JP3766040B2 - Method for producing cytosine nucleoside compound - Google Patents

Description

（上記式（Ｉ）中、Ｘは炭素原子または窒素原子を、Ｙは水素原子、ハロゲン原子または低級アルキル基を示す。）
で表される化合物を用いることができる。その具体例としては、例えばアザシトシン及び５−フルオロシトシンを挙げることができる。
【００２５】
更に、上記のシトシンヌクレオシドホスホリラーゼ活性を有する酵素は、この酵素を有する微生物の菌体またはその培養液、あるいはこれらから得られた粗酵素抽出液、粗酵素溶液、粗酵素調製物および精製酵素調製物など酵素調製物の形態で反応系中に供給することができる。
【００２６】
これらの菌体あるいは酵素調製物としては、シトシンデアミナーゼ活性およびシチジンデアミナーゼ活性がないか、あるいは存在していてもシトシンヌクレオシドホスホリラーゼ活性を利用したシトシンヌクレオシド化合物の製造が可能である程度に低いものであることが好ましい。例えば、シトシンデアミナーゼ活性およびシチジンデアミナーゼ活性よりもシトシンヌクレオシドホスホリラーゼ活性が高い菌体及び酵素調製物を好適に用いることができる。
【００２７】
このような菌体及び酵素調製物の一例としては、これらの有するシトシンデアミナーゼ活性およびシチジンデアミナーゼ活性を低減化処理によって低下させたものを挙げることができる。
【００２８】
上記のデアミナーゼ活性の失活または低減化のための処理が施された微生物の菌体は、例えばシトシンヌクレオシドホスホリラーゼ活性を有する微生物の菌体を有機溶媒を含む水に接触させて、シトシンデアミナーゼ活性およびシチジンデアミナーゼ活性を選択的に減少させて得ることができる。この有機溶媒としては、アルコール類などの極性溶媒を利用することができる。この有機溶媒としては、例えばメチルアルコール、エチルアルコール、プロピルアルコール、ブチルアルコール、ジオキサン及びアセトンの中から選ばれる１種以上の有機溶媒が利用できる。
【００２９】
更に、有機溶媒によりシトシンデアミナーゼ活性およびシチジンデアミナーゼ活性を失活または低減化させる際に、水中の有機溶媒濃度を２０容量％以上とすることが好ましい。
【００３０】
一方、デアミナーゼ活性の失活または低減化のための処理が施された微生物の菌体は、シトシンヌクレオシドホスホリラーゼ活性を有する微生物の菌体を水性媒体中でシトシンデアミナーゼ活性およびシチジンデアミナーゼ活性を選択的に減少させる温度で加熱処理して得ることもできる。この加熱処理の条件としては、５０℃以上で１０分間以上４０時間以内の条件を採用することができる。
【００３１】
一方、デアミナーゼ活性の失活または低減化のための処理を行う際に、ペントース−１−リン酸を存在させることで、維持すべきシトシンヌクレオシドホスホリラーゼ活性をより効果的に維持することが可能となる。その際のペントース−１−リン酸の濃度は１ｍＭ以上１００ｍＭ以下とすることが好ましい。
【００３２】
【発明の実施の形態】
本発明においてシトシンヌクレオシドホスホリラーゼとは、シトシンまたはシトシン誘導体を基質としてシトシンヌクレオシド化合物を生成する活性を有する酵素を指しており、この要件を満たす限り動物、植物、微生物の何れの起源であっても構わない。シトシンヌクレオシドホスホリラーゼなる酵素は当該分野においては一般的に知られていない。そのため、本発明においてのみかかる用語を上記の通り規定して用いる。このようなヌクレオシドホスホリラーゼの具体例としては、エシェリシア・コリ（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）等のエシェリシア（Ｅｓｃｈｅｒｉｃｈｉａ）属に含まれる微生物の従来のプリンヌクレオシドホスホリラーゼとして知られている酵素でシトシンヌクレオシドホスホリラーゼ活性も有するものを好適な例として挙げることができる。具体的な例として、エシェリシア・コリ（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）のプリンヌクレオシドホスホリラーゼのＤＮＡ塩基配列を配列番号３に、該塩基配列より翻訳されるアミノ酸配列を配列番号：４に例示した。また、近年の遺伝子工学の進歩により塩基配列の一部を失活、挿入、置換によりアミノ酸配列を改変することが容易となった。かかる技術水準に鑑み、該塩基配列の一部を、所望とする酵素活性に影響を及ぼさない範囲内、例えば酵素活性を維持または向上できる範囲内で、改変してアミノ酸配列を改変したものも本発明のシトシンヌクレオシドホスホリラーゼに包含されるものとする。例えば、配列番号：４に示したアミノ酸配列に、目的とする酵素活性に影響を及ぼさない範囲内で２〜３個のアミノ酸の欠失、置換または付加が行われたものや、配列番号：３の塩基配列に目的とする酵素活性に影響を及ぼさず、かつ、その相補配列がストリンジェントな条件下でハイブリダイズし得る範囲内での欠失、置換または付加等の変異を生じさせた塩基配列によってコードされたアミノ酸配列を有するものを用いることができる。
【００３３】
この様に、プリンヌクレオシドスホリラーゼ活性を有すると、プリン型ヌクレオシド化合物とシトシンヌクレオシド化合物を１種類の酵素で製造することが可能となり、ヌクレオシド類の工業的な製造には大変有利である。
【００３４】
本発明の方法に用いるシトシンヌクレオシドホスホリラーゼ活性を有する酵素は、該酵素を有する微生物の菌体、この菌体またはその培養液から調製された酵素調製物などの形態で反応系に供給することができる。なお、この酵素調製物には、菌体またはその培養液の各種処理物、酵素抽出物、酵素抽出物を用いた粗精製品及び単離精製品などが含まれる。
【００３５】
これらの微生物の菌体や酵素調製物としては、市販のものや各種方法を利用して調製したものを用いることができる。例えば、この酵素活性を有する調製物としては市販の酵素、該酵素活性を有する微生物菌体、及び菌体処理物またはそれらの固定化物などが使用できる。菌体処理物とは、例えばアセトン乾燥菌体や機械的破壊、超音波破砕、凍結融解処理、加圧減圧処理、浸透圧処理、自己消化、細胞壁分解処理、界面活性剤処理などにより調製した菌体破砕物などであり、また、必要に応じて硫安沈殿やアセトン沈殿、カラムクロマトグラフィーにより精製を重ねたものを用いても良い。
【００３６】
なお、シトシンヌクレオシドホスホリラーゼ活性を有する微生物は、シトシンまたはシトシン誘導体を基質としてシトシンヌクレオシド化合物を生成するヌクレオシドホスホリラーゼを発現しているものであれば、特に限定されない。このような微生物は、例えば、一般的なヌクレオシドホスホリラーゼを発現しているものから選択することができる。そのようなヌクレオシドホスホリラーゼを生産する微生物の具体例としては、エシェリシア・コリ（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）等のエシェリシア（Ｅｓｃｈｅｒｉｃｈｉａ）属に含まれる微生物を好適な例として挙げることができる。
【００３７】
近年の分子生物学および遺伝子工学の進歩により、上述の微生物株のプリンヌクレオシドホスホリラーゼの分子生物学的な性質やアミノ酸配列等を解析することにより、該蛋白質の遺伝子を該微生物株より取得し、該遺伝子および発現に必要な制御領域が挿入された組換えプラスミドを構築し、これを任意の宿主に導入し、該蛋白質を発現させた遺伝子組換え菌を作出することが可能となり、かつ、比較的容易にもなった。かかる技術水準に鑑み、このようなヌクレオシドホスホリラーゼの遺伝子を任意の宿主に導入した遺伝子組換え菌も本発明のヌクレオシドホスホリラーゼを発現している微生物に包含されるものとする。
【００３８】
ここでいう発現に必要な制御領域とは、プロモーター配列（転写を制御するオペレーター配列を含む）・リボゾーム結合配列（ＳＤ配列）・転写終結配列等を示している。プロモーター配列の具体例としては、大腸菌由来のトリプトファンオペロンのｔｒｐプロモーター・ラクトースオペロンのｌａｃプロモーター・ラムダファージ由来のＰＬプロモーター及びＰＲプロモーターや、枯草菌由来のグルコン酸合成酵素プロモーター（ｇｎｔ）・アルカリプロテアーゼプロモーター（ａｐｒ）・中性プロテアーゼプロモーター（ｎｐｒ）・α−アミラーゼプロモーター（ａｍｙ）等が挙げられる。また、ｔａｃプロモーターのように独自に改変・設計された配列も利用できる。リボゾーム結合配列としては、大腸菌由来または枯草菌由来の配列が挙げられるが、大腸菌や枯草菌等の所望の宿主内で機能する配列であれば特に限定されるものではない。たとえば、１６ＳリボゾームＲＮＡの３’末端領域に相補的な配列が４塩基以上連続したコンセンサス配列をＤＮＡ合成により作成してこれを利用してもよい。転写終結配列は必ずしも必要ではないが、ρ因子非依存性のもの、例えばリポプロテインターミネーター・ｔｒｐオペロンターミネーター等が利用できる。これら制御領域の組換えプラスミド上での配列順序は、５’末端側上流からプロモーター配列、リボゾーム結合配列、ヌクレオシドホスホリラーゼをコードする遺伝子、転写終結配列の順に並ぶことが望ましい。
【００３９】
ここでいうプラスミドの具体例としては、大腸菌中での自律複製可能な領域を有しているｐＢＲ３２２、ｐＵＣ１８、Ｂｌｕｅｓｃｒｉｐｔ ＩＩ ＳＫ（＋）、ｐＫＫ２２３−３、ｐＳＣ１０１や、枯草菌中での自律複製可能な領域を有しているｐＵＢ１１０、ｐＴＺ４、ｐＣ１９４、ρ１１、φ１、φ１０５等をベクターとして利用することができる。また、２種類以上の宿主内での自律複製が可能なプラスミドの例として、ｐＨＶ１４、ＴＲｐ７、ＹＥｐ７及びｐＢＳ７をベクターとして利用することができる。
【００４０】
ここでいう任意の宿主には、後述の実施例のように大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）が代表例として挙げられるが、とくに大腸菌に限定されるのものではなく枯草菌（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）等のバチルス属菌、酵母や放線菌等の他の微生物菌株も含まれる。
【００４１】
本発明におけるシトシンヌクレオシド化合物とはリン酸化糖と核酸塩基としてシトシンまたはシトシン誘導体がＮ−グリコシド結合で結合した化合物のことである。その代表例を挙げると、例えばシチジン、デオキシシチジン、ジデオキシシチジン、アザシチジン、デオキシアザシチジン、５−フルオロシチジン及び５−フルオロデオキシシチジンなどを挙げられるが、これらに限定されるものではない。
【００４２】
本発明におけるシトシン誘導体とは、シトシン構造部分を有し、このシトシン構造部分がシトシンヌクレオシドホスホリラーゼ活性の作用によりシトシンヌクレオシド構造に変化し得るものである。このシトシン誘導体の好ましい例としは、前記一般式（Ｉ）で表されるシトシン誘導体を挙げることができる。前記一般式（Ｉ）におけるＹとしての低級アルキル基としては、炭素数１〜４のアルキル基、例えば、メチル基、エチル基、ｎ−プロピル基、ｉ−プロピル基、ｎ−ブチル基、sec−ブチル基及びtert−ブチル基を挙げることができる。これらのシトシン誘導体の中では、例えば、アザシトシン、５−フルオロシトシン、５―メチルシトシンなどが特に好ましい。
【００４３】
