JPH07147975A - Artificial fused enzyme - Google Patents
Artificial fused enzymeInfo
- Publication number
- JPH07147975A JPH07147975A JP5298279A JP29827993A JPH07147975A JP H07147975 A JPH07147975 A JP H07147975A JP 5298279 A JP5298279 A JP 5298279A JP 29827993 A JP29827993 A JP 29827993A JP H07147975 A JPH07147975 A JP H07147975A
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- Prior art keywords
- leu
- cytochrome
- yeast
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- ser
- Prior art date
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- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、人工融合酵素およびそ
の製造方法、その人工融合酵素遺伝子、その人工融合酵
素遺伝子を含むプラスミドならびに酵母内発現プラスミ
ド、その人工融合酵素遺伝子が導入された酵母菌株およ
びその利用に関するものである。TECHNICAL FIELD The present invention relates to an artificial fusion enzyme and a method for producing the same, an artificial fusion enzyme gene thereof, a plasmid containing the artificial fusion enzyme gene and a yeast expression plasmid, and a yeast strain into which the artificial fusion enzyme gene is introduced. And its use.
【０００２】[0002]
【従来の技術】ミトコンドリア型チトクロム P450 モノ
オキシゲナーゼは P450 、アドレノドキシン（以下、AD
X と記す。) 、およびNADPH-アドレノドキシン還元酵素
（以下、 ADRと記す。) の３つの成分からなり、これら
がミトコンドリア内膜およびマトリックスにおいて電子
伝達系を形成し、NADPH からの電子がフェレドキシン還
元酵素およびフェレドキシンを経由し、チトクロム P45
0 へ伝達されることにより１原子酸素添加活性が発揮さ
れる。本発明者らは以前にシグナル配列を改変すること
により細胞内局在性をミクロソーム型に改変したラット
P450 c25（以下、改変ラットP450 c25と記す。）、ウシ
副腎ADR および ウシ副腎ADX の三者を同時に酵母内に
おいて発現することに成功した。この場合、改変ラット
P450 c25はミクロソーム膜に存在するが、一方、ウシ副
腎ADR およびウシ副腎ADX は細胞質に存在する。このた
め菌体内においてP450活性を発揮するには、ミクロソー
ム膜と細胞質の間に電子伝達系が構築される必要があっ
た。2. Description of the Related Art Mitochondrial cytochrome P450 monooxygenase is P450, adrenodoxin (hereinafter referred to as AD
Write X. ), And NADPH-adrenodoxin reductase (hereinafter referred to as ADR), which form an electron transfer system in the inner mitochondrial membrane and matrix, and the electrons from NADPH are ferredoxin reductase and ferredoxin. Via Cytochrome P45
The one-atom oxygen addition activity is exhibited by being transmitted to 0. The present inventors have previously modified the intracellular localization to a microsomal rat by modifying the signal sequence.
We succeeded in expressing P450 c25 (hereinafter referred to as modified rat P450 c25), bovine adrenal ADR and bovine adrenal ADX in yeast at the same time. In this case, the modified rat
P450 c25 is present in the microsomal membrane, whereas bovine adrenal ADR and bovine adrenal ADX are present in the cytoplasm. Therefore, in order to exert P450 activity in the cells, it was necessary to construct an electron transfer system between the microsomal membrane and the cytoplasm.
【０００３】[0003]
【発明が解決しようとする課題】しかしながら、チトク
ロム P450 を利用するバイオリアクターを用いて、有用
物質の酸化反応プロセス、または産業廃水中の有害物質
の酸化的除去等へ実用的応用する場合、チトクロム P45
0 の電子伝達系を単純化し、１つの酵素として活性を発
揮させることが望まれていた。However, when a bioreactor utilizing cytochrome P450 is practically applied to the oxidation reaction process of useful substances or the oxidative removal of harmful substances in industrial wastewater, cytochrome P45 is used.
It has been desired to simplify the electron transfer system of 0 and exert its activity as one enzyme.
【０００４】[0004]
【課題を解決するための手段】本発明者らは、上記の状
況を鑑み、すぐれた活性を有する人工融合酵素を見い出
すべく、鋭意検討を重ねた結果、Ｎ末端側にミトコンド
リア型の成熟チトクロム P450 をコードするアミノ酸配
列を有し、かつＣ末端側にある種のチトクロムP450 を
還元する役割を有する酵素をコードするアミノ酸配列を
有する人工融合酵素をコードする人工融合酵素遺伝子を
構築し、この遺伝子に係る酵母内発現ベクター、さらに
該酵母内発現ベクター導入酵母菌株を得て、そして酵母
菌株に人工融合酵素を生産させることに成功した。生産
された人工融合酵素は、単一分子内に電子伝達と基質の
酸化の両機能を有しており、すぐれた性質を有する人工
的な新酵素であることを確認し、本発明を完成した。す
なわち、本発明はＮ末端側にミトコンドリア型の成熟チ
トクロム P450 をコードするアミノ酸配列を有し、かつ
Ｃ末端側にミクロソーム型チトクロム P450を還元する
役割を有するNADPH-チトクロム P450 還元酵素をコード
するアミノ酸配列を有する人工融合酵素およびその製造
方法、その人工融合酵素遺伝子、その人工融合酵素遺伝
子を含むプラスミドならびに酵母内発現プラスミド、そ
の人工融合酵素遺伝子が導入された酵母菌株およびその
利用を提供するものである。[Means for Solving the Problems] In view of the above situation, the present inventors have conducted extensive studies in order to find an artificial fusion enzyme having excellent activity. As a result, the N-terminal side mitochondrial mature cytochrome P450 is found. Was constructed, and an artificial fusion enzyme gene encoding an artificial fusion enzyme having an amino acid sequence encoding an enzyme having a role of reducing certain cytochrome P450s on the C-terminal side was constructed. The present inventors have succeeded in obtaining such an expression vector in yeast, a yeast strain into which the expression vector in yeast has been introduced, and allowing the yeast strain to produce an artificial fusion enzyme. It was confirmed that the produced artificial fusion enzyme has both functions of electron transfer and substrate oxidation in a single molecule and is an artificial new enzyme having excellent properties, and completed the present invention. . That is, the present invention has an amino acid sequence encoding a mitochondrial mature cytochrome P450 on the N-terminal side and an amino acid sequence encoding NADPH-cytochrome P450 reductase having a role of reducing microsomal cytochrome P450 on the C-terminal side. And an artificial fusion enzyme having the same, a method for producing the artificial fusion enzyme, a plasmid containing the artificial fusion enzyme gene and a yeast expression plasmid, a yeast strain into which the artificial fusion enzyme gene is introduced, and use thereof are provided. .
【０００５】本発明において用いられるミトコンドリア
型の成熟チトクロム P450 をコードする塩基配列を有す
る遺伝子としては、特に限定されることなく、種々の分
子種、たとえば、チトクロム P450 scc 、チトクロム P
450 11β、チトクロム P450cc24等の遺伝子をあげるこ
とができ、好ましくはラット肝チトクロム P450 c25の
遺伝子等があげられる。これら遺伝子は、たとえば、Pr
oc. Natl. Acad. Sci.USA 81,4647-4651、J. Biochem.
102,559-568 、FEBS Lett. 278,195-198等に記載される
通常の遺伝子工学的方法に準じて単離・調製することが
でき、特にラット肝チトクロム P450 c25 の遺伝子の場
合は、たとえば特開平４-349887 号に記載されるpAMS25
から通常の方法により容易に取得することもできる。な
お、本願中で用いられる“ミトコンドリア型の成熟チト
クロム P450 ”とは、後述するミトコンドリア型のチト
クロム P450 からＮ末端側に存在するミトコンドリア型
のシグナルペプチドを除いた残りのタンパク質、すなわ
ち１原子酸素添加活性を有する酵素本体部分を意味して
いる。The gene having a nucleotide sequence encoding a mitochondrial mature cytochrome P450 used in the present invention is not particularly limited and various molecular species such as cytochrome P450 scc and cytochrome P can be used.
Examples include genes such as 450 11 β and cytochrome P450 cc24, and preferably the gene for rat liver cytochrome P450 c25. These genes are, for example, Pr
oc. Natl. Acad. Sci. USA 81,4647-4651, J. Biochem.
102,559-568, FEBS Lett. 278,195-198, etc., and can be isolated and prepared according to the usual genetic engineering method. Particularly, in the case of the rat liver cytochrome P450 c25 gene, for example, JP-A-4- PAMS25 described in 349887
Can also be easily obtained by a usual method. The term "mitochondrial mature cytochrome P450" used in the present application means the remaining protein except the mitochondrial signal peptide present on the N-terminal side from the mitochondrial cytochrome P450 described later, that is, one-atom oxygen addition activity. Means the enzyme main body portion having.
【０００６】本発明において用いられるミクロソーム型
チトクロム P450 を還元する役割を有するNADPH-チトク
ロム P450 還元酵素をコードする塩基配列を有する遺伝
子としては、特に限定されることなく、たとえば、動
物、植物、カビ、酵母等の真核生物由来のNADPH-チトク
ロム P450 還元酵素等のフラビンモノヌクレオチド、フ
ラビンアデニンジヌクレオチドおよびNADPH 結合領域を
含む遺伝子をあげることができ、好ましくは酵母NADPH-
チトクロム P450 還元酵素の遺伝子等があげられる。こ
れら遺伝子は、たとえば、特開昭62-19085号等に記載さ
れる通常の遺伝子工学的方法に準じて単離することがで
き、特に酵母NADPH-チトクロム P450 還元酵素の遺伝子
の場合は、たとえば特開昭62-325527 号に記載されるpG
YRから通常の方法により容易に取得することもできる。The gene having a nucleotide sequence encoding NADPH-cytochrome P450 reductase having a role of reducing microsomal cytochrome P450 used in the present invention is not particularly limited and includes, for example, animals, plants, molds, NADPH-derived cytochrome P450 reductase-derived flavin mononucleotides such as yeast, flavin adenine dinucleotide, and a gene containing a NADPH binding region can be mentioned, and preferably yeast NADPH-
Examples include cytochrome P450 reductase gene. These genes can be isolated, for example, according to the usual genetic engineering methods described in JP-A No. 62-19085, and particularly in the case of the gene for yeast NADPH-cytochrome P450 reductase, for example, PG described in Kaisho 62-325527
It can also be easily obtained from YR by a usual method.
