【発明の詳細な説明】〔産業の利用分野〕本発明は、フェノールオキシダーゼ産生、分泌能を有す
る白色腐朽菌〔特にアラゲカワラタケ（Ｃｏｒｉｏｌｕ
ｓ ｈｉｒｓｕｔｕｓ ＩＦＯ ４９１７））のメッセ
ンジャーＲＮＡ （以下、ｍＲＮＡと略す）由来のフェ
ノールオキシダーゼ遺伝子を発現ベクターに挿入してな
る組換えＤＮＡ、これにより形質転換された大腸菌形質
転換株、フェノールオキシダーゼを含む該菌株の培養物
、およびフェノールオキシダーゼの製造方法に関する。Detailed Description of the Invention [Field of Industrial Application] The present invention is directed to the use of white rot fungi [particularly Coriolu
A recombinant DNA obtained by inserting a phenol oxidase gene derived from the messenger RNA (hereinafter abbreviated as mRNA) of S. hirsutus IFO 4917)) into an expression vector, an E. coli transformed strain transformed with the recombinant DNA, and the strain containing phenol oxidase. and a method for producing phenol oxidase.
フェノールオキシダーゼは分子状酸素の存在下でフェノ
ール類を酸化し、０−キノンあるいはｐ−キノンを生成
する酵素であり、補欠分子団として銅を含むことが知ら
れている。フェノールオキシダーゼは、動植物界に広く
分布しているが特に白色腐朽菌と呼ばれる一群の菌類の
生産するフェノールオキシダーゼは産業上有用であると
考えられる．白色腐朽菌は木材等のリグノセルロース物質中のリグニ
ンを分解する能力が高いことが知られており、この白色
腐朽菌をリグノセルロース物質に接種、培養し、リグニ
ンの一部を分解させパルプを製造するバイオロジカルパ
ルピングの試みがなされている（特開昭５０−４６９０
３号）。しかし、白色腐朽菌はリグニンを分解するだけ
でなく、パルプの原料となるセルロースやヘミセルロー
スをも分解する能力を有しており、バルプ収率の低下と
いう問題点を持っている。また、白色腐朽菌のリグニン
分解が二次代謝的に生育後期に起こるため時間がかかる
という問題点もあった。Phenol oxidase is an enzyme that oxidizes phenols in the presence of molecular oxygen to produce 0-quinone or p-quinone, and is known to contain copper as a prosthetic group. Phenol oxidase is widely distributed in the animal and plant kingdoms, but phenol oxidase produced by a group of fungi called white-rot fungi is considered to be particularly useful industrially. White rot fungi are known to have a high ability to decompose lignin in lignocellulosic materials such as wood, and these white rot fungi are inoculated into lignocellulosic materials, cultured, and a portion of the lignin is decomposed to produce pulp. Attempts have been made to develop biological pulping (Japanese Patent Application Laid-Open No. 1983-4690).
No. 3). However, white rot fungi have the ability not only to decompose lignin but also to decompose cellulose and hemicellulose, which are raw materials for pulp, and have the problem of reducing pulp yield. Another problem was that the decomposition of lignin by white-rot fungi occurs late in growth due to secondary metabolism, so it takes time.
白色腐朽菌のリグニン分解力は、白色腐朽菌が生産分泌
するフェノールオキシダーゼによるものが大きいと考え
られており、その遺伝子をクローニングする試みも行な
われており、本発明者等は先にアラゲカワラタケのフェ
ノールオキシダーゼ遺伝子をイントロンを含む塩基配列
として染色体から単離し（特願昭６３−１７５２３５号
）、またイントロンを含まないｃＤＮＡをｍＲＮＡから
単離したが（特願昭６３−１７５２３６号）、これらに
おいて、ベクターは遺伝子のクローニングのために用い
られているものであり、アラゲカワラタケ以外の異種生
物においてフェノールオキシダーゼ遺伝子を発現した例
は見当らない。一方、白色腐朽菌のフェノールオキシダ
ーゼと類似の活性を持っているラッカーゼについては、
ノイロスボラ・クラッサ（Ｎｅｕｒｏ−ｓｐｏｒａ ｃ
ｒａｓｓａ）のラッカーゼ遺伝子のクローニング（Ｕ．
Ｓ．Ｇｅｒｍａｎｎ＋ Ｋ．Ｌｅｒｃｈ；（１９８８）
Ｊ．Ｂｉｏｌ．Ｃｈｅｗ＋．％ｉ３． ８８５−５９６
）が報告されているが、そのアミノ酸配列はアラゲカワ
ラタケ由来のフェノールオキシダーゼのアミノ酸配列と
は異なるものであり、ノイロスポラ・クラッサのラッカ
ーゼによるリグニン分解についても、まだ報告されてい
ない。The lignin decomposition ability of white-rot fungi is thought to be largely due to the phenol oxidase produced and secreted by white-rot fungi, and attempts have been made to clone the gene for this. The phenol oxidase gene was isolated from chromosomes as a nucleotide sequence containing introns (Japanese Patent Application No. 175,235/1982), and cDNA without introns was isolated from mRNA (Japanese Patent Application No. 175,236/1980); Vectors are used for gene cloning, and there have been no examples of expression of the phenol oxidase gene in a heterologous organism other than C. versicolor. On the other hand, regarding laccase, which has similar activity to phenol oxidase of white rot fungi,
Neurospora crassa (Neuro-spora c
Cloning of the laccase gene of U. rassa)
S. German+K. (1988)
J. Biol. Chew+. %i3. 885-596
) has been reported, but its amino acid sequence is different from that of phenol oxidase derived from C. versicolor, and lignin degradation by Neurospora crassa laccase has not yet been reported.
［発明が解決しようとする課題］本発明は、前述の従来の問題点を解消し、フェノールオ
キシダーゼ遺伝子を白色腐朽菌以外の異種生物の発現ベ
クターに挿入し、フェノールオキシダーゼだけを生産す
る新規生物を提供することを目的とするものである。[Problems to be Solved by the Invention] The present invention solves the above-mentioned conventional problems, inserts the phenol oxidase gene into an expression vector of a heterologous organism other than white rot fungi, and creates a new organism that produces only phenol oxidase. The purpose is to provide
自然界におけるリグニンの住分解は、数種の酵素が関与
していると考えられているが白色腐朽菌が生産するフェ
ノールオキシダーゼはその中で中心的役割を果たすと考
えられており、リグニン分解の研究においても必須の酵
素となっている．したがって、リグニン分解能力だけを
効率的に発現する新規生物としてフェノールオキシダー
ゼ生産能力を付与した生物が望まれている。Several types of enzymes are thought to be involved in the biodegradation of lignin in nature, and phenol oxidase produced by white rot fungi is thought to play a central role. It is also an essential enzyme. Therefore, a novel organism that efficiently expresses only the ability to decompose lignin and is endowed with the ability to produce phenol oxidase is desired.