本発明におけるリン酸化糖とは、ポリヒドロキシアルデヒドまたはポリヒドロキシケトンおよびその誘導体の１位にリン酸がエステル結合したもののことである。その好ましい代表例を挙げると、例えばリボース１−リン酸、２´−デオキシリボース１−リン酸、２´，３´−ジデオキシリボース１−リン酸、アラビノース１−リン酸、ジオキソラン糖−リン酸などが挙げられる。
【００４４】
ここでいうポリヒドロキシアルデヒドまたはポリヒドロキシケトンとは、天然物由来のものとしては、Ｄ−アラビノース、Ｌ−アラビノース、Ｄ−キシロース、Ｌ−リキソーズ、Ｄ−リボースのようなアルドペントース、Ｄ−キシルロース、Ｌ−キシルロース、Ｄ−リブロースのようなケトペントース、Ｄ−２−デオキシリボース、Ｄ−２，３−ジデオキシリボースのようなデオキシ糖類を挙げることができるが、これらに限定されるものではない。
【００４５】
これらリン酸化糖は、ヌクレオシドホスホリラーゼの作用によりヌクレオシド化合物の加リン酸分解反応を行って製造する方法（Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．Ｖｏｌ．１８４、４３７、１９５０）や、アノマー選択的な化学合成法等によっても調製することができる。また、ＷＯ ０１／１４５６６に記載の酵素的なデオキリボースー１−リン酸の合成方法によっても調整することができる。（ロッシュの特許のdRP合成ルートも記載。）
本発明において、シトシンデアミナーゼ活性およびシチジンデアミナーゼ活性の低減化処理とは、ヌクレオシドホスホリラーゼを失活させず、シトシンデアミナーゼ活性およびシチジンデアミナーゼ活性を失活または低減化させることができれば特に限定されないが、例えば該微生物の菌体、培養液またはそれらの処理物を有機溶媒中にさらしたり、加熱処理を施すことを好適な例として挙げることができる。
【００４６】
本発明において、有機溶媒とはシトシンデアミナーゼ活性およびシチジンデアミナーゼ活性を失活させることが可能な溶媒であれば特に限定されないが、具体的にはメチルアルコール、エチルアルコール、プロピルアルコール、ブチルアルコール、ジオキサン、テトラヒドロフラン、メチルエチルケトン及びアセトン等の極性溶媒や１−ヘキサノール、２−メチル−１−ペンタノール、４−メチル−２−ペンタノール、２−エチル−１−ブタノール、１−ヘプタノール、２−ヘプタノール、３−ヘプタノール、１−オクタノール、２−オクタノール、１−ノナノール等のアルコール類、酢酸プロピル、酢酸ブチル、酢酸イソブチル、酢酸sec−ブチル、酢酸ペンチル、酢酸イソペンチル、酢酸シクロヘキシル、酢酸ベンジル等のエステル類、ペンタン、ヘキサン、２−メチルヘキサン、２，２−ジメチルブタン、２，３−ジメチルブタン、シクロヘキサン、メチルシクロヘキサン、ヘプタン、シクロヘプタン、オクタン、シクロオクタン、イソオクタン、ノナン、デカン、ドデカン、石油エーテル、石油ベンジン、リグロイン、工業ガソリン、灯油、ベンゼン、トルエン、キシレン、エチルベンゼン、プロピルベンゼン、クメン、メシチレン、ナフタレン等の炭化水素類、ジクロロメタン、クロロホルム、四塩化炭素、ジクロロエタン、クロロベンゼン、ジクロロベンゼン等のハロゲン化炭化水素類、クレゾール、キシレノール等のフェノール類、メチルイソブチルケトン、２−ヘキサノン等のケトン類、ジプロピルエーテル、ジイソプロピルエーテル、ジフェニルエーテル、ジベンジルエーテル等のエーテル類が挙げられ、ペンタン、ヘキサン、ヘプタン、オクタン、シクロペンタン、シクロヘキサン、シクロヘプタン、シクロオクタン、トルエン、エチルベンゼン等の炭化水素類が挙げられる。また、Ｎ，Ｎ−ジメチルホルムアミド、Ｎ，Ｎ−ジメチルアセトアミド、Ｎ，Ｎ−ジメチルベンズアミド、Ｎ−メチル−２−ピロリドン、Ｎ−メチルホルムアミド、Ｎ−エチルホルムアミド、Ｎ−メチルアセトアミド、ホルムアミド、アセチルアミド、安息香酸アミド等のアミド化合物や、ウレア、Ｎ，Ｎ’−ジメチルウレア、テトラメチルウレア、Ｎ，Ｎ−ジメチルイミダゾリジノン等のウレア化合物が挙げられ、ジメチルスルホキシド、ジエチルスルホキシド、ジフェニルスルホキシド等のスルホキシド化合物が挙げられる。
【００４７】
本発明でいうデアミナーゼ活性の失活または低減化のための有機溶媒処理とは、ヌクレオシドホスホリラーゼを失活させず、シトシンデアミナーゼ活性およびシチジンデアミナーゼ活性を失活または低減化させることができれば特に限定されないが、例えば該微生物の菌体、培養液またはそれらの処理物をｐＨは通常４．０以上１０．０以下であり、好ましくは６．０以上９．０以下で、有機溶媒を水中濃度として、例えば１０容量％以上、好ましくは２０容量％以上、より好ましくは３０容量％以上の濃度で、通常０℃以上の温度、好ましくは２０℃、更には５０℃以上で、８０℃以下の温度で、通常１０分間以上で４０時間以下、より好ましくは１時間以上２０時間以下の時間で静置または懸濁するような方法を挙げることができる。
【００４８】
また、処理液中にリン酸化糖を１ｍＭ以上好ましくは１００ｍＭ以下添加することにより、ヌクレオシドホスホリラーゼをより安定化させることができる。
【００４９】
また、反応液中にこれらの有機溶媒を添加することによっても同様の効果を得ることが可能である。
【００５０】
本発明でいう熱処理とはヌクレオシドホスホリラーゼを失活させず、シトシンデアミナーゼ活性およびシチジンデアミナーゼ活性を失活または低減化させることができれば特に限定されないが、例えば、該微生物の菌体、培養液またはそれらの処理物を水性媒体中でｐＨは通常４．０以上１０．０以下であり、好ましくは６．０以上９．０以下で、温度は通常５０℃以上、好ましくは６０℃以上８０℃以下で１０分間以上、より好ましくは３０分間以上静置または懸濁するような方法を挙げることができる。加熱時間は４０時間以下が更に好ましい。また、処理液中にリン酸化糖を１ｍＭ以上、好ましくは１０ｍＭ以上１００ｍＭ以下添加することにより、ヌクレオシドホスホリラーゼをより安定化させることができる。
【００５１】
本発明でいうシトシンデアミナーゼおよびシチジンデアミナーゼの両酵素活性を有さない微生物とは、例えばＢａｃｉｌｌｕｓ属細菌ではシチジンデアミナーゼのみ存在し、シトシンデアミナーゼの存在は知られていない。この様にＢａｃｉｌｌｕｓ属細菌のシチジンデアミナーゼ欠損株を用いることが可能である。例えばＢａｃｉｌｌｕｓ Ｇｅｎｅｔｉｃ Ｓｔｏｃｋ Ｃｅｎｔｅｒ（ＢＧＣＳ）より入手が可能なＢａｃｉｌｌｕｓ ｓｕｂｔｉｌｕｓ １Ａ４７９などが例示できる。このような菌株にヌクレオシドホスホリラーゼを発現させた菌体を用いることも可能である。
【００５２】
本発明でいうホスファターゼの欠損株とは、例えばアルカリホスファターゼの欠損株としては大腸菌Ｋ−１２ ＤＨ５αを例示することができる。この様な菌株に、シトシンヌクレオシドホスホリラーゼを発現させた菌体を用いることも可能である。
【００５３】
この様な菌体を加熱処理する事により酸性ホスファターゼを失活または低減化することが可能であり、更に高い反応収率を達成することができる。例えば、該微生物の菌体、培養液またはそれらの処理物を水性媒体中でｐＨは通常４．０以上１０．０以下であり、好ましくは６．０以上９．０以下で、温度は通常５０℃以上、好ましくは６０℃以上８０℃以下で１０分間以上、より好ましくは３０分間以上静置または懸濁するような方法を挙げることができる。
【００５４】
本発明でいうシトシンヌクレオシドホスホリラーゼ活性を阻害する物質を低減化または除去するとは、反応を阻害する物質を除去または低減化できれば特に限定されないが、例えば菌体を超音波破砕などにより酵素液を調整し、該酵素液に極性溶媒を加えることでシトシンヌクレオシドホスホリラーゼを沈殿として得る方法や硫酸アンモニウムなどを加える塩析により沈殿として集める方法、或いはイオン交換樹脂等で精製する方法等によりシトシンヌクレオシドホスホリラーゼ活性を阻害する物質を除去または低減化することが可能である。
【００５５】
本発明におけるシトシンヌクレオシド化合物の合成反応は、シトシンまたはシトシン誘導体とリン酸化糖を基質としてシトシンヌクレオシド化合物を合成できるヌクレオシドホスホリラーゼを発現している微生物であって、シトシンまたはシトシン誘導体やシトシンヌクレオシド化合物を分解する活性を失活、あるいは欠損した微生物の菌体、培養液それらの処理物を用い、適切なｐＨや温度などの反応条件を選べばよいが通常はｐＨ４〜１０、温度１０〜８０℃の範囲で行うことができる。反応に使用するリン酸化糖とシトシンまたはシトシン誘導体の濃度は０．１〜１０００ｍＭ程度が適当であり、両者のモル比は添加するシトシンまたはシトシン誘導体の比率をリン酸化糖またはその塩に対して０．１〜１０倍モル量で行える。反応転化率を考えれば０．９５倍モル量程度が好ましい。
【００５６】
また、反応液中に生成するリン酸をトラップする目的でリン酸と難溶性の金属塩類を存在させたり、イオン交換樹脂等の担体を存在させることで反応収率を高めることも可能である。
【００５７】
反応液よりシトシンヌクレオシド化合物を採取する方法は、水等の溶媒に対する溶解度差を利用したり、イオン交換や吸着樹脂を用いる方法で行うことができる。
【００５８】
反応液中に残存する微量のシトシンをシトシンデアミナーゼの発現している微生物を用い、シチジンデアミナーゼの低減化または失活処理を施すか、或いはシチジンデアミナーゼ欠損株にシトシンデアミナーゼを発現させた微生物を用い、反応液中に存在させることで微量のシトシンをウラシルに変換することができる。該反応液を陽イオン交換樹脂に通すことで未反応のシトシンが変換されたウラシルやシトシンヌクレオシド化合物の分解物であるウラシルヌクレオシド化合物は吸着せず、シトシンヌクレオシド化合物のみを吸着させることで容易に精製することができる。
【００５９】
【実施例】
以下に実施例で本発明を説明するが、本発明はこれら実施例によって何等制限されるものではない。
［分析法］
生成したヌクレオシド化合物はすべて高速液体クロマトグラフィーにより定量した。分析条件は以下による。
カラム；Ｄｅｖｅｌｏｓｉｌ ＯＤＳ−ＭＧ−５ ４．６×２５０ｍｍ（野村化学）
カラム温度；４０℃
ポンプ流速；１．０ｍｌ／ｍｉｎ．
検出；ＵＶ２５４ｎｍ、
溶離液；５０ｍＭリン酸１カリウム：メタノール＝８：１（Ｖ／Ｖ）
参考例１（特開平１−６０３９６公報の再現性試験）
特開平１−６０３９６公報の実施例１に従ってデオキシシチジンの合成を検討した。即ち、実施例１記載の菌株２４株の中から表１に示す７寄託株を取り寄せた。酵母エキス０．５ｇ／ｄｌ、ペプトン１．０ｇ／ｄｌ、肉エキス１．０ｇ／ｄｌおよびＮａＣｌ０．５ｇ／ｄｌを含む培地（ｐＨ７．０）５０ｍｌを５００ｍｌ容肩付フラスコに分注し殺菌した。この培地に、ブイヨン寒天培地にて３０℃，１６時間前培養した表１に示す微生物を１白金耳ずつ接種し、３０℃にて１６時間振とう培養した。得られた培養液より菌体を遠心分離により分離した後、０．０５Ｍリン酸バッファー（ｐＨ７．０）で洗浄し、更に遠心分離することにより洗浄菌体を調製した。
【００６０】
上記洗浄菌体を２０ｍＭの２’−デオキシリボース−１−リン酸と２０ｍＭのシトシンを含む０．０５Ｍトリスバッファー（ｐＨ７．２）１０ｍｌに５ｇ／ｄｌになる様に添加し、６０℃，２４時間反応させた。反応液を希釈しＨＰＬＣで分析した結果、何れも基質であるシトシンは分解されほとんど消失されていたが、デオキシシチジンは検出されなかった。
【００６１】
【表１】

【００６２】
参考例２（ＰＮＰクローニングと発現株作製、対照菌体の作製）
大腸菌染色体ＤＮＡを次のようにして調製した。
【００６３】