【０００７】上記のミトコンドリア型の成熟チトクロム
P450 をコードする塩基配列を有する遺伝子とミクロソ
ーム型チトクロム P450 を還元する役割を有するNADPH-
チトクロム P450 還元酵素をコードする塩基配列を有す
る遺伝子を接続し、単一の遺伝子とし、ミトコンドリア
型の成熟チトクロム P450 の有する１原子酸素添加活性
及びミクロソーム型チトクロム P450 を還元する役割を
有するNADPH-チトクロム P450 還元酵素の有するNADPH
からの還元力供給能を同一分子内に有する人工融合酵素
をコードする人工融合酵素遺伝子を構築するには、たと
えば、ミトコンドリア型のシグナルペプチドをコードす
る塩基配列を有するチトクロム P450 遺伝子（以下、ミ
トコンドリア型のチトクロム P450 遺伝子と記す。）の
５′末端側に存在するミトコンドリア型のシグナルペプ
チドをコードする塩基配列を除いたチトクロム P450 遺
伝子をＮ末端側に、かつミクロソーム型チトクロム P45
0を還元する役割を有するNADPH-チトクロム P450 還元
酵素をコードする塩基配列を有する遺伝子をＣ末端側に
なるように配列し、そして接続部分には適当なリンカー
を用いてフレームがずれないように通常の遺伝子工学的
方法を用いて接続する方法等をあげることができる。な
お、ミトコンドリア型のチトクロム P450 遺伝子の５′
末端側に存在するミトコンドリア型のシグナルペプチド
をコードする塩基配列を酵母内で機能するミクロソーム
型のシグナルペプチドに変換したチトクロム P450 遺伝
子をＮ末端側に、かつミクロソーム型チトクロム P450
を還元する役割を有するNADPH-チトクロム P450 還元酵
素をコードする塩基配列を有する遺伝子をＣ末端側にな
るように配列し、そして接続部分には適当なリンカーを
用いてフレームがずれないように通常の遺伝子工学的方
法を用いて接続する方法等もあげることができる。[0007] Mitochondrial mature cytochrome
A gene having a nucleotide sequence encoding P450 and NADPH- having a role of reducing microsomal cytochrome P450
NADPH-cytochrome P450 which has a function of reducing the microsomal cytochrome P450 and the one-atom oxygenation activity of mitochondrial mature cytochrome P450 by connecting genes having a nucleotide sequence encoding cytochrome P450 reductase into a single gene NADPH possessed by reductase
To construct an artificial fusion enzyme gene encoding an artificial fusion enzyme having the ability to supply reducing power from the same molecule, for example, a cytochrome P450 gene having a nucleotide sequence encoding a mitochondrial type signal peptide (hereinafter referred to as mitochondrial type The cytochrome P450 gene of 5) is located at the N-terminal side of the cytochrome P450 gene excluding the nucleotide sequence encoding the mitochondrial-type signal peptide existing at the 5'end side of the
A gene having a nucleotide sequence encoding NADPH-cytochrome P450 reductase having a role of reducing 0 is arranged at the C-terminal side, and an appropriate linker is used at the connecting portion so that the frame is not displaced normally. The method of connecting using the genetic engineering method can be mentioned. 5'of the mitochondrial cytochrome P450 gene
A cytochrome P450 gene in which a nucleotide sequence encoding a mitochondrial signal peptide existing at the terminal side is converted into a microsomal signal peptide that functions in yeast is at the N-terminal side and a microsomal cytochrome P450 gene.
A gene having a nucleotide sequence encoding NADPH-cytochrome P450 reductase, which has a role of reducing the amino acid sequence, is arranged at the C-terminal side, and an appropriate linker is used at the connecting portion to prevent out-of-frame The method of connecting using a genetic engineering method etc. can also be mentioned.
【０００８】酵母内で発現させるためのプロモーターと
しては、通常の酵母発現系において用いられるプロモー
ターであれば特に制限されるものではないが、たとえば
酵母アルコール脱水素酵素遺伝子のプロモーター（以
下、ADH プロモーターと記す。）、グリセルアルデヒド
−３リン酸脱水素酵素（以下、GAPDH プロモーターと記
す。）、フォスフォグリセリン酸キナーゼ（以下、PGK
プロモーターと記す。）等をあげることができる。な
お、ADH プロモーターは、たとえば酵母ADH1プロモータ
ーおよび同ターミネーターを保持する酵母発現ベクター
pAAH5 〔WashingtonResearch Fundation から入手可
能、Ammerer ら、Method in Enzymology、101part（p.1
92-201)〕から通常の遺伝子操作方法により調製するこ
とができる。酵母ADH1プロモーターは、Washington Res
earch Fundation の米国特許出願第299,733 に含まれて
おり、米国において、工業的、商業目的で使用する場合
は、権利者からの権利許諾を必要とする。The promoter for expression in yeast is not particularly limited as long as it is a promoter used in ordinary yeast expression systems. For example, a promoter of yeast alcohol dehydrogenase gene (hereinafter referred to as ADH promoter and ), Glyceraldehyde-3-phosphate dehydrogenase (hereinafter referred to as GAPDH promoter), phosphoglycerate kinase (hereinafter referred to as PGK).
Described as a promoter. ) And so on. The ADH promoter is, for example, a yeast expression vector containing the yeast ADH1 promoter and the terminator.
pAAH5 [Available from Washington Research Fundation, Ammerer et al., Method in Enzymology, 101part (p. 1
92-201)], and can be prepared by a usual gene manipulation method. The yeast ADH1 promoter is a product of the Washington Res
Included in earch Fundation's US Patent Application No. 299,733, for use in the United States for industrial and commercial purposes requires permission from the right holder.
【０００９】上記の酵母内で発現させるためのプロモー
ターおよび前記の人工融合酵素遺伝子を含む酵母内発現
プラスミドは通常の遺伝子組み換え方法を用いて構築す
ることができる。たとえば、前記の人工融合酵素遺伝子
を特開平2-211880号公報等に記載されるADH プロモータ
ーとターミネーターを保有する酵母発現ベクターpAAH5N
のHind III部位に挿入することにより構築する方法等を
あげることができる。こうして得られた酵母内発現プラ
スミドを、たとえばアルカリ金属(LiCl)を用いる方法、
プロトプラスト法等の通常の方法によって酵母菌株に導
入する。[0009] The yeast expression plasmid containing the above-mentioned promoter for expression in yeast and the above artificial fusion enzyme gene can be constructed using a conventional gene recombination method. For example, a yeast expression vector pAAH5N having the ADH promoter and terminator described in Japanese Patent Laid-Open No. 2-211880 is used as the artificial fusion enzyme gene.
The method of constructing by inserting into the Hind III site of The yeast expression plasmid thus obtained, for example, a method using alkali metal (LiCl),
It is introduced into the yeast strain by a conventional method such as the protoplast method.
【００１０】このようにして得られた酵母内発現プラス
ミド導入酵母菌株を培養することにより、本発明人工融
合酵素を製造することができる。培養は、通常の培養方
法により行うことができる。なお、培地としては酵母の
生育が可能な組成であればよいが、本発明の酵母内発現
プラスミドの脱落を防ぐために、選択圧を加えることが
好ましい。たとえば、特定のアミノ酸を要求する酵母変
異株を宿主として用いて、その特定のアミノ酸を合成す
る機能を有するマーカー遺伝子を本発明の酵母内発現プ
ラスミドに組み込み、そしてこのプラスミドが導入され
た酵母菌株をその特定のアミノ酸を含まない最小培地で
培養することにより、該プラスミドの脱落を防ぎ、効率
よく目的遺伝子を発現させることもできる。The artificial fusion enzyme of the present invention can be produced by culturing the yeast strain into which the expression plasmid in yeast thus obtained is introduced. The culture can be performed by a usual culture method. The medium may have any composition that allows yeast to grow, but it is preferable to apply selective pressure to prevent the yeast expression plasmid of the present invention from falling off. For example, using a yeast mutant strain that requires a specific amino acid as a host, a marker gene having the function of synthesizing the specific amino acid is incorporated into the yeast expression plasmid of the present invention, and the yeast strain into which this plasmid has been introduced is By culturing in a minimal medium that does not contain the specific amino acid, it is possible to prevent loss of the plasmid and efficiently express the target gene.
【００１１】本発明において用いられる酵母菌株として
は、たとえばサッカロミセス(Saccharomyces) 属、シゾ
サッカロミセス(Schizosaccharomyces）属、カンディダ
(Candida）属に属する酵母等があげることができる。好
ましくはサッカロミセス・セレビシェーAH22株（ATCC38
626)、サッカロミセス・セレビシェーSHY ３株、サッカ
ロミセス・セレビシェーNA87-11A株等をあげられる。Examples of yeast strains used in the present invention include Saccharomyces, Schizosaccharomyces, and Candida.
Examples thereof include yeasts belonging to the genus (Candida). Preferably Saccharomyces cerevisiae AH22 strain (ATCC38
626), Saccharomyces cerevisiae SHY 3 strain, Saccharomyces cerevisiae NA87-11A strain and the like.
【００１２】このようにして製造された本発明人工融合
酵素は、培養菌体から通常の方法により抽出・精製する
ことができる。たとえば、菌体をザイモリアーゼ等の溶
菌酵素で処理し、スフェロプラスト化した後、これを超
音波処理、フレンチプレス、ガラスビーズを用いる機械
的方法で破砕し、ミクロソーム画分を調製する。これか
らDEAE−セルロースカラムクロマトグラフィー等のイオ
ン交換カラムクロマトグラフィーやNADPH-チトクロム P
450 還元酵素部分の特性を使用した2'，5'-ADPセファロ
ース4Bカラムクロマトグラフィー等の通常の方法により
精製することができる。The artificial fusion enzyme of the present invention thus produced can be extracted and purified from the cultured cells by a conventional method. For example, the microbial cells are treated with a lysing enzyme such as zymolyase to form spheroplasts, which are then crushed by a mechanical method using ultrasonic treatment, French press, glass beads to prepare a microsome fraction. From this, ion exchange column chromatography such as DEAE-cellulose column chromatography and NADPH-cytochrome P
It can be purified by an ordinary method such as 2 ', 5'-ADP Sepharose 4B column chromatography using the property of 450 reductase moiety.