（ｌ）〈フェノールオキシダーゼ遺伝子の調製〉本発明
において、発現ベクターに挿入されるフェノールオキシ
ダーゼ遺伝子は、例えば前記の特願昭６３−１７５２３
６号に記載したものであって、下記のアミノ酸配列をコ
ードするフェノールオキシダーゼ遺伝子（ＩＩ）である
か、ＰｈｅＴｒｐＴｙｒＨｉｓｓｅｒＨｉｓＬｅｕｓｅｒＴ
ｈｒＧｌｎＴｙｒｌ；ｙｓＡｓｐＬｉｔｙＬｅｕＶａｌ
ＡｓｎＡｓｐＧｌｎ（但し、式中３９６ｘはＰｒｏまたは＾ｌａを表わす。(l) <Preparation of phenol oxidase gene> In the present invention, the phenol oxidase gene inserted into the expression vector can be
6 and encodes the following amino acid sequence, or PheTrpTyrHisserHisLeuserT
hrGlnTyrl;ysAspLityLeuVal
AsnAspGln (However, in the formula, 396x represents Pro or ^la.
）あるいは、上記アミノ酸配列をコードするＤＮＡにハ
イブリッドするＤＮＡ配列であって、天然、合成、もし
くは半合成によって得られるものであり、上記アミノ酸
配列をコードするＤＮＡ配列に対して、ヌクレオチドの
置換、ヌクレオチドの挿入及びヌクレオチド配列の逆位
その他の変異によって関連づけられており、かつ、フェ
ノールオキシダーゼ活性を有するポリベプチドをコード
するＤＮＡからなるフェノールオキシダーゼ遺伝子であ
る。) or a DNA sequence that hybridizes to the DNA encoding the above amino acid sequence, which is obtained naturally, synthetically, or semi-synthetically, and which is a nucleotide substitution or nucleotide The phenoloxidase gene is related by insertions, inversions of the nucleotide sequence, and other mutations, and consists of DNA encoding a polypeptide having phenoloxidase activity.
さらに、具体的には、フェノールオキシダーゼ遺伝子（
ＩＩ）が次式（ＯＪ−ＰＯＭ５）Ｖａ Ｉ Ｖａ ＩＡ
ｒｇＳｅｒＡ ｌ ａＧ １ｙＳｅｒＴ ｈｒＶａ ｌ
１’ｙｒＡｓｎｒｙｒＡｓ ｐＡｓｎｒｒｏＧＣＣＡ
ＴＴＧＧＧＣＣＣＡＣＣＧＣＴＧＡＣＣＴＣＡＣＣＡＴ
ＣＴＣＣＡＡＴＧＣＣＧＣＧＴＣＧＧＡＴＴＴＧＣＧＧ
ＧＴＧＧＴＡＴＣＡＡＣＴＣＧＧＣＣＡＴＣＣＴＧＣＧ
Ｃまたは次式（ＯＪ−ＰＯＭ２）ＧＣＣＡＴＴＧＧＧＣＣＣＡＣＣＧＣＴＧＡＣＣＴＣＡ
ＣＣＡＴＣＴＣＣＡＡＴＧＣＣＧＣＧＴＣＧＧＡＴＴＴ
ＧＣＧＧＧＴＧＧＴＡＴＣＡＡＣＴＣＧＧＣＣＡＴＣＣ
ＴＧＣＧＣのＤＮＡ配列を有するものが挙げられる。Furthermore, specifically, the phenol oxidase gene (
II) is the following formula (OJ-POM5) Va I Va IA
rgSerA l aG 1ySerT hrVa l
1'yrAsnryrAs pAsnrroGCCA
TTGGGCCCACCGCTGACCTCACCAT
CTCCAATGCCGCGTCGGATTTGCGG
GTGGTATCAACTCGGCCATCCTGCG
C or the following formula (OJ-POM2) GCCATTGGGCCCACCGCTGACCTCA
CCATCTCCAATGCCGCGTCGGATTT
GCGGGTGGTATCAACTCGGCCATCC
Examples include those having the DNA sequence of TGCGC.
ＴＣＧＧＴＡＡＣＴＡＣＴＧＧＡＴＴＣＧＣＧＣＣＡＡ
ＣＣＣＣＡＡＣＴＴＣＧＧＣＡＡなお、フェノールオキ
シダーゼ遺伝子が（ＯＪ−ＰＯＭ ５）である場合、上
記アミノ酸配列の３９０ＸはＡｌａとなり、また、フェ
ノールオキシダーゼ遺伝子が（ＯＪ−ＰＯＭ ２）であ
る場合、上記アミノ酸配列の３９０ｘはＰｒｏ となる
。TCGGTAACTACTGGATTCGCGCCAA
CCCCAAACTTCGGCAAIn addition, when the phenol oxidase gene is (OJ-POM 5), 390X of the above amino acid sequence becomes Ala, and when the phenol oxidase gene is (OJ-POM 2), 390X of the above amino acid sequence becomes Pro. Become.
（２） ＜ ５ ’末端の非翻訳領域の除去〉クローニ
ングしたフェノールオキシダーゼ遺伝子（Ｉ［）を含む
プラスミドをもつ大腸菌形質転換株は寄託されており、
（ＯＪ−ＰＯＭ２ ；微工研菌寄第１００５５号，ＯＪ
−ＰＯＭ ５ ；微工研菌寄第１００６１号）、これら
のプラスミドはフェノールオキシダーゼの構造遺伝子以
外の領域を含む（第１図、第２図参照）。(2) <Removal of untranslated region at the 5'end> An E. coli transformed strain carrying a plasmid containing the cloned phenol oxidase gene (I[) has been deposited.
(OJ-POM2; Microtechnical Research Institute No. 10055, OJ
-POM 5 ; Microtechnical Research Institute No. 10061), these plasmids contain regions other than the structural gene of phenol oxidase (see Figures 1 and 2).
大腸菌等の異種微生物を用いて発現生産させる場合、で
きるだけタンパク質をコードしていない領域を取り除く
ことが好ましい。取り除く方法としてはエキソヌクレア
ーゼｍ （Ｈｅｎｉｋｏｆｆ，Ｓ，Ｇｅｎｅ，２８，３
５１−３５９． （１９８４） ）やＢＡＬ３１ヌクレ
アーゼ（Ｇｕｏ．Ｌ，−Ｈ．ｅｔ ａｌ．Ｍｅｔｈｏｄ
ｓ ｉｎ Ｅｎｚｙｓ＋ｏｌｏｇＬ １００＋ ６０−
９６（１９８３） ３などの修飾酵素で非翻訳領域を削
除する方法により除去することが出来る。When performing expression production using a heterologous microorganism such as E. coli, it is preferable to remove regions that do not encode proteins as much as possible. Exonuclease m (Henikoff, S. Gene, 28, 3
51-359. (1984) ) and BAL31 nuclease (Guo. L, -H. et al. Method
s in Enzys+ologL 100+ 60-
96 (1983) 3, etc., by deleting the untranslated region.
（３）＜発現ベクターへの連結〉大ｌＩ！１１１発現ベクターとしては大腸菌ベクターＣ
ｏＩＥＩ，ｐＭＢｌ系などに発現に関与するプロモータ
ー領域を、その下流にタンパク質合成に関与するＳＤ領
域を、さらに翻訳開始点ＡＴＧを含むもので、その下流
に幾つかの制限酵素サイトを持つものであればいずれも
用いることが出来るが、ｐＫＫ２２３−３（ファルマシ
ア社製） 、ｐＫＫ２３３−２ （ファルマシア社製）
、ｐＰＬ−ｌａｍｂｄａ （ファルマシア社製）等が
例示される．これらの大腸菌発現ベクターの翻訳開始点ＡＴＧの下流
の適当な制限酵素サイトを用いて、５゜末端の非翻訳領
域を除いたフェノールオキシダーゼ遺伝子を翻訳のフレ
ームが一致するように、ベクター側の制限酵素で切断し
たＤＮＡ断片およびリンカーＤＮＡと連結し、フェノー
ルオキシダーゼ遺伝子に関する発現プラスミドを構築す
ることが出来る．（４）＜大腸菌への導入〉上記の組換えプラスミドを導入する宿主大腸菌はいずれ
の株も用いることが出来るが、公知のＫ１２株系ＨＢＩ
ＯＩ株、ＪＭ１０９株、Ｃ ６００株などが例示される
。(3) <Connection to expression vector> Big lI! 111 expression vector is E. coli vector C.
