and Miuraの方法
〔Biochim.Biophys. Acta 72, 619(1963) 参照〕により
染色体ＤＮＡを分離し、これを制限酵素 (BamHI あるい
は BglII) で切断後、ベクタープラスミド pUC18に挿し
組換え体ＤＮＡのライブラリーを作成した。(1) Preparation of chromosomal DNA of Corynebacterium sp. N-1074 and preparation of DNA library: Method of Saito and Miura from Corynebacterium sp. N-1074 [Biochim. Biophys. Acta 72, 619 (1963) Chromosomal DNA was isolated by the method described in (4), cut with a restriction enzyme (BamHI or BglII), and inserted into vector plasmid pUC18 to prepare a recombinant DNA library.
【０００９】(2) 形質転換体の作成および組換え体ＤＮ
Ａの選別：工程(1) で調製した組換え体ライブラリーに
よる形質転換体を宿主生物としてE. coli JM109 株を用
いて塩化カルシウム法〔J. Mol. Biol. 53, 154 (197
0)〕により作成し、その中からハロヒドリンエポキシダ
ーゼ活性を示すようになったものを選別した。選別は以
下のようにして行った。アンピシリン(100μg/ml) とＩ
ＰＴＧ(1ml) を含むＬＢ寒天培地(1％バクトトリプト
ン、0.5 ％バクトイーストエキス、0.5 ％ＮａＣｌ、1.
5 ％寒天）に作成した形質転換体のコロニーを形成させ
た。10mMトリス−塩酸緩衝液(pH 7.5)、0.02％ブロモク
レゾールパープル、１％１，３−ジクロロ−２−プロパ
ノールを染み込ませたロ紙にコロニーを移し、室温にて
数時間放置した。ハロヒドリンエポキシダーゼ活性を持
つコロニーは塩酸を遊離しコロニー付近の pH は低下
し、pH指示薬であるブロモクレゾールパープルは青紫色
から黄色に変化するため、肉眼観察によりハロヒドリン
エポキシダーゼ遺伝子を持つ株を選別することができ
る。(2) Preparation of transformant and recombinant DN
Selection of A: Calcium chloride method [J. Mol. Biol. 53, 154 (197) using E. coli JM109 strain with the transformant prepared by the recombinant library prepared in step (1) as a host organism.
0)] and selected from them were those showing halohydrin epoxidase activity. The selection was performed as follows. Ampicillin (100 μg / ml) and I
LB agar medium containing PTG (1 ml) (1% bactotryptone, 0.5% bacto yeast extract, 0.5% NaCl, 1.
A colony of the prepared transformant was formed on 5% agar). The colonies were transferred to a paper impregnated with 10 mM Tris-hydrochloric acid buffer (pH 7.5), 0.02% bromocresol purple and 1% 1,3-dichloro-2-propanol, and left at room temperature for several hours. Colonies with halohydrin epoxidase activity release hydrochloric acid, the pH in the vicinity of the colonies drops, and the pH indicator bromocresol purple changes from blue-purple to yellow, so it has the halohydrin epoxidase gene by visual observation. The strain can be selected.
【００１０】これらの形質転換株が実際にハロヒドリン
エポキシダーゼ活性を有しているかどうかは次のように
して調べることができる。これらの株をアンピシリン
(50μg/ml) とＩＰＴＧ(1mM) を含むＬＢ培地(1％バク
トトリプトン、0.5 ％バクトイーストエキス、0.5 ％Ｎ
ａＣｌ）にて37℃で一夜培養する。菌体を50mMトリス−
硫酸緩衝液(pH 8)で２回洗浄後、１％１，３−ジクロロ
プロパノールを含む1Mトリス−硫酸緩衝液(pH 8)に懸濁
し、20℃にてインキュベートした。一定時間後、生成す
るエピクロルヒドリンをガスクロマトグラフィーにて定
量した。こうして得られた形質転換株から再びプラスミ
ドＤＮＡを取り出し、選別された２種の目的のプラスミ
ドを得た。これらのプラスミドをpST001およびpST005、
ならびにこれらのプラスミドが導入された形質転換体を
JM109/pST001 およびJM109/pST005と称する。Whether these transformants actually have halohydrin epoxidase activity can be examined as follows. Ampicillin these strains
(50 μg / ml) and IPTG (1 mM) in LB medium (1% bactotryptone, 0.5% bacto yeast extract, 0.5% N)
aCl) overnight at 37 ° C. 50mM Tris-
After washing twice with a sulfate buffer (pH 8), the cells were suspended in 1M Tris-sulfate buffer (pH 8) containing 1% 1,3-dichloropropanol and incubated at 20 ° C. After a certain period of time, the produced epichlorohydrin was quantified by gas chromatography. From the transformant thus obtained, the plasmid DNA was extracted again to obtain two selected plasmids of interest. These plasmids were designated pST001 and pST005,
And transformants into which these plasmids have been introduced
Referred to as JM109 / pST001 and JM109 / pST005.
【００１１】(3) 制限酵素地図の作成とハロヒドリンエ
ポキシダーゼ遺伝子の位置の決定：工程(2) で得られた
プラスミドについて制限酵素地図を作成した。その後、
より小さなＤＮＡ断片を持つプラスミドを作成した。こ
れらのプラスミドによって工程(2) と同様にして形質転
換された株のハロヒドリンエポキシダーゼ活性の有無に
よって目的遺伝子の含まれている箇所を決定した。この
過程で、pST001のBamHI-Bgl 1.3Kb 断片を含むpST015
(pUC118ベクター）およびpST005のBamHI-PstI1.1Kb 断
片を含む pST111(pUC118ベクター）プラスミドを作成し
た（図１）。これらのプラスミドが導入された形質転換
体をJM109/pST015およびJM109/pST111 と称する。(3) Construction of restriction enzyme map and determination of position of halohydrin epoxidase gene: A restriction enzyme map was prepared for the plasmid obtained in step (2). afterwards,
A plasmid with a smaller DNA fragment was created. The site containing the target gene was determined by the presence or absence of halohydrin epoxidase activity of the strain transformed with these plasmids in the same manner as in step (2). In the process, pST015 containing the BamHI-Bgl 1.3 Kb fragment of pST001
(pUC118 vector) and pST111 (pUC118 vector) plasmid containing the BamHI-PstI1.1Kb fragment of pST005 were constructed (FIG. 1). Transformants into which these plasmids were introduced are called JM109 / pST015 and JM109 / pST111.
【００１２】(4) 塩基配列の決定：工程(3) で得られた
プラスミドpST015およびpST111のハロヒドリンエポキシ
ダーゼ遺伝子に関する部分のＤＮＡの塩基配列を決定し
た（配列番号：５および配列番号：６）。なお、ここで
得られた形質転換体 JM109/pST001 、JM109/pST005、JM
109/pST015 およびJM109/pST111は、工業技術院微生物
工業技術研究所（微工研）に、それぞれ微工研菌寄第11
961 号、微工研菌寄第11962 号、微工研菌寄第12064 号
および微工研菌寄第12065 号として寄託されている。(4) Determination of nucleotide sequence: The nucleotide sequences of the DNAs of the plasmids pST015 and pST111 obtained in step (3) relating to the halohydrin epoxidase gene were determined (SEQ ID NO: 5 and SEQ ID NO: 6). ). The transformants JM109 / pST001, JM109 / pST005, JM
109 / pST015 and JM109 / pST111 were sent to the Institute of Microbial Science and Technology of the Institute of Industrial Science and Technology (Microtechnical Laboratory), respectively.
They have been deposited as 961, No. 11962, Mikken, No. 12064, No. 12064, and No. 12065, Mikken.
【００１３】本発明の形質転換微生物の培養は、通常は
液体培養で行われるが、固体培養によっても行うことが
できる。培地としては、例えばＬＢ培地が用いられる。
培養は10〜50℃の温度で、pH 2〜11の範囲で行われる。
微生物の生育を促進させるために通気攪拌を行ってもよ
い。Cultivation of the transformed microorganism of the present invention is usually carried out by liquid culture, but it can also be carried out by solid culture. As the medium, for example, LB medium is used.
Culturing is performed at a temperature of 10 to 50 ° C. and a pH range of 2 to 11.
Aeration and agitation may be carried out to promote the growth of microorganisms.
【００１４】培養により得られた形質転換微生物は、培
養液あるいは遠心分離などにより得た菌体の懸濁液に基
質を添加する方法、菌体処理物（例えば菌体破砕物、粗
酵素・精製酵素等の菌体抽出物等）あるいは常法により
固定化した菌体または菌体処理物等の懸濁液に基質を添
加する方法、微生物の培養時に基質を培養液に添加して
培養と同時に反応を行う方法等により、シアン化アルカ
リの存在下に、前記一般式〔Ｉ〕で示される１，２−エ
ポキシ化合物に作用させて、これを前記一般式〔II〕で
示される３−ヒドロキシニトリル化合物に変換すること
ができる。The transformed microorganisms obtained by culturing can be obtained by adding a substrate to a culture solution or a suspension of cells obtained by centrifugation or the like, treated cells (for example, disrupted cells, crude enzyme / purification). A method of adding a substrate to a suspension of a bacterial cell extract such as an enzyme or the like or a suspension of a bacterial cell or a treated product of a bacterial cell immobilized by a conventional method, and adding the substrate to a culture solution at the time of culturing a microorganism and simultaneously culturing By the reaction method or the like, the 1,2-epoxy compound represented by the general formula [I] is allowed to act in the presence of alkali cyanide to give the 3-hydroxynitrile represented by the general formula [II]. It can be converted into a compound.
【００１５】一般式〔Ｉ〕で示される１，２−エポキシ
化合物は、例えば、１，２−エポキシプロパン、１，２
−エポキシブタン、１，２−エポキシヘキサン等であ
る。また、シアン化アルカリは、シアン化カリウム、シ
アン化ナトリウム等である。反応液中の基質濃度は特に
限定するものではないが、 0.1〜10(W/V) ％が好まし
く、また、シアン化アルカリの使用量は、通常基質の１
〜３倍量（モル）である。基質は反応液に一括して加え
るかあるいは分割添加することができる。反応温度は５
〜50℃、反応 pH は４〜10の範囲で行うことが好まし
い。反応時間は基質等の濃度、菌体濃度あるいはその他
の反応条件等によって変わるが、通常１〜120 時間で終
了するように条件を設定するのが好ましい。The 1,2-epoxy compound represented by the general formula [I] is, for example, 1,2-epoxypropane, 1,2-epoxypropane.
-Epoxybutane, 1,2-epoxyhexane and the like. The alkali cyanide is potassium cyanide, sodium cyanide, or the like. The substrate concentration in the reaction solution is not particularly limited, but is preferably 0.1 to 10 (W / V)%, and the amount of alkali cyanide used is usually 1
~ 3 times the amount (mole). The substrate can be added to the reaction solution all at once or in portions. Reaction temperature is 5
It is preferable to carry out at -50 ° C and a reaction pH in the range of 4-10. The reaction time varies depending on the concentration of the substrate and the like, the cell concentration or other reaction conditions, but it is preferable to set the conditions so that the reaction is usually completed in 1 to 120 hours.
【００１６】かくして、反応液中に生成、蓄積した３−
ヒドロキシニトリル化合物は公知の方法を用いて採取お
よび精製することができる。例えば、反応液から遠心分
離などの方法を用いて菌体を除いた後、酢酸エチルなど
の溶媒で抽出を行い、減圧下に溶媒を除去することによ
り３−ヒドロキシニトリル化合物のシロップを得ること
ができる。また、これらのシロップを減圧下に蒸留する
ことによりさらに精製することもできる。Thus, the 3-produced and accumulated in the reaction solution
The hydroxynitrile compound can be collected and purified using a known method. For example, a syrup of a 3-hydroxynitrile compound can be obtained by removing cells from a reaction solution by using a method such as centrifugation, extracting with a solvent such as ethyl acetate, and removing the solvent under reduced pressure. it can. Further, these syrups can be further purified by distillation under reduced pressure.