【００１３】本発明の人工融合酵素または該酵素を発現
する酵母菌株をバイオリアクターとして用いて、有用物
質の酸化反応プロセス、または産業廃水中の有害物質の
酸化的除去等に利用することができる。たとえば、本発
明の人工融合酵素または該酵素を発現する酵母菌株をス
テロイド化合物に作用させることにより該化合物を水酸
化させることができる。ステロイド化合物としては、た
とえば、ビタミンＤ3、１α−ヒドロキシビタミンＤ3
ならびに２５−ヒドロキシビタミンＤ3 等のビタミンＤ
3 誘導体、および５β−コレスタン ３α，７α，１２
α−トリオール（以下、THC と記す。）等をあげること
ができ、目的とする水酸化ステロイド化合物に応じて適
宜望ましいミトコンドリア型の成熟チトクロム P450 を
選択することができる。The artificial fusion enzyme of the present invention or the yeast strain expressing the enzyme can be used as a bioreactor for the oxidation reaction process of useful substances or the oxidative removal of harmful substances in industrial wastewater. For example, the artificial fusion enzyme of the present invention or a yeast strain expressing the enzyme can be reacted with a steroid compound to hydroxylate the compound. Examples of the steroid compound include vitamin D 3 , 1α-hydroxyvitamin D 3
And vitamin D such as 25-hydroxyvitamin D 3
3 derivatives, and 5β-cholestane 3α, 7α, 12
Examples thereof include α-triol (hereinafter referred to as THC) and the like, and a desired mitochondrial mature cytochrome P450 can be appropriately selected according to the target hydroxysteroid compound.
【００１４】以下、実施例についてさらに詳しく説明す
るが、本発明はこれらの実施例になんら限定されるもの
ではない。Examples will be described below in more detail, but the present invention is not limited to these examples.
【００１５】実施例１ ミトコンドリア型のチトクロム
P450 遺伝子の取得および酵母内発現プラスミドの構築 ミトコンドリア型のチトクロム P450 遺伝子の５′端末
側に存在するミトコンドリア型のシグナルペプチドをコ
ードする塩基配列を酵母内で機能するミクロソーム型の
シグナルペプチド（チトクロムP45017αのＮ末端15アミ
ノ酸残基）に変換したラット肝チトクロム P450 c25 の
cDNAを含むpAMS25（特開平４-349887 号）を鋳型にし、
配列番号１および２で示されるプライマーを用いて PCR
法によりBamHI-XhoI断片（約0.24kb）を得た。この断片
とpAMS25から得た HindIII-BamHI断片（約1.３kb）を同
時に市販のベクターBluescriptのHindIII 、XhoI部位に
挿入しpBMS25を得た。一方、酵母NADPH-P450還元酵素を
含むpBYR717(H)（特開平２−451 号）を PvuIIで消化
し、これに配列番号３で示されるXhoIリンカーを挿入
し、PvuII 部位をXhoI部位に変えた後、XhoIとHindIII
で同時消化し、約2.１kbの断片を得た。この断片と上記
のpBMS25から得たHindIII-XhoI断片（約1.５kb）を同時
にベクターpAAH５（特開昭57-159489 号）のHindIII 部
位へ挿入することにより酵母内発現プラスミドpAFC25R
(図１参照）を得た。よって、該人工融合酵素は1169ア
ミノ酸残基から成り、その構造はＮ末端から酵母内で機
能するミクロソーム型のシグナルペプチドに交換したラ
ット肝チトクロム P450 c25 をコードする全アミノ酸配
列(516残基：配列番号４において１番目から１５番目ま
でがミクロソーム型のシグナルペプチド部分であり、１
６番目から５０１番目までがミトコンドリア型の成熟チ
トクロム P450 部分である）、リンカーに由来する配列
（３残基：配列番号４において５０２番目から５０４番
目にある GCT-CGA-GCT)、酵母NADPH-チトクロム P450
還元酵素Ｎ末端42番目からＣ末端（650 残基：配列番号
４において５０５番目から６５０番目までの部分）と続
いている（配列番号４および図４参照）。Example 1 Mitochondrial cytochrome
Acquisition of P450 gene and construction of expression plasmid in yeast Microsomal signal peptide (cytochrome P450 17 that functions in yeast with a nucleotide sequence encoding a mitochondrial signal peptide existing on the 5'terminal side of mitochondrial cytochrome P450 gene) (N-terminal 15 amino acid residues of α) of rat liver cytochrome P450 c25
Using pAMS25 containing cDNA (JP-A-4-349887) as a template,
PCR using the primers shown in SEQ ID NOS: 1 and 2
A BamHI-XhoI fragment (about 0.24 kb) was obtained by the method. This fragment and the HindIII-BamHI fragment (about 1.3 kb) obtained from pAMS25 were simultaneously inserted into the HindIII and XhoI sites of the commercially available vector Bluescript to obtain pBMS25. On the other hand, pBYR717 (H) containing yeast NADPH-P450 reductase (Japanese Patent Laid-Open No. 2-451) was digested with PvuII, and the XhoI linker represented by SEQ ID NO: 3 was inserted thereinto to change the PvuII site into an XhoI site. Later, XhoI and HindIII
The fragment was co-digested with to obtain a fragment of about 2.1 kb. This fragment and the HindIII-XhoI fragment (about 1.5 kb) obtained from pBMS25 described above were simultaneously inserted into the HindIII site of the vector pAAH5 (JP-A-57-159489) to express the yeast expression plasmid pAFC25R.
(See FIG. 1) was obtained. Therefore, the artificial fusion enzyme consists of 1169 amino acid residues, the structure of which is the entire amino acid sequence encoding the rat liver cytochrome P450 c25 (the 516 residue: sequence, which is replaced with a microsomal signal peptide that functions in yeast from the N-terminus). In No. 4, 1 to 15 are microsomal signal peptide parts,
6th to 501st is a mitochondrial mature cytochrome P450 portion), a sequence derived from a linker (3 residues: GCT-CGA-GCT located from 502nd to 504th in SEQ ID NO: 4), yeast NADPH-cytochrome P450
Reductase N-terminal 42nd to C-terminal (650 residues: a portion from 505th to 650th in SEQ ID NO: 4) are continued (see SEQ ID NO: 4 and FIG. 4).
【００１６】実施例２ 酵母内発現プラスミドpAFC25R
の酵母内への導入 1.０mlの YPD培地（１％(w/v) 酵母エキス、２％(w/v)
ポリペプトン、２％(w/v) グルコース）にサッカロミセ
ス・セレビシェーAH22株（ATCC 38626）を植菌し、30℃
で18時間振盪した後、１mlの酵母培養液を遠心分離（50
00×ｇ、10分間）により集菌した。得られた菌体を1.０
mlの0.２M LiCl溶液に懸濁した後、再度遠心分離（5000
×ｇ、10分間）し、得られたペレットに20μｌの1M LiC
l 溶液、30μｌの70％ポリエチレングリコール4000（和
光純薬工業社）溶液、約1.０μｇの実施例１において得
られた各種の酵母内発現プラスミドをおのおの単独で含
む10μｌの溶液を添加した。これを十分に混合した後、
30℃で１時間インキュベートし、さらに140 μｌの滅菌
水を加えて攪拌した。この溶液をSD合成培地プレート
〔2.０％(w/v) グルコース、0.67％(w/v) 窒素源アミノ
酸不含（Nitrogen base w/o amino acids, Difco製）、
20μg/mlヒスチジン2.０％(w/v) 寒天〕上に蒔き、30℃
で３日間インキュベートし、上記の酵母内発現プラスミ
ドを保有する形質転換酵母菌株を選択した。このように
して、酵母内で人工融合酵素を発現させる酵母菌体を作
製した。Example 2 Expression plasmid pAFC25R in yeast
Introduction of yeast into yeast 1.0 ml YPD medium (1% (w / v) yeast extract, 2% (w / v)
Polypeptone, 2% (w / v) glucose) was inoculated with Saccharomyces cerevisiae AH22 strain (ATCC 38626) at 30 ° C.
After shaking for 18 hours at room temperature, 1 ml of yeast culture solution was centrifuged (50
The cells were collected at 00 × g for 10 minutes. The bacterial cells obtained were 1.0
After suspending in 0.2 ml of 0.2M LiCl solution, centrifuge again (5000
Xg for 10 minutes) and add 20 μl of 1M LiC to the resulting pellet.
l solution, 30 μl of 70% polyethylene glycol 4000 (Wako Pure Chemical Industries, Ltd.) solution, and 10 μl of a solution containing about 1.0 μg of each of the various yeast expression plasmids obtained in Example 1 alone. After mixing this well,
After incubating at 30 ° C. for 1 hour, 140 μl of sterilized water was further added and stirred. This solution was added to an SD synthetic medium plate [2.0% (w / v) glucose, 0.67% (w / v) nitrogen source amino acid-free (Nitrogen base w / o amino acids, manufactured by Difco),
20 μg / ml histidine 2.0% (w / v) agar], 30 ℃
After incubating for 3 days in the above, a transformed yeast strain carrying the above yeast expression plasmid was selected. In this way, yeast cells expressing the artificial fusion enzyme in yeast were prepared.
【００１７】実施例３ 酵母内で発現した人工融合酵素
の定量 人工融合酵素を発現した酵母の培養液〔合成培地（８％
(w/v) グルコース、5.４％(w/v) 窒素源アミノ酸不含
（Nitrogen base w/o amino acids, Difco製）、160 μ
g/mlヒスチジン）、菌体濃度2.０×107 菌体／ml〕200
mlを集菌し、10mlの100mM リン酸カリウム緩衝液（pH7.
０）に懸濁した後、遠心分離（5000×ｇ、10分間）し
た。得られたペレットを新たに2.０mlの 100mMリン酸カ
リウム緩衝液（pH7.０）に懸濁し、２本のキュベットに
1.０mlずつ分注した。サンプル側のキュベットに一酸化
炭素を吹き込んだ後、両キュベット内にジチオナイト５
−10mgを添加し、攪拌したのち400-500nm の差スペクト
ルを測定し、人工融合酵素濃度を算出した〔Δε(450nm
− 490nm) ＝91mM−１cm−１〕。菌体あたりの人工融合
酵素の発現量は約1.５×105 分子であった。Example 3 Quantification of artificial fusion enzyme expressed in yeast A culture solution of yeast expressing the artificial fusion enzyme [synthesis medium (8%
(w / v) glucose, 5.4% (w / v) nitrogen source amino acid-free (Nitrogen base w / o amino acids, manufactured by Difco), 160 μ
g / ml histidine), cell concentration 2.0 x 10 7 cells / ml] 200
Collect 10 ml of 100 mM potassium phosphate buffer (pH 7.
After suspending in (0), it was centrifuged (5000 × g, 10 minutes). The pellet thus obtained was resuspended in 2.0 ml of 100 mM potassium phosphate buffer (pH 7.0) and placed in two cuvettes.