A promoter region involved in expression such as oIEI or pMBl system, an SD region involved in protein synthesis downstream thereof, a translation initiation point ATG, and several restriction enzyme sites downstream thereof. Either pKK223-3 (manufactured by Pharmacia) or pKK233-2 (manufactured by Pharmacia) can be used.
, pPL-lambda (manufactured by Pharmacia), etc. Using an appropriate restriction enzyme site downstream of the translation start point ATG of these E. coli expression vectors, the phenol oxidase gene, excluding the untranslated region at the 5° end, is inserted into the restriction enzyme on the vector side so that the translation frame matches. It is possible to construct an expression plasmid for the phenol oxidase gene by ligating the DNA fragment cut with and linker DNA. (4) <Introduction into E. coli> Any strain of E. coli can be used as the host E. coli into which the above recombinant plasmid is introduced, but the known K12 strain HBI
Examples include OI strain, JM109 strain, and C600 strain.
上記のプラスミドを宿主大腸菌に形質転換する方法は、
例えば塩化カルシウム法、塩化ルビジウム法（Ｍａｎｉ
ａｔｉｓら、”Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ＋
Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ”（１９８２
））など既存の技術によって行なうことが出来る。形質
転換株の選抜は、各々の発現ベクターにある薬剤耐性な
どのマーカーを利用して行なうことが出来る。例えば発
現ベクターとしてｐＫＫ２２３−３，ρＫＫ２３３−２
を用いる場合には、形質転換株をアンピシリンを含むＬ
Ｂ寒天培地上に塗布、培養することにより形質転換株を
選抜することが出来る。The method for transforming the above plasmid into host E. coli is as follows:
For example, calcium chloride method, rubidium chloride method (Mani
atis et al., “Molecular Cloning+
A Laboratory Manual” (1982
)). Selection of transformed strains can be performed using markers such as drug resistance included in each expression vector. For example, pKK223-3, ρKK233-2 as an expression vector.
When using L, the transformed strain is treated with L containing ampicillin.
Transformed strains can be selected by coating and culturing on B agar medium.
ｐＫＫ２３３−２のＴｒｃプロモーターの下流にフレー
ムがあうように旧ｎｄＩ［Ｉリンカーを介してフェノー
ルオキシダーゼ遺伝子ＯＪ−ＰＯＭ５の構造遺伝子をつ
ないで得られたｐＫＫＰＯ５で形質転換した大腸菌Ｅ．
ｃｏｌｉＪＭ１０９／ｐＫＫＰＯ５は微生物工業技術研
究所に寄託した（寄託番号ＦＥＲＭ Ｐ−１０５７６
）。Escherichia coli E. coli was transformed with pKKPO5, which was obtained by ligating the structural gene of the phenol oxidase gene OJ-POM5 downstream of the Trc promoter of pKK233-2 through the old ndI[I linker so that it matched the frame.
coli JM109/pKKPO5 was deposited with the Microbial Technology Research Institute (deposit number FERM P-10576).
).
（５）〈フェノールオキシダーゼの生産〉上記のように
して得られたフェノールオキシダーゼ遺伝子（■）を含
む大腸菌の発現プラスミドで形質転換した大腸歯形質転
換株を大腸菌の生育可能な適当な培地で培養することに
より、その培養物中にフェノールオキシダーゼを多量に
産生ずることができた。更に培地に銅イオンを添加する
とフェノールオキシダーゼの生産量が増大する。(5) <Production of phenol oxidase> The colon tooth transformant strain transformed with the E. coli expression plasmid containing the phenol oxidase gene (■) obtained as above is cultured in an appropriate medium in which E. coli can grow. As a result, a large amount of phenol oxidase could be produced in the culture. Furthermore, addition of copper ions to the medium increases the amount of phenol oxidase produced.
ｐＫＫ２２３−３． ｐＫＫ２３３−２を用いる場合に
は培地中にイソプロビルーβ一Ｄ−チオガラクトシド等
の大腸菌ラクトースオペロンの誘導物質を培地に添加す
ることにより、生産量の増大が図られる。pKK223-3. When pKK233-2 is used, the production amount can be increased by adding an inducer of the Escherichia coli lactose operon, such as isoprobil-β-D-thiogalactoside, to the medium.
フェノールオキシダーゼ遺伝子にコードされるタンパク
質は、大腸菌の培養物を超音波処理またはフレンチプレ
ス処理などにより、細胞を破砕し、アラゲカワラタケの
フェノールオキシダーゼに対する抗体を用いて検出した
。この様にして得られたフェノールオキシダーゼは通常
活性が無いかまたは低いが、レドックス系など一般に行
なわれる巻戻し操作により活性のあるタンパク質を得る
ことが出来る。例えば６Ｍグアニジン塩酸に溶解後、２
Ｍグアニジン塩酸溶液とし、銅イオンを含む溶液中でグ
ルタチオン処理することによりフェノールオキシダーゼ
の活性を上昇せしめることができる。The protein encoded by the phenol oxidase gene was detected by disrupting the cells of an E. coli culture by sonication or French press treatment, and using an antibody against the phenol oxidase of C. versicolor. The phenol oxidase obtained in this way usually has no or low activity, but an active protein can be obtained by a commonly performed unwinding operation such as a redox system. For example, after dissolving in 6M guanidine hydrochloride, 2
The activity of phenol oxidase can be increased by treating it with glutathione in a solution containing M guanidine hydrochloride and copper ions.
以下、実施例により本発明をさらに詳細に説明するが、
下記の実施例は本発明を制限するものではない。Hereinafter, the present invention will be explained in more detail with reference to Examples.
The examples below do not limit the invention.
また、特に断わりがない限りＴ．Ｍａｎｉａｔｉｓ ＆
ε．Ｆ．Ｆｒｉ ｔｓｃｈらのｒＭｏｌｅｃｕｌａｒ
Ｃｌｏｎｉｎｇ （１９８２）ＪおよびＳ．Ｌ．Ｂｅｒ
ｇｅｒ ＆＾．Ｒ．Ｘｉｓ＋ａ＋ｅｌのｒ Ｍｅｔｈｏ
ｄｓ ｉｎＥｎｚｙｍｏｌｏｇｙ Ｖｏ１．１５２．（
１９８７） Ｊ等に記載されている実験操作に従って行
った。Also, unless otherwise specified, T. Maniatis &
ε. F. rMolecular of Fritsch et al.
Cloning (1982) J and S. L. Ber
ger ＆＾． R. Xis+a+el's rMetho
ds inEnzymology Vol1.152. (
1987) according to the experimental procedure described in J. et al.