【００１７】[0017]
【発明の効果】本発明によれば、遺伝子組換えの方法で
クローン化されたハロヒドリンエポキシダーゼ遺伝子が
菌体内に多数存在する形質転換微生物の使用により、シ
アン化アルカリの存在下、１，２−エポキシ化合物から
３−ヒドロキシニトリル化合物を効率よく製造すること
が可能である。INDUSTRIAL APPLICABILITY According to the present invention, the use of a transformed microorganism in which a large number of halohydrin epoxidase genes cloned by the method of gene recombination are present in the cells, It is possible to efficiently produce a 3-hydroxynitrile compound from a 2-epoxy compound.
【００１８】[0018]
実施例１ アンピシリン (50μg/ml) と1mM ＩＰＴＧを含むＬＢ培
地18 lにJM109/pST001を接種し37℃にて16時間振盪培養
を行った。得られた培養液から遠心分離により菌体を回
収し、100mM トリス−硫酸緩衝液(pH 8.0)50mlで洗浄
後、同緩衝液に懸濁し菌体懸濁液を調製した。菌体を超
音波破砕した後、遠心分離して沈澱物を除去し、上清を
菌体抽出液とした。常法にて硫安分画を行い、ＤＥＡＥ
−セファセル、Phenyl−セファロ−スおよび Octyl−セ
ファロ−ス（ファルマシア製）を用いたカラムクロマト
グラフィ−によって酵素を精製した。400mM のトリス−
硫酸緩衝液(pH 8.0)にシアン化カリウムを200mM になる
ように溶かした後、1Nの硫酸でpHを8.0 に調整し、この
溶液25mlに精製酵素溶液（タンパク濃度：30mg/ml)0.1m
l と400mM の１，２−エポキシブタン溶液25mlを加え、
20℃で２時間反応した。反応液をガスクロマトグラフィ
−で分析したところ、100mM の３−ヒドロキシバレロニ
トリルが生成していた。なお、本反応生成物は、反応液
から酢酸エチルで抽出することによって単離した後、Ｎ
ＭＲ、赤外吸光スペクトルおよびマススペクトルによる
分析から、３−ヒドロキシバレロニトリルであることを
確認した。Example 1 JM109 / pST001 was inoculated into 18 liters of LB medium containing ampicillin (50 μg / ml) and 1 mM IPTG, and shake culture was carried out at 37 ° C. for 16 hours. The cells were collected from the obtained culture solution by centrifugation, washed with 50 ml of 100 mM Tris-sulfate buffer (pH 8.0), and then suspended in the same buffer to prepare a cell suspension. The cells were ultrasonically disrupted and then centrifuged to remove the precipitate, and the supernatant was used as a cell extract. Ammonium sulphate fractionation is performed by the usual method, and DEAE
The enzyme was purified by column chromatography using -Sephacel, Phenyl-Sepharose and Octyl-Sepharose (Pharmacia). 400 mM Tris-
Dissolve potassium cyanide in a sulfuric acid buffer solution (pH 8.0) to a concentration of 200 mM, adjust the pH to 8.0 with 1N sulfuric acid, and add 25 ml of this solution to 0.1 m of purified enzyme solution (protein concentration: 30 mg / ml).
l and 25 ml of 400 mM 1,2-epoxybutane solution,
The reaction was carried out at 20 ° C for 2 hours. When the reaction liquid was analyzed by gas chromatography, it was found that 100 mM 3-hydroxyvaleronitrile was produced. The reaction product was isolated from the reaction solution by extraction with ethyl acetate,
From analysis by MR, infrared absorption spectrum and mass spectrum, it was confirmed to be 3-hydroxyvaleronitrile.
【００１９】実施例２ １，２−エポキシブタンの代わりに１，２−エポキシプ
ロパンを使用して実施例１と同様の反応を行ったとこ
ろ、71mMの３−ヒドロキシブチロニトリルが生成した。
本反応生成物は実施例１と同様にして同定した。Example 2 When 1,2-epoxypropane was used instead of 1,2-epoxybutane and the same reaction as in Example 1 was performed, 71 mM of 3-hydroxybutyronitrile was produced.
The reaction product was identified in the same manner as in Example 1.
【００２０】実施例３ 実施例１と同様にして調製した培地(100ml) に、それぞ
れJM109/pST015およびJM109/pST111を接種し、37℃にて
16時間培養を行った。これらの培養液をそれぞれ遠心分
離して菌体を集め、100mM トリス−硫酸緩衝液(pH 8.0)
50mlで洗浄後、同緩衝液25mlに懸濁し菌体懸濁液を調製
した。400mM のトリス−硫酸緩衝液(pH8.0)にシアン化
カリウムを200mM となるように溶かした後、1Nの硫酸で
pHを8.0に調整した溶液50mlを作成し、この溶液に上記
菌体懸濁液と200mM の１，２−エポキシブタン溶液をそ
れぞれ25ml加え、20℃で30分(JM109/pST015)および20℃
で15分(JM109/pST111)反応させた。反応液をガスクロマ
トグラフィ−で分析したところ、それぞれ11.1mM(JM109
/pST015)および7.2mM(JM109/pST111) の３−ヒドロキシ
バレロニトリルが生成していた。Example 3 A medium (100 ml) prepared in the same manner as in Example 1 was inoculated with JM109 / pST015 and JM109 / pST111, respectively, at 37 ° C.
Culture was performed for 16 hours. Collect these cells by centrifuging each of these cultures, and use 100 mM Tris-sulfate buffer (pH 8.0).
After washing with 50 ml, the cells were suspended in 25 ml of the same buffer to prepare a cell suspension. Dissolve potassium cyanide to 200 mM in 400 mM Tris-sulfate buffer (pH 8.0) and add 1 N sulfuric acid.
Prepare 50 ml of a solution with pH adjusted to 8.0, add 25 ml of the cell suspension and 200 mM 1,2-epoxybutane solution to this solution, and add at 20 ° C for 30 minutes (JM109 / pST015) and 20 ° C.
For 15 minutes (JM109 / pST111). When the reaction solution was analyzed by gas chromatography, it was found to be 11.1 mM (JM109
/ pST015) and 7.2 mM (JM109 / pST111) of 3-hydroxyvaleronitrile were formed.
【００２１】実施例４ １，２−エポキシブタンの代わりに１，２−エポキシプ
ロパン使用して実施例３と同様の反応を行ったところ、
それぞれ3.6mM(JM109/pST015) および6.2mM(JM109/pST1
11) の３−ヒドロキシブチロニトリルが生成していた。Example 4 When 1,2-epoxypropane was used instead of 1,2-epoxybutane and the same reaction as in Example 3 was carried out,
3.6 mM (JM109 / pST015) and 6.2 mM (JM109 / pST1) respectively
3-hydroxybutyronitrile of 11) was produced.
【００２２】[0022]
図１は組換え体プラスミドpST001、pST005、pST015およ
びpST111の制限酵素地図を示す。FIG. 1 shows a restriction map of recombinant plasmids pST001, pST005, pST015 and pST111.
【００２３】配列番号：１ 配列の長さ：244 配列の型：アミノ酸 トポロジ−：直鎖状 配列の種類：ペプチド 起源 生物名：コリネバクテリウム(Corynebacterium) 株名：N-1074 配列： Met Lys Ile Ala Leu Val Thr His Ala Arg His Phe Ala Gly Pro Ala 1 5 10 15 Ala Val Glu Ala Leu Thr Arg Asp Gly Tyr Thr Val Val Cys His Asp 20 25 30 Ala Thr Phe Ala Asp Ala Ala Glu Arg Gln Arg Phe Glu Ser Glu Asn 35 40 45 Pro Gly Thr Val Ala Leu Ala Glu Gln Lys Pro Glu Arg Leu Val Asp 50 55 60 Ala Thr Leu Gln His Gly Glu Ala Ile Asp Thr Ile Val Ser Asn Asp 65 70 75 80 Tyr Ile Pro Arg Pro Met Asn Arg Leu Pro Ile Glu Gly Thr Ser Glu 85 90 95 Ala Asp Ile Arg Gln Val Phe Glu Ala Leu Ser Ile Phe Pro Ile Leu 100 105 110 Leu Leu Gln Ser Ala Ile Ala Pro Leu Arg Ala Ala Gly Gly Ala Ser 115 120 125 Val Ile Phe Ile Thr Ser Ser Val Gly Lys Lys Pro Leu Ala Tyr Asn 130 135 140 Pro Leu Tyr Gly Pro Ala Arg Ala Ala Thr Val Ala Leu Val Glu Ser 145 150 155 160 Ala Ala Lys Thr Leu Ser Arg Asp Gly Ile Leu Leu Tyr Ala Ile Gly 165 170 175 Pro Asn Phe Phe Asn Asn Pro Thr Tyr Phe Pro Thr Ser Asp Trp Glu 180 185 190 Asn Asn Pro Glu Leu Arg Glu Arg Val Glu Arg Asp Val Pro Leu Gly 195 200 205 Arg Leu Gly Arg Pro Asp Glu Met Gly Ala Leu Ile Thr Phe Leu Ala 210 215 220 Ser Arg Arg Ala Ala Pro Ile Val Gly Gln Phe Phe Ala Phe Thr Gly 225 230 235 240 Gly Tyr Leu Pro SEQ ID NO: 1 Sequence length: 244 Sequence type: Amino acid Topology :: Linear Sequence type: Peptide Origin organism name: Corynebacterium strain name: N-1074 Sequence: Met Lys Ile Ala Leu Val Thr His Ala Arg His Phe Ala Gly Pro Ala 1 5 10 15 Ala Val Glu Ala Leu Thr Arg Asp Gly Tyr Thr Val Val Cys His Asp 20 25 30 Ala Thr Phe Ala Asp Ala Ala Glu Arg Gln Arg Phe Glu Ser Glu Asn 35 40 45 Pro Gly Thr Val Ala Leu Ala Glu Gln Lys Pro Glu Arg Leu Val Asp 50 55 60 Ala Thr Leu Gln His Gly Glu Ala Ile Asp Thr Ile Val Ser Asn Asp 65 70 75 80 Tyr Ile Pro Arg Pro Met Asn Arg Leu Pro Ile Glu Gly Thr Ser Glu 85 90 95 Ala Asp Ile Arg Gln Val Phe Glu Ala Leu Ser Ile Phe Pro Ile Leu 100 105 110 Leu Leu Gln Ser Ala Ile Ala Pro Leu Arg Ala Ala Gly Gly Ala Ser 115 120 125 Val Ile Phe Ile Thr Ser Ser Val Gly Lys Lys Pro Leu Ala Tyr Asn 130 135 140 Pro Leu Tyr Gly Pro Ala Arg Ala Ala Thr Val Ala Leu Val Glu Ser 145 150 155 160 Ala Ala Lys Th r Leu Ser Arg Asp Gly Ile Leu Leu Tyr Ala Ile Gly 165 170 175 Pro Asn Phe Phe Asn Asn Pro Thr Tyr Phe Pro Thr Ser Asp Trp Glu 180 185 190 Asn Asn Pro Glu Leu Arg Glu Arg Val Glu Arg Asp Val Pro Leu Gly 195 200 205 Arg Leu Gly Arg Pro Asp Glu Met Gly Ala Leu Ile Thr Phe Leu Ala 210 215 220 Ser Arg Arg Ala Ala Pro Ile Val Gly Gln Phe Phe Ala Phe Thr Gly 225 230 235 240 Gly Tyr Leu Pro