Dispensed 1.0 ml each. After blowing carbon monoxide into the sample side cuvette, dithionite 5 was placed in both cuvettes.
After adding -10 mg and stirring, the difference spectrum at 400-500 nm was measured to calculate the artificial fusion enzyme concentration [Δε (450 nm
-490 nm) = 91 mM-1 cm-1]. The expression level of the artificial fusion enzyme per cell was about 1.5 × 10 5 molecules.
【００１８】実施例４ 酵母ミクロソーム画分の調製 実施例２によって作製された各種の酵母菌体おのおのの
培養液（合成培地、菌体濃度約2.０×107 菌体/ml)2.０
ｌを集菌し、該菌体を 400mlの緩衝液Ａ（10mMTris-HCl
(pH7.５),２M ソルビトール，0.１mM DTT, 0.１mM EDT
A)に懸濁した後、0.３mg／mlの濃度になるようにザイモ
リエイス（Zymolyase 100T；生化学工業社))を加え、30
℃で60分間インキュベートした。遠心分離（5000×ｇ、
10分間）して得られたスフェロプラストを 100mlの緩衝
液Ａに懸濁した後、再び遠心分離（5000×ｇ、10分間）
した。同じ遠心分離操作をもう一度繰り返してスフェロ
プラストの洗浄を行った後、スフェロプラストを 200ml
の緩衝液（10mM Tris-HCl(pH 7.５),0.65M ソルビトー
ル，0.１mM DTT, 0.１mM EDTA,1.０mMフェニルメチルス
ルホニルフロリド）に懸濁し、該懸濁液をテフロンホモ
ジナイザーを用いてホモジナイズした。このようにして
得られた菌破砕物を遠心分離（3000×ｇ、５分間）して
上清を回収した。沈殿は再度20mlの同緩衝液に懸濁し、
該懸濁液をテフロンホモジナイザーを用いてホモジナイ
ズした。このようにして得られた菌破砕物を遠心分離
（3000×ｇ、５分間）して上清を回収した。この上清と
先に回収された上清を併せた上清を遠心分離（10,000×
ｇ、20分間）して、上清を得た。このようにして得られ
た上清をさらに超遠心分離（120,000 ×ｇ、７０分間）
して、沈殿を回収した。該沈殿に0.１M のリン酸カリウ
ム緩衝液（pH7.４）を10ml添加し、懸濁することによ
り、ミクロソーム画分を得た。Example 4 Preparation of Yeast Microsome Fraction A culture solution of each of the various yeast cells prepared in Example 2 (synthetic medium, cell concentration: about 2.0 × 10 7 cells / ml) 2.0
1 was collected, and the cells were mixed with 400 ml of buffer A (10 mM Tris-HCl).
(pH7.5), 2M sorbitol, 0.1mM DTT, 0.1mM EDT
After suspending in A), add zymolyase (Zymolyase 100T; Seikagaku Corporation)) to a concentration of 0.3 mg / ml, and add 30
Incubated at 60 ° C for 60 minutes. Centrifuge (5000 xg,
The spheroplasts obtained after 10 minutes) were suspended in 100 ml of buffer A and then centrifuged again (5000 xg, 10 minutes).
did. Repeat the same centrifugation operation once more to wash the spheroplasts, and then add 200 ml of spheroplasts.
Buffer (10 mM Tris-HCl (pH 7.5), 0.65 M sorbitol, 0.1 mM DTT, 0.1 mM EDTA, 1.0 mM phenylmethylsulfonyl fluoride), and the suspension was added to a Teflon homogenizer. Used to homogenize. The crushed product thus obtained was centrifuged (3000 × g, 5 minutes) to collect the supernatant. The precipitate is suspended again in 20 ml of the same buffer,
The suspension was homogenized using a Teflon homogenizer. The crushed product thus obtained was centrifuged (3000 × g, 5 minutes) to collect the supernatant. The supernatant containing this supernatant and the previously collected supernatant was centrifuged (10,000 x
g, 20 minutes) to obtain a supernatant. The supernatant thus obtained is further subjected to ultracentrifugation (120,000 xg, 70 minutes).
Then, the precipitate was collected. A microsome fraction was obtained by adding 10 ml of 0.1 M potassium phosphate buffer (pH 7.4) to the precipitate and suspending it.
【００１９】実施例５ 人工融合酵素発現酵母菌株のミ
クロソーム画分におけるステロイド化合物水酸化活性の
測定 実施例４によって作製された人工融合酵素発現酵母菌株
およびベクターpAAH5のみを含む菌株のミクロソーム画
分50μｌを1.０mM EDTA を含む 100mM Tris-HCl 緩衝液
（pH7.８）に混合し、これに終濃度 100μＭになるよう
にステロイド化合物（１α−ヒドロキシビタミンＤ3 ま
たは3 H-THC ）を添加して全量を２mlに調製した。この
反応混合液を37℃で２分間インキュベートした後、該反
応液に終濃度0.５mMになるようにNADPH を加え、反応を
開始した。５分および10分後に、それぞれ反応液0.５ml
を分取し、これに２mlのジクロロメタンを加え、よく攪
拌した後、遠心分離（5000×ｇ、10分間）した。分離し
た層からジクロロメタンを回収し、該回収物をHPLCによ
り分析した。以下に分析条件を示す。 １．カラム：μBondapak C18 (φ４ｘ300mm 、ウォータ
ーズ社） ２．溶出条件：０〜５分 80％アセトニトリル水溶液 ５〜15分 80％〜100 ％の直線濃度勾配 15〜20分 100 ％アセトニトリル ３．流 速：1.０ml／min ４．検 出：１α−ヒドロキシビタミンＤ3 の場合， 2
65nmにおける吸光度検出器：3H-THCの場合，放射性検出
器 その結果、人工融合酵素発現酵母菌株のミクロソーム画
分においてステロイド水酸化物（１α，25−ジヒドロキ
シビタミンＤ3 または27−ヒドロキシ 3 H-THC) が検出
された。代謝回転速度は、１α−ヒドロキシビタミンＤ
3 を基質とした場合、約５mol/mol 融合酵素／min 、 3
H-THCを基質とした場合、約50mol/mol融合酵素／min
であった。一方、ベクターpAAH5 のみを含む酵母菌株の
ミクロソーム画分ではステロイド水酸化物は検出されな
かった。Example 5 Measurement of Steroid Compound Hydroxylation Activity in Microsomal Fraction of Artificial Fusion Enzyme Expressing Yeast Strain 50 μl of the artificial fusion enzyme expressing yeast strain prepared in Example 4 and the microsome fraction of the strain containing only the vector pAAH5 Mix with 100 mM Tris-HCl buffer (pH 7.8) containing 1.0 mM EDTA, and add a steroid compound (1α-hydroxyvitamin D 3 or 3 H-THC) to this to make a final concentration of 100 μM. Was prepared to 2 ml. After incubating the reaction mixture at 37 ° C. for 2 minutes, NADPH was added to the reaction to a final concentration of 0.5 mM to start the reaction. After 5 and 10 minutes, 0.5 ml of reaction solution
Was collected, 2 ml of dichloromethane was added thereto, and the mixture was stirred well and then centrifuged (5000 xg, 10 minutes). Dichloromethane was recovered from the separated layers and the recovered material was analyzed by HPLC. The analysis conditions are shown below. 1. Column: μBondapak C18 (φ4x300mm, Waters) 2. Elution conditions: 0 to 5 minutes 80% acetonitrile aqueous solution 5 to 15 minutes 80% to 100% linear concentration gradient 15 to 20 minutes 100% acetonitrile 3. Flow rate: 1.0 ml / min 4. Detection: 2 for 1α-hydroxyvitamin D 3
Absorbance detector at 65 nm: radioactive detector in the case of 3H-THC As a result, steroid hydroxide (1α, 25-dihydroxyvitamin D 3 or 27-hydroxy 3 H-THC was detected in the microsomal fraction of yeast strain expressing artificial fusion enzyme. ) Was detected. Turnover rate is 1α-hydroxyvitamin D
When 3 is used as a substrate, about 5 mol / mol fusion enzyme / min, 3
When H-THC is used as a substrate, about 50 mol / mol fusion enzyme / min
Met. On the other hand, no steroid hydroxide was detected in the microsomal fraction of the yeast strain containing only the vector pAAH5.
【００２０】[0020]
【発明の効果】本発明の人工融合酵素遺伝子が導入され
た酵母菌株のミクロソーム画分は、ミトコンドリア型の
成熟チトクロム P450 の有する１原子酸素添加反応にお
ける活性を著しく高めた。そして本発明人工融合酵素は
チトクロムＰ４５０の電子伝達系を単純化した一つの酵
素であり、有用物質の酸化反応プロセスまたは産業廃水
中の有害物質の酸化的除去等へ実用的応用を可能にす
る。INDUSTRIAL APPLICABILITY The microsomal fraction of the yeast strain into which the artificial fusion enzyme gene of the present invention has been introduced markedly increased the activity of the mitochondrial mature cytochrome P450 in the one-atom oxygenation reaction. The artificial fusion enzyme of the present invention is one enzyme that simplifies the electron transfer system of cytochrome P450, and enables practical application to the oxidation reaction process of useful substances or the oxidative removal of harmful substances in industrial wastewater.