以下、実施例により本発明を詳細に説明する．〔実施例
〕（１） ＤＮＡプローブのＡＤＮＡプローブの合成は、アミダイト法により、ＤＮＡ
合成機（日本ゼオン．ＧｅｎｅｔＡ − ｍ ）を用い
て行なった．３種の白色腐朽菌〔アラゲカワラタケ（ＩＦＯ ４９１
７）．カワラタケ（ＩＦＯ ３０３４０），カイガラタ
ケ（ＩＰＯ Ｂ７１４））から精製したフェノールオキ
シダーゼのＮ末端からのアミノ酸配列をエドマン分解法
で２５段目まで分析した結果を次に示す。Hereinafter, the present invention will be explained in detail with reference to Examples. [Example] (1) DNA probe A The DNA probe was synthesized using the amidite method.
This was carried out using a synthesizer (Nippon Zeon GenetA-m). Three types of white rot fungi [IFO 491
7). The results of an analysis of the amino acid sequence from the N-terminus of phenol oxidase purified from C. versicolor (IFO 30340) and C. versicolor (IPO B714)) up to the 25th stage using the Edman degradation method are shown below.
カツラタケＡｌａ−＾ｒｇ−Ｇｌｎ−Ａｌａ−Ｖａｌ−カイガラタ
ケＡｌａ−Ａｒｇ−Ｇｌｎ−Ａｌａ−Ｖａｌ−上記配列の
１７段目のＰｒｏから２５段目のＶａｌ に対応するよ
うに、次のＤＮＡプロープを合成した。Katsuratake Ala-^rg-Gln-Ala-Val-Scale mushroom Ala-Arg-Gln-Ala-Val-The following DNA probes were synthesized to correspond to Pro at the 17th line to Val at the 25th line of the above sequence. .
但しＩはデオキシイノシン。However, I is deoxyinosine.
また、３種の白色腐朽菌のフェノールオキシダーゼをＢ
ｒＣＮで分解し、逆相系高速液体クロマトグラフィー〔
溶出条件，カラム： Ｐｈｅｎｙｌ−５ＰＷ ＲＰ（東
洋ソーダ社製）， 溶出液２０％アセトニトリル／０．
ｌ％ＴＦＡから７５％アセトリトリル／０．１％ＴＦ＾
への濃度勾配溶出，室温〕で分離したポリペプチドのア
ミノ酸配列をエドマン分解法で分析した結果を次に示す
。In addition, B
Decomposition with rCN and reverse phase high performance liquid chromatography [
Elution conditions, column: Phenyl-5PW RP (manufactured by Toyo Soda Co., Ltd.), eluent 20% acetonitrile/0.
1% TFA to 75% acetotrile/0.1% TF^
The amino acid sequence of the polypeptide separated by concentration gradient elution at room temperature was analyzed using the Edman degradation method.The results are shown below.
アラゲカ１５タケ Ｍｅｔ−Ａｌａ−Ｐｈｅ−Ａ
ｓｎ−Ｐｈｅカワラタケ Ｍｅｔ−Ａｌａ
−Ｐｈｅ−Ａｓｎ−Ｐｈｅカイｉラタケ Ｍ
ｅｔ−Ａｌａ−Ｐｈｅ−Ａｓｎ−Ｐｈｅ上記アミノ酸配
列に対応するように次のＤＮＡプローブを合成した。Arageka 15 Bamboo Met-Ala-Phe-A
sn-Phe Versicolor Met-Ala
-Phe-Asn-Phe Chiratake M
et-Ala-Phe-Asn-Phe The following DNA probe was synthesized to correspond to the above amino acid sequence.
ＧＧ（；Ｉ；以上の結果から白色腐朽菌が生産、分泌するフェノール
オキシダーゼのアミノ酸配列の相同性は非常に高く、本
発明で使用するＤＮＡプローブを用いることにより、い
かなる白色腐朽菌のフェノールオキシダーゼ遺伝子（Ｉ
Ｉ）をもクローニングすることができる．したがって以
下の実施例では、アラゲカワラタケのフェノールオキシ
ダーゼ遺伝子（ｎ）のクローニング方法について説明す
る。GG(;I; From the above results, the homology of the amino acid sequences of phenol oxidase produced and secreted by white rot fungi is very high. By using the DNA probe used in the present invention, it is possible to identify the phenol oxidase gene of any white rot fungi. (I
I) can also be cloned. Therefore, in the following example, a method for cloning the phenol oxidase gene (n) of C. versicolor will be described.
（２）且Ｋ八夏坦袈アラゲカワラタケ（ＩＦＯ ４９１７）を１１のＹＰＤ
培地（酵母エキス１０ｇ／ ｌ ，ポリペプトン２０ｇ
／ ｌ ，グルコース２０ｇ／ ｆｔ ）が入った５ｌ
容三角フラスコに植菌し、２７℃，６日間振盪培養した
．培養液中にフェノールオキシダーゼを生産，分泌して
いることを確認した後、集菌し、液体窒素中で凍結した
結果、約２０ｇの凍結菌体を得た。(2) And K. Yasatsudankesa Aragekawaratake (IFO 4917) with 11 YPD
Medium (yeast extract 10g/l, polypeptone 20g
/l, glucose 20g/ft)
The cells were inoculated into an Erlenmeyer flask and cultured with shaking at 27°C for 6 days. After confirming that phenol oxidase was produced and secreted in the culture solution, the bacteria were collected and frozen in liquid nitrogen, resulting in approximately 20 g of frozen bacterial cells.
Ｂｒｏｄａ等の方法により、凍結菌体５ｇから１１■の
全ＲＮＡを抽出した．凍結菌体５ｇを液体窒素を加えた
１００−のりーリングブレンダーで破砕し、３倍量のＴ
ＮＳ緩衝液Ｃ１％ｔｒｉ−ｉｓｏ−Ｐｒｏｐｙｌｎａｐ
ｈｔｈａｌｅｎｅ ｓｕｌｐｈｏｎｉｃ ａｃｉｄ，
２００ｍＭ Ｔｒｉｓ−ＨＣＩ， ２５ｍＭＥＧＴ
Ａ （ｐＨ７．８），２５０ｍＭ ＮａＣ１ ）に溶か
した。遠心分離によりペレットを除き、上清ｌｍＡにつ
き、０．５ｇのフェノールを加え、５〜１５℃に保ち溶
解した。全てのフェノールが溶けたら％量のクロロホル
ムを加え、遠心分離後、上清を回収し、クロロホルムで
２回抽出した後、エタノール沈澱により全ＲＮＡ１１ｍ
ｇを回収した。11 μg of total RNA was extracted from 5 g of frozen bacterial cells by the method of Broda et al. Crush 5 g of frozen bacterial cells with a 100-ml ring blender containing liquid nitrogen, and add 3 times the amount of T.
NS buffer C1% tri-iso-Propylnap
hthalene sulfonic acid,
200mM Tris-HCI, 25mMEGT
A (pH 7.8), 250mM NaCl). The pellet was removed by centrifugation, and 0.5 g of phenol was added to each supernatant lmA, and the mixture was maintained at 5 to 15°C to dissolve. When all the phenol has dissolved, add 1% chloroform, centrifuge, collect the supernatant, extract twice with chloroform, and precipitate with ethanol to obtain 11 m of total RNA.
g was collected.