【００２４】配列番号：２ 配列の長さ：235 配列の型：アミノ酸 トポロジ−：直鎖状 配列の種類：ペプチド 起源 生物名：コリネバクテリウム(Corynebacterium) 株名：N-1074 配列： Met Ala Asn Gly Arg Lys Arg Glu Met Ala Asn Gly Arg Leu Ala Gly 1 5 10 15 Lys Arg Val Leu Leu Thr Asn Ala Asp Ala Tyr Met Gly Glu Ala Thr 20 25 30 Val Gln Val Phe Glu Glu Glu Gly Ala Glu Val Ile Ala Asp His Thr 35 40 45 Asp Leu Thr Lys Val Gly Ala Ala Glu Glu Val Val Glu Arg Ala Gly 50 55 60 His Ile Asp Val Leu Val Ala Asn Phe Ala Val Asp Ala His Phe Gly 65 70 75 80 Val Thr Val Leu Glu Thr Asp Glu Glu Leu Trp Gln Thr Ala Tyr Glu 85 90 95 Thr Ile Val His Pro Leu His Arg Ile Cys Arg Ala Val Leu Pro Gln 100 105 110 Phe Tyr Glu Arg Asn Lys Gly Lys Ile Val Val Tyr Gly Ser Ala Ala 115 120 125 Ala Met Arg Tyr Gln Glu Gly Ala Leu Ala Tyr Ser Thr Ala Arg Phe 130 135 140 Ala Gln Arg Gly Tyr Val Thr Ala Leu Gly Pro Glu Ala Ala Arg His 145 150 155 160 Asn Val Asn Val Asn Phe Ile Ala Gln His Trp Thr Gln Asn Lys Glu 165 170 175 Tyr Phe Trp Pro Glu Arg Ile Ala Thr Asp Glu Phe Lys Glu Asp Met 180 185 190 Ala Arg Arg Val Pro Leu Gly Arg Leu Ala Thr Ala Arg Glu Asp Ala 195 200 205 Leu Leu Ala Leu Phe Leu Ala Ser Asp Glu Ser Asp Phe Ile Val Gly 210 215 220 Lys Ser Ile Glu Phe Asp Gly Gly Trp Ala Thr 225 230 235 SEQ ID NO: 2 Sequence length: 235 Sequence type: Amino acid Topology :: Linear Sequence type: Peptide Origin organism name: Corynebacterium strain name: N-1074 Sequence: Met Ala Asn Gly Arg Lys Arg Glu Met Ala Asn Gly Arg Leu Ala Gly 1 5 10 15 Lys Arg Val Leu Leu Thr Asn Ala Asp Ala Tyr Met Gly Glu Ala Thr 20 25 30 Val Gln Val Phe Glu Glu Glu Gly Ala Glu Val Ile Ala Asp His Thr 35 40 45 Asp Leu Thr Lys Val Gly Ala Ala Glu Glu Val Val Glu Arg Ala Gly 50 55 60 His Ile Asp Val Leu Val Ala Asn Phe Ala Val Asp Ala His Phe Gly 65 70 75 80 Val Thr Val Leu Glu Thr Asp Glu Glu Leu Trp Gln Thr Ala Tyr Glu 85 90 95 Thr Ile Val His Pro Leu His Arg Ile Cys Arg Ala Val Leu Pro Gln 100 105 110 Phe Tyr Glu Arg Asn Lys Gly Lys Ile Val Val Tyr Gly Ser Ala Ala 115 120 125 Ala Met Arg Tyr Gln Glu Gly Ala Leu Ala Tyr Ser Thr Ala Arg Phe 130 135 140 Ala Gln Arg Gly Tyr Val Thr Ala Leu Gly Pro Glu Ala Ala Arg His 145 150 155 160 Asn Val Asn V al Asn Phe Ile Ala Gln His Trp Thr Gln Asn Lys Glu 165 170 175 Tyr Phe Trp Pro Glu Arg Ile Ala Thr Asp Glu Phe Lys Glu Asp Met 180 185 190 Ala Arg Arg Val Pro Leu Gly Arg Leu Ala Thr Ala Arg Glu Asp Ala 195 200 205 Leu Leu Ala Leu Phe Leu Ala Ser Asp Glu Ser Asp Phe Ile Val Gly 210 215 220 Lys Ser Ile Glu Phe Asp Gly Gly Trp Ala Thr 225 230 235
【００２５】配列番号：３ 配列の長さ：732 配列の型：核酸 鎖の数：一本鎖 トポロジ−：直鎖状 配列の種類：Genomic DNA 起源 生物名：コリネバクテリウム(Corynebacterium) 株名：N-1074 配列： ATG AAG ATC GCC CTC GTG ACT CAT GCA CGG CAT TTT GCA GGC CCC GCC 48 GCC GTC GAG GCG CTT ACG CGG GAT GGC TAT ACC GTG GTT TGC CAC GAC 96 GCG ACG TTC GCT GAT GCA GCT GAA CGA CAG CGT TTC GAG TCG GAG AAC 144 CCG GGC ACC GTC GCG CTC GCC GAG CAG AAG CCC GAG CGT CTG GTC GAC 192 GCC ACG CTG CAG CAC GGG GAA GCG ATC GAC ACG ATC GTC TCG AAC GAT 240 TAC ATT CCG CGC CCG ATG AAT CGG CTC CCG ATC GAG GGA ACG AGC GAG 288 GCC GAC ATC CGA CAG GTG TTC GAG GCG CTC AGC ATC TTC CCG ATC CTG 336 CTC CTG CAG TCG GCC ATC GCG CCG CTA CGG GCT GCA GGC GGC GCC TCC 384 GTT ATC TTC ATC ACG TCC TCA GTT GGC AAG AAG CCG CTC GCC TAC AAC 432 CCT CTC TAT GGG CCC GCG CGC GCC GCT ACC GTC GCG CTT GTC GAA TCG 480 GCA GCG AAG ACG CTG TCC CGT GAC GGA ATC TTG CTC TAC GCG ATC GGT 528 CCG AAC TTC TTC AAC AAC CCG ACG TAC TTC CCG ACG TCG GAT TGG GAG 576 AAC AAC CCC GAG CTC CGG GAG CGT GTC GAG CGG GAC GTG CCG CTC GGT 624 CGC CTC GGC CGT CCG GAC GAG ATG GGT GCG CTG ATC ACC TTC CTC GCT 672 TCG CGT CGT GCA GCG CCC ATC GTG GGG CAG TTC TTC GCT TTC ACC GGT 720 GGC TAT CTG CCC 732SEQ ID NO: 3 Sequence length: 732 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Genomic DNA Origin organism name: Corynebacterium strain name: N-1074 Sequence: ATG AAG ATC GCC CTC GTG ACT CAT GCA CGG CAT TTT GCA GGC CCC GCC 48 GCC GTC GAG GCG CTT ACG CGG GAT GGC TAT ACC GTG GTT TGC CAC GAC 96 GCG ACG TTC GCT GAT GCA GCT GAA CGA CAG CGT TTC GAG TCG GAG AAC 144 CCG GGC ACC GTC GCG CTC GCC GAG CAG AAG CCC GAG CGT CTG GTC GAC 192 GCC ACG CTG CAG CAC GGG GAA GCG ATC GAC ACG ATC GTC TCG AAC GAT 240 TAC ATT CCG CGC CCG ATG AAT CGG CTC CTC ATC GAG GGA ACG AGC GAG 288 GCC GAC ATC CGA CAG GTG TTC GAG GCG CTC AGC ATC TTC CCG ATC CTG 336 CTC CTG CAG TCG GCC ATC GCG CCG CTA CGG GCT GCA GGC GGC GCC TCC 384 GTT ATC TTC ATC ACG TCC TCA GTT AAG AAG CCG CTC GCC TAC AAC 432 CCT CTC TAT GGG CCC GCG CGC GCC GCT ACC GTC GCG CTT GTC GAA TCG 480 GCA GCG AAG ACG CTG TCC CGT GAC GGA ATC TTG CTC TAC GCG ATC GGT 528 CCG AAC TTC TTC AAC AAC CCG ACG TAC TTC CCG ACG TCG GAT TGG GAG 576 AAC AAC CCC GAG CTC CGG GAG CGT GTC GAG CGG GAC GTG CCG CTC GGT 624 CGC CTC GGC CGT CCG GAC GAG ATG GGT GCG CTG ATC ACC TTC CTC GCT TCG CGT CGT GCA GCG CCC ATC GTG GGG CAG TTC TTC GCT TTC ACC GGT 720 GGC TAT CTG CCC 732
【００２６】配列番号：４ 配列の長さ：705 配列の型：核酸 鎖の数：一本鎖 トポロジ−：直鎖状 配列の種類：Genomic DNA 起源 生物名：コリネバクテリウム(Corynebacterium) 株名：N-1074 配列： ATG GCT AAC GGA AGG AAA AGG GAA ATG GCT AAC GGA AGA CTG GCA GGC 48 AAG CGG GTC CTA CTC ACG AAC GCC GAT GCC TAC ATG GGT GAG GCC ACG 96 GTC CAG GTG TTC GAG GAG GAG GGC GCA GAG GTC ATC GCT GAC CAC ACC 144 GAC TTG ACG AAG GTC GGC GCG GCG GAG GAG GTC GTC GAG AGG GCT GGG 192 CAC ATC GAT GTC CTG GTG GCC AAC TTC GCG GTC GAC GCC CAC TTC GGG 240 GTG ACC GTG CTG GAG ACC GAC GAG GAG CTG TGG CAG ACG GCC TAC GAG 288 ACC ATC GTG CAC CCG CTG CAT CGG ATC TGC CGT GCG GTG CTC CCG CAG 336 TTC TAC GAG CGG AAC AAG GGC AAG ATC GTT GTC TAC GGA AGT GCC GCA 384 GCG ATG CGG TAC CAG GAA GGT GCG CTG GCC TAC AGC ACG GCG CGT TTC 432 GCT CAG CGC GGG TAC GTC ACC GCC CTC GGT CCC GAG GCA GCG AGG CAC 480 AAC GTC AAC GTG AAC TTC ATC GCC CAG CAC TGG ACC CAA AAC AAG GAG 528 TAC TTC TGG CCC GAG CGC ATC GCC ACC GAC GAG TTC AAG GAG GAT ATG 576 GCG CGC CGA GTT CCC CTG GGT CGG CTC GCG ACT GCC CGA GAG GAC GCG 624 CTG CTC GCG TTG TTC CTG GCC TCG GAC GAG AGT GAC TTC ATC GTC GGC 672 AAG TCG ATC GAG TTC GAC GGC GGC TGG GCC ACC 705SEQ ID NO: 4 Sequence length: 705 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Genomic DNA Origin organism name: Corynebacterium strain name: N-1074 Sequence: ATG GCT AAC GGA AGG AAA AGG GAA ATG GCT AAC GGA AGA CTG GCA GGC 48 AAG CGG GTC CTA CTC ACG AAC GCC GAT GCC TAC ATG GGT GAG GCC ACG 96 GTC CAG GTG TTC GAG GAG GAG GGC GCA GAG GTC ATC GCT GAC CAC ACC 144 GAC TTG ACG AAG GTC GGC GCG GCG GAG GAG GTC GTC GAG AGG GCT GGG 192 CAC ATC GAT GTC CTG GTG GCC AAC TTC GCG GTC GAC GCC CAC TTC GGG 240 GTG ACC GTG CTG GAG ACC GAC GAG GAG CTG TGG CAG ACG GCC TAC GAG 288 ACC ATC GTG CAC CCG CTG CAT CGG ATC TGC CGT GCG GTG CTC CCG CAG 336 TTC TAC GAG CGG AAC AAG GGC AAG ATC GTT GTC TAC GGA AGT GCC GCA 384 GCG ATG CGG TAC CAG GAA GGT GCC TAC AGC ACG GCG CGT TTC 432 GCT CAG CGC GGG TAC GTC ACC GCC CTC GGT CCC GAG GCA GCG AGG CAC 480 AAC GTC AAC GTG AAC TTC ATC GCC CAG CAC TGG ACC CAA AAC AAG GAG 528 TAC TTC TGG CCC GAG CGC ATC GCC ACC GAC GAG TTC AAG GAG GAT ATG 576 GCG CGC CGA GTT CCC CTG GGT CGG CTC GCG ACT GCC CGA GAG GAC GCG 624 CTG CTC GCG TTG TTC CTG GCC TCG GAC GAG AGT GAC TTC ATC GTC GTC AAG TCG ATC GAG TTC GAC GGC GGC TGG GCC ACC 705