【００２１】配列番号：１ 配列の長さ：３０ 配列の型：核酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ 配列 TCCGGGATCCAACACCCATTTGGCTCTGTG 30SEQ ID NO: 1 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence TCCGGGATCCAACACCCATTTGGCTCTGTG 30
【００２２】配列番号：２ 配列の長さ：３５ 配列の型：核酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ 配列 GCTTCTCGAGCCTGTCTCTGCAGAAAGCGTAGGCT 35SEQ ID NO: 2 Sequence Length: 35 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence GCTTCTCGAGCCTGTCTCTGCAGAAAGCGTAGGCT 35
【００２３】配列番号：３ 配列の長さ：１２ 配列の型：核酸 鎖の数：二本鎖 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ 配列 CAGCTCGAGCTG 12SEQ ID NO: 3 Sequence Length: 12 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence CAGCTCGAGCTG 12
【００２４】配列番号：４ 配列の長さ： 配列の型：核酸 鎖の数：二本鎖 トポロジー：直鎖状 配列の種類：他の核酸 配列 ATG TGG CTG CTC CTG GCT GTC TTT CTG CTC ACC CTC GCC TAT TTA GCG 48 Met Trp Leu Leu Leu Ala Val Phe Leu Leu Thr Leu Ala Tyr Leu Ala 1 5 10 15 ATC CCT GCA GCC CTC CGG GAT CAC GAG AGC ACG GAG GGT CCA GGA ACA 96 Ile Pro Ala Ala Leu Arg Asp His Glu Ser Thr Glu Gly Pro Gly Thr 20 25 30 GGT CAA GAC CGA CCG CGC CTG CGG AGT CTG GCG GAG CTT CCG GGA CCC 144 Gly Gln Asp Arg Pro Arg Leu Arg Ser Leu Ala Glu Leu Pro Gly Pro 35 40 45 GGA ACG CTA CGC TTT TTA TTC CAG CTA TTT CTA CGA GGC TAT GTG CTG 192 Gly Thr Leu Arg Phe Leu Phe Gln Leu Phe Leu Arg Gly Tyr Val Leu 50 55 60 CAC TTG CAC GAG CTC CAG GCG CTG AAC AAG GCC AAG TAG GGC CCA ATG 240 His Leu His Glu Leu Gln Ala Leu Asn Lys Ala Lys Tyr Gly Pro Met 65 70 75 80 TGG ACA ACC ACC TTT GGG ACT CGC ACC AAT GTG AAT CTG GCT AGC GCC 288 Trp Thr Thr Thr Phe Gly Thr Arg Thr Asn Val Asn Leu Ala Ser Ala 85 90 95 CCG CTC TTG GAG CAA GTG ATG AGA CAG GAG GGC AAG TAG CCC ATA AGA 336 Pro Leu Leu Glu Gln Val Met Arg Gln Glu Gly Lys Tyr Pro Ile Arg 100 105 110 GAC AGC ATG GAG CAG TGG AAG GAG CAC CAG GAC CAC AAA GGC CTC TCC 384 Asp Ser Met Glu Gln Trp Lys Glu His Arg Asp His Lys Gly Leu Ser 115 120 125 TAT GGG ATC TTC ATC ACA CAA GGA CAG CAG TGG TAC CAT CTG CGT CAT 432 Tyr Gly Ile Phe Ile Thr Gln Gly Gln Gln Trp Tyr His Leu Arg His 130 135 140 AGT TTG AAT CAG CGG ATG CTG AAG CCT GCT GAG GCA GCC CTC TAC ACA 480 Ser Leu Asn Gln Arg Met Leu Lys Pro Ala Glu Ala Ala Leu Tyr Thr 145 150 155 160 GAT GCC TTA AAC GAG GTC ATC AGT GAC TTT ATT GCC CGG CTG GAC CAG 528 Asp Ala Leu Asn Glu Val Ile Ser Asp Phe Ile Ala Arg Leu Asp Gln 165 170 175 GTG CGG ACA GAG AGT GCA TCA GGG GAT CAG GTG CCA GAT GTG GCA CAT 576 Val Arg Thr Glu Ser Ala Ser Gly Asp Gln Val Pro Asp Val Ala His 180 185 190 CTT CTC TAC CAC CTT GCC TTG GAA GCC ATC TGC TAT ATC CTG TTT GAG 624 Leu Leu Tyr His Leu Ala Leu Glu Ala Ile Cys Tyr Ile Leu Phe Glu 195 200 205 AAA AGG GTT GGC TGC CTG GAG CCC TCC ATC CCT GAG GAC ACC GCC ACC 672 Lys Arg Val Gly Cys Leu Glu Pro Ser Ile Pro Glu Asp Thr Ala Thr 210 215 220 TTC ATC AGA TCT GTT GGA CTC ATG TTC AAG AAC TCA GTC TAT GTC ACT 720 Phe Ile Arg Ser Val Gly Leu Met Phe Lys Asn Ser Val Tyr Val Thr 225 230 235 240 TTC CTT CCC AAG TGG TCT CGG CCT CTG CTG CCC TTT TGG AAG CGA TAC 768 Phe Leu Pro Lys Trp Ser Arg Pro Leu Leu Pro Phe Trp Lys Arg Tyr 245 250 255 ATG AAT AAC TGG GAT AAC ATT TTC TCC TTC GGG GAG AAG ATG ATT CAT 816 Met Asn Asn Trp Asp Asn Ile Phe Ser Phe Gly Glu Lys Met Ile His 260 265 270 CAA AAA GTC CAG GAG ATA GAA GCC CAG CTA CAG GCG GCT GGG CCA GAT 864 Gln Lys Val Gln Glu Ile Glu Ala Gln Leu Gln Ala Ala Gly Pro Asp 275 280 285 GGG GTC CAG GTA TCT GGC TAC CTG CAC TTC CTG CTG ACT AAG GAA TTG 912 Gly Val Gln Val Ser Gly Tyr Leu His Phe Leu Leu Thr Lys Glu Leu 290 295 300 CTC AGT CCT CAA GAG ACT GTC GGC ACC TTT CCT GAG CTG ATC TTG GCT 960 Leu Ser Pro Gln Glu Thr Val Gly Thr Phe Pro Glu Leu Ile Leu Ala 305 310 315 320 GGG GTA GAC ACG ACA TCC AAT ACA CTG ACC TGG GCC CTG TAT CAC CTT 1008 Gly Val Asp Thr Thr Ser Asn Thr Leu Thr Trp Ala Leu Tyr His Leu 325 330 335 TCA AAG AAC CCA GAG ATC CAG GAA GCC TTG CAC AAG GAA GTG ACT GGT 1056 Ser Lys Asn Pro Glu Ile Gln Glu Ala Leu His Lys Glu Val Thr Gly 340 345 350 GTG GTA CCC TTC GGG AAG GTG CCC CAG AAC AAG GAC TTT GCC CAC ATG 1104 Val Val Pro Phe Gly Lys Val Pro Gln Asn Lys Asp Phe Ala His Met 355 360 365 CCC CTG CTA AAA GCT GTG ATT AAG GAG ACC CTG CGC CTC TAC CCT GTG 1152 Pro Leu Leu Lys Ala Val Ile Lys Glu Thr Leu Arg Leu Tyr Pro Val 370 375 380 GTT CCC ACA AAC TCC CGG ATC ATC ACA GAA AAG GAA ACT GAA ATT AAT 1200 Val Pro Thr Asn Ser Arg Ile Ile Thr Glu Lys Glu Thr Glu Ile Asn 385 390 395 400 GGC TTC CTC TTC CCT AAG AAT ACA CAG TTT GTG TTA TGC CAC TAC GTG 1248 Gly Phe Leu Phe Pro Lys Asn Thr Gln Phe Val Leu Cys His Tyr Val 405 410 415 GTG TCC CGA GAT CCC AGT GTC TTT CCT GAG CCC GAG AGC TTC CAG CCT 1296 Val Ser Arg Asp Pro Ser Val Phe Pro Glu Pro Glu Ser Phe Gln Pro 420 425 430 CAG CGA TGG CTG AGG AAG AGA GAG GAC GAT AAC TCC GGG ATC CAA CAC 1344 His Arg Trp Leu Arg Lys Arg Glu Asp Asp Asn Ser Gly Ile Gln His 435 440 445 CCA TTT GGC TCT GTG CCC TTT GGC TAT GGG GTT CGG TCC TGC CTG GGT 1392 Pro Phe Gly Ser Val Pro Phe Gly Tyr Gly Val Arg Ser Cys Leu Gly 450 455 460 CGC AGG ATT GCA GAA CTG GAG ATG CAA CTC CTG CTG TCA AGG CTG ATA 1440 Arg Arg Ile Ala Glu Leu Glu Met Gln Leu Leu Leu Ser Arg Leu Ile 465 470 475 480 CAA AAG TAT GAG GTG GTC CTG TCT CCC GGG ATG GGA GAA GTG AAG TCT 1488 Gln Lys Tyr Glu Val Val Leu Ser Pro Gly Met Gly Glu Val Lys Ser 485 490 495 GTG TCC CGC ATC GTC CTG GTT CCC AGC AAG AAG GTG AGC CTA CGC TTT 1536 Val Ser Arg Ile Val Leu Val Pro Ser Lys Lys Val Ser Leu Arg Phe 500 505 510 CTG CAG AGA CAG GCT CGA GCT GCT GTC AGC TCG GGC AAC AGA GAC ATT 1584 Leu Gln Arg Gln Ala Arg Ala Ala Val Ser Ser Gly Asn Arg Asp Ile 515 520 525 GCT CAG GTG GTG ACC GAA AAC AAC AAG AAC TAC TTG GTG TTG TAT GCG 1632 Ala Gln Val Val Thr Glu Asn Asn Lys Asn Tyr Leu Val Leu Tyr Ala 530 535 540 TCG CAG ACT GGG ACT GCC GAG GAT TAC GCC AAA AAG TTT TCC AAG GAG 1680 Ser Gln Thr Gly Thr Ala Glu Asp Tyr Ala Lys Lys Phe Ser Lys Glu 545 550 555 560 CTG GTG GCC AAG TTC AAC CTA AAC GTG ATG TGC GCA GAT GTT GAG AAC 1728 Leu Val Ala Lys Phe Asn Leu Asn Val Met Cys Ala Asp Val Glu Asn 565 570 575 TAC GAC TTT GAG TCG CTA AAC GAT GTG CCC GTC ATA GTC TCG ATT TTT 1776 Tyr Asp Phe Glu Ser Leu Asn Asp Val Pro Val Ile Val Ser Ile Phe 580 585 590 ATC TCT ACA TAT GGT GAA GGA GAC TTC CCC GAC GGG GCG GTC AAC TTT 1824 Ile Ser Thr Tyr Gly Glu Gly Asp Phe Pro Asp Gly Ala Val Asn Phe 595 600 605 GAA GAC TTT ATT TGT AAT GCG GAA GCG GGT GCA CTA TCC AAC CTG AGG 1872 Glu Asp Phe Ile Cys Asn Ala Glu Ala Gly Ala Leu Ser Asn Leu Arg 610 615 620 TAT AAT ATG TTT