また、市販のＲＮＡ抽出キット〔アマシャム・ジャパン
■社製〕を用いた場合は、３ｇの凍結菌体から６．７■
の全ＲＮＡを抽出することができた．上述の２方法で回
収した全ＲＮＡは、フェノールオキシダーゼのＤＮＡプ
ロープによるノーザン・フ゛口・冫ト・ハイフ゛リダイ
ゼーション法（Ｔｈｏ−ｖａｓ．Ｐ．Ｓ．，Ｐｒｏｃ．
Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ ７７． ５２０
１ （１９８０））により、フェノールオキシダーゼ遺
伝子（ＩＩ）由来のＲＮＡを含むことを確認した。In addition, when using a commercially available RNA extraction kit (manufactured by Amersham Japan), 6.7 μg was extracted from 3 g of frozen bacterial cells.
We were able to extract the total RNA of. The total RNA recovered by the above two methods was subjected to Northern PCR using a phenoloxidase DNA probe (Tho-vas.P.S., Proc.
Natl. Acad. Sci. USA 77. 520
1 (1980)), it was confirmed that it contained RNA derived from the phenol oxidase gene (II).
全ＲＮＡ５■からオリゴ（ｄＴ）セルロース力ラムを使
用するＭａｎｉａｔｉｓ等の方法［ （Ｍａｎｉａｔｉ
ｓ他編，Ｍｏｌｅ−ｃｕｌａｒ Ｃｌｏｎｉｎｇ，Ａ
ＬａｂｏｒａＬｒｙ Ｍａｎｕａｌ＋ １９７〜１９９
（１９Ｂ２））を用いてポリ （八）ｍＲＮＡの分離
・精製を行ない、約１００μｇのポリ （Ａ） ｍ Ｒ
Ｎ Ａを精製した．実施例３（３）又旦Ｎ人夏金底白色腐朽菌アラゲカワラタケ由来ポリ （Ａ） ｍ Ｒ
ＮＡよりｃＤＮＡの合成は、ＧｕｂｌｅｒとＨｏｆｆｍ
ａｎの方法（Ｕ．Ｇｕｂｉｅｒ ＆ Ｂ．Ｊ．ｌＩｏｆ
ｆｍａｎ ； Ｇｅｎｅ，２５，２６３〜２６９（１９
８３） 参照）に従い、アマシャム・ジャパン製ｃＤＮ
Ａ合成キットを用いておこなった。The method of Maniatis et al. [(Maniati
Edited by S et al., Mole-cular Cloning, A
LaboraLry Manual+ 197-199
(19B2)) was used to separate and purify poly(8)mRNA, and about 100 μg of poly(A)mR
NA was purified. Example 3 (3) Poly (A) m R derived from the white rot fungus Arage kawaratake
Synthesis of cDNA from NA is performed by Gubler and Hoffm.
The method of an (U. Guvier & B.J. lIof
fman; Gene, 25, 263-269 (19
cDNA manufactured by Amersham Japan according to
A synthesis kit was used.
５μｇのポリ （Ａ）ｍＲＮＡに５０ユニットのヒト胎
児由来ＲＮａｓｅ阻害酵素（ＨＰＲＩ）の存在下５μｇ
のオリゴ（ｄＴ） １２〜１８（ファルマシア社製２７
−７８５８０１）を加え１００ユニットの逆転写酵素を
４２゜Ｃで１．５時間反応させて約３０％の収率で１本
鎖ｃＤＮＡを合成した．この反応液に４ユニットの大腸
菌リボヌクレアーゼＨと１１５ユニットの大腸菌ＤＮＡ
ポリメラーゼ■を加え１２゜Ｃで１時間，２２゜Ｃで１
時間反応させた後７０゜Ｃで１０分間放置して酵素を失
活させた．その後１０ユニットのＴ４ＤＮＡポリメラ−
ゼを加え３７゜Ｃで１０分間反応させて、約９５％の収
率で２本鎖ｃＤＮＡを得た。5 μg of poly(A) mRNA in the presence of 50 units of human fetal RNase inhibitor enzyme (HPRI)
Oligo (dT) 12-18 (27 manufactured by Pharmacia)
-785801) and reacted with 100 units of reverse transcriptase at 42°C for 1.5 hours to synthesize single-stranded cDNA with a yield of about 30%. Add 4 units of E. coli ribonuclease H and 115 units of E. coli DNA to this reaction solution.
Add polymerase■ and incubate at 12°C for 1 hour, then at 22°C for 1 hour.
After reacting for an hour, the mixture was left at 70°C for 10 minutes to inactivate the enzyme. Then 10 units of T4 DNA polymer
A double-stranded cDNA was obtained with a yield of about 95%.
（４） ｃ Ｄ Ｎ Ａ−イブー奪−の市販のλｇｔ
ｌｌクローニングシステム〔アマシャム・ジャパン■社
製〕を用いて、ｃＤＮＡライブラリーを構築した．１００μｇの２本鎖ｃＤＮＡを２０ユニットのＥｃｏＲ
Ｉメチラーゼを３７゜Ｃで１時間反応させた後、Ｅｃｏ
ＲＩリンカーを結合させた。これに１６ユニフトのＥｃ
ｏＲＩを加え３７゜Ｃで２時間反応させた後、Ｓｅｐｈ
ａｒｏｓｅ ＣＬ４Ｂカラムを通し、純化した。λｇｔ
ｌｌアーム１μｇとの連結反応後、ｉｎ ｖｉｔｒｏパ
ッケージング（Ａ．Ｂｅｃｋｅｒ ＆ Ｍ．Ｇｏｌ
ｄＨＰｒｏｃ．Ｎａｔｌ，八ｃａｄ．Ｓｃｉ．ＵＳＡ＋
７２＋５８１ （１９７５）参照〕を行ない、１０
６個の組換え体λファージを得て、アラゲカワラタケの
ｍＲＮＡ由来のｃＤＮＡライブラリーとした．（５）フェノールオキシ ーゼ ■）のクローニ
ング実施例４で得られたアラゲカワラタケのｍＲＮＡ由来の
ｃＤＮＡライブラリーを大腸菌Ｙ１０９０株に感染させ
、プラークを形成させた。(4) Commercially available λgt of cDNA-Ibuu
A cDNA library was constructed using the ll cloning system (manufactured by Amersham Japan). 100 μg of double-stranded cDNA was added to 20 units of EcoR.
After reacting I methylase at 37°C for 1 hour,
A RI linker was attached. In addition to this, 16 units of Ec
After adding oRI and reacting at 37°C for 2 hours, Seph
It was purified by passing through an arose CL4B column. λgt
After ligation reaction with 1 μg of ll arm, in vitro packaging (A. Becker & M. Gol
dHProc. Natl, 8 cad. Sci. USA+
72+581 (1975)] and 10
Six recombinant λ phages were obtained and used as a cDNA library derived from mRNA of C. versicolor. (5) Cloning of phenoloxise ①) The cDNA library derived from the mRNA of C. versicolor obtained in Example 4 was infected with E. coli strain Y1090 to form plaques.
フェノールオキシダーゼ遺伝子（ＩＩ）を含むクローン
は、放射性同位元素〔γノ”Ｐ）ＡＴＰ〔アマシャム・
ジャパン■社製ＰＢ１０１６８ ）とＴ４ボリヌクレオ
チドキナーゼ〔宝酒造■社製２０２ＬＡ〕を用いて標識
化（Ｒｉｃｈａｒｄｓｏｎ， Ｃ．Ｃ．（１９６５）＋
Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ
．， ５４，１５８〜１６１参照〕した実施例１の合成
ＤＮＡプローブを用いてＢｅｎ ｔｏｎとＤａｖｉｓの
プラークハイプリダイゼーション法〔Ｗ．Ｄ．Ｂｅｎｔ
ｏｎ ＆ Ｒ．Ｗ． Ｄａｖｉｓ； Ｓｃｉｅｎｃｅ．
１９６ １８０（１９７７）参照〕に従って２種の
クローンを選別した。The clone containing the phenol oxidase gene (II) was isolated from the radioactive isotope [γ-P)ATP [Amersham.