【００２７】配列番号：５ 配列の長さ：829 配列の型：核酸 鎖の数：一本鎖 トポロジ−：直鎖状 配列の種類：Genomic DNA 起源 生物名：コリネバクテリウム(Corynebacterium) 株名：N-1074 配列： GAATTCCAGA ACCAATTGAG AGGAAATGAA CA ATG AAG ATC GCC CTC GTG ACT 53 Met Lys Ile Ala Leu Val Thr 1 5 CAT GCA CGG CAT TTT GCA GGC CCC GCC GCC GTC GAG GCG CTT ACG CGG 101 His Ala Arg His Phe Ala Gly Pro Ala Ala Val Glu Ala Leu Thr Arg 10 15 20 GAT GGC TAT ACC GTG GTT TGC CAC GAC GCG ACG TTC GCT GAT GCA GCT 149 Asp Gly Tyr Thr Val Val Cys His Asp Ala Thr Phe Ala Asp Ala Ala 25 30 35 GAA CGA CAG CGT TTC GAG TCG GAG AAC CCG GGC ACC GTC GCG CTC GCC 197 Glu Arg Gln Arg Phe Glu Ser Glu Asn Pro Gly Thr Val Ala Leu Ala 40 45 50 55 GAG CAG AAG CCC GAG CGT CTG GTC GAC GCC ACG CTG CAG CAC GGG GAA 245 Glu Gln Lys Pro Glu Arg Leu Val Asp Ala Thr Leu Gln His Gly Glu 60 65 70 GCG ATC GAC ACG ATC GTC TCG AAC GAT TAC ATT CCG CGC CCG ATG AAT 293 Ala Ile Asp Thr Ile Val Ser Asn Asp Tyr Ile Pro Arg Pro Met Asn 75 80 85 CGG CTC CCG ATC GAG GGA ACG AGC GAG GCC GAC ATC CGA CAG GTG TTC 341 Arg Leu Pro Ile Glu Gly Thr Ser Glu Ala Asp Ile Arg Gln Val Phe 90 95 100 GAG GCG CTC AGC ATC TTC CCG ATC CTG CTC CTG CAG TCG GCC ATC GCG 389 Glu Ala Leu Ser Ile Phe Pro Ile Leu Leu Leu Gln Ser Ala Ile Ala 105 110 115 CCG CTA CGG GCT GCA GGC GGC GCC TCC GTT ATC TTC ATC ACG TCC TCA 437 Pro Leu Arg Ala Ala Gly Gly Ala Ser Val Ile Phe Ile Thr Ser Ser 120 125 130 135 GTT GGC AAG AAG CCG CTC GCC TAC AAC CCT CTC TAT GGG CCC GCG CGC 485 Val Gly Lys Lys Pro Leu Ala Tyr Asn Pro Leu Tyr Gly Pro Ala Arg 140 145 150 GCC GCT ACC GTC GCG CTT GTC GAA TCG GCA GCG AAG ACG CTG TCC CGT 533 Ala Ala Thr Val Ala Leu Val Glu Ser Ala Ala Lys Thr Leu Ser Arg 155 160 165 GAC GGA ATC TTG CTC TAC GCG ATC GGT CCG AAC TTC TTC AAC AAC CCG 581 Asp Gly Ile Leu Leu Tyr Ala Ile Gly Pro Asn Phe Phe Asn Asn Pro 170 175 180 ACG TAC TTC CCG ACG TCG GAT TGG GAG AAC AAC CCC GAG CTC CGG GAG 629 Thr Tyr Phe Pro Thr Ser Asp Trp Glu Asn Asn Pro Glu Leu Arg Glu 185 190 195 CGT GTC GAG CGG GAC GTG CCG CTC GGT CGC CTC GGC CGT CCG GAC GAG 677 Arg Val Glu Arg Asp Val Pro Leu Gly Arg Leu Gly Arg Pro Asp Glu 200 205 210 215 ATG GGT GCG CTG ATC ACC TTC CTC GCT TCG CGT CGT GCA GCG CCC ATC 725 Met Gly Ala Leu Ile Thr Phe Leu Ala Ser Arg Arg Ala Ala Pro Ile 220 225 230 GTG GGG CAG TTC TTC GCT TTC ACC GGT GGC TAT CTG CCC TAACCCGCGC 774 Val Gly Gln Phe Phe Ala Phe Thr Gly Gly Tyr Leu Pro 235 240 CGGTACGGCA ACAGGAAGGA CTGTCTGACA CGGTTCGTCC TCCCAACGCG CCGGC 829SEQ ID NO: 5 Sequence length: 829 Sequence type: Nucleic acid Number of strands: Single strand Topology-: Linear Sequence type: Genomic DNA Origin organism name: Corynebacterium strain name: N-1074 Sequence: GAATTCCAGA ACCAATTGAG AGGAAATGAA CA ATG AAG ATC GCC CTC GTG ACT 53 Met Lys Ile Ala Leu Val Thr 1 5 CAT GCA CGG CAT TTT GCA GGC CCC GCC GCC GTC GAG GCG CTT ACG CGG 101 His Ala Arg His Phe Ala Gly Pro Ala Ala Val Glu Ala Leu Thr Arg 10 15 20 GAT GGC TAT ACC GTG GTT TGC CAC GAC GCG ACG TTC GCT GAT GCA GCT 149 Asp Gly Tyr Thr Val Val Cys His Asp Ala Thr Phe Ala Asp Ala Ala 25 30 35 GAA CGA CAG CGT TTC GAG TCG GAG AAC CCG GGC ACC GTC GCG CTC GCC 197 Glu Arg Gln Arg Phe Glu Ser Glu Asn Pro Gly Thr Val Ala Leu Ala 40 45 50 55 GAG CAG AAG CCC GAG CGT CTG GTC GAC GCC ACG CTG CAG CAC GGG GAA 245 Glu Gln Lys Pro Glu Arg Leu Val Asp Ala Thr Leu Gln His Gly Glu 60 65 70 GCG ATC GAC ACG ATC GTC TCG AAC GAT TAC ATT CCG CGC CCG ATG AAT 293 Ala Ile Asp Thr Ile Val Ser Asn Asp Tyr Ile Pro Arg Pro Met Asn 75 80 85 CGG CTC CCG ATC GAG GGA ACG AGC GAG GCC GAC ATC CGA CAG GTG TTC 341 Arg Leu Pro Ile Glu Gly Thr Ser Glu Ala Asp Ile Arg Gln Val Phe 90 95 100 GAG GCG CTC AGC ATC TTC CCG ATC CTG CTC CTG CAG TCG GCC ATC GCG 389 Glu Ala Leu Ser Ile Phe Pro Ile Leu Leu Leu Gln Ser Ala Ile Ala 105 110 115 CCG CTA CGG GCT GCA GGC GGC GCC TCC GTT ATC TTC ATC ACG TCC TCA 437 Pro Leu Arg Ala Ala Gly Gly Ala Ser Val Ile Phe Ile Thr Ser Ser 120 125 130 135 GTT GGC AAG AAG CCG CTC GCC TAC AAC CCT CTC TAT GGG CCC GCG CGC 485 Val Gly Lys Lys Pro Leu Ala Tyr Asn Pro Leu Tyr Gly Pro Ala Arg 140 145 150 GCC GCT ACC GTC GCG CTT GTC GAA TCG GCA GCG AAG ACG CTG TCC CGT 533 Ala Ala Thr Val Ala Leu Val Glu Ser Ala Ala Lys Thr Leu Ser Arg 155 160 165 GAC GGA ATC TTG CTC TAC GCG ATC GGT CCG AAC TTC TTC AAC AAC CCG 581 Asp Gly Ile Leu Leu Tyr Ala Ile Gly Pro Asn Phe Phe Asn Asn Pro 170 175 180 ACG TAC TTC CCG ACG TCG GAT TGG GAG AAC AAC CCC GAG CTC CGG GAG 629 Thr Tyr Phe Pro Thr Ser Asp Trp Glu Asn Asn Pro Glu Leu Arg Glu 185 190 195 CGT GTC GAG CGG GAC GTG CCG CTC GGT CGC CTC GGC CGT CCG GAC GAG 677 Arg Val Glu Arg Asp Val Pro Leu Gly Arg Leu Gly Arg Pro Asp Glu 200 205 210 215 ATG GGT GCG CTG ATC ACC TTC CTC GCT TCG CGT CGT GCA GCG CCC ATC 725 Met Gly Ala Leu Ile Thr Phe Leu Ala Ser Arg Arg Ala Ala Pro Ile 220 225 230 GTG GGG CAG TTC TTC GCT TTC ACC GGT GGC TAT CTG CCC TAACCCGCGC 774 Val Gly Gln Phe Phe Ala Phe Thr Gly Gly Tyr Leu Pro 235 240 CGGTACGGCA ACAGGAAGGA CTGTCTGACA CGGTTCGTCC TCCCAACGCG CCGGC 829
【００２８】配列番号：６ 配列の長さ：843 配列の型：核酸 鎖の数：一本鎖 トポロジ−：直鎖状 配列の種類：Genomic DNA 起源 生物名：コリネバクテリウム(Corynebacterium) 株名：N-1074 配列： GTCGACTAGA GAAGGTATTC CGACTGCTGC GGTGCCTGGC ACCGCAGCAA AAGATTCAAG 60 GATTCTCGAA GAAAGGAAAA GGGAA ATG GCT AAC GGA AGG AAA AGG GAA ATG 112 Met Ala Asn Gly Arg Lys Arg Glu Met 1 5 GCT AAC GGA AGA CTG GCA GGC AAG CGG GTC CTA CTC ACG AAC GCC GAT 160 Ala Asn Gly Arg Leu Ala Gly Lys Arg Val Leu Leu Thr Asn Ala Asp 10 15 20 25 GCC TAC ATG GGT GAG GCC ACG GTC CAG GTG TTC GAG GAG GAG GGC GCA 208 Ala Tyr Met Gly Glu Ala Thr Val Gln Val Phe Glu Glu Glu Gly Ala 30 35 40 GAG GTC ATC GCT GAC CAC ACC GAC TTG ACG AAG GTC GGC GCG GCG GAG 256 Glu Val Ile Ala Asp His Thr Asp Leu Thr Lys Val Gly Ala Ala Glu 45 50 55 GAG GTC GTC GAG AGG GCT GGG CAC ATC GAT GTC CTG GTG GCC AAC TTC 304 Glu Val Val Glu Arg Ala Gly His Ile Asp Val Leu Val Ala Asn Phe 60 65 70 GCG GTC GAC GCC CAC TTC GGG GTG ACC GTG CTG GAG ACC GAC GAG GAG 352 Ala Val Asp Ala His Phe Gly Val Thr Val Leu Glu Thr Asp Glu Glu 75 80 85 CTG TGG CAG ACG GCC TAC GAG ACC ATC GTG CAC CCG CTG CAT CGG ATC 400 Leu Trp Gln Thr Ala Tyr Glu Thr Ile Val His Pro Leu His Arg Ile 90 95 100 105 TGC CGT GCG GTG CTC CCG CAG TTC TAC GAG CGG AAC AAG GGC AAG ATC 448 Cys Arg Ala Val Leu Pro Gln Phe Tyr Glu Arg Asn Lys Gly Lys Ile 110 115 120 GTT GTC TAC GGA AGT GCC GCA GCG ATG CGG TAC CAG GAA GGT GCG CTG 496 Val Val Tyr Gly Ser Ala Ala Ala Met Arg Tyr Gln Glu Gly Ala Leu 125 130 135 GCC TAC AGC ACG GCG CGT TTC GCT CAG CGC GGG TAC GTC ACC GCC CTC 544 Ala Tyr Ser Thr Ala Arg Phe Ala Gln Arg Gly Tyr Val Thr Ala Leu 140 145 150 GGT CCC GAG GCA GCG AGG CAC AAC GTC AAC GTG AAC TTC ATC GCC CAG 592 Gly Pro Glu Ala Ala Arg His Asn Val Asn Val Asn Phe Ile Ala Gln 155 160 165 CAC TGG ACC CAA AAC AAG GAG TAC TTC TGG CCC GAG CGC ATC GCC ACC 640 His Trp Thr Gln Asn Lys Glu Tyr Phe Trp Pro Glu Arg Ile Ala Thr 170 175 180 185 GAC GAG TTC AAG GAG GAT ATG GCG CGC CGA GTT CCC CTG GGT CGG CTC 688 