GGT CTG AGG AAT TCT ACT TAT GAA TTC TTT AAT GGT 1920 Tyr Asn Met Phe Gly Leu Gly Asn Ser Thr Tyr Glu Phe Phe Asn Gly 625 630 635 640 GCC GCC AAG AAG GCC GAG AAG CAT CTC TCC GCC GCG GGC GCT ATC AGA 1968 Ala Ala Lys Lys Ala Glu Lys His Ile Ser Ala Ala Gly Ala Ile Arg 645 650 655 CTA GGC AAG CTC GGT GAA GCT GAT GAT GGT GCA GGA ACT ACA GAC GAA 2016 Leu Gly Lys Leu Gly Glu Ala Asp Asp Gly Ala Gly Thr Thr Asp Glu 660 665 670 GAT TAC ATG GCC TGG AAG GAC TCC ATC CTG GAG GTT TTG AAA GAC GAA 2064 Asp Tyr Met Ala Trp Lys Asp Ser Ile Leu Glu Val Leu Lys Asp Glu 675 680 685 CTG CAT TTG GAC GAA CAG GAA GCC AAG TTC ACC TCT CAA TTC CAG TAC 2112 Leu His Leu Asp Glu Gln Glu Ala Lys Phe Thr Ser Gln Phe Gln Tyr 690 695 700 ACT GTG TTG AAC GAA ATC ACT GAC TCC ATG TCG CTT GGT GAA CCC TCT 2160 Thr Val Leu Asn Glu Ile Thr Asp Ser Met Ser Leu Gly Glu Pro Ser 705 710 715 720 GCT CAC TAT TTG CCC TCG CAT CAG TTG AAC CGC AAC GCA GAC GGC ATC 2208 Ala His Tyr Leu Pro Ser His Gln Leu Asn Arg Asn Ala Asp Gly Ile 725 730 735 CAA TTG GGT CCC TTC GAT TTG TCT CAA CCG TAT ATT GCA CCC ATC GTG 2256 Gln Leu Gly Pro Phe Asp Leu Ser Gln Pro Tyr Ile Ala Pro Ile Val 740 745 750 AAA TCT CGC GAA CTG TTC TCT TCC AAT GAC CGT AAT TGC ATC CAC TCT 2304 Lys Ser Arg Glu Leu Phe Ser Ser Asn Asp Arg Asn Cys Ile His Ser 755 760 765 GAA TTT GAC TTG TCC GGC TCT AAC ATC AAG TAC TCC ACT GGT GAC CAT 2352 Glu Phe Asp Leu Ser Gly Ser Asn Ile Lys Tyr Ser Thr Gly Asp His 770 775 780 CTT GCT GTT TGG CCT TCC AAC CCA TTG GAA AAG GTC GAA CAG TTC TTA 2400 Leu Ala Val Trp Pro Ser Asn Pro Leu Glu Lys Val Glu Gln Phe Leu 785 790 795 800 TCC ATA TTC AAC CTG GAC CCT GAA ACC ATT TTT GAC TTG AAG CCC CTG 2448 Ser Ile Phe Asn Leu Asp Pro Glu Thr Ile Phe Asp Leu Lys Pro Leu 805 810 815 GAT CCC ACC GTC AAA GTG CCC TTC CCA ACG CCA ACT ACT ATT GGC GCT 2496 Asp Pro Thr Val Lys Val Pro Phe Pro Thr Pro Thr Thr Ile Gly Ala 820 825 830 GCT ATT AAA CAC TAT TTG GAA ATT ACA GGA CCT GTC TCC AGA CAA TTG 2544 Ala Ile Lys His Tyr Leu Glu Ile Thr Gly Pro Val Ser Arg Gln Leu 835 840 845 TTT TCA TCT TTG ATT CAG TTC GCC CCC AAC GCT GAC GTC AAG GAA AAA 2592 Phe Ser Ser Leu Ile Gln Phe Ala Pro Asn Ala Asp Val Lys Glu Lys 850 855 860 TTG ACT CTG CTT TCG AAA GAC AAG GAC CAA TTC GCC GTC GAG ATA ACC 2640 Leu Thr Leu Leu Ser Lys Asp Lys Asp Gln Phe Ala Val Glu Ile Thr 865 870 875 880 TCC AAA TAT TTC AAC ATC GCA GAT GCT CTG AAA TAT TTG TCT GAT GGC 2688 Ser Lys Tyr Phe Asn Ile Ala Asp Ala Leu Lys Tyr Leu Ser Asp Gly 885 890 895 GCC AAA TGG GAC AAC GTA CCC ATG CAA TTC TTG GTC GAA TCA GTT CCC 2736 Ala Lys Trp Asp Asn Val Pro Met Gln Phe Leu Val Glu Ser Val Pro 900 905 910 CAA ATG ACT CCT CGT TAC TAC TCT ATC TCT TCC TCT TCT CTG TCT GAA 2784 Gln Met Thr Pro Arg Tyr Try Ser Ile Ser Ser Ser Ser Leu Ser Glu 915 920 925 AAG CAA ACC GTC CAT GTC ACC TCC ATT GTG GAA AAC TTT CCT AAC CCA 2832 Lys Gln Thr Val His Val Thr Ser Ile Val Glu Asn Phe Pro Asn Pro 930 935 940 GAA TTG CCT GAT GCT CCT CCA GGT GTT GGT GTT ACG ACT AAC TTG TTA 2880 Glu Leu Pro Asp Ala Pro Pro Gly Val Gly Val Thr Thr Asn Leu Leu 945 950 955 960 AGA AAC ATT CAA TTG GCT CAA AAC AAT GTT AAC ATT GCC GAA ACT AAC 2928 Arg Asn Ile Gln Leu Ala Gln Asn Asn Val Asn Ile Ala Glu Thr Asn 965 970 975 CTA CCT GTT CAC TAC GAT TTA AAT GGC CCA CGT AAA CTT TTC GCC AAT 2976 Leu Pro Val His Tyr Asp Leu Asn Gly Pro Arg Lys Leu Phe Ala Asn 980 985 990 TAC AAA TTG CCC GTC CAC GTT CGT CGT TCT AAC TTC AGA TTG CCT TCC 3024 Tyr Lys Leu Pro Val His Val Arg Arg Ser Asn Phe Arg Leu Pro Ser 995 1000 1005 AAC CCT TCC ACC CCA GTT ATC ATG ATC GGT CCA GGT ACC GGT GTT GCC 3072 Asn Pro Ser Thr Pro Val Ile Met Ile Gly Pro Gly Thr Gly Val Ala 1010 1015 1020 CCA TTC CGT GGG TTT ATC AGA GAG CGT GTC GCG TTC CTG GAA TCA CAA 3120 Pro Phe Arg Gly Phe Ile Arg Glu Arg Val Ala Phe Leu Glu Ser Gln 1025 1030 1035 1040 AAG AAG GGC GGT AAC AAC GTT TCG CTA GGT AAG CAT ATA CTG TTT TAT 3168 Lys Lys Gly Gly Asn Asn Val Ser Leu Gly Lys His Ile Leu Phe Tyr 1045 1050 1055 GGA TCC CGT AAC ACT GAT GAT TTC TTG TAC CAG GAC GAA TGG CCA GAA 3216 Gly Ser Arg Asn Thr Asp Asp Phe Leu Tyr Gln Asp Glu Trp Pro Glu 1060 1065 1070 TAC GCC AAA AAA TTG GAT GGT TCG TTC GAA ATG GTC GTG GCC CAT TCC 3264 Tyr Ala Lys Lys Leu Asp Gly Ser Phe Glu Met Val Val Ala His Ser 1075 1080 1085 AGG TTG CCA AAC ACC AAA AAA GTT TAT GTT CAA GAT AAA TTA AAG GAT 3312 Arg Leu Pro Asn Thr Lys Lys Val Tyr Val Gln Asp Lys Leu Lys Asp 1090 1095 1100 TAC GAA GAC CAA GTA TTT GAA ATG ATT AAC AAC GGT GCA TTT ATC TAC 3360 Tyr Glu Asp Gln Val Phe Glu Met Ile Asn Asn Gly Ala Phe Ile Tyr 1105 1110 1115 1120 GTC TGT GGT GAT GCA AAG GGT ATG GCC AAG GGT GTG TCA ACC GCA TTG 3408 Val Cys Gly Asp Ala Lys Gly Met Ala Lys Gly Val Ser Thr Ala Leu 1125 1130 1135 GTT GGC ATC TTA TCC CGT GGT AAA TCC ATT ACC ACT GAT GAA GCA ACA 3456 Val Gly Ile Leu Ser Arg Gly Lys Ser Ile Thr Thr Asp Glu Ala Thr 1140 1145 1150 GAG CTA ATC AAG ATG CTC AAG ACT TCA GGT AGA TAC CAA GAA GAT GTC 3504 Glu Leu Ile Lys Met Leu Lys Thr Ser Gly Arg Tyr Gln Glu Asp Val 1155 1160 1165 TGG TAA 3510 Trp *** 1169SEQ ID NO: 4 Sequence length: Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Other nucleic acid Sequence ATG TGG CTG CTC CTG GCT GTC TTT CTG CTC ACC CTC GCC TAT TTA GCG 48 Met Trp Leu Leu Leu Ala Val Phe Leu Leu Thr Leu Ala Tyr Leu Ala 1 5 10 15 ATC CCT GCA GCC CTC CGG GAT CAC GAG AGC ACG GAG GGT CCA GGA ACA 96 Ile Pro Ala Ala Leu Arg Asp His Glu Ser Thr Glu Gly Pro Gly Thr 20 25 30 GGT CAA GAC CGA CCG CGC CTG CGG AGT CTG GCG GAG CTT CCG GGA CCC 144 Gly Gln Asp Arg Pro Arg Leu Arg Ser Leu Ala Glu Leu Pro Gly Pro 35 40 45 GGA ACG CTA CGC TTT TTA TTC CAG CTA TTT CTA CGA GGC TAT GTG CTG 192 Gly Thr Leu Arg Phe Leu Phe Gln Leu Phe Leu Arg Gly Tyr Val Leu 50 55 60 CAC TTG CAC GAG CTC CAG GCG CTG AAC AAG GCC AAG TAG GGC CCA ATG 240 His Leu His Glu Leu Gln Ala Leu Asn Lys Ala Lys Tyr Gly Pro Met 65 70 75 80 TGG ACA ACC ACC TTT GGG ACT CGC ACC AAT GTG AAT CTG GCT AGC GCC 288 Trp Thr Thr Thr Phe Gly Thr Arg Thr Asn Val Asn Leu Ala Se r Ala 85 90 95 CCG CTC TTG GAG CAA GTG ATG AGA CAG GAG GGC AAG TAG CCC ATA AGA 336 Pro Leu Leu Glu Gln Val Met Arg Gln Glu Gly Lys Tyr Pro Ile Arg 100 105 110 GAC AGC ATG GAG CAG TGG AAG GAG CAC CAG GAC CAC AAA GGC CTC TCC 384 Asp Ser Met Glu Gln Trp Lys Glu His Arg Asp His Lys Gly Leu Ser 115 120 125 TAT GGG ATC TTC ATC ACA CAA GGA CAG CAG TGG TAC CAT CTG CGT CAT 432 Tyr Gly Ile Phe Ile Thr Gln Gly Gln Gln Trp Tyr His Leu Arg His 130 135 140 AGT TTG AAT CAG CGG ATG