Labeling (Richardson, C.C. (1965) +
Proc. Natl. Acad. Sci. U. S. A
．． The plaque hybridization method of Benton and Davis [W. D. Bent
on & R. W. Davis; Science.
196 180 (1977)], two types of clones were selected.
フェノールオキシダーゼ遺伝子（ＩＩ）を含むλｇ ｔ
ｌｌファージからのＤＮＡの精製は、ＴｈｏｍａｓとＤ
ａｖｉｓの方法［Ｍ．Ｔｈｏｍａｓ ＆ Ｒ，Ｗ．Ｄａ
ｖｉｓ ； Ｊｏｕｒｎａｌｏｆ Ｍｏｌｅｃｕｌａｒ
Ｂｉｏｌｏｇｙ＋ ９１，３１５（１９７４）参照〕
により行なった。λg t containing phenol oxidase gene (II)
Purification of DNA from phage was performed by Thomas and D.
avis method [M. Thomas & R.W. Da
vis; Journal of Molecular
See Biology+ 91, 315 (1974)]
This was done by
（５）で得られたフェノールオキシダーゼ遺伝子（ｎ）
を含む２クローンλｇｔｌｌＤＮＡを５μｇ用いて、制
限酵素ＥｃｏＲＩ （宝酒造製）１０ユニットで３７℃
、２時間消化し、フェノールオキシダーゼ遺伝子（ＩＩ
）を含む領域１．９ｋｂの断片をＤＮＡセル（第−化学
薬品製）を用いて回収した。該Ｄ Ｎ Ａ断片を大腸菌
プラスミドベクターｐＵｃ１９またはｐＵｃ１１８のＥ
ｃｏＲＩ サイトに挿入した。Phenoloxidase gene (n) obtained in (5)
Using 5 μg of 2 clones λgtll DNA containing
, digested for 2 hours, and extracted the phenol oxidase gene (II
) was recovered using a DNA cell (manufactured by Dai-Kyakuyaku). The DNA fragment was inserted into E. coli plasmid vector pUc19 or pUc118.
inserted into the coRI site.
ｐＵｃ１９にサブクローニングしたクローンをＯＪ−Ｐ
ＯＭ ５とし、ｐｌＪｃ１１８にサブクローニングした
クローンをＯＪ−ＰＯＭ ２として、それぞれ微生物工
業技術研究所に寄託した（ＯＪ−ＰＯＭ ２；微工研菌
寄第１００５５号、ＯＪ−ＰＯＭ ５；微工研菌寄第１
００６１号）。The clone subcloned into pUc19 was transferred to OJ-P.
The clone subcloned into OM 5 and plJc118 was named OJ-POM 2 and deposited with the National Institute of Microbial Technology (OJ-POM 2; Microbiological Research Institute No. 10055; OJ-POM 5; Microbiological Research Institute). 1st
No. 0061).
また、得られ′たプラスミドをｐＵｃＰＯ２（ＯＪ−Ｐ
ＯＭ ２）、ｐＵｃＰＯ５（ＯＪ−ＰＯＭ ５）とした
。In addition, the obtained plasmid was pUcPO2 (OJ-P
OM 2) and pUcPO5 (OJ-POM 5).
それぞれの塩基配列および翻訳されてできるフェノール
オキシダーゼのアミノ酸配列を第１図（ＯＪ−ＰＯＭ
５）、第２図（ＯＪ−ＰＯＭ ２）ニ示した。The respective base sequences and the translated amino acid sequence of phenol oxidase are shown in Figure 1 (OJ-POM
5), as shown in Figure 2 (OJ-POM 2).
（７）非皿訳皿菫■徐去（６）で得られたプラスミドｐＵｃＰＯ５、１０μｇを
、制限酵素ＢａｍＨＩ［０ユニット）およびＰｓｔｌ（
１０ユニット）（宝酒造製）で、３７゜Ｃ、２時間消化
し、エタノ？ル沈澱により塩などの不純物を除去した。(7) 10 μg of the plasmid pUcPO5 obtained in (6) was added to the restriction enzyme BamHI [0 units] and Pstl (
10 units) (manufactured by Takara Shuzo) at 37°C for 2 hours and digested with ethano? Impurities such as salts were removed by precipitation.
得られた沈澱物をエキソヌクレアーゼ■緩衝液（５０ｍ
ＭＴｒｉｓ−ＨＣＩ （ｐＨ８．０）． １００ｍＭ
ＮａＣｌ． ５ｍＭ ＭｇＣ１ｚ，１０ｍＭ ２−メ
ルカプトエタノール）１００ｔＩｆに溶解し、エキソヌ
クレアーゼ■（宝酒造製）１８０ユニット添加し、３７
゜Ｃに保温した。２０秒毎に１０ｕｌずつサンプリング
し、１００ｕｆのＭｕｎｇ−ｂｅａｎヌクレアーゼ緩衝
液（４０ｍＭ Ｎａ−ａｃｅｔａｔｅ （ｐＨ４．５Ｌ
． ｌｏｏｍＭ ＮａＣ１．２ｍＭ ＺｎＣ１ｚ＋ ｔ
ｏ％Ｇｌｙｃｅｒｏｆ）に順次加えてエキソヌクレアー
ゼ■の反応を停止させた。サンプリングがすべて終了し
た後、６５゛Ｃで１０分間熱失活を行った．３７゜Ｃで
保温し、Ｍｕｎｇ−ｂｅａｎヌクレアーゼ（宝酒造製）
２５ユニットを加え、３０分間放置した後、フェノール
処理し、Ｍｕｎｇ−ｂｅａｎヌクレアーゼ失活させた後
、エタノール沈澱を行った。沈澱物を５０，ｚｆのＫｌ
ｅｎｏ−緩衝液（７ｓＭ Ｔｒｉｓ−ＨＣＩ （ｐＨ７
．５）．０．１ｍＭ ＨＤＴＡ． ２０ｓ＋Ｍ
ＮａＣ１．７ａ＋Ｍ ＭｇＣｌ■ ｄＡＴＰ， ｄ
ＧＴＰ，ｄＣＴＰ， ｄＴＴＰ各々０．１ｍＭ）に溶解
し、Ｋｌｅｎｏｗ−ｅｎｚｙｍｅ（宝酒造製）２ユニッ
トを加え、３７゜Ｃで１５分間保温した．２．５倍量の
エタノールを加え遠心して、沈澱を回収し、沈澱物を４
０ｔｔｌ！のＴＥ緩衝液（１０ｍＭ Ｔｒｉｓ−ＨＣＩ
（ｐ Ｈ８．０）， １ｍＭ ＥＤＴＡ）に溶解した
。The obtained precipitate was diluted with exonuclease ■ buffer (50 m
MTris-HCI (pH 8.0). 100mM
NaCl. Dissolve in 100tIf of 5mM MgC1z, 10mM 2-mercaptoethanol, add 180 units of exonuclease (manufactured by Takara Shuzo),
It was kept warm at °C. Sample 10ul every 20 seconds and add 100uf of Mung-bean nuclease buffer (40mM Na-acetate (pH 4.5L)
．． roomM NaC1.2mM ZnC1z+t
% Glycerof) to stop the exonuclease reaction. After all sampling was completed, heat inactivation was performed at 65°C for 10 minutes. Insulate at 37°C and incubate with Mung-bean nuclease (Takara Shuzo).