Asp Glu Phe Lys Glu Asp Met Ala Arg Arg Val Pro Leu Gly Arg Leu 190 195 200 GCG ACT GCC CGA GAG GAC GCG CTG CTC GCG TTG TTC CTG GCC TCG GAC 736 Ala Thr Ala Arg Glu Asp Ala Leu Leu Ala Leu Phe Leu Ala Ser Asp 205 210 215 GAG AGT GAC TTC ATC GTC GGC AAG TCG ATC GAG TTC GAC GGC GGC TGG 784 Glu Ser Asp Phe Ile Val Gly Lys Ser Ile Glu Phe Asp Gly Gly Trp 220 225 230 GCC ACC TGAGAGACGT CACAGCCCCC TCGGGCAGGC GCTCGTCGTC GTTGTAGCTG CAG 843 Ala Thr 235SEQ ID NO: 6 Sequence length: 843 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Genomic DNA Origin organism name: Corynebacterium strain name: N-1074 Sequence: GTCGACTAGA GAAGGTATTC CGACTGCTGC GGTGCCTGGC ACCGCAGCAA AAGATTCAAG 60 GATTCTCGAA GAAAGGAAAA GGGAA ATG GCT AAC GGA AGG AAA AGG GAA ATG 112 Met Ala Asn Gly Arg Lys Arg Glu Met 1 5 GCT CTC ACA GGA AGA GCC GAT 160 Ala Asn Gly Arg Leu Ala Gly Lys Arg Val Leu Leu Thr Asn Ala Asp 10 15 20 25 GCC TAC ATG GGT GAG GCC ACG GTC CAG GTG TTC GAG GAG GAG GGC GCA 208 Ala Tyr Met Gly Glu Ala Thr Val Gln Val Phe Glu Glu Glu Gly Ala 30 35 40 GAG GTC ATC GCT GAC CAC ACC GAC TTG ACG AAG GTC GGC GCG GCG GAG 256 Glu Val Ile Ala Asp His Thr Asp Leu Thr Lys Val Gly Ala Ala Glu 45 50 55 GAG GTC GTC GAG AGG GCT GGG CAC ATC GAT GTC CTG GTG GCC AAC TTC 304 Glu Val Val Glu Arg Ala Gly His Ile Asp Val Leu Val Ala Asn Phe 60 65 70 GCG G TC GAC GCC CAC TTC GGG GTG ACC GTG CTG GAG ACC GAC GAG GAG 352 Ala Val Asp Ala His Phe Gly Val Thr Val Leu Glu Thr Asp Glu Glu 75 80 85 CTG TGG CAG ACG GCC TAC GAG ACC ATC GTG CAC CCG CTG CAT CGG ATC 400 Leu Trp Gln Thr Ala Tyr Glu Thr Ile Val His Pro Leu His Arg Ile 90 95 100 105 TGC CGT GCG GTG CTC CCG CAG TTC TAC GAG CGG AAC AAG GGC AAG ATC 448 Cys Arg Ala Val Leu Pro Gln Phe Tyr Glu Arg Asn Lys Gly Lys Ile 110 115 120 GTT GTC TAC GGA AGT GCC GCA GCG ATG CGG TAC CAG GAA GGT GCG CTG 496 Val Val Tyr Gly Ser Ala Ala Ala Met Arg Tyr Gln Glu Gly Ala Leu 125 130 135 GCC TAC AGC ACG GCG CGT TTC GCT CAG CGC GGG TAC GTC ACC GCC CTC 544 Ala Tyr Ser Thr Ala Arg Phe Ala Gln Arg Gly Tyr Val Thr Ala Leu 140 145 150 GGT CCC GAG GCA GCG AGG CAC AAC GTC AAC GTG AAC TTC ATC GCC CAG 592 Gly Pro Glu Ala Ala Arg His Asn Val Asn Val Asn Phe Ile Ala Gln 155 160 165 CAC TGG ACC CAA AAC AAG GAG TAC TTC TGG CCC GAG CGC ATC GCC ACC 640 His Trp Thr Gln Asn Lys Glu Tyr Phe Trp Pro Glu Arg Ile Ala Thr 170 175 1 80 185 GAC GAG TTC AAG GAG GAT ATG GCG CGC CGA GTT CCC CTG GGT CGG CTC 688 Asp Glu Phe Lys Glu Asp Met Ala Arg Arg Val Pro Leu Gly Arg Leu 190 195 200 GCG ACT GCC CGA GAG GAC GCG CTG CTC GCG TTG TTC CTG GCC TCG GAC 736 Ala Thr Ala Arg Glu Asp Ala Leu Leu Ala Leu Phe Leu Ala Ser Asp 205 210 215 GAG AGT GAC TTC ATC GTC GGC AAG TCG ATC GAG TTC GAC GGC GGC TGG 784 Glu Ser Asp Phe Ile Val Gly Lys Ser Ile Glu Phe Asp Gly Gly Trp 220 225 230 GCC ACC TGAGAGACGT CACAGCCCCC TCGGGCAGGC GCTCGTCGTC GTTGTAGCTG CAG 843 Ala Thr 235
─────────────────────────────────────────────────────
─────────────────────────────────────────────────── ───
【手続補正書】[Procedure amendment]
【提出日】平成５年３月２２日[Submission date] March 22, 1993
【手続補正１】[Procedure Amendment 1]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】発明の詳細な説明[Name of item to be amended] Detailed explanation of the invention
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【発明の詳細な説明】Detailed Description of the Invention
【０００１】[0001]
【産業上の利用分野】本発明は、１，３−ジハロ−２−
プロパノールをエピハロヒドリンに変換する活性および
その逆反応を触媒する活性を有する酵素（以下ハロヒド
リンエポキシダーゼと略す）遺伝子ＤＮＡをベクタープ
ラスミドに連結した組換え体プラスミドを宿主微生物に
導入した形質転換微生物による、３−ヒドロキシニトリ
ル化合物の製造法に関する。３−ヒドロキシニトリル化
合物は、種々の医薬品や生理活性物質の合成原料として
有用な物質として知られている。This invention relates to 1,3-dihalo-2-
An enzyme having an activity of converting propanol to epihalohydrin and an activity of catalyzing its reverse reaction (hereinafter abbreviated as halohydrin epoxidase) by a transformed microorganism in which a recombinant plasmid in which a gene DNA is ligated to a vector plasmid is introduced into a host microorganism. , A method for producing a 3-hydroxynitrile compound. The 3-hydroxynitrile compound is known as a substance useful as a raw material for synthesizing various drugs and physiologically active substances.
【０００２】[0002]
【発明の背景】脱ハロゲン化酵素の作用によりエピハロ
ヒドリンから４−ハロ−３−ヒドロキシブチロニトリル
を製造する方法は、先に、本発明者らの一部により見出
されている（特願平１−１８５９９２号明細書参照）。
しかし、これら脱ハロゲン化酵素を有する微生物の触媒
能力は高くなく、工業的に用いる場合には満足できるも
のではない。BACKGROUND OF THE INVENTION A method for producing 4-halo-3-hydroxybutyronitrile from epihalohydrin by the action of a dehalogenase has been previously found by a part of the present inventors (Japanese Patent Application No. Hei 10 (1999) -135242). No. 1-185992).
However, the catalytic ability of microorganisms having these dehalogenating enzymes is not high, and they are not satisfactory for industrial use.
【０００３】[0003]
【発明が解決しようとする課題】遺伝子組換えの方法で
クローン化された脱ハロゲン化酵素遺伝子によるエピハ
ロヒドリンから４−ハロ−３−ヒドロキシブチロニトリ
ルへの変換反応では、菌体内に多数の遺伝子を存在させ
ることができるため微生物の触媒能力を従来の方法に比
して飛躍的に増大させることが期待できる。このような
状況下、本発明者らの一部は、エピハロヒドリンを４−
ハロ−３−ヒドロキシブチロニトリルに変換する酵素が
ハロヒドリンエポキシダーゼであることを見い出し、さ
らに微生物由来のハロヒドリンエポキシダーゼ酵素遺伝
子ＤＮＡをベクタープラスミドに挿入して組換え体プラ
スミドを得、この組換え体プラスミドを導入した形質転
換体によりハロヒドリンエポキシダーゼを発現させるこ
とに成功した。[Problems to be Solved by the Invention] In the conversion reaction of epihalohydrin to 4-halo-3-hydroxybutyronitrile by the dehalogenase gene cloned by the method of gene recombination, a large number of genes are Since it can be present, it can be expected to dramatically increase the catalytic ability of microorganisms as compared with conventional methods. Under these circumstances, some of the present inventors have reported that epihalohydrin has a 4-
It was found that the enzyme converting into halo-3-hydroxybutyronitrile is halohydrin epoxidase, and further, halohydrin epoxidase enzyme gene DNA derived from a microorganism was inserted into a vector plasmid to obtain a recombinant plasmid, We succeeded in expressing a halohydrin epoxidase by the transformant introduced with this recombinant plasmid.
【０００４】[0004]
【問題を解決するための手段】本発明は、この形質転換
体によるハロヒドリンエポキシダーゼ酵素反応が、エピ
ハロヒドリン以外の１，２−エポキシ化合物にも適用で
き、３−ヒドロキシニトリル化合物の製造に有用できる
ことを見い出し、本発明を完成させた。すなわち、本発
明は、微生物由来のハロヒドリンエポキシダーゼ酵素遺
伝子ＤＮＡをベクタープラスミドに連結した組換え体プ
ラスミドにより形質転換された形質転換微生物の培養
液、菌体または菌体処理物を、シアン化アルカリの存在
下で、下記一般式〔Ｉ〕で示される１，２−エポキシ化
合物に作用させ、これを下記一般式（ＩＩ〕で示される
３−ヒドロキシニトリル化合物に変換せしめることを特
徴とする３−ヒドロキシニトリル化合物の製造法、であ
る。INDUSTRIAL APPLICABILITY According to the present invention, the halohydrin epoxidase enzymatic reaction by this transformant can be applied to 1,2-epoxy compounds other than epihalohydrin, and is useful for the production of 3-hydroxynitrile compounds. They found what they could do and completed the present invention. That is, the present invention is for cyanating a culture solution, cells or treated cells of a transformed microorganism transformed with a recombinant plasmid in which a halohydrin epoxidase enzyme gene DNA derived from a microorganism is ligated to a vector plasmid. In the presence of an alkali, it is allowed to act on a 1,2-epoxy compound represented by the following general formula [I], and this is converted into a 3-hydroxynitrile compound represented by the following general formula (II). A method for producing a hydroxynitrile compound.