エシェリヒア・コリＫ−１２／ＸＬ−１０株（Ｓｔｒａｔａｇｅｎｅ社）を５０ｍｌのＬＢ培地に接種し、３７℃で一夜培養した後集菌し、リゾチーム１ｍｇ／ｍｌを含む溶菌液で溶菌した。溶菌液をフェノール処理した後、通常の方法によりエタノール沈殿によりＤＮＡを沈殿させた。生じたＤＮＡの沈殿は、ガラス棒に巻き付けて回収した後、洗浄し、ＰＣＲに用いた。
【００６４】
ＰＣＲ用のプライマーには、エシェリヒア・コリの既知のプリンヌクレオシドホスホリラーゼ（以下ＰＮＰと表記する）をコードするｄｅｏＤ遺伝子の塩基配列（ＧｅｎＢａｎｋ ａｃｃｅｓｓｉｏｎ Ｎｏ． ＡＥ０００５０８（コード領域は塩基番号１１５３１−１２２５０）に基づいて設計した配列番号１及び２に示す塩基配列を有するオリゴヌクレオチド（北海道システム・サイエンス株式会社に委託して合成した）を用いた。これらのプライマーの５’末端付近及び３’末端付近には、それぞれＥｃｏＲＩ及びＨｉｎｄＩＩＩの制限酵素認識配列を有する。
【００６５】
制限酵素ＨｉｎｄＩＩＩで完全に消化した前記大腸菌染色体ＤＮＡ ６ｎｇ／μｌ及びプライマー各３μＭを含む０．１ｍｌのＰＣＲ反応液を用いて、変性：９６℃、１分間、アニーリング：５５℃、１分間、伸長反応：７４℃、１分間からなる反応サイクルを、３０サイクルの条件でＰＣＲを行った。
【００６６】
上記反応産物及びプラスミドｐＵＣ１８（宝酒造（株））を、ＥｃｏＲＩ及びＨｉｎｄＩＩＩで消化し、ライゲーション・ハイ（東洋紡（株））を用いて連結した後、得られた組換えプラスミドを用いて、エシェリヒア・コリＤＨ５αを形質転換した。形質転換株を、アンピシリン（Ａｍ）５０μｇ／ｍｌ及びＸ−Ｇａｌ（５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシド）を含むＬＢ寒天培地で培養し、Ａｍ耐性で且つ白色コロニーとなった形質転換株を得た。
【００６７】
このようにして得られた形質転換株よりプラスミドを抽出し、目的のＤＮＡ断片が挿入されたプラスミドを、ｐＵＣ−ＰＮＰ７３と命名した。ｐＵＣ−ＰＮＰ７３に導入されたＤＮＡ断片の塩基配列を通常の塩基配列の決定法に従い塩基配列を確認した。得られた塩基配列を配列番号３に、塩基配列より翻訳されたアミノ酸配列を配列番号４に示した。本酵素のサブユニットの分子量は約２６０００であり、６量体として活性発現していることが知られている。本酵素の至適温度は約７０℃であり、至適ｐＨは約７．０から７．５である。こうして得られた形質転換体を、エシェリヒア・コリ ＭＴ−１０９０５と名づけた。
【００６８】
エシェリヒア・コリ ＭＴ−１０９０５株をＡｍ５０μｇ／ｍｌを含むＬＢ培地１００ｍＬで３７℃・１晩振とう培養した。培養液を１３０００ｒｐｍで１０ｍｉｎ遠心分離し、得られた菌体を２０ｍＬの１００ｍＭトリス塩酸緩衝液（ｐＨ８．０）に懸濁した。懸濁液を再度１３０００ｒｐｍで１０ｍｉｎ遠心分離し、得られた菌体を２ｍＬの１００ｍＭトリス塩酸緩衝液（ｐＨ８．０）、１０ｍＭの２´−デオキシリボース１−リン酸ジ（モノシクロヘキシルアンモニウム）塩（ＳＩＧＭＡ製）に懸濁し、−２０℃にて凍結保存した。
【００６９】
プラスミドｐＵＣ１８（宝酒造（株））を用いて、エシェリヒア・コリＤＨ５αを形質転換した。形質転換株をｐＵＣ１８／ＤＨ５αと名づけた。ｐＵＣ１８／ＤＨ５α株をＡｍ５０μｇ／ｍｌを含むＬＢ培地１００ｍＬで３７℃・１晩振とう培養した。培養液を１３０００ｒｐｍで１０ｍｉｎ遠心分離し、得られた菌体を２０ｍＬの１００ｍＭトリス塩酸緩衝液（ｐＨ８．０）に懸濁した。懸濁液を再度１３０００ｒｐｍで１０ｍｉｎ遠心分離し、得られた菌体を２ｍＬの１００ｍＭトリス塩酸緩衝液（ｐＨ８．０）、１０ｍＭの２´−デオキシリボース１−リン酸ジ（モノシクロヘキシルアンモニウム）塩（ＳＩＧＭＡ製）に懸濁し、−２０℃にて凍結保存した。
【００７０】
比較例１（ＰＮＰを組換えた大腸菌でのデオキシシチジン合成）
２０ｍＭの２´−デオキシリボース１−リン酸ジ（モノシクロヘキシルアンモニウム）塩（ＳＩＧＭＡ製）、２０ｍＭのシトシン（和光純薬製、特級）、１００ｍＭのトリス塩酸緩衝液（ｐＨ８．０）、参考例２で得られたｐＵＣ１８／ＤＨ５α株の菌体懸濁液０．１ｍＬからなる反応液１．０ｍｌを５０℃で２０時間反応させた。基質を入れずに同様に保温したものを比較例とした。反応液を希釈した後分析したところデオキシシチジンは検出されなかった。図１（Ａ）に比較例、図１（Ｂ）に反応液のＨＰＬＣの分析チャートを示した。
【００７１】
比較例２（ＰＮＰを組み換えていない大腸菌のデオキシシチジン合成）
２０ｍＭの２´−デオキシリボース１−リン酸ジ（モノシクロヘキシルアンモニウム）塩（ＳＩＧＭＡ製）、２０ｍＭのシトシン（和光純薬製、特級）、１００ｍＭのトリス塩酸緩衝液（ｐＨ８．０）、参考例２で得られたＭＴ―１０９０５株の菌体懸濁液０．１ｍＬからなる反応液１．０ｍｌを５０℃で２０時間反応させた。基質を入れずに同様に保温したものを比較例とした。反応液を希釈した後分析したところ、１０ｍＭのデオキシウリヂン、３ｍＭのウラシルが生成していたがデオキシシチジンは検出されなかった。図２（Ａ）に比較例、図２（Ｂ）に反応液のＨＰＬＣの分析チャートを示した。
【００７２】
実施例１（有機溶媒処理）
参考例２のＭＴ−１０９０５株の懸濁液にメタノール（ＭｅＯＨ）を表２に示すような濃度で添加して３０℃にて１時間保温した。以下シチジンデアミナーゼはｃｄｄと表記する。ｃｄｄの活性測定は１００ｍＭトリス塩酸緩衝液（ｐＨ８．０）、２０ｍＭシチジンの反応液１．０ｍｌに菌体懸濁液を加えて５０℃で２ｈｒ反応させることにより測定した。上述の菌体懸濁液にＭｅＯＨを添加せずに、３０℃にて１時間保温したものを１００％として相対値で示した。
【００７３】
ＰＮＰの活性は２０ｍＭの２´−デオキシリボース１−リン酸ジ（モノシクロヘキシルアンモニウム）塩（ＳＩＧＭＡ製）、５．４ｍＭのアデニン（和光純薬製、特級）、１００ｍＭのトリス塩酸緩衝液（ｐＨ８．０）反応液１．０ｍｌに菌体を添加し５０℃で１５分反応させることにより測定した。デオキシアデノシンの生成量が２ｍＭ以下の生成量である菌体量を用い反応を行った。上述の菌体懸濁液にＭｅＯＨを添加せずに３０℃にて１時間保温したものを１００％として相対値で示した。結果を表２に示した通り、ＰＮＰ活性はほとんど低下しなかったが、ｃｄｄ活性はほとんど失活していた。
【００７４】
【表２】

【００７５】
実施例２（有機溶媒処理）
参考例２のＭＴ−１０９０５株の懸濁液に表３に示すような有機溶媒を添加し、３０℃にて１時間保温した。結果は表３に示した通り、ＰＮＰの活性はほとんど低下しなかったが、ｃｄｄの活性はほとんど失活していた。
【００７６】
【表３】

【００７７】
実施例３（加熱処理）
参考例２のＭＴ−１０９０５株の懸濁液を６０℃にて保温した。以下シトシンデアミナーゼはｃｏｄと表記する。ｃｏｄ活性は１００ｍＭトリス塩酸緩衝液（ｐＨ８．０）、２０ｍＭシトシンに菌体懸濁液を添加して５０℃で２ｈｒ反応させることにより測定した。無処理の菌体を添加した時のシトシン分解量を１００％として、残存活性を相対値で表した。結果を表４に示した通り、ＰＮＰの活性はほとんど低下しなかったが、ｃｏｄの活性はほとんど失活していた。
【００７８】
【表４】

【００７９】
実施例４（加熱処理＋有機溶媒処理）
実施例３で得た加熱処理２０ｈｒの菌体を実施例１と同じ方法でＭｅＯＨ処理を行った。実施例３の２０ｈｒ処理の菌体活性を１００％としたときの相対値として表示した。結果を表５に示す通り、ＰＮＰの活性はほとんど低下しなかったが、ｃｏｄとｃｄｄの活性はほとんど失活していた。
【００８０】
【表５】

【００８１】
実施例５（実施例４の処理を施した組換えＰＮＰ発現菌によるデオキシシチジンの合成）
２０ｍＭの２´−デオキシリボース１−リン酸ジ（モノシクロヘキシルアンモニウム）塩（ＳＩＧＭＡ製）、２０ｍＭのシトシン（和光純薬製、特級）、１００ｍＭのトリス塩酸緩衝液（ｐＨ８．０）、実施例４で得られた５０％ＭｅＯＨでの処理菌体液０．１ｍｌからなる反応液１．０ｍｌを５０℃で２０時間反応させた。反応液を希釈した後分析したところ、１０．７ｍＭのデオキシシチジン、０．２ｍＭのデオキシウリヂン、０．１ｍＭのウラシルが生成していた。その時の反応収率は５３％であった。図２（Ｃ）に反応液のＨＰＬＣの分析チャートを示した。
【００８２】
実施例６（実施例４の処理を施した大腸菌によるデオキシシチジンの合成）
２０ｍＭの２´−デオキシリボース１−リン酸ジ（モノシクロヘキシルアンモニウム）塩（ＳＩＧＭＡ製）、２０ｍＭのシトシン（和光純薬製、特級）、１００ｍＭのトリス塩酸緩衝液（ｐＨ８．０）、参考例２で得られたｐＵＣ１８／ＤＨ５αの菌体懸濁液を実施例４と同様の方法で５０％ＭｅＯＨ処理をした菌体懸濁液０．１ｍｌからなる反応液１．０ｍｌを５０℃で２０時間反応させた。反応液を希釈した後分析したところ、０．１４ｍＭのデオキシシチジン、０．１７ｍＭのデオキシウリヂンが生成していた。その時の反応収率は０．７％であった。図１（Ｃ）に反応液のＨＰＬＣの分析チャートを示した。
【００８３】
実施例７（実施例４の処理を施した組換えＰＮＰ発現菌によるシチジンの合成）
２０ｍＭのリボース１−リン酸ジ（シクロヘキシルアンモニウム）塩（ＳＩＧＭＡ製）、２０ｍＭのシトシン（和光純薬製、特級）、１００ｍＭのトリス塩酸緩衝液（ｐＨ８．０）、実施例４で得られた５０％ＭｅＯＨ処理の菌体懸濁液０．１ｍｌからなる反応液１．０ｍｌを５０℃で２０時間反応させた。反応液を希釈した後分析したところ、５ｍＭのシチジン、０．１ｍＭのウリヂン、０．１ｍＭのウラシルが生成していた。その時の反応収率は２５％であった。
【００８４】
実施例８（ＰＮＰの精製と精製ＰＮＰによるデオキシシチジンの合成）
実施例４で得られた菌体を１０ｍＬの１０ｍＭトリス塩酸緩衝液（ｐＨ７．５）に懸濁し、超音波破砕機で菌体を破砕した。次いで破砕液を遠心分離により粗酵素液を調整し、５０ｍＭトリス塩酸緩衝液（ｐＨ７．５）で平衡化したＤＥＡＥ−トヨパール（トーソー）３ｃｍ×１０ｃｍのカラムに加え、５０ｍＭＮａＣｌから５００ｍＭＮａＣｌのリニアグラジエントで溶出し、活性画分を回収した。溶離液を７０％硫安飽和で沈殿として回収し、１０ｍＭトリス塩酸緩衝液（ｐＨ７．５）に対して透析した。１０ｍＭトリス塩酸緩衝液（ｐＨ７．５）で平衡化したハイドロキシアパタイトカラム（３ｃｍ×１５ｃｍ）に透析液を加え、１０ｍＭトリス塩酸緩衝液（ｐＨ７．５）から５０ｍＭトリス塩酸緩衝液（ｐＨ７．５）で溶出し、活性画分を回収した。酵素液を７０％硫安飽和で沈殿として回収し、１ｍＬの１０ｍＭトリス塩酸緩衝液（ｐＨ７．５）に溶解し、１０ｍＭトリス塩酸緩衝液（ｐＨ７．５）に対して透析し精製ＰＮＰを２ｍＬを得た。こうして得た精製ＰＮＰはＳＤＳ−ポリアクリルアミド電気泳動により単一のバンドであることを確認した。結果を図３に示した。 ２０ｍＭの２´−デオキシリボース１−リン酸ジ（モノシクロヘキシルアンモニウム）塩（ＳＩＧＭＡ製）、２０ｍＭのシトシン（和光純薬製、特級）、１００ｍＭのトリス塩酸緩衝液（ｐＨ８．０）、精製酵素０．１ｍｌからなる反応液１．０ｍｌを５０℃で２０時間反応させた。反応液を希釈した後分析したところ、１１．５ｍＭのデオキシシチジンが生成し、その時の反応収率は５７．５％であった。
【００８５】
実施例９（実施例４の処理を施した組換えＰＮＰ発現菌によるアザシチジンの合成）
２０ｍＭのリボース１−リン酸ジ（シクロヘキシルアンモニウム）塩（ＳＩＧＭＡ製）、２０ｍＭのアザシトシン（ＳＩＧＭＡ製）、１００ｍＭのトリス塩酸緩衝液（ｐＨ８．０）、実施例４で得られた５０％ＭｅＯＨ処理の菌体懸濁液０．１ｍｌからなる反応液１．０ｍｌを５０℃で２０時間反応させた。反応液を希釈した後分析したところ、３．２ｍＭのアザシチジンが生成していた。その時の反応収率は１６％であった。