CTG AAG CCT GCT GAG GCA GCC CTC TAC ACA 480 Ser Leu Asn Gln Arg Met Leu Lys Pro Ala Glu Ala Ala Leu Tyr Thr 145 150 155 160 GAT GCC TTA AAC GAG GTC ATC AGT GAC TTT ATT GCC CGG CTG GAC CAG 528 Asp Ala Leu Asn Glu Val Ile Ser Asp Phe Ile Ala Arg Leu Asp Gln 165 170 175 GTG CGG ACA GAG AGT GCA TCA GGG GAT CAG GTG CCA GAT GTG GCA CAT 576 Val Arg Thr Glu Ser Ala Ser Gly Asp Gln Val Pro Asp Val Ala His 180 185 190 CTT CTC TAC CAC CTT GCC TTG GAA GCC ATC TGC TAT ATC CTG TTT GAG 624 Leu Leu Tyr His Leu Ala Leu Glu Ala Ile Cys Tyr I le Leu Phe Glu 195 200 205 AAA AGG GTT GGC TGC CTG GAG CCC TCC ATC CCT GAG GAC ACC GCC ACC 672 Lys Arg Val Gly Cys Leu Glu Pro Ser Ile Pro Glu Asp Thr Ala Thr 210 215 220 TTC ATC AGA TCT GTT GGA CTC ATG TTC AAG AAC TCA GTC TAT GTC ACT 720 Phe Ile Arg Ser Val Gly Leu Met Phe Lys Asn Ser Val Tyr Val Thr 225 230 235 240 TTC CTT CCC AAG TGG TCT CGG CCT CTG CTG CCC TTT TGG AAG CGA TAC 768 Phe Leu Pro Lys Trp Ser Arg Pro Leu Leu Pro Phe Trp Lys Arg Tyr 245 250 255 ATG AAT AAC TGG GAT AAC ATT TTC TCC TTC GGG GAG AAG ATG ATT CAT 816 Met Asn Asn Trp Asp Asn Ile Phe Ser Phe Gly Glu Lys Met Ile His 260 265 270 CAA AAA GTC CAG GAG ATA GAA GCC CAG CTA CAG GCG GCT GGG CCA GAT 864 Gln Lys Val Gln Glu Ile Glu Ala Gln Leu Gln Ala Ala Gly Pro Asp 275 280 285 GGG GTC CAG GTA TCT GGC TAC CTG CAC TTC CTG CTG ACT AAG GAA TTG 912 Gly Val Gln Val Ser Gly Tyr Leu His Phe Leu Leu Thr Lys Glu Leu 290 295 300 CTC AGT CCT CAA GAG ACT GTC GGC ACC TTT CCT GAG CTG ATC TTG GCT 960 Leu Ser Pro Gln Glu Thr Val Gly Thr P he Pro Glu Leu Ile Leu Ala 305 310 315 320 GGG GTA GAC ACG ACA TCC AAT ACA CTG ACC TGG GCC CTG TAT CAC CTT 1008 Gly Val Asp Thr Thr Ser Asn Thr Leu Thr Trp Ala Leu Tyr His Leu 325 330 335 TCA AAG AAC CCA GAG ATC CAG GAA GCC TTG CAC AAG GAA GTG ACT GGT 1056 Ser Lys Asn Pro Glu Ile Gln Glu Ala Leu His Lys Glu Val Thr Gly 340 345 350 GTG GTA CCC TTC GGG AAG GTG CCC CAG AAC AAG GAC TTT GCC CAC ATG 1104 Val Val Pro Phe Gly Lys Val Pro Gln Asn Lys Asp Phe Ala His Met 355 360 365 CCC CTG CTA AAA GCT GTG ATT AAG GAG ACC CTG CGC CTC TAC CCT GTG 1152 Pro Leu Leu Lys Ala Val Ile Lys Glu Thr Leu Arg Leu Tyr Pro Val 370 375 380 GTT CCC ACA AAC TCC CGG ATC ATC ACA GAA AAG GAA ACT GAA ATT AAT 1200 Val Pro Thr Asn Ser Arg Ile Ile Thr Glu Lys Glu Thr Glu Ile Asn 385 390 395 400 GGC TTC CTC TTC CCT AAG AAT ACA CAG TTT GTG TTA TGC CAC TAC GTG 1248 Gly Phe Leu Phe Pro Lys Asn Thr Gln Phe Val Leu Cys His Tyr Val 405 410 415 GTG TCC CGA GAT CCC AGT GTC TTT CCT GAG CCC GAG AGC TTC CAG CCT 1296 Val Ser Arg As p Pro Ser Val Phe Pro Glu Pro Glu Ser Phe Gln Pro 420 425 430 CAG CGA TGG CTG AGG AAG AGA GAG GAC GAT AAC TCC GGG ATC CAA CAC 1344 His Arg Trp Leu Arg Lys Arg Glu Asp Asp Asn Ser Gly Ile Gln His 435 440 445 CCA TTT GGC TCT GTG CCC TTT GGC TAT GGG GTT CGG TCC TGC CTG GGT 1392 Pro Phe Gly Ser Val Pro Phe Gly Tyr Gly Val Arg Ser Cys Leu Gly 450 455 460 CGC AGG ATT GCA GAA CTG GAG ATG CAA CTC CTG CTG TCA AGG CTG ATA 1440 Arg Arg Ile Ala Glu Leu Glu Met Gln Leu Leu Leu Ser Arg Leu Ile 465 470 475 480 CAA AAG TAT GAG GTG GTC CTG TCT CCC GGG ATG GGA GAA GTG AAG TCT 1488 Gln Lys Tyr Glu Val Val Leu Ser Pro Gly Met Gly Glu Val Lys Ser 485 490 495 GTG TCC CGC ATC GTC CTG GTT CCC AGC AAG AAG GTG AGC CTA CGC TTT 1536 Val Ser Arg Ile Val Leu Val Pro Ser Lys Lys Val Ser Leu Arg Phe 500 505 510 CTG CAG AGA CAG GCT CGA GCT GCT GTC AGC TCG GGC AAC AGA GAC ATT 1584 Leu Gln Arg Gln Ala Arg Ala Ala Val Ser Ser Gly Asn Arg Asp Ile 515 520 525 GCT CAG GTG GTG ACC GAA AAC AAC AAG AAC TAC TTG GTG TTG TAT GCG 1632 Ala Gln Val Val Glu Asn Asn Lys Asn Tyr Leu Val Leu Tyr Ala 530 535 540 540 TCG CAG ACT GGG ACT GCC GAG GAT TAC GCC AAA AAG TTT TCC AAG GAG 1680 Ser Gln Thr Gly Thr Ala Glu Asp Tyr Ala Lys Lys Phe Ser Lys Glu 545 550 555 560 CTG GTG GCC AAG TTC AAC CTA AAC GTG ATG TGC GCA GAT GTT GAG AAC 1728 Leu Val Ala Lys Phe Asn Leu Asn Val Met Cys Ala Asp Val Glu Asn 565 570 575 TAC GAC TTT GAG TCG CTA AAC GAT GTG CCC GTC ATA GTC TCG ATT TTT 1776 Tyr Asp Phe Glu Ser Leu Asn Asp Val Pro Val Ile Val Ser Ile Phe 580 585 590 ATC TCT ACA TAT GGT GAA GGA GAC TTC CCC GAC GGG GCG GTC AAC TTT 1824 Ile Ser Thr Tyr Gly Glu Gly Asp Phe Pro Asp Gly Ala Val Asn Phe 595 600 605 GAA GAC TTT ATT TGT AAT GCG GAA GCG GGT GCA CTA TCC AAC CTG AGG 1872 Glu Asp Phe Ile Cys Asn Ala Glu Ala Gly Ala Leu Ser Asn Leu Arg 610 615 620 TAT AAT ATG TTT GGT CTG AGG AAT TCT ACT TAT GAA TTC TTT AAT GGT 1920 Tyr Asn Met Phe Gly Leu Gly Asn Ser Thr Tyr Glu Phe Phe Asn Gly 625 630 635 640 GCC GCC AAG AAG GCC GAG AAG CAT CTC TCC GCC GCG GGC GCT ATC AGA 1968 Ala Ala Lys Lys Ala Glu Lys His Ile Ser Ala Ala Gly Ala Ile Arg 645 650 655 CTA GGC AAG CTC GGT GAA GCT GAT GAT GGT GCA GGA ACT ACA GAC GAA 2016 Leu Gly Lys Leu Gly Glu Ala Asp Asp Gly Ala Gly Thr Thr Asp Glu 660 665 670 GAT TAC ATG GCC TGG AAG GAC TCC ATC CTG GAG GTT TTG AAA GAC GAA 2064 Asp Tyr Met Ala Trp Lys Asp Ser Ile Leu Glu Val Leu Lys Asp Glu 675 680 685 CTG CAT TTG GAC GAA CAG GAA GCC AAG TTC ACC TCT CAA TTC CAG TAC 2112 Leu His Leu Asp Glu Gln Glu Ala Lys Phe Thr Ser Gln Phe Gln Tyr 690 695 700 ACT GTG TTG AAC GAA ATC ACT GAC TCC ATG TCG CTT GGT GAA CCC TCT 2160 Thr Val Leu Asn Glu Ile Thr Asp Ser Met Ser Leu Gly Glu Pro Ser 705 710 715 720 GCT CAC TAT TTG CCC TCG CAT CAG TTG AAC CGC AAC GCA GAC GGC ATC 2208 Ala His Tyr Leu Pro Ser His Gln Leu Asn Arg Asn Ala Asp Gly Ile 725 730 735 CAA TTG GGT CCC TTC GAT TTG TCT CAA CCG TAT ATT GCA CCC ATC GTG 2256 Gln Leu Gly Pro Phe Asp Leu Ser Gln Pro Tyr Ile Ala Pro Ile Val 740 745 750 AAA TCT CGC GAA CTG T TC TCT TCC AAT GAC CGT AAT TGC ATC CAC TCT 2304 Lys Ser Arg Glu Leu Phe Ser Ser Asn Asp Arg Asn Cys Ile His Ser 755 760 765 GAA TTT GAC TTG TCC GGC TCT AAC ATC AAG TAC TCC ACT GGT GAC CAT 2352 Glu Phe Asp Leu Ser Gly Ser Asn Ile Lys Tyr Ser Thr Gly Asp His 770 775 780 CTT GCT GTT TGG CCT TCC AAC CCA TTG GAA AAG GTC GAA CAG TTC TTA 2400 Leu Ala Val Trp Pro Ser Asn Pro Leu Glu Lys Val Glu Gln Phe Leu 785 790 795 800 TCC ATA TTC AAC CTG GAC CCT GAA ACC ATT TTT GAC TTG AAG CCC CTG 2448 Ser Ile Phe Asn Leu Asp Pro Glu Thr Ile Phe Asp Leu Lys Pro Leu 805 810 815 GAT CCC ACC GTC AAA GTG CCC TTC CCA ACG CCA ACT ACT ATT GGC GCT 2496 Asp Pro Thr Val Lys Val Pro Phe Pro Thr Pro Thr Thr Ile Gly Ala 820 825 830 GCT ATT AAA CAC TAT TTG GAA ATT ACA GGA CCT GTC TCC AGA CAA TTG 2544 Ala Ile Lys His Tyr Leu Glu Ile Thr Gly Pro Val Ser Arg Gln Leu 835 840 845 TTT TCA TCT TTG ATT CAG TTC GCC