After adding 25 units and leaving the mixture for 30 minutes, it was treated with phenol to inactivate Mung-bean nuclease, and then ethanol precipitated. The precipitate was heated to 50, zf Kl.
eno-buffer (7sM Tris-HCI (pH 7)
．． 5). 0.1mM HDTA. 20s+M
NaC1.7a+M MgCl■ dATP, d
GTP, dCTP, and dTTP (0.1 mM each) were added, 2 units of Klenow-enzyme (manufactured by Takara Shuzo) were added, and the mixture was incubated at 37°C for 15 minutes. Add 2.5 times the volume of ethanol and centrifuge to collect the precipitate.
0ttl! of TE buffer (10mM Tris-HCI
(pH 8.0), 1mM EDTA).
そのうち５μ！を用いてライゲーシッン反応を１６゜Ｃ
，一晩行ない、大腸菌ＪＭ１０９に形質転換を行った．
得られた大腸菌形質転換株のうちプラスミドの大きさが
やや短くなっている１ＧクローンをＬＢ培地（Ｂａｃｔ
ｏ−ｔｒｙｐｔｏｎ １％， Ｂａｃｔｏ−Ｙｅａｓｔ
−ｅｘｔｒａｃｔＯ．５％． ＮａＣ１ １％）で３７
゜Ｃ．一晩、培養し常法のアルカリ抽出により、組換え
ＤＮＡプラスミドを調製した。５゛上流の非翻訳領域約
５０塩基が除去された組換えＤＮＡの塩基配列を、ダイ
デオキシ法により決定した。5μ of that! The ligation reaction was carried out at 16°C using
, and transformed into E. coli JM109 overnight.
Among the E. coli transformants obtained, a 1G clone with a slightly shorter plasmid size was cultured in LB medium (Bact
o-trypton 1%, Bacto-Yeast
-extractO. 5%. 37 with NaC1 1%)
゜C. After culturing overnight, a recombinant DNA plasmid was prepared by conventional alkaline extraction. The base sequence of the recombinant DNA from which approximately 50 bases of the untranslated region upstream of the DNA were removed was determined by the dideoxy method.
１６クローンのうち１クローンの塩基配列は、第４図に
示す通りであって、発現ベクターｐＫＫ２３３−２のβ
−ガラクトシダーゼ遺伝子の開始コドンからのフレーム
が、フェノールオキシダーゼのフレームと一致するもの
であった。The nucleotide sequence of one clone among the 16 clones is as shown in FIG.
- The frame from the start codon of the galactosidase gene matched the frame of phenol oxidase.
（８） に るＤＮＡ の（７）で得られ
た組換え体ＤＮ・Ａブラスミド１０μｇをｌＯユニット
のＥｃｏＲＩで切断し、エタノール沈澱により得られる
沈澱物をＸｌｅｎｏ一緩衝液１００μｌに溶解した。２
ユニットのＫｌｅｎｏｗ−ｅｎｚｙｍｅを加え、３７゜
Ｃで３０分反応し、フェノールークロロホルムで反応を
停止した．エタノール沈澱によりＤＮＡを回収し沈澱物
を１０χ７４ＤＮＡリガーゼ緩衝液５ｕｌに溶解した．
次にｇ ｏｖｅｒのｔｌｉｎｄｌｌ！リンカー（宝酒造
製）１μｇを含むように加え、さらに５μｉのＴ４ＤＮ
Ａリガーゼ（１．５００ユニット）を加えた後、滅菌蒸
留水を加えて５０μｌとし１６゜Ｃで１晩反応させた。(8) 10 μg of the recombinant DNA/A plasmid obtained in step (7) was digested with 10 units of EcoRI, and the precipitate obtained by ethanol precipitation was dissolved in 100 μl of Xleno buffer. 2
A unit of Klenow-enzyme was added, the reaction was carried out at 37°C for 30 minutes, and the reaction was stopped with phenol-chloroform. DNA was recovered by ethanol precipitation, and the precipitate was dissolved in 5 ul of 10x74 DNA ligase buffer.
Next up is tryndll! Added 1 μg of linker (manufactured by Takara Shuzo) and further added 5 μi of T4DN.
After adding A ligase (1.500 units), sterile distilled water was added to make 50 μl, and the mixture was reacted overnight at 16°C.
つぎに、ｌＯ×旧ｎｄｌｌｌ緩衝液１０μ２及び１０ユ
ニットの旧ｎｄ （宝酒造製）を加え滅菌蒸留水を加え
て１００μ２とし３７゜Ｃで２時間切断した。Next, 10 .mu.2 of 10.times.old ndlll buffer and 10 units of old nd (manufactured by Takara Shuzo) were added, and sterilized distilled water was added to make the volume 100 .mu.2, followed by cutting at 37.degree. C. for 2 hours.
その後アガロースゲル電気泳動により目的とするＤＮＡ
断片約１．８ｋｂを得た．これを断片−１とする（第３
図）．（９） ベ ーの大腸菌発現ベ
クターｐＫＫ２３３−２（ファルマシア製）５μｇを含
む旧ｎｄｌ［［５衝液にＩＯＵの旧ｎｄｌｌ［（宝酒造
製）を加えて３７゜Ｃで２時間切断した。その後、アル
カリ・フォスファターゼ（Ｅ．ｃｏｌｉ Ｃ７５）（宝
酒造製）を０．５Ｕ加え、６５゜Ｃで３０分反応し、フ
ェノールークロロホルムにより反応を停止し、エタノー
ル沈澱によりベクターＤＮＡを精製した。これを断片−
２とする。つぎに、（８）で得られた断片一ｌと断片−
２とをＴ４ＤＮＡリガーゼを用いて連結し、フェノール
オキシダーゼに関する発現プラスミドｐＫＫＰＯ５を構
築した（第４図）。この組換えプラスミドを大腸菌ＪＭ
１０９株にカルシウム法により導入し、目的とする形質
転換株を得た。該形質転換体は微生物工業技術研究所に
寄託した（微工研菌寄第１０５７６号）。Then, target DNA is extracted by agarose gel electrophoresis.
A fragment of approximately 1.8 kb was obtained. Let this be Fragment-1 (3rd
figure). (9) IOU of old ndll (manufactured by Takara Shuzo) was added to a solution containing 5 μg of the E. coli expression vector pKK233-2 (manufactured by Pharmacia) and cleaved at 37°C for 2 hours. Thereafter, 0.5 U of alkaline phosphatase (E. coli C75) (manufactured by Takara Shuzo) was added, the reaction was carried out at 65°C for 30 minutes, the reaction was stopped with phenol-chloroform, and the vector DNA was purified by ethanol precipitation. A fragment of this-
Set it to 2. Next, fragment 1 obtained in (8) and fragment -
2 was ligated with T4 DNA ligase to construct an expression plasmid pKKPO5 for phenol oxidase (Fig. 4). This recombinant plasmid was transferred to E. coli JM
It was introduced into strain 109 by the calcium method to obtain the desired transformed strain. The transformant was deposited with the Institute of Microbial Technology (Feikoken Bacterium No. 10576).