【００８６】
実施例１０（実施例４の処理を施した組換えＰＮＰ発現菌によるフルオロシチジンの合成）
２０ｍＭのリボース１−リン酸ジ（シクロヘキシルアンモニウム）塩（ＳＩＧＭＡ製）、２０ｍＭの５−フルオロシトシン（ＳＩＧＭＡ製）、１００ｍＭのトリス塩酸緩衝液（ｐＨ８．０）、実施例４で得られた５０％ＭｅＯＨ処理の菌体懸濁液０．１ｍｌからなる反応液１．０ｍｌを５０℃で２０時間反応させた。反応液を希釈した後分析したところ、２．４ｍＭの５−フルオロシチジンが生成していた。その時の反応収率は１２％であった。
【００８７】
実施例１１（実施例４の処理を施した組換えＰＮＰ発現菌によるメチルシチジンの合成）
２０ｍＭのリボース１−リン酸ジ（シクロヘキシルアンモニウム）塩（ＳＩＧＭＡ製）、２０ｍＭの５−メチルシトシン（ＳＩＧＭＡ製）、１００ｍＭのトリス塩酸緩衝液（ｐＨ８．０）、実施例４で得られた５０％ＭｅＯＨ処理の菌体懸濁液０．１ｍｌからなる反応液１．０ｍｌを５０℃で２０時間反応させた。反応液を希釈した後分析したところ、２．1ｍＭの５−メチルシチジンが生成していた。その時の反応収率は１０．５％であった。
【００８８】
実施例１２（分解活性欠損株にＰＮＰを組込んだ菌体によりデオキシシチジンを合成）
（１）大腸菌ＰＮＰを枯草菌で発現させる大腸菌‐枯草菌シャトルベクターｐＰＮＰ０４、及びｐＰＮＰ０５の作製
ｐＵＣ−ＰＮＰ７３を鋳型として、配列番号５と配列番号６の合成オリゴヌクレオチドプライマーを用いたＰＣＲ法により大腸菌プリンヌクレオシドホスホリラーゼ遺伝子を増幅し、０．８ｋｂのＤＮＡ断片Ａを得た。次に、特開昭６０−２１０９８６公報に記載のプラスミドｐＮＰ１５０（ＦＥＲＭ ＢＰ‐４２５）を鋳型として、配列番号７と配列番号８の合成オリゴヌクレオチドプライマーを用いたＰＣＲ法によりバチルス・アミロリキファシエンス（Ｂａｃｉｌｌｕｓ ａｍｙｌｏｌｉｑｕｅｆａｃｉｅｎｓ）の中性プロテアーゼ由来の転写プロモーター、及び翻訳調節部位を含む領域を増幅し、１．０ｋｂのＤＮＡ断片Ｂを得た。続いて、ＤＮＡ断片ＡとＤＮＡ断片Ｂの重複領域同士を用いてアニーリングし、この生成断片を鋳型として配列番号６、及び配列番号７の合成オリゴヌクレオチドプライマーを用いたＰＣＲ法により増幅し、約１．８ｋｂのＤＮＡ断片Ｃを得た。このＤＮＡ断片Ｃを制限酵素ＥｃｏＲＩとＨｉｎｄＩＩＩで消化し、インサートフラグメントとした。ベクターＤＮＡは、枯草菌・大腸菌シャトルベクターｐＲＢ３７３、及びｐＲＢ３７４をＢａｃｉｌｌｕｓ Ｇｅｎｅｔｉｃ Ｓｔｏｃｋ Ｃｅｎｔｅｒ（ＢＧＳＣ；ＯＨ）より入手し、それぞれを制限酵素ＥｃｏＲＩとＨｉｎｄＩＩＩで消化し、ベクターフラグメントを調製した。このベクターフラグメントを上記インサートフラグメントの過剰量存在下でライゲートし、大腸菌プリンヌクレオシドホスホリラーゼ発現シャトルベクターｐＰＮＰ０４、及びｐＰＮＰ０５を作製した。
（２）枯草菌へのｐＰＮＰ０４、及びｐＰＮＰ０５の導入、および形質転換体を用いたデオキシシチジンの合成
枯草菌としては、シチジンデアミナーゼ活性を有さないバチルス・ズブチリス（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）１Ａ４７９株及びバチルス・ズブチリス（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）１Ａ４８０株をＢａｃｉｌｌｕｓ Ｇｅｎｅｔｉｃ Ｓｔｏｃｋ Ｃｅｎｔｅｒ（ＢＧＳＣ；ＯＨ）より入手して用いた。
【００８９】
（１）で得られたプラスミドｐＰＮＰ０４、及びｐＰＮＰ０５による形質転換はＣｈａｎｇのプロトプラスト法（Ｃｈａｎｇ．Ｓ ａｎｄ Ｃｏｈｅｎ，Ｓ．Ｎ．；Ｍｏｌ．Ｇｅｎ．Ｇｅｎｅｔ． １６８， １１１（１９７８））に従って実施し、形質転換体１Ａ４８０（ｐＰＮＰ０４）、１Ａ４８０（ｐＰＮＰ０５）及び１Ａ４７９（ｐＰＮＰ０４）及び１Ａ４７９（ｐＰＮＰ０５）を得た。
【００９０】
次に、１Ａ４８０（ｐＰＮＰ０４）株についてデオキシシチジン合成活性を以下のように評価した。
【００９１】
１Ａ４８０（ｐＰＮＰ０４）を２倍濃度のＬ培地２０ｍＬで３５℃、１７時間培養した。この培養液１．５ｍＬを遠心して得られた菌体を−２０℃で凍結保存した。２４ｍＭの２´−デオキシリボース１−リン酸ジ（モノシクロヘキシルアンモニウム）塩（ＳＩＧＭＡ製）、２０ｍＭのシトシン（和光純薬製、特級）、１００ｍＭのトリス塩酸緩衝液（ｐＨ８．０）よりなる反応液１ｍｌを凍結菌体に加えて５０℃にて振とうした。１．５時間及び１８時間後にサンプリングを行ない、水で２０倍に希釈、遠心して菌体を除去した後に上清をＨＰＬＣにて分析したところ、１．５時間で１．４〜１．５ｍＭ、１８時間で６．４〜７．０ｍＭのデオキシシチジンを蓄積し、基質及び生成物の分解物は認められなかった。
【００９２】
１Ａ４８０（ｐＰＮＰ０５）、１Ａ４７９（ｐＰＮＰ０４）及び１Ａ４７９（ｐＰＮＰ０５）についてもデオキシシチジン合成を測定した結果、１Ａ４８０（ｐＰＮＰ０４）株とほぼ同等のデオキシシチジン合成能力を保持していることが明らかとなった。
【００９３】
実施例１３（シトシンヌクレオシドホスホリラーゼ活性を保持したアミノ酸置換体の取得）
参考例２で得られたｐＵＣ−ＰＮＰ７３のプラスミドＤＮＡを鋳型として、ＳＴＲＡＴＡＧＥＮＥ社のＱｕｉｃｋＣｈａｎｇｅ Site-Directed Mutagenesis Kitを用いて変異を導入した。以降単にキットと呼ぶ。以下の実施例では、基本的にキットの原理および操作方法を踏襲した。
【００９４】
３０ｍｌの試験管に１０ｍｌのＬＢ液体培地を調製し、１２１℃・２０分間のオートクレーブにより滅菌した。この培地に終濃度が１００μｇ／ｍｌとなるようにアンピシリンを添加した後、参考例２で得られたＭＴ−１０９０５株を一白菌耳植菌し、３７℃・３００ｒｐｍにて約２０時間培養した。該培養液１ｍｌを適当な遠心チューブに分取した後、遠心分離（１５０００ｒｐｍ×５分）により該菌体を分離した。続いてアルカリＳＤＳ抽出法により該菌体よりｐＵＣ−ＰＮＰ７３のプラスミドＤＮＡを調製した。
【００９５】
ｐＵＣ−ＰＮＰ７３のプラスミドＤＮＡを鋳型として配列番号９と１０、１１と１２、１３と１４、１５と１６、１７と１８、１９と２０、２１と２２、２３と２４、２５と２６、２７と２８、２９と３０、３１と３２、３３と３４、３５と３６、３７と３８、３９と４０、４１と４２、４３と４４、４５と４６のＤＮＡをプライマーとしてＰＣＲを実施した。ＰＣＲ反応液の組成を表６に示した。増幅条件を表７に示した。
【００９６】
上記ＰＣＲ増幅反応液に制限酵素ＤｐｎＩを４ｕｎｉｔ加え、３７℃にて１ｈｒ保温した。この反応液１μＬを大腸菌Ｋ−１２ＤＨ５αのコンピテントセル（東洋紡）１００μＬに加え、氷中で３０分間保持した。４２℃の恒温水槽に３０秒間浸した後、コンピテントセルに付属するNZY+培地０．９ｍＬを加え３７℃で１時間振とうした。上記培養液をＬＢ寒天培地にアンピシリンを１００μｇ／ｍＬとなるように加えた培地上に塗抹し、３７℃で２０時間保温しコロニーを形成させた。
【００９７】
該コロニーより任意に選別した５クローンを各一白菌耳ずつ植菌し、Ａｍ５０μｇ／ｍｌを含むＬＢ培地１００ｍＬで３７℃・１晩振とう培養した。培養液を１３０００ｒｐｍで１０ｍｉｎ遠心分離し、得られた菌体を２０ｍＬの１００ｍＭトリス塩酸緩衝液（ｐＨ８．０）に懸濁した。懸濁液を再度１３０００ｒｐｍで１０ｍｉｎ遠心分離し、得られた菌体を２ｍＬの１００ｍＭトリス塩酸緩衝液（ｐＨ８．０）、１０ｍＭの２´−デオキシリボース１−リン酸ジ（モノシクロヘキシルアンモニウム）塩（ＳＩＧＭＡ製）に懸濁し、実施例４と同じ方法で菌体の処理を施し、実施例５と同じ方法でデオキシシチジンの合成反応を行った。ＨＰＬＣ分析によりデオキシシチジンを測定した結果、５クローン中４クローンでデオキシシチジンの生成が検出され、シトシンヌクレオシドホスホリラーゼ活性を保持していることが確認された。
【００９８】
シトシンヌクレオシドホスホリラーゼ活性の測定に供した上記培養液の残部１ｍｌより該４クローンの菌体をそれぞれ分離し、アルカリＳＤＳ抽出法により各クローンのプラスミドＤＮＡを調製した。ＤＮＡ断片の塩基配列を通常の塩基配列の決定法に従い塩基配列を確認した。活性は、実施例１と同じ方法で測定した。結果の一覧を表８に示した。
【００９９】
【表６】

【０１００】
【表７】

【０１０１】
【表８】

【０１０２】
参考例３（大腸菌Ｋ―１２ Ｗ３１１０株への形質転換）
参考例２で得たプラスミドｐＵＣ−ＰＮＰ７３を通常の方法に従って大腸菌Ｋ−１２ Ｗ３１１０株（ＡＴＣＣ２７３２５）に形質転換した。得られた形質転換体をＭＴ−１０９４８と名づけた。参考例２と同様の方法で培養した形質転換体の活性は大腸菌Ｋ−１２ ＤＨ５α株への形質転換体の２倍の活性を有していた。
【０１０３】
実施例１４（反応阻害物質の除去：極性溶媒による沈殿）
参考例２と同じ方法で培養したＭＴ−１０９４８菌体を超音波破砕機で破砕した。破砕液と同容量のアセトンを加えて沈殿を遠心分離により沈殿を除いた。次に上記遠心上清液に上記の半分のアセトンを加え沈殿を遠心分離により回収した。回収沈殿は真空乾燥機により乾固した。乾燥沈殿を２ｍＬの１００ｍＭトリス塩酸緩衝液（ｐＨ８．０）、１０ｍＭの２´−デオキシリボース１−リン酸ジ（モノシクロヘキシルアンモニウム）塩（ＳＩＧＭＡ製）に溶解し、実施例４と同じ方法で処理した。アセトンで分画した酵素液２ｍＬをシトシン（６．９６ｇ）、２´−デオキシリボース１−リン酸２アンモニウム塩（１８．７ｇ）、水酸化マグネシウム（６．２ｇ）を水（１０５．３ｇ）に加え、５０℃で２４ｈｒ反応した。反応終了後、ＨＰＬＣで分析した所、２'−デオキシシチジンを１１．４ｇ（８０％）得た。比較例としてアセトン処理前の菌体液２ｍＬ上記と同様に反応したところ２'−デオキシシチジンを８．５ｇ（６０％）得た。
【０１０４】
実施例１５（反応阻害物質の除去：硫酸アンモニウムによる沈殿）
参考例２と同じ方法で培養したＭＴ−１０９４８菌体を超音波破砕機で破砕した。破砕液に粉砕した硫酸アンモニウムをゆっくりと加えていき、硫安飽和４０％まで添加した。氷水中で１時間ゆっくり攪拌し、遠心分離により沈殿を除いた。遠心上精液に粉砕した硫酸アンモニウムを硫安飽和７０％まで添加し、同様に氷水中で１時間ゆっくり攪拌し、遠心分離により沈殿を得た。沈殿を２ｍＬの１００ｍＭトリス塩酸緩衝液（ｐＨ８．０）、１０ｍＭの２´−デオキシリボース１−リン酸ジ（モノシクロヘキシルアンモニウム）塩（ＳＩＧＭＡ製）に溶解した。実施例４と同じ方法で処理し、実施例１４と同じ方法で反応したところ、２'−デオキシシチジンを１１．０ｇ（８０％）得た。
【０１０５】
実施例１６（反応阻害物質の除去：精製酵素は実施例８の酵素）
実施例８と同様の方法で得た酵素液２ｍＬを用いて実施例１４と同様の反応を行ったところ、２'−デオキシシチジンを１１．０ｇ（８０％）得た。
【０１０６】
実施例１７（ｐｈｏＡ欠損株の効果）
実施例４で得た菌体２ｍｌを用い、実施例１４と同じ反応を行ったところ２'−デオキシシチジンを１１．０ｇ（８０％）得た。一方、ＭＴ−１０９４８株を実施例４と同じ方法で処理したところ活性はＭＴ−１０９０５の２倍検出されたが、実施例１４と同じ反応を行ったところ２'−デオキシシチジンは８．５ｇ（６０％）しか得られなかった。
【０１０７】
実施例１８