CCC AAC GCT GAC GTC AAG GAA AAA 2592 Phe Ser Ser Leu Ile Gln Phe Ala Pro Asn Ala Asp Val Lys Glu Lys 850 855 860 T TG ACT CTG CTT TCG AAA GAC AAG GAC CAA TTC GCC GTC GAG ATA ACC 2640 Leu Thr Leu Leu Ser Lys Asp Lys Asp Gln Phe Ala Val Glu Ile Thr 865 870 875 880 TCC AAA TAT TTC AAC ATC GCA GAT GCT CTG AAA TAT TTG TCT GAT GGC 2688 Ser Lys Tyr Phe Asn Ile Ala Asp Ala Leu Lys Tyr Leu Ser Asp Gly 885 890 895 GCC AAA TGG GAC AAC GTA CCC ATG CAA TTC TTG GTC GAA TCA GTT CCC 2736 Ala Lys Trp Asp Asn Val Pro Met Gln Phe Leu Val Glu Ser Val Pro 900 905 910 CAA ATG ACT CCT CGT TAC TAC TCT ATC TCT TCC TCT TCT CTG TCT GAA 2784 Gln Met Thr Pro Arg Tyr Try Ser Ile Ser Ser Ser Leu Ser Glu 915 920 925 AAG CAA ACC GTC CAT GTC ACC TCC ATT GTG GAA AAC TTT CCT AAC CCA 2832 Lys Gln Thr Val His Val Thr Ser Ile Val Glu Asn Phe Pro Asn Pro 930 935 940 GAA TTG CCT GAT GCT CCT CCA GGT GTT GGT GTT ACG ACT AAC TTG TTA 2880 Glu Leu Pro Asp Ala Pro Pro Gly Val Gly Val Thr Thr Asn Leu Leu 945 950 955 960 AGA AAC ATT CAA TTG GCT CAA AAC AAT GTT AAC ATT GCC GAA ACT AAC 2928 Arg Asn Ile Gln Leu Ala Gln Asn Asn Val Asn Ile Ala Gl u Thr Asn 965 970 975 CTA CCT GTT CAC TAC GAT TTA AAT GGC CCA CGT AAA CTT TTC GCC AAT 2976 Leu Pro Val His Tyr Asp Leu Asn Gly Pro Arg Lys Leu Phe Ala Asn 980 985 990 TAC AAA TTG CCC GTC CAC GTT CGT CGT TCT AAC TTC AGA TTG CCT TCC 3024 Tyr Lys Leu Pro Val His Val Arg Arg Ser Asn Phe Arg Leu Pro Ser 995 1000 1005 AAC CCT TCC ACC CCA GTT ATC ATG ATC GGT CCA GGT ACC GGT GTT GCC 3072 Asn Pro Ser Thr Pro Val Ile Met Ile Gly Pro Gly Thr Gly Val Ala 1010 1015 1020 CCA TTC CGT GGG TTT ATC AGA GAG CGT GTC GCG TTC CTG GAA TCA CAA 3120 Pro Phe Arg Gly Phe Ile Arg Glu Arg Val Ala Phe Leu Glu Ser Gln 1025 1030 1035 1040 AAG AAG GGC GGT AAC AAC GTT TCG CTA GGT AAG CAT ATA CTG TTT TAT 3168 Lys Lys Gly Gly Asn Asn Val Ser Leu Gly Lys His Ile Leu Phe Tyr 1045 1050 1055 GGA TCC CGT AAC ACT GAT GAT TTC TTG TAC CAG GAC GAA TGG CCA GAA 3216 Gly Ser Arg Asn Thr Asp Asp Phe Leu Tyr Gln Asp Glu Trp Pro Glu 1060 1065 1070 TAC GCC AAA AAA TTG GAT GGT TCG TTC GAA ATG GTC GTG GCC CAT TCC 3264 Tyr Ala Lys Lys Leu Asp Gly Ser Phe Glu Met Val Val Ala His Ser 1075 1080 1085 AGG TTG CCA AAC ACC AAA AAA GTT TAT GTT CAA GAT AAA TTA AAG GAT 3312 Arg Leu Pro Asn Thr Lys Lys Val Tyr Val Gln Asp Lys Leu Lys Asp 1090 1095 1100 TAC GAA GAC CAA GTA TTT GAA ATG ATT AAC AAC GGT GCA TTT ATC TAC 3360 Tyr Glu Asp Gln Val Phe Glu Met Ile Asn Asn Gly Ala Phe Ile Tyr 1105 1110 1115 1120 GTC TGT GGT GAT GCA AAG GGT ATG GCC AAG GGT GTG ACC GCA TTG 3408 Val Cys Gly Asp Ala Lys Gly Met Ala Lys Gly Val Ser Thr Ala Leu 1125 1130 1135 GTT GGC ATC TTA TCC CGT GGT AAA TCC ATT ACC ACT GAT GAA GCA ACA 3456 Val Gly Ile Leu Ser Arg Gly Lys Ser Ile Thr Thr Asp Glu Ala Thr 1140 1145 1150 GAG CTA ATC AAG ATG CTC AAG ACT TCA GGT AGA TAC CAA GAA GAT GTC 3504 Glu Leu Ile Lys Met Leu Lys Thr Ser Gly Arg Tyr Gln Glu Asp Val 1155 1160 1165 TGG TAA 3510 Trp * ** 1169
【図１】人工融合酵素遺伝子を含む酵母内発現プラスミ
ドpAFC25R の構築方法を示す図である。FIG. 1 is a diagram showing a method for constructing a yeast expression plasmid pAFC25R containing an artificial fusion enzyme gene.
【図２】人工融合酵素の構造を示す図である。FIG. 2 is a diagram showing the structure of an artificial fusion enzyme.
フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｃ１２Ｒ 1:865） (Ｃ１２Ｎ 1/19 Ｃ１２Ｒ 1:865） Continuation of the front page (51) Int.Cl. 6 Identification code Office reference number FI technical display area C12R 1: 865) (C12N 1/19 C12R 1: 865)
Claims (9)
ロム P450 をコードするアミノ酸配列を有し、かつＣ末
端側にミクロソーム型チトクロム P450 を還元する役割
を有するNADPH-チトクロム P450 還元酵素をコードする
アミノ酸配列を有する人工融合酵素。1. An amino acid sequence having an amino acid sequence encoding a mitochondrial mature cytochrome P450 on the N-terminal side and an NADPH-cytochrome P450 reductase having a role of reducing microsomal cytochrome P450 on the C-terminal side. An artificial fusion enzyme having:
ロム P450 c25 をコードする塩基配列を有し、かつＣ末
端側にミクロソーム型チトクロム P450 を還元する役割
を有する酵母NADPH-チトクロム P450 還元酵素をコード
する塩基配列を有する人工融合酵素遺伝子。2. A yeast NADPH-cytochrome P450 reductase having a nucleotide sequence encoding a mitochondrial mature cytochrome P450 c25 on the N-terminal side and having a role of reducing microsomal cytochrome P450 on the C-terminal side. An artificial fusion enzyme gene having a base sequence.
よび請求項２記載の遺伝子を含む酵母内発現プラスミ
ド。4. A yeast expression plasmid containing a promoter for expression in yeast and the gene according to claim 2.
株。6. A yeast strain into which the gene according to claim 2 has been introduced.
25R)。7. A Saccharomyces cerevisiae AH22 (pAFC
25R).
入された酵母菌株を培養することを特徴とする請求項１
記載の人工融合酵素の製造方法。8. A yeast strain into which the yeast expression plasmid according to claim 4 has been introduced is cultured.
A method for producing the artificial fusion enzyme described.
６記載の酵母菌株をステロイド化合物に作用されること
により、該化合物を水酸化させる方法。9. A method for hydroxylating the artificial fusion enzyme according to claim 1 or the yeast strain according to claim 6 by acting on a steroid compound.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5298279A JPH07147975A (en) | 1993-11-29 | 1993-11-29 | Artificial fused enzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5298279A JPH07147975A (en) | 1993-11-29 | 1993-11-29 | Artificial fused enzyme |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH07147975A true JPH07147975A (en) | 1995-06-13 |
Family
ID=17857579
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP5298279A Pending JPH07147975A (en) | 1993-11-29 | 1993-11-29 | Artificial fused enzyme |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH07147975A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013512663A (en) * | 2009-12-03 | 2013-04-18 | ディーエスエム アイピー アセッツ ビー．ブイ． | Production of non-yeast sterols by yeast. |
| CN114774297A (en) * | 2022-03-25 | 2022-07-22 | 湖北工业大学 | Recombinant saccharomyces cerevisiae for producing T-cadinol and application thereof |
-
1993
- 1993-11-29 JP JP5298279A patent/JPH07147975A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013512663A (en) * | 2009-12-03 | 2013-04-18 | ディーエスエム アイピー アセッツ ビー．ブイ． | Production of non-yeast sterols by yeast. |
| CN114774297A (en) * | 2022-03-25 | 2022-07-22 | 湖北工业大学 | Recombinant saccharomyces cerevisiae for producing T-cadinol and application thereof |
| CN114774297B (en) * | 2022-03-25 | 2023-09-12 | 湖北工业大学 | Recombinant saccharomyces cerevisiae for producing T-juniper alcohol and application thereof |
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