【０００５】[0005]
【化２】〔Ｒは炭素数１〜４のアルキル基を表す〕Embedded image [R represents an alkyl group having 1 to 4 carbon atoms]
【０００６】本発明における形質転換微生物は、ハロヒ
ドリンエポキシダーゼ遺伝子ＤＮＡをベクタープラスミ
ドに連結した組換え体プラスミドにより形質転換された
微生物であり、この組換え体プラスミドとして、（１）
配列番号：１で示されるアミノ酸配列またはその一部の
配列を有しハロヒドリンエポキシダーゼ活性を有するポ
リペプチドをコードするＤＮＡ配列、（２）配列番号：
２で示されるアミノ酸配列またはその一部の配列を有し
ハロヒドリンエポキシダーゼ活性を有するポリペプチド
をコードするＤＮＡ配列、（３）上記（１）項のハロヒ
ドリンエポキシダーゼ活性を有するポリペプチドをコー
ドするＤＮＡ配列が、配列番号：３で示されるＤＮＡ配
列またはその一部の配列からなるもの、（４）上記
（２）項のハロヒドリンエポキシダーゼ活性を有するポ
リペプチドをコードするＤＮＡ配列が、配列番号：４で
示されるＤＮＡ配列またはその一部の配列からなるも
の、の少なくとも一つを含むものが挙げられる。The transformed microorganism in the present invention is a microorganism transformed with a recombinant plasmid in which halohydrin epoxidase gene DNA is ligated to a vector plasmid, and the recombinant plasmid is (1)
A DNA sequence encoding a polypeptide having the halohydrin epoxidase activity, which has the amino acid sequence represented by SEQ ID NO: 1 or a part thereof, (2) SEQ ID NO:
A DNA sequence encoding a polypeptide having the halohydrin epoxidase activity, which has the amino acid sequence shown in 2 or a partial sequence thereof, and (3) the polypeptide having the halohydrin epoxidase activity of (1) above. Wherein the DNA sequence encoding the above is a DNA sequence represented by SEQ ID NO: 3 or a partial sequence thereof, (4) a DNA sequence encoding a polypeptide having halohydrin epoxidase activity according to (2) above. Which comprises at least one of the DNA sequence shown in SEQ ID NO: 4 or a partial sequence thereof.
【０００７】以下に本発明を具体的に説明する。本発明
で使用し得る形質転換微生物におけるＤＮＡ供与体微生
物としては、コリネバクテリウムｓｐ．Ｎ−１０７４
（微工研条寄第２６４３号）、ミクロバクテリウムｓ
ｐ．Ｎ−４７０１（微工研条寄第２６４４号）等が挙げ
られ、その菌学的性質はそれぞれ特開平２−２９１２８
０号公報に記載されている。ベクターとしては、プラス
ミドベクター（例えばｐＵＣ１８ 、ｐＵＣ１９、ｐＵ
Ｃ１１８、ｐＵＣ１１９等）、ファージベクター（例え
ばλｇｔ１１等）のいずれでもよい。また、形質転換に
用いられる宿主微生物としては、エシェリシア コリ
（Ｅ．ｃｏｌｉ）ＪＭ１０５株あるいは同ＪＭ１０９株
が挙げられるが、特に、これらに限定されるものではな
く、他の宿主生物を用いることもできる。一例として、
コリネバクテリウムｓｐ．Ｎ−１０７４（微工研条寄第
２６４３号）のハロヒドリンエポキシダーゼ遺伝子の
Ｅ．ｃｏｌｉ ＪＭ１０９株 へのクローニングを、以
下に示す。The present invention will be specifically described below. Examples of the DNA donor microorganism in the transformed microorganism that can be used in the present invention include Corynebacterium sp. N-1074
(Microtechnology Research Institute No. 2643), Microbacterium s
p. N-4701 (Microtechnical Laboratory Article No. 2644) and the like, and their mycological properties are described in JP-A-2-29128, respectively.
No. 0 publication. The vector may be a plasmid vector (eg pUC18, pUC19, pU).
C118, pUC119 etc.) or a phage vector (eg λgt11 etc.). Examples of the host microorganism used for transformation include E. coli JM105 strain and JM109 strain, but the host microorganism is not particularly limited to these and other host organisms can be used. .. As an example,
Corynebacterium sp. Of the halohydrin epoxidase gene of N-1074 (Ministry of Industrial Science and Technology No. 2643). Cloning into E. coli strain JM109 is shown below.
【０００８】（１）コリネバクテリウムｓｐ．Ｎ−１０
７４染色体ＤＮＡの調製とＤＮＡライブラリーの作成：
コリネバクテリウムｓｐ．Ｎ−１０７４からＳａｉｔｏ
ａｎｄ Ｍｉｕｒａの方法〔Ｂｉｏｃｈｉｍ．Ｂｉｏ
ｐｈｙｓ．Ａｃｔａ ７２，６１９（１９６３）参照〕
により染色体ＤＮＡを分離し、これを制限酵素（Ｂａｍ
ＨＩあるいはＢｇｌＩＩ）で切断後、ベクタープラスミ
ドｐＵＣ１８に挿し組換え体ＤＮＡのライブラリーを作
成した。(1) Corynebacterium sp. N-10
Preparation of 74 chromosomal DNA and preparation of DNA library:
Corynebacterium sp. N-1074 to Saito
and Miura's method [Biochim. Bio
phys. Acta 72, 619 (1963)].
The chromosomal DNA is separated by using a restriction enzyme (Bam
After cutting with HI or BglII), it was inserted into vector plasmid pUC18 to prepare a recombinant DNA library.
【０００９】（２）形質転換体の作成および組換え体Ｄ
ＮＡの選別：工程（１）で調製した組換え体ライブラリ
ーによる形質転換体を宿主生物としてＥ．ｃｏｌｉ Ｊ
Ｍ１０９株を用いて塩化カルシウム法〔Ｊ．Ｍｏｌ．Ｂ
ｉｏｌ．５３，１５４（１９７０）〕により作成し、そ
の中からハロヒドリンエポキシダーゼ活性を示すように
なったものを選別した。選別は以下のようにして行っ
た。アンピシリン（１００μｇ／ｍｌ）とＩＰＴＧ（１
ｍｌ）を含むＬＢ寒天培地（１％バクトトリプトン、
０．５％バクトイーストエキス、０．５％ＮａＣ１、
１．５％寒天）に作成した形質転換体のコロニーを形成
させた。１０ｍＭトリス−塩酸緩衝液（ｐＨ７．５）、
０．０２％ブロモクレゾールパープル、１％１，３−ジ
クロロ−２−プロパノールを染み込ませたロ紙にコロニ
ーを移し、室温にて数時間放置した。ハロヒドリンエポ
キシダーゼ活性を持つコロニーは塩酸を遊離しコロニー
付近のｐＨは低下し、ｐＨ指示薬であるブロモクレゾー
ルパープルは青紫色から黄色に変化するため、肉眼観察
によりハロヒドリンエポキシダーゼ遺伝子を持つ株を選
別することができる。(2) Preparation of transformant and recombinant D
Selection of NA: E. coli using the transformant prepared by the recombinant library prepared in step (1) as a host organism. coli J
Calcium chloride method [J. Mol. B
iol. 53, 154 (1970)], and among them, those showing halohydrin epoxidase activity were selected. The selection was performed as follows. Ampicillin (100 μg / ml) and IPTG (1
LB agar medium (1% bactotryptone,
0.5% Bacto yeast extract, 0.5% NaCl1,
The formed transformant colonies were formed on 1.5% agar. 10 mM Tris-HCl buffer (pH 7.5),
The colonies were transferred to a paper filter soaked with 0.02% bromocresol purple and 1% 1,3-dichloro-2-propanol and left at room temperature for several hours. Colonies with halohydrin epoxidase activity release hydrochloric acid, the pH in the vicinity of the colonies decreases, and the pH indicator bromocresol purple changes from blue-purple to yellow, so it has the halohydrin epoxidase gene by visual observation. The strain can be selected.
【００１０】これらの形質転換株が実際にハロヒドリン
エポキシダーゼ活性を有しているかどうかは次のように
して調べることができる。これらの株をアンピシリン
（５０μｇ／ｍｌ）とＩＰＴＧ（１ｍＭ）を含むＬＢ培
地（１％バクトトリプトン、０．５％バクトイーストエ
キス、０．５％ＮａＣｌ）にて３７℃で一夜培養する。
菌体を５０ｍＭトリス−硫酸緩衝液（ｐＨ８）で２回洗
浄後、１％１，３−ジクロロプロパノールを含む１Ｍト
リス−硫酸緩衝液（ｐＨ８）に懸濁し、２０℃にてイン
キューベートした。一定時間後、生成するエピクロルヒ
ドリンをガスクロマトグラフィーにて定量した。こうし
て得られた形質転換株から再びプラスミドＤＮＡを取り
出し、選別された２種の目的のプラスミドを得た。これ
らのプラスミドをｐＳＴ００１およびｐＳＴ００５、な
らびにこれらのプラスミドが導入された形質転換体をＪ
Ｍ１０９／ｐＳＴ００１およびＪＭ１０９／ｐＳＴ００
５と称する。Whether these transformants actually have halohydrin epoxidase activity can be examined as follows. These strains are cultured overnight at 37 ° C. in LB medium (1% bactotryptone, 0.5% bacto yeast extract, 0.5% NaCl) containing ampicillin (50 μg / ml) and IPTG (1 mM).
The cells were washed twice with 50 mM Tris-sulfate buffer (pH 8), suspended in 1M Tris-sulfate buffer (pH 8) containing 1% 1,3-dichloropropanol, and incubated at 20 ° C. After a certain period of time, the produced epichlorohydrin was quantified by gas chromatography. From the transformant thus obtained, the plasmid DNA was extracted again to obtain two selected plasmids of interest. These plasmids were designated as pST001 and pST005, and transformants introduced with these plasmids were designated as JST.
M109 / pST001 and JM109 / pST00
It is called 5.
【００１１】（３）制限酵素地図の作成とハロヒドリン
エポキシダーゼ遺伝子の位置の決定：工程（２）で得ら
れたプラスミドについて制限酵素地図を作成した。その
後、より小さなＤＮＡ断片を持つプラスミドを作成し
た。これらのプラスミドによって工程（２）と同様にし
て形質転換された株のハロヒドリンエポキシダーゼ活性
の有無によって目的遺伝子の含まれている箇所を決定し
た。この過程で、ｐＳＴ００１のＢａｍＨＩ−Ｂｇｌ
１．３Ｋｂ断片を含むｐＳＴ０１５（ｐＵＣ１１８ベク
ター）およびｐＳＴ００５のＢａｍＨＩ−ＰｓｔＩ１．
１Ｋｂ断片を含むｐＳＴ１１１（ｐＵＣ１１８ベクタ
ー）プラスミドを作成した（図１）。 これらのプラス
ミドが導入された形質転換体をＪＭ１０９／ｐＳＴ０１
５およびＪＭ１０９／ｐＳＴ１１１と称する。(3) Construction of restriction enzyme map and determination of position of halohydrin epoxidase gene: A restriction enzyme map was prepared for the plasmid obtained in step (2). Then, a plasmid having a smaller DNA fragment was prepared. The site containing the target gene was determined by the presence or absence of halohydrin epoxidase activity of the strain transformed with these plasmids in the same manner as in step (2). In the process, BamHI-Bgl of pST001
PST015 (pUC118 vector) containing the 1.3 Kb fragment and BamHI-PstI1.