シトシン６．９６ｇ（６２．６ｍｍｏｌ）、２´−デオキシリボース１−リン酸２アンモニウム塩１８．７ｇ（７５．４ｍｍｏｌ）、水酸化マグネシウム７．５８ｇ（１３２ｍｍｏｌ）、参考例３で調整した凍結菌体（２．０ｇ）を水（９６．４ｇ）、シクロヘキサン４．８ｇの混合液に加え、酢酸にて反応液のｐＨを８．８にコントロールしながら４５℃で１８ｈｒ反応した。反応終了後、ＨＰＬＣで分析した所、目的物である、２'−デオキシシチジンを１０．０３ｇ（７０．５モル％／シトシン）得た。この時、副生物であるウラシルは０・７３ｇ（１０．４モル％／シトシン）、２'−デオキシウリジンは１．８０ｇ（１２．６モル％／シトシン）であった。
【０１０８】
実施例１９
実施例１８と同様の条件で、菌体処理に用いる溶媒、反応時に添加する溶媒を変えて２'−デオキシシチジンの合成を行なった時の結果を表９及び表１０に示す。
【０１０９】
【表９】

【０１１０】
【表１０】

【０１１１】
実施例２０
２´−デオキシシチジンの合成
純水２０ｇに強塩基性アニオン交換樹脂（ＭＰ５００ レバチッド：交換容量１．１ｅｑ／Ｌ）２０ｍｌ（総交換容量 ２４ｍｍｏｌ）と２−デオキシリボース１−リン酸ジ（アンモニウム）塩（４．９６ｇ、２０ｍｍｏｌ）を加え、室温下、３０分間攪拌した。３０分後、実施例４と同じ方法で調整した酵素液（０．５ｍｌ）とシトシン（２．１１ｇ、１９ｍｍｏｌ）加え、攪拌下、５０℃で１０時間反応した。１０時間後に反応マスをＨＰＬＣにて分析した結果、所望の２´−デオキシシチジンを反応収率８０％で得た。
【０１１２】
【発明の効果】
ヌクレオシドホスホリラーゼを用いるヌクレオシド化合物の製造は、穏和な条件で位置、立体特異的にヌクレオシド化合物を製造することが可能であり、工業的な製造法の確立が望まれているがこれまでヌクレオシドホスホリラーゼによる工業的なシトシンヌクレオシド化合物の合成方法は知られていなかった。
【０１１３】
本発明によればシトシンに反応性を有するヌクレオシドホスホリラーゼを用い、ペントース−１−リン酸とシトシンまたはシトシン誘導体からのシトシンヌクレオシド化合物の製造が可能となる。その際、基質あるいは生成物の分解活性を特異的に減少させる方法を提供することで、効率よくシトシンヌクレオシド化合物を製造できる。
【０１１４】
【配列表】

【図面の簡単な説明】
【図１】（Ａ）は比較例１におけるＨＰＬＣの分析チャートであり、（Ｂ）は比較例１の反応液のＨＰＬＣの分析チャートであり、（Ｃ）は実施例６のＨＰＬＣの分析チャートである。
【図２】（Ａ）は比較例２における比較例におけるＨＰＬＣの分析チャートであり、（Ｂ）は比較例２の反応液のＨＰＬＣの分析チャートであり、（Ｃ）は実施例５のＨＰＬＣの分析チャートである。
【図３】実施例８にけるＳＤＳ−ポリアクリルアミド電気泳動の結果を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing a cytosine nucleoside compound useful as a raw material for synthesis of pharmaceuticals and the like. More specifically, an enzyme having cytosine nucleoside phosphorylase activity or a microbial cell having the enzyme activity, an enzyme preparation obtained by treating the microbial cell or a culture solution thereof, and the like, and pentose-1-phosphate The present invention relates to a method for producing a cytosine nucleoside compound from cytosine or a cytosine derivative.
[0002]
[Prior art]
Nucleoside phosphorylase is a general term for enzymes that phosphorylate the N-glycoside bond of nucleoside in the presence of phosphoric acid, and catalyzes a reaction represented by the following formula when ribonucleoside is taken as an example.
[0003]
Ribonucleoside + phosphate (salt) → nucleobase + ribose 1-phosphate
The enzymes, which are roughly classified into purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase, are widely distributed in the living world and are present in tissues such as mammals, birds and fish, yeasts and bacteria. This enzymatic reaction is reversible, and the synthesis of various nucleosides using reverse reactions has been known for some time. For example, from 2′-deoxyribose 1-phosphate and a nucleobase (thymine, adenine or guanine) to thymidine (JP-A-01-104190), 2′-deoxyadenosine (JP-A-11-137290) or 2 ′ A method for producing deoxyguanosine (Japanese Patent Laid-Open No. 11-137290) is known. Thus, the production of a nucleoside compound using a nucleoside phosphorylase can be produced in a position and stereospecific manner under mild conditions, and the synthesis of many nucleoside compounds has been studied.
[0004]
Japanese Patent Laid-Open No. 1-60396 discloses a method for producing deoxycytidine from deoxyribose-1-phosphate and cytosine by a reaction using the cell itself as a catalyst. However, the method of this publication is a reaction using the cells themselves as a catalyst, and the presence of cytosine nucleoside phosphorylase itself has not been confirmed. For example, deoxycytidine accumulated in the microbial cell is eluted from the microbial cell during the reaction, or the cytosine added as a substrate is transferred to the deoxynucleoside base in the microbial cell by nucleoside deoxyribosyltransferase in the microbial cell. It is also possible that deoxycytidine was detected. In order to clarify these points, the present inventors ordered 7 strains deposited in the examples of the publication and examined whether deoxycytidine is produced from deoxyribose-1-phosphate and cytosine in the same manner as in the examples. Tested. As a result, no deoxycytidine was detected in the reaction solution in any reaction using any strain, and it was not confirmed that the strain itself had an activity corresponding to cytosine nucleoside phosphorylase.
[0005]
On the other hand, JP-A-3-12786 reports a novel nucleoside phosphorylase that acts on both purine and pyrimidine bases, although it is described that it acts on deoxyuridine, deoxycytidine, and deoxythymidine as pyrimidine bases. The specification does not disclose any substrate specificity. The publication has already been withdrawn, and its activity cannot be confirmed. However, the inventors of the publication have purified nucleoside phosphorylase from a strain having the same name as the microorganism disclosed in the publication, and characterized its characteristics. Applied and Environmental Microbiology, Vol. 56, PP3830-3834 (1990). The document states that this enzyme has no activity against cytosine, cytidine and deoxycytidine.
[0006]