００）フェノールオキシ ーゼの（９）で得られた形質転換株１白金耳をＬＢ培地２ｄに
接種して、３７゜Ｃで１晩振盪培養し、１００−の１ｍ
Ｍの塩化第二銅を含むＬＢ培地に該前培養液をＩＩｎ１
加え、３７゜Ｃで培養を行い、対数後期に、最終濃度１
ｍＭになるようにイソプロピルーβ一Ｄ−チオガラクト
シド（ＩＰＴＧ）を添加し、酵素生産を誘導した．
ＩＰＴＧ添加６時間後に菌体を集菌して、超音波破砕に
より全タンパク質試料を調製した．この全タンパク質試
料を用いてＳＯＳ−ＰＡＧＥを行い、ニトロセルロース
フィルター（アマシャム社製Ｈｙｂｏｎｄ−Ｃ）にトラ
ンスファーした．さらにアマシャム社製プロテイング検
出キットを用いて抗フェノールオキシダーゼウサギ血清
で検出した。アミノ酸配列より推定される分子量５３，
０００とほぼ一致する大きさのバンドが検出され、フェ
ノールオキシダーゼ遺伝子（■）にコードされるタンパ
ク質の生産が確められた。00) A platinum loop of the transformed strain 1 obtained in (9) of phenoloxise was inoculated into 2 d of LB medium, cultured overnight at 37°C with shaking, and 1 m of 100-
The preculture solution was transferred to LB medium containing cupric chloride of M.IIn1.
In addition, culture was carried out at 37°C, and at the late logarithmic stage, a final concentration of 1
Isopropyl-β-D-thiogalactoside (IPTG) was added at a concentration of mM to induce enzyme production.
Six hours after IPTG addition, the bacterial cells were collected and a total protein sample was prepared by ultrasonic disruption. SOS-PAGE was performed using this total protein sample and transferred to a nitrocellulose filter (Hybond-C manufactured by Amersham). Further, detection was performed using anti-phenol oxidase rabbit serum using a protein detection kit manufactured by Amersham. Molecular weight estimated from the amino acid sequence: 53,
A band with a size almost identical to 000 was detected, confirming the production of a protein encoded by the phenol oxidase gene (■).
ｐｕｃ ＰＯ２ニツｕＮてもｐｕｃ ＰＯ５と同様に上
記（７）〜００）を行ない、フェノールオキシダーゼ遺
伝子（旧（ＯＪ−ＰＯＭ ２）にコードされるタンパク
質の生産を確かめた．〔発明の効果〕本発明においては、白色腐朽菌由来のフェノールオキシ
ダーゼ遺伝子を白色腐朽菌以外の異種生物に発現せしめ
たことにより、本発明の形質転換株はセルラーゼ、ヘミ
セルラーゼが混入することなくフェノールオキシダーゼ
のみを生圧丁ることができる．したがって、フェノール
オキシダーゼを含むその培養物はパルプ収率の低下をま
ねくことなくバイオロジカルパルビング、工場廃水の脱
色、木材糖化の前処理に利用でき、木材糖化の前処理に
利用できるものである．The above (7) to 00) were carried out for puc PO2 Nitsu uN in the same manner as for puc PO5, and the production of the protein encoded by the phenol oxidase gene (formerly (OJ-POM 2)) was confirmed. [Effects of the Invention] The present invention By expressing the phenol oxidase gene derived from white-rot fungi in a heterologous organism other than white-rot fungi, the transformed strain of the present invention can produce only phenol oxidase without contamination with cellulase or hemicellulase. Therefore, its culture containing phenol oxidase can be used for biological pulping, decolorization of industrial wastewater, and pretreatment of wood saccharification without causing a decrease in pulp yield; It is something.
第１図，第２図は、それぞれｐＵｃ ＰＯ５， ｐｕｃ
ＰＯ２のアラゲカワラヌケフェノールオキシダーゼ遺
伝子■の塩基配列および翻訳されてできると考えられる
アミノ酸配列を示す図である。図中、一・・一−・−・
−↓は発現ベクターに組み込む際、エキソヌクレアーゼ
で除去した部分を示す．第３図はフェノールオキシダー
ゼ遺伝子を含む断片−１の作成工程を示す図である。第
４図はフェノールオキシダーゼ遺伝子に関する発現ベク
ターの構築工程及びその連結部位の塩基配列とアミノ酸
配列を示す図である。Ｇｌｒ＋ＡｌａＴｒｐｓ＊ｒＡｓｐＬｅｕｃｙｓＰｒｏ
ＩｌｅＴｙｒＡｓｐＡｌａＬｅｕＡｓｐｙａｌＡｓｎ＋
ＬｓｐＧｌｎｚｘｚ第図１ｌａＡｇｐＧｌｙｌｌｌｓＡ＊ｐＬｅｕＴｈｒｌ Ｉ
ｓｌｌｅＧＩｕＶａｌＡｇｐ５ｓｒ１１ｅＡｓｎ５ｅｒ
ＧｌｎＰｒｏＬｅｕＶａｌＶｉ１▲ｓｐ５ａｒｌｌｅＧ
ＩｎｌｌｅＰｈｅ▲ｌａＡＩｍ第図（３）Ｇ ｌａＡ ＩａＴｒｐＳｅｒＡｓｐＬａｕＣｙｓＰｒ
ａ Ｉ Ｉ ｅＴＷｒＡｓｐＡ ｌａＬｅｕＡｓｐ Ｖ
ａ ｌＡｍｎＡｓｐ（ｉ ＋獅嘯■■第図第図Figures 1 and 2 show pUc PO5 and puc, respectively.
FIG. 2 is a diagram showing the nucleotide sequence and the amino acid sequence thought to be produced by translation of the PO2 Arugae nigra phenol oxidase gene (2). In the figure, 1・・1−・−・
−↓ indicates the part removed with exonuclease when integrating into the expression vector. FIG. 3 is a diagram showing the steps for creating fragment-1 containing the phenol oxidase gene. FIG. 4 is a diagram showing the steps for constructing an expression vector for the phenol oxidase gene and the nucleotide sequence and amino acid sequence of its connection site. Glr+AlaTrps*rAspLeucysPro
IleTyrAspAlaLeuAspyalAsn+
LspGlnzxzFigure 1laAgpGlyllsA*pLeuThrl I
slleGIuValAgp5sr11eAsn5er
GlnProLeuValVi1▲sp5arlleG
InllePhe▲laAImFigure (3) G laA IaTrpSerAspLauCysPr
a I I eTWrAspA laLeuAsp V
a lAmnAsp(i + shi嘯■■ Figure Figure Figure
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5795289AJPH02238885A (en) | 1989-03-13 | 1989-03-13 | Phenol oxidase genetically recombinant DNA, microorganisms transformed with the recombinant DNA, culture thereof, and method for producing phenol oxidase |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5795289AJPH02238885A (en) | 1989-03-13 | 1989-03-13 | Phenol oxidase genetically recombinant DNA, microorganisms transformed with the recombinant DNA, culture thereof, and method for producing phenol oxidase |
| Publication Number | Publication Date |
|---|---|
| JPH02238885Atrue JPH02238885A (en) | 1990-09-21 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP5795289APendingJPH02238885A (en) | 1989-03-13 | 1989-03-13 | Phenol oxidase genetically recombinant DNA, microorganisms transformed with the recombinant DNA, culture thereof, and method for producing phenol oxidase |
| Country | Link |
|---|---|
| JP (1) | JPH02238885A (en) |
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