A pST111 (pUC118 vector) plasmid containing a 1 Kb fragment was prepared (FIG. 1). Transformants into which these plasmids were introduced were designated as JM109 / pST01.
5 and JM109 / pST111.
【００１２】（４）塩基配列の決定：工程（３）で得ら
れたプラスミドｐＳＴ０１５およびｐＳＴ１１１のハロ
ヒドリンエポキシダーゼ遺伝子に関する部分のＤＮＡの
塩基配列を決定した（配列番号：５および配列番号：
６）。なお、ここで得られた形質転換体ＪＭ１０９／ｐ
ＳＴ００１、ＪＭ１０９／ｐＳＴ００５、ＪＭ１０９／
ｐＳＴ０１５およびＪＭ１０９／ｐＳＴ１１１は、工業
技術院微生物工業技術研究所（微工研）に、それぞれ微
工研菌寄第１１９６１号、微工研菌寄第１１９６２号、
微工研菌寄第１２０６４号および微工研菌寄第１２０６
５号として寄託されている。(4) Determination of nucleotide sequence: The nucleotide sequence of the DNA of the portion of the plasmids pST015 and pST111 relating to the halohydrin epoxidase gene obtained in step (3) was determined (SEQ ID NO: 5 and SEQ ID NO:
6). The transformant JM109 / p obtained here was used.
ST001, JM109 / pST005, JM109 /
pST015 and JM109 / pST111 were obtained from the Institute of Microbiology and Industrial Technology, Institute of Industrial Science and Technology (Microtechnical Laboratory), respectively.
MICRO LABORATORY No. 12064 and MICRO LABORATORY BATTERY 1206
Deposited as No. 5.
【００１３】本発明の形質転換微生物の培養は、通常は
液体培養で行われるが、固体培養によっても行うことが
できる。培地としては、例えばＬＢ培地が用いられる。
培養は１０〜５０℃の温度で、ｐＨ２〜１１の範囲で行
われる。微生物の生育を促進させるために通気攪拌を行
ってもよい。Cultivation of the transformed microorganism of the present invention is usually carried out by liquid culture, but it can also be carried out by solid culture. As the medium, for example, LB medium is used.
Culturing is performed at a temperature of 10 to 50 ° C. and a pH of 2 to 11. Aeration and agitation may be carried out to promote the growth of microorganisms.
【００１４】培養により得られた形質転換微生物は、培
養液あるいは遠心分離などにより得た菌体の懸濁液に基
質を添加する方法、菌体処理物（例えば菌体破砕物、粗
酵素・精製酵素等の菌体抽出物等）あるいは常法により
固定化した菌体または菌体処理物等の懸濁液に基質を添
加する方法、微生物の培養時に基質を培養液に添加して
培養と同時に反応を行う方法等により、シアン化アルカ
リの存在下に、前記一般式〔Ｉ〕で示される１，２−エ
ポキシ化合物に作用させて、これを前記一般式〔ＩＩ〕
で示される３−ヒドロキシニトリル化合物に変換するこ
とができる。The transformed microorganisms obtained by culturing can be obtained by adding a substrate to a culture solution or a suspension of cells obtained by centrifugation or the like, treated cells (for example, disrupted cells, crude enzyme / purification). A method of adding a substrate to a suspension of a bacterial cell extract such as an enzyme or the like or a suspension of a bacterial cell or a treated product of a bacterial cell immobilized by a conventional method, and adding the substrate to a culture solution at the time of culturing a microorganism and simultaneously culturing The 1,2-epoxy compound represented by the general formula [I] is allowed to act on the 1,2-epoxy compound represented by the general formula [I] in the presence of alkali cyanide by a reaction method or the like.
Can be converted to a 3-hydroxynitrile compound represented by.
【００１５】一般式〔Ｉ〕で示される１，２−エポキシ
化合物は、例えば、１，２−エポキシプロパン、１，２
−エポキシブタン、１，２−エポキシヘキサン等であ
る。また、シアン化アルカリは、シアン化カリウム、シ
アン化ナトリウム等である。反応液中の基質濃度は特に
限定するものではないが、０．１〜１０（Ｗ／Ｖ）％が
好ましく、また、シアン化アルカリの使用量は、通常基
質の１〜３倍量（モル）である。基質は反応液に一括し
て加えるかあるいは分割添加することができる。反応温
度は５〜５０℃、反応ｐＨは４〜１０の範囲で行うこと
が好ましい。反応時間は基質等の濃度、菌体濃度あるい
はその他の反応条件等によって変わるが、通常１〜１２
０時間で終了するように条件を設定するのが好ましい。The 1,2-epoxy compound represented by the general formula [I] is, for example, 1,2-epoxypropane, 1,2-epoxypropane.
-Epoxybutane, 1,2-epoxyhexane and the like. The alkali cyanide is potassium cyanide, sodium cyanide, or the like. The substrate concentration in the reaction solution is not particularly limited, but is preferably 0.1 to 10 (W / V)%, and the amount of alkali cyanide used is usually 1 to 3 times the amount (mol) of the substrate. Is. The substrate can be added to the reaction solution all at once or in portions. The reaction temperature is preferably 5 to 50 ° C. and the reaction pH is preferably 4 to 10. The reaction time varies depending on the concentration of the substrate and the like, the cell concentration or other reaction conditions, but it is usually 1 to 12
It is preferable to set the conditions so that it ends in 0 hours.
【００１６】かくして、反応液中に生成、蓄積した３−
ヒドロキシニトリル化合物は公知の方法を用いて採取お
よび精製することができる。例えば、反応液から遠心分
離などの方法を用いて菌体を除いた後、酢酸エチルなど
の溶媒で抽出を行い、減圧下に溶媒を除去することによ
り３−ヒドロキシニトリル化合物のシロップを得ること
ができる。また、これらのシロップを減圧下に蒸留する
ことによりさらに精製することもできる。Thus, the 3-produced and accumulated in the reaction solution
The hydroxynitrile compound can be collected and purified using a known method. For example, a syrup of a 3-hydroxynitrile compound can be obtained by removing cells from a reaction solution by using a method such as centrifugation, extracting with a solvent such as ethyl acetate, and removing the solvent under reduced pressure. it can. Further, these syrups can be further purified by distillation under reduced pressure.
【００１７】[0017]
【発明の効果】本発明によれば、遺伝子組換えの方法で
クローン化されたハロヒドリンエポキシダーゼ遺伝子が
菌体内に多数存在する形質転換微生物の使用により、シ
アン化アルカリの存在下、１，２−エポキシ化合物から
３−ヒドロキシニトリル化合物を効率よく製造すること
が可能である。INDUSTRIAL APPLICABILITY According to the present invention, the use of a transformed microorganism in which a large number of halohydrin epoxidase genes cloned by the method of gene recombination are present in the cells, It is possible to efficiently produce a 3-hydroxynitrile compound from a 2-epoxy compound.
【００１８】[0018]
【実施例】 実施例１ アンピシリン（５０μｇ／ｍｌ）と１ｍＭ ＩＰＴＧを
含むＬＢ培地１８ｌにＪＭ１０９／ｐＳＴ００１を接種
し３７℃にて１６時間振盪培養を行った。得られた培養
液から遠心分離により菌体を回収し、１００ｍＭ トリ
ス−硫酸緩衝液（ｐＨ８．０）５０ｍｌで洗浄後、同緩
衝液に懸濁し菌体懸濁液を調製した。菌体を超音波破砕
した後、遠心分離して沈澱物を除去し、上清を菌体抽出
液とした。常法にて硫安分画を行い、ＤＥＡＥ−セファ
セル、Ｐｈｅｎｙｌ−セファロースおよびＯｃｔｙｌ−
セファロース（ファルマシア製）を用いたカラムクロマ
トグラフィーによって酵素を精製した。４００ｍＭのト
リス−硫酸緩衝液（ｐＨ８．０）にシアン化カリウムを
２００ｍＭになるように溶かした後、１Ｎの硫酸でｐＨ
を８．０に調整し、この溶液２５ｍｌに精製酵素溶液
（タンパク濃度：３０ｍｇ／ｍｌ）０．１ｍｌと４００
ｍＭの１，２−エポキシブタン溶液２５ｍｌを加え、２
０℃で２時間反応した。反応液をガスクロマトグラフィ
ーで分析したところ、１００ｍＭの３−ヒドロキシバレ
ロニトリルが生成していた。なお、本反応生成物は、反
応液から酢酸エチルで抽出することによって単離した
後、ＮＭＲ、赤外吸光スペクトルおよびマススペクトル
による分析から、３−ヒドロキシバレロニトリルである
ことを確認した。Example 1 JM109 / pST001 was inoculated into 18 L of LB medium containing ampicillin (50 μg / ml) and 1 mM IPTG, and shake culture was performed at 37 ° C. for 16 hours. The cells were collected from the obtained culture solution by centrifugation, washed with 50 ml of 100 mM Tris-sulfate buffer (pH 8.0), and then suspended in the same buffer to prepare a cell suspension. The cells were ultrasonically disrupted and then centrifuged to remove the precipitate, and the supernatant was used as a cell extract. Ammonium sulphate fractionation was carried out by a conventional method, and DEAE-Sephacel, Phenyl-Sepharose and Octyl-
The enzyme was purified by column chromatography using Sepharose (Pharmacia). Dissolve potassium cyanide to 200 mM in 400 mM Tris-sulfate buffer (pH 8.0), and add 1N sulfuric acid to adjust the pH.
Was adjusted to 8.0, and 25 ml of this solution was added with 0.1 ml of purified enzyme solution (protein concentration: 30 mg / ml) and 400 ml.
Add 25 ml of mM 1,2-epoxybutane solution and add 2
The reaction was carried out at 0 ° C for 2 hours. When the reaction liquid was analyzed by gas chromatography, 100 mM of 3-hydroxyvaleronitrile was produced. The reaction product was isolated from the reaction solution by extraction with ethyl acetate, and then confirmed to be 3-hydroxyvaleronitrile by NMR, infrared absorption spectrum, and mass spectrum analysis.
【００１９】実施例２ １，２−エポキシブタンの代わりに１，２−エポキシプ
ロパンを使用して実施例１と同様の反応を行ったとこ
ろ、７１ｍＭの３−ヒドロキシブチロニトリルが生成し
た。本反応生成物は実施例１と同様にして同定した。Example 2 When 1,2-epoxypropane was used in place of 1,2-epoxybutane and the same reaction as in Example 1 was carried out, 71 mM 3-hydroxybutyronitrile was produced. The reaction product was identified in the same manner as in Example 1.