Except for the above, neither purine nucleoside phosphorylase nor pyrimidine nucleoside phosphorylase has been reported that cytosine or a cytosine derivative serves as a substrate as a nucleobase. For example, Method. Enzymology, Vol. 51, PP437-442 (1978) describes that thymidine phosphorylase, a kind of pyrimidine nucleoside phosphorylase of Salmonella typhimurium, has no reactivity with deoxycytidine. In addition, in J. Biol. Chem. , Vol. 248, no. 6, PP 2040-2043 (1973) describes that Salmonella typhimurium purine nucleoside phosphorylase does not show activity on pyrimidine nucleosides, and uridine, cytidine, deoxyuridine, and deoxycytidine are exemplified.
[0007]
Considering these reports comprehensively, although there was knowledge to predict the presence of an enzyme corresponding to the cytosine nucleoside phosphorylase according to the present invention, in any of those prior art documents, the existence was confirmed and actually this There are no reports of the enzyme being acquired.
[0008]
[Problems to be solved by the invention]
Nucleoside compounds using nucleoside phosphorylase can be produced in a position-stereospecific manner under mild conditions, and various nucleoside compounds have been extensively studied. Alternatively, no isolated and purified nucleoside phosphorylase capable of synthesizing a cytosine nucleoside compound using a cytosine derivative and a phosphorylated saccharide as a substrate has been reported.
[0009]
Further, according to the study by the present inventors, since microorganisms generally have cytosine deaminase activity and cytidine deaminase activity, microorganisms themselves are used, or enzyme preparations prepared from cells or cultures thereof are used. When these deaminase activities remain, cytosine or a cytosine derivative as a substrate and a deamino form of a cytosine nucleoside compound as a reaction product are synthesized, and it is difficult to accumulate cytosine nucleoside compounds efficiently. It becomes.
[0010]
That is, an object of the present invention is to provide an amino acid sequence of a nucleoside phosphorylase capable of synthesizing a cytosine nucleoside compound. Furthermore, a recombinant plasmid containing the gene, a transformant containing the recombinant plasmid, a method for producing the enzyme using the transformed strain, and a method for producing a cytosine nucleoside compound using the transformed strain are provided. That is. Furthermore, cytosine deaminase and cytidine deaminase activities can be reduced while maintaining the activity of nucleoside phosphorylase, or cytosine nucleoside can be efficiently used by using a microorganism lacking both enzymes and expressing a cytosine nucleoside phosphorylase. It is to provide a method for producing a compound.
[0011]
In addition, the cytosine nucleoside compound can be efficiently produced by reducing the activity of the phosphatase of the phosphorylated saccharide as a raw material, or by using a microorganism in which cytosine nucleoside phosphorylase is expressed in a microorganism lacking the enzyme It is in providing the method of manufacturing.
[0012]
When a cytosine nucleoside compound is produced, especially when it is used as a raw material for pharmaceuticals, a very small amount of by-products is a serious problem. Separation of by-product nucleosides in the purification process increases the purification load and reduces the recovery rate of cytosine nucleoside compounds, which is a serious problem in industrial production. When used for such purposes, it is necessary to almost completely deactivate cytosine deaminase activity and cytidine deaminase activity from the enzyme preparation.
[0013]
[Means for Solving the Problems]
As a result of intensive studies to solve these problems, the present inventors have found that purine nucleoside phosphorylase, which catalyzes a reaction using a purine base as a substrate, is surprisingly a pyrimidine base cytosine or cytosine derivative and pentose-1. -It has been found to catalyze the reaction of producing a cytosine nucleoside compound from phosphoric acid.
[0014]
The presence of this purine nucleoside phosphorylase found by the present inventors indicates the presence of cytosine nucleoside phosphorylase.
[0015]
On the other hand, the present inventors tried a reaction to produce a cytosine nucleoside compound from cytosine or a cytosine derivative and pentose-1-phosphate using a microorganism having this enzyme activity, and most of the products were cytosine or cytosine. It was a deamino form of a derivative and a cytosine nucleoside compound, and the target cytosine nucleoside compound could not be accumulated efficiently. As a result of extensive studies, the formation of this deamino body is due to the action of deaminase. The cell having this deaminase activity is stirred or left in an organic solvent, or the cell is heated. In addition, the activity of cytosine deaminase and cytidine deaminase can be inactivated, and in addition, both of these treatments deactivate cytosine nucleoside phosphorylase to be maintained by the presence of phosphate or phosphorylated sugar in the treatment solution. It was found that can be suppressed more effectively.