【００２０】実施例３ 実施例１と同様にして調製した培地（１００ｍｌ）に、
それぞれＪＭ１０９／ｐＳＴ０１５およびＪＭ１０９／
ｐＳＴ１１１を接種し、３７℃にて１６時間培養を行っ
た。これらの培養液をそれぞれ遠心分離して菌体を集
め、１００ｍＭトリス−硫酸緩衝液（ｐＨ８．０）５０
ｍｌで洗浄後、同緩衝液２５ｍｌに懸濁し菌体懸濁液を
調製した。４００ｍＭのトリス−硫酸緩衝液（ｐＨ８．
０）にシアン化カリウムを２００ｍＭとなるように溶か
した後、１Ｎの硫酸でｐＨを８．０に調整した溶液５０
ｍｌを作成し、この溶液に上記菌体懸濁液と２００ｍＭ
の１，２−エポキシブタン溶液をそれぞれ２５ｍｌ加
え、２０℃で３０分（ＪＭ１０９／ｐＳＴ０１５）およ
び２０℃で１５分（ＪＭ１０９／ｐＳＴ１１１）反応さ
せた。反応液をガスクロマトグラフィーで分析したとこ
ろ、それぞれ１１．１ｍＭ（ＪＭ１０９／ｐＳＴ０１
５）および７．２ｍＭ（ＪＭ１０９／ｐＳＴ１１１）の
３−ヒドロキシバレロニトリルが生成していた。Example 3 In a medium (100 ml) prepared in the same manner as in Example 1,
JM109 / pST015 and JM109 / respectively
It was inoculated with pST111 and cultured at 37 ° C. for 16 hours. These cultures were centrifuged to collect the cells, and 100 mM Tris-sulfate buffer (pH 8.0) 50
After washing with ml, the cells were suspended in 25 ml of the same buffer to prepare a cell suspension. 400 mM Tris-sulfate buffer (pH 8.
0) potassium cyanide was dissolved to 200 mM, and the pH was adjusted to 8.0 with 1N sulfuric acid.
Make up ml, and add the above-mentioned cell suspension and 200 mM to this solution.
25 ml of each 1,2-epoxybutane solution was added and reacted at 20 ° C. for 30 minutes (JM109 / pST015) and at 20 ° C. for 15 minutes (JM109 / pST111). When the reaction solution was analyzed by gas chromatography, it was found to be 11.1 mM (JM109 / pST01).
5) and 7.2 mM (JM109 / pST111) of 3-hydroxyvaleronitrile were produced.
【００２１】実施例４ １，２−エポキシブタンの代わりに１，２−エポキシプ
ロパン使用して実施例３と同様の反応を行ったところ、
それぞれ３．６ｍＭ（ＪＭ１０９／ｐＳＴ０１５）およ
び６．２ｍＭ（ＪＭ１０９／ｐＳＴ１１１）の３−ヒド
ロキシブチロニトリルが生成していた。Example 4 When 1,2-epoxypropane was used instead of 1,2-epoxybutane and the same reaction as in Example 3 was carried out,
3.6 mM (JM109 / pST015) and 6.2 mM (JM109 / pST111) of 3-hydroxybutyronitrile were produced, respectively.
【００２２】[0022]
【配列表】 [Sequence list]
【００２３】 [0023]
【００２４】 [0024]
【００２５】 [0025]
【００２６】 [0026]
【００２７】 [0027]
【手続補正２】[Procedure Amendment 2]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】図面の簡単な説明[Name of item to be corrected] Brief explanation of the drawing
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【図面の簡単な説明】[Brief description of drawings]
【図１】組換え体プラスミドｐＳＴ００１、ｐＳＴ００
５、ｐＳＴ０１５およびｐＳＴ１１１の制限酵素地図を
示す。FIG. 1 Recombinant plasmids pST001, pST00
5 shows a restriction map of 5, pST015 and pST111.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.5 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｃ１２Ｒ 1:19） (Ｃ１２Ｎ 1/21 Ｃ１２Ｒ 1:19） (Ｃ１２Ｎ 9/00 Ｃ１２Ｒ 1:19） (Ｃ１２Ｎ 15/52 Ｃ１２Ｒ 1:15） (72)発明者 中村 哲二 神奈川県横浜市鶴見区大黒町10番１号 日 東化学工業株式会社中央研究所内 (72)発明者 水無 渉 神奈川県横浜市鶴見区大黒町10番１号 日 東化学工業株式会社中央研究所内 (72)発明者 湯 不二夫 神奈川県横浜市鶴見区大黒町10番１号 日 東化学工業株式会社中央研究所内─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 5 Identification code Internal reference number FI Technical display location C12R 1:19) (C12N 1/21 C12R 1:19) (C12N 9/00 C12R 1:19) (C12N 15/52 C12R 1:15) (72) Inventor Tetsuji Nakamura 10-1 Daikokucho, Tsurumi-ku, Yokohama-shi, Kanagawa NITTO CHEMICAL INDUSTRIES CO., LTD. (72) Inventor Wataru Mizunashi, Yokohama-shi, Kanagawa Central Research Institute, Nitto Chemical Co., Ltd. 10-1 Daikokucho, Tsurumi-ku (72) Inventor Fujio Yu 10-10 Daikokucho, Tsurumi-ku, Yokohama-shi, Kanagawa Central Research Laboratory, Nitto Chemical Co., Ltd.
Claims (5)
ゼ酵素遺伝子ＤＮＡをベクタープラスミドに連結した組
換え体プラスミドにより形質転換された形質転換微生物
の培養液、菌体または菌体処理物を、シアン化アルカリ
の存在下で、下記一般式〔Ｉ〕で示される１，２−エポ
キシ化合物に作用させ、これを下記一般式〔II〕で示さ
れる３−ヒドロキシニトリル化合物に変換せしめること
を特徴とする３−ヒドロキシニトリル化合物の製造法。 【化１】 〔R は炭素数１〜４のアルキル基を表す〕1. A culture broth, a microbial cell or a treated product of a transformed microorganism transformed with a recombinant plasmid obtained by ligating a halohydrin epoxidase enzyme gene DNA derived from a microorganism to a vector plasmid is treated with an alkali cyanide. In the presence of a compound represented by the following general formula [I], the 1,2-epoxy compound is allowed to act on the 1,2-epoxy compound to convert it into a 3-hydroxynitrile compound represented by the following general formula [II]. Process for producing hydroxynitrile compound. [Chemical 1] [R represents an alkyl group having 1 to 4 carbon atoms]
ＤＮＡが、配列番号：１で示されるアミノ酸配列または
その一部の配列を有しハロヒドリンエポキシダーゼ活性
を有するポリペプチドをコ−ドするＤＮＡ配列からなる
請求項１記載の３−ヒドロキシニトリル化合物の製造
法。2. A DNA sequence encoding a polypeptide having a halohydrin epoxidase activity, wherein the halohydrin epoxidase enzyme gene DNA has the amino acid sequence represented by SEQ ID NO: 1 or a partial sequence thereof. The method for producing a 3-hydroxynitrile compound according to claim 1, which comprises
ＤＮＡが、配列番号：２で示されるアミノ酸配列または
その一部の配列を有しハロヒドリンエポキシダーゼ活性
を有するポリペプチドをコ−ドするＤＮＡ配列からなる
請求項１記載の３−ヒドロキシニトリル化合物の製造
法。3. A DNA sequence for coding a polypeptide having halohydrin epoxidase activity, wherein the halohydrin epoxidase enzyme gene DNA has the amino acid sequence represented by SEQ ID NO: 2 or a part of the sequence thereof. The method for producing a 3-hydroxynitrile compound according to claim 1, which comprises
るポリペプチドをコードするＤＮＡ配列が、配列番号：
３で示されるＤＮＡ配列またはその一部の配列からなる
請求項２記載の３−ヒドロキシニトリル化合物の製造
法。4. A DNA sequence encoding a polypeptide having halohydrin epoxidase activity is SEQ ID NO :.
The method for producing a 3-hydroxynitrile compound according to claim 2, which comprises the DNA sequence shown in 3 or a partial sequence thereof.
るポリペプチドをコードするＤＮＡ配列が、配列番号：
４で示されるＤＮＡ配列またはその一部の配列からなる
請求項３記載の３−ヒドロキシニトリル化合物の製造
法。5. A DNA sequence encoding a polypeptide having halohydrin epoxidase activity is SEQ ID NO :.
The method for producing a 3-hydroxynitrile compound according to claim 3, which comprises the DNA sequence represented by 4 or a partial sequence thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3062597A JP3026367B2 (en) | 1991-03-04 | 1991-03-04 | Method for producing 3-hydroxynitrile compound by a microorganism transformed with a recombinant plasmid having halohydrin epoxidase gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3062597A JP3026367B2 (en) | 1991-03-04 | 1991-03-04 | Method for producing 3-hydroxynitrile compound by a microorganism transformed with a recombinant plasmid having halohydrin epoxidase gene |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH05317066A true JPH05317066A (en) | 1993-12-03 |
| JP3026367B2 JP3026367B2 (en) | 2000-03-27 |
Family
ID=13204897
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3062597A Expired - Lifetime JP3026367B2 (en) | 1991-03-04 | 1991-03-04 | Method for producing 3-hydroxynitrile compound by a microorganism transformed with a recombinant plasmid having halohydrin epoxidase gene |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3026367B2 (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6384263B1 (en) | 2000-08-04 | 2002-05-07 | E. I. Du Pont De Nemours And Company | Process for making 3-hydroxyalkanelnitriles and conversion of the 3-hydroxyalkanelnitrile to an hydroxyaminoalkane |
| JP2006325519A (en) * | 2005-05-27 | 2006-12-07 | Mitsubishi Rayon Co Ltd | Method for producing 4-halo-3-hydroxybutyronitrile and bacterium used therefor |
| JP2007049932A (en) * | 2005-08-18 | 2007-03-01 | Mitsubishi Rayon Co Ltd | Transformed body of bacterium belonging to genus rhodococcus and having halohydrinepoxidase activity |
| WO2007071599A3 (en) * | 2005-12-21 | 2007-08-16 | Basf Ag | A process for the production of an optically enriched tertiary alcohol |
| WO2008056827A1 (en) | 2006-11-09 | 2008-05-15 | Mitsubishi Rayon Co., Ltd. | Process for production of betaine |
| WO2008108466A1 (en) | 2007-03-07 | 2008-09-12 | Mitsubishi Rayon Co., Ltd. | Improved halohydrin epoxidase |
-
1991
- 1991-03-04 JP JP3062597A patent/JP3026367B2/en not_active Expired - Lifetime
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6384263B1 (en) | 2000-08-04 | 2002-05-07 | E. I. Du Pont De Nemours And Company | Process for making 3-hydroxyalkanelnitriles and conversion of the 3-hydroxyalkanelnitrile to an hydroxyaminoalkane |
| JP2006325519A (en) * | 2005-05-27 | 2006-12-07 | Mitsubishi Rayon Co Ltd | Method for producing 4-halo-3-hydroxybutyronitrile and bacterium used therefor |
| JP4716784B2 (en) * | 2005-05-27 | 2011-07-06 | 三菱レイヨン株式会社 | Process for producing 4-halo-3-hydroxybutyronitrile and microorganism used therefor |
| JP2007049932A (en) * | 2005-08-18 | 2007-03-01 | Mitsubishi Rayon Co Ltd | Transformed body of bacterium belonging to genus rhodococcus and having halohydrinepoxidase activity |
| WO2007071599A3 (en) * | 2005-12-21 | 2007-08-16 | Basf Ag | A process for the production of an optically enriched tertiary alcohol |
| US8198069B2 (en) | 2005-12-21 | 2012-06-12 | Basf Se | Method of producing an optically enriched tertiary alcohol from an epoxide using halohydrin dehalogenase |
| WO2008056827A1 (en) | 2006-11-09 | 2008-05-15 | Mitsubishi Rayon Co., Ltd. | Process for production of betaine |
| WO2008108466A1 (en) | 2007-03-07 | 2008-09-12 | Mitsubishi Rayon Co., Ltd. | Improved halohydrin epoxidase |
| EP2518152A2 (en) | 2007-03-07 | 2012-10-31 | Mitsubishi Rayon Co., Ltd. | Improved halohydrin epoxidase |
| JP5226509B2 (en) * | 2007-03-07 | 2013-07-03 | 三菱レイヨン株式会社 | Improved halohydrin epoxidase |
| US8637272B2 (en) | 2007-03-07 | 2014-01-28 | Mitsubishi Rayon Co., Ltd. | Halohydrin epoxidase |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3026367B2 (en) | 2000-03-27 |
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