[0016]
According to the above-described method, it is possible to specifically reduce both the substrate and product degradation activities while maintaining the activity of the nucleoside phosphorylase, and the deaminase activity is deactivated or reduced as described above. It has been found that by using bacterial cells, a cytosine nucleoside compound can be produced from cytosine or a cytosine derivative and a phosphorylated saccharide with little by-product, and the present invention has been completed.
[0017]
According to the deaminase deactivation or reduction treatment of the present invention, the activity of cytosine nucleoside phosphorylase can be almost completely maintained, and the degradation activity can be almost completely deactivated. Furthermore, the present inventors have also found that a cytosine nucleoside compound can be efficiently produced by expressing the enzyme in a microorganism lacking both enzyme activities of cytosine deaminase and cytidine deaminase, and the present invention has been completed.
[0018]
In addition, the present inventors have found that the reaction yield decreases in the presence of an enzyme activity for dephosphorylating the phosphorylated saccharide as a raw material. It has been found that the enzyme activity can be selectively removed by adding a polar solvent to the cell disruption of cells having the enzyme activity. Alternatively, the enzyme activity can be removed by purifying cytosine nucleoside phosphorylase with a carrier such as fractionation by salting out or ion exchange resin. It has also been found that a cytosine nucleoside compound can be efficiently produced by using such a treated product, and the present invention has been completed.
Furthermore, the present inventors have also found that a cytosine nucleoside phosphorylase can be efficiently expressed by expressing a cytosine nucleoside phosphorylase in a microorganism lacking the enzyme activity, and the present invention has been completed.
[0019]
ADVANTAGE OF THE INVENTION According to this invention, the efficient synthesis | combining method of the cytosine nucleoside compound by cytosine nucleoside phosphorylase which could not be achieved conventionally can be provided.
[0020]
That is, the present invention is as follows.
[0021]
The method for producing a cytosine nucleoside compound according to the present invention comprises a step of obtaining a cytosine nucleoside compound by reacting a phosphorylated saccharide with cytosine or a cytosine derivative in the presence of an enzyme having cytosine nucleoside phosphorylase activity. And
[0022]
As the above enzyme, an enzyme having purine nucleoside phosphorylase activity can be used, and an enzyme preparation containing an enzyme having purine nucleoside phosphorylase activity can be suitably used in the method for producing a cytosine nucleoside compound according to the present invention. In addition, as an enzyme which has such purine nucleoside phosphorylase activity, the enzyme derived from colon_bacillus | E._coli can be mentioned, for example.
[0023]
Examples of the cytosine derivative include the following formula (I):
[0024]
[Chemical 2]

(In the above formula (I), X represents a carbon atom or a nitrogen atom, and Y represents a hydrogen atom, a halogen atom or a lower alkyl group.)
The compound represented by these can be used. Specific examples thereof include azacytosine and 5-fluorocytosine.
[0025]
Furthermore, the enzyme having cytosine nucleoside phosphorylase activity described above is a microorganism cell or a culture solution of the microorganism having this enzyme, or a crude enzyme extract, a crude enzyme solution, a crude enzyme preparation and a purified enzyme preparation obtained therefrom. Etc., and can be supplied into the reaction system in the form of an enzyme preparation.
[0026]
These bacterial cells or enzyme preparations have no cytosine deaminase activity and cytidine deaminase activity, or are low enough to produce cytosine nucleoside compounds using cytosine nucleoside phosphorylase activity even if they exist. Is preferred. For example, bacterial cells and enzyme preparations that have higher cytosine nucleoside phosphorylase activity than cytosine deaminase activity and cytidine deaminase activity can be preferably used.
[0027]
As an example of such a microbial cell and enzyme preparation, what reduced these cytosine deaminase activities and cytidine deaminase activity by the reduction process can be mentioned.
[0028]
The microbial cell subjected to the treatment for deactivation or reduction of the deaminase activity described above is obtained by, for example, contacting a microbial cell having cytosine nucleoside phosphorylase activity with water containing an organic solvent, thereby causing cytosine deaminase activity and It can be obtained by selectively reducing cytidine deaminase activity. As the organic solvent, polar solvents such as alcohols can be used. As the organic solvent, for example, one or more organic solvents selected from methyl alcohol, ethyl alcohol, propyl alcohol, butyl alcohol, dioxane and acetone can be used.
[0029]
Furthermore, when deactivating or reducing cytosine deaminase activity and cytidine deaminase activity with an organic solvent, the concentration of the organic solvent in water is preferably 20% by volume or more.
[0030]
On the other hand, microbial cells that have been treated for deactivation or reduction of deaminase activity are selectively treated with cytosine deaminase activity and cytidine deaminase activity in an aqueous medium by microbial cells having cytosine nucleoside phosphorylase activity. It can also be obtained by heat treatment at a decreasing temperature. As conditions for this heat treatment, conditions of 50 ° C. or more and 10 minutes or more and 40 hours or less can be employed.
[0031]
On the other hand, when performing treatment for deactivation or reduction of deaminase activity, the presence of pentose-1-phosphate makes it possible to more effectively maintain cytosine nucleoside phosphorylase activity to be maintained. . In this case, the concentration of pentose-1-phosphate is preferably 1 mM or more and 100 mM or less.
[0032]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, cytosine nucleoside phosphorylase refers to an enzyme having an activity of producing a cytosine nucleoside compound using cytosine or a cytosine derivative as a substrate, and may be of any origin such as an animal, a plant, and a microorganism as long as this requirement is satisfied. Absent. The enzyme cytosine nucleoside phosphorylase is not generally known in the art. Therefore, such terms are defined and used only in the present invention as described above. Specific examples of such nucleoside phosphorylases include those known as conventional purine nucleoside phosphorylases of microorganisms contained in the genus Escherichia such as Escherichia coli, which also have cytosine nucleoside phosphorylase activity. It can be mentioned as a suitable example. As a specific example, the DNA base sequence of purine nucleoside phosphorylase of Escherichia coli is exemplified in SEQ ID NO: 3, and the amino acid sequence translated from the base sequence is exemplified in SEQ ID NO: 4. In addition, recent advances in genetic engineering have made it easy to alter amino acid sequences by inactivating, inserting, or substituting part of the base sequence. In view of such a technical level, a part of the base sequence is modified within a range that does not affect the desired enzyme activity, for example, within a range in which the enzyme activity can be maintained or improved, and the amino acid sequence is modified. It is intended to be encompassed by the cytosine nucleoside phosphorylase of the invention. For example, the amino acid sequence shown in SEQ ID NO: 4 has been deleted, substituted, or added within a range that does not affect the target enzyme activity, or SEQ ID NO: 3 Nucleotide sequence that does not affect the target enzyme activity and that has a mutation such as deletion, substitution or addition within the range that its complementary sequence can hybridize under stringent conditions Those having the amino acid sequence encoded by can be used.
[0033]
Thus, having a purine nucleoside sphorylase activity makes it possible to produce a purine-type nucleoside compound and a cytosine nucleoside compound with a single enzyme, which is very advantageous for industrial production of nucleosides.
[0034]
The enzyme having cytosine nucleoside phosphorylase activity used in the method of the present invention can be supplied to the reaction system in the form of a microbial cell having the enzyme, an enzyme preparation prepared from this microbial cell or a culture solution thereof. . The enzyme preparation includes various processed products of the microbial cells or the culture solution thereof, enzyme extract, crude purified product using the enzyme extract, isolated purified product, and the like.
[0035]
As the cells and enzyme preparations of these microorganisms, commercially available products and those prepared using various methods can be used. For example, as a preparation having this enzyme activity, commercially available enzymes, microbial cells having the enzyme activity, treated cells of the cells, or immobilization products thereof can be used. Processed bacterial cells include, for example, acetone-dried bacterial cells and bacteria prepared by mechanical disruption, ultrasonic disruption, freeze-thaw treatment, pressure / pressure reduction treatment, osmotic pressure treatment, self-digestion, cell wall degradation treatment, surfactant treatment, etc. A crushed body or the like, and if necessary, those obtained by repeated purification by ammonium sulfate precipitation, acetone precipitation, or column chromatography may be used.
[0036]
The microorganism having cytosine nucleoside phosphorylase activity is not particularly limited as long as it expresses a nucleoside phosphorylase that produces a cytosine nucleoside compound using cytosine or a cytosine derivative as a substrate. Such a microorganism can be selected, for example, from those expressing a general nucleoside phosphorylase. Specific examples of microorganisms that produce such nucleoside phosphorylase include microorganisms belonging to the genus Escherichia such as Escherichia coli.
[0037]
By analyzing the molecular biological properties and amino acid sequences of purine nucleoside phosphorylases of the above-mentioned microorganism strains due to recent advances in molecular biology and genetic engineering, the gene of the protein is obtained from the microorganism strain, It is possible to construct a recombinant plasmid in which a gene and a control region necessary for expression are inserted, introduce this into an arbitrary host, and create a genetically modified bacterium that expresses the protein, It became easy. In view of the state of the art, a genetically modified bacterium in which such a nucleoside phosphorylase gene is introduced into an arbitrary host is also included in the microorganism expressing the nucleoside phosphorylase of the present invention.
[0038]
The control region necessary for expression herein refers to a promoter sequence (including an operator sequence that controls transcription), a ribosome binding sequence (SD sequence), a transcription termination sequence, and the like. Specific examples of the promoter sequence include trp promoter of tryptophan operon derived from E. coli, lac promoter of lactose operon, PL promoter and PR promoter derived from lambda phage, gluconate synthase promoter (gnt) and alkaline protease promoter derived from Bacillus subtilis. (Apr), neutral protease promoter (npr), α-amylase promoter (amy), and the like. In addition, a uniquely modified and designed sequence such as the tac promoter can also be used. Examples of the ribosome binding sequence include sequences derived from Escherichia coli or Bacillus subtilis, but are not particularly limited as long as the sequence functions in a desired host such as Escherichia coli or Bacillus subtilis. For example, a consensus sequence in which a sequence complementary to the 3 ′ end region of 16S ribosomal RNA is continuous by 4 or more bases may be prepared by DNA synthesis and used. A transcription termination sequence is not always necessary, but a ρ-factor-independent sequence such as a lipoprotein terminator, a trp operon terminator, etc. can be used. The sequence of these control regions on the recombinant plasmid is preferably arranged in the order of promoter sequence, ribosome binding sequence, gene encoding nucleoside phosphorylase, and transcription termination sequence from the 5 ′ end upstream.
[0039]
Specific examples of the plasmid herein include pBR322, pUC18, Bluescript II SK (+), pKK223-3, pSC101 having autonomously replicable regions in E. coli, and autonomous replication in Bacillus subtilis. PUB110, pTZ4, pC194, ρ11, φ1, φ105, etc. having various regions can be used as vectors. Further, as examples of plasmids capable of autonomous replication in two or more types of hosts, pHV14, TRp7, YEp7 and pBS7 can be used as vectors.
[0040]
As an arbitrary host mentioned here, Escherichia coli is exemplified as a representative example as will be described later. However, it is not particularly limited to Escherichia coli, and is not limited to Escherichia coli, and Bacillus subtilis or other Bacillus bacteria. Other microbial strains such as yeast and actinomycetes are also included.
[0041]
The cytosine nucleoside compound in the present invention is a compound in which cytosine or a cytosine derivative is bound as a nucleobase with an N-glycoside bond. Typical examples thereof include, but are not limited to, cytidine, deoxycytidine, dideoxycytidine, azacitidine, deoxyazacytidine, 5-fluorocytidine and 5-fluorodeoxycytidine.
[0042]
The cytosine derivative in the present invention has a cytosine structure part, and this cytosine structure part can be changed into a cytosine nucleoside structure by the action of cytosine nucleoside phosphorylase activity. Preferable examples of this cytosine derivative include the cytosine derivatives represented by the general formula (I). As the lower alkyl group as Y in the general formula (I), an alkyl group having 1 to 4 carbon atoms such as a methyl group, an ethyl group, an n-propyl group, an i-propyl group, an n-butyl group, sec- Mention may be made of butyl and tert-butyl groups. Among these cytosine derivatives, for example, azacytosine, 5-fluorocytosine, 5-methylcytosine and the like are particularly preferable.
[0043]
The phosphorylated saccharide in the present invention is one in which phosphoric acid is ester-bonded to the 1-position of polyhydroxyaldehyde or polyhydroxyketone and its derivative. Preferred examples thereof include ribose 1-phosphate, 2′-deoxyribose 1-phosphate, 2 ′, 3′-dideoxyribose 1-phosphate, arabinose 1-phosphate, dioxolane sugar-phosphate, and the like. Is mentioned.
[0044]
The polyhydroxyaldehyde or polyhydroxyketone as used herein is derived from natural products such as D-arabinose, L-arabinose, D-xylose, L-lyxose, aldopentose such as D-ribose, D-xylulose, Examples include, but are not limited to, ketopentoses such as L-xylulose and D-ribulose, and deoxysaccharides such as D-2-deoxyribose and D-2,3-dideoxyribose.
[0045]
These phosphorylated saccharides can be produced by subjecting a nucleoside compound to a phosphorolysis reaction by the action of nucleoside phosphorylase (J. Biol. Chem. Vol. 184, 437, 1950), anomeric selective chemical synthesis methods, etc. Can also be prepared. It can also be prepared by the enzymatic deoxyribose-1-phosphate synthesis method described in WO 01/14566. (Also includes Roche's patented dRP synthesis route.)
In the present invention, the reduction treatment of cytosine deaminase activity and cytidine deaminase activity is not particularly limited as long as it can inactivate or reduce cytosine deaminase activity and cytidine deaminase activity without inactivating nucleoside phosphorylase. As a suitable example, it is possible to expose the microbial cells, culture solution or treated product thereof to an organic solvent or heat treatment.
[0046]
In the present invention, the organic solvent is not particularly limited as long as it is a solvent capable of deactivating cytosine deaminase activity and cytidine deaminase activity. Specifically, methyl alcohol, ethyl alcohol, propyl alcohol, butyl alcohol, dioxane, Polar solvents such as tetrahydrofuran, methyl ethyl ketone and acetone, 1-hexanol, 2-methyl-1-pentanol, 4-methyl-2-pentanol, 2-ethyl-1-butanol, 1-heptanol, 2-heptanol, 3- Alcohols such as heptanol, 1-octanol, 2-octanol, 1-nonanol, esters such as propyl acetate, butyl acetate, isobutyl acetate, sec-butyl acetate, pentyl acetate, isopentyl acetate, cyclohexyl acetate, benzyl acetate, Tan, hexane, 2-methylhexane, 2,2-dimethylbutane, 2,3-dimethylbutane, cyclohexane, methylcyclohexane, heptane, cycloheptane, octane, cyclooctane, isooctane, nonane, decane, dodecane, petroleum ether, petroleum Benzene, ligroin, industrial gasoline, kerosene, hydrocarbons such as benzene, toluene, xylene, ethylbenzene, propylbenzene, cumene, mesitylene, naphthalene, halogenated carbonization such as dichloromethane, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, dichlorobenzene Hydrogens, phenols such as cresol and xylenol, ketones such as methyl isobutyl ketone and 2-hexanone, dipropyl ether, diisopropyl ether, diphenyl ether, diben Include ethers such as ether is pentane, hexane, heptane, octane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, toluene, hydrocarbons ethylbenzene. N, N-dimethylformamide, N, N-dimethylacetamide, N, N-dimethylbenzamide, N-methyl-2-pyrrolidone, N-methylformamide, N-ethylformamide, N-methylacetamide, formamide, acetylamide Amide compounds such as benzoic acid amide, and urea compounds such as urea, N, N′-dimethylurea, tetramethylurea, N, N-dimethylimidazolidinone, and the like, such as dimethyl sulfoxide, diethyl sulfoxide, diphenyl sulfoxide, etc. Examples thereof include sulfoxide compounds.
[0047]
The treatment with an organic solvent for deactivation or reduction of deaminase activity in the present invention is not particularly limited as long as nucleoside phosphorylase is not inactivated and cytosine deaminase activity and cytidine deaminase activity can be inactivated or reduced. For example, the pH of the microbial cell, culture solution or treated product thereof is usually 4.0 or more and 10.0 or less, preferably 6.0 or more and 9.0 or less, and the organic solvent is in water concentration. At a concentration of 10% by volume or more, preferably 20% by volume or more, more preferably 30% by volume or more, usually at a temperature of 0 ° C. or more, preferably 20 ° C., more preferably 50 ° C. or more, and a temperature of 80 ° C. or less, Examples thereof include a method of standing or suspending for 10 minutes or more and 40 hours or less, more preferably 1 hour or more and 20 hours or less.
[0048]
Further, nucleoside phosphorylase can be further stabilized by adding phosphorylated saccharide to the treatment solution in an amount of 1 mM or more, preferably 100 mM or less.
[0049]
The same effect can be obtained by adding these organic solvents to the reaction solution.
[0050]
The heat treatment referred to in the present invention is not particularly limited as long as nucleoside phosphorylase is not inactivated and cytosine deaminase activity and cytidine deaminase activity can be inactivated or reduced. For example, the microorganism cells, culture solution or their The pH of the treated product in an aqueous medium is usually 4.0 or more and 10.0 or less, preferably 6.0 or more and 9.0 or less, and the temperature is usually 50 ° C. or more, preferably 60 ° C. or more and 80 ° C. or less. Examples of the method include allowing to stand or suspend for 30 minutes or more, more preferably for 30 minutes or more. The heating time is more preferably 40 hours or less. In addition, nucleoside phosphorylase can be further stabilized by adding phosphorylated saccharide to the treatment solution in an amount of 1 mM or more, preferably 10 mM or more and 100 mM or less.
[0051]
In the present invention, for example, a microorganism not having both enzyme activities of cytosine deaminase and cytidine deaminase is cytidine deaminase in bacteria belonging to the genus Bacillus, and the presence of cytosine deaminase is not known. Thus, it is possible to use a cytidine deaminase-deficient strain of Bacillus genus bacteria. For example, Bacillus subtilus 1A479 available from Bacillus Genetic Stock Center (BGCS) can be exemplified. It is also possible to use bacterial cells in which nucleoside phosphorylase is expressed in such a strain.
[0052]
Examples of the phosphatase-deficient strain in the present invention include Escherichia coli K-12 DH5α as an alkaline phosphatase-deficient strain. It is also possible to use bacterial cells in which cytosine nucleoside phosphorylase is expressed in such a strain.
[0053]
By heat-treating such cells, acid phosphatase can be inactivated or reduced, and a higher reaction yield can be achieved. For example, the pH of the microbial cell, culture solution or treated product thereof in an aqueous medium is usually 4.0 or more and 10.0 or less, preferably 6.0 or more and 9.0 or less, and the temperature is usually 50. Examples of the method include allowing to stand or suspend at or above, preferably 60 to 80 ° C. for 10 minutes or more, more preferably 30 minutes or more.
[0054]
The reduction or removal of a substance that inhibits cytosine nucleoside phosphorylase activity as used in the present invention is not particularly limited as long as the substance that inhibits the reaction can be removed or reduced, but for example, an enzyme solution is prepared by ultrasonically crushing bacterial cells. Inhibiting cytosine nucleoside phosphorylase activity by a method of obtaining cytosine nucleoside phosphorylase as a precipitate by adding a polar solvent to the enzyme solution, a method of collecting as a precipitate by salting out by adding ammonium sulfate or the like, or a method of purification using an ion exchange resin or the like It is possible to remove or reduce material.
[0055]
The synthesis reaction of the cytosine nucleoside compound in the present invention is a microorganism expressing a nucleoside phosphorylase capable of synthesizing a cytosine nucleoside compound using cytosine or a cytosine derivative and a phosphorylated sugar as a substrate, and decomposes the cytosine or cytosine derivative or cytosine nucleoside compound. It is sufficient to select the reaction conditions such as pH and temperature using the cells of the microorganisms that have been inactivated or deficient in the activity, culture broth, and processed products thereof. Can be done. The concentration of phosphorylated saccharide and cytosine or cytosine derivative used in the reaction is suitably about 0.1 to 1000 mM, and the molar ratio of both is 0 to the ratio of added cytosine or cytosine derivative to phosphorylated saccharide or salt thereof. .1 to 10 times the molar amount. Considering the reaction conversion rate, a molar amount of about 0.95 times is preferable.
[0056]
It is also possible to increase the reaction yield by making phosphoric acid and a hardly soluble metal salt exist for the purpose of trapping phosphoric acid produced in the reaction solution, or by making a carrier such as an ion exchange resin.
[0057]
The method for collecting the cytosine nucleoside compound from the reaction solution can be performed by using a difference in solubility in a solvent such as water, or by a method using ion exchange or an adsorbing resin.
[0058]
Using a microorganism in which cytosine deaminase is expressed in a trace amount of cytosine remaining in the reaction solution, reducing or inactivating cytidine deaminase, or using a microorganism in which cytosine deaminase is expressed in a cytidine deaminase-deficient strain, A small amount of cytosine can be converted into uracil by being present in the reaction solution. By passing the reaction solution through a cation exchange resin, uracil converted from unreacted cytosine and uracil nucleoside compounds, which are degradation products of cytosine nucleoside compounds, are not adsorbed, but they are easily purified by adsorbing only cytosine nucleoside compounds. can do.
[0059]
【Example】
EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.
[Analysis method]
All the produced nucleoside compounds were quantified by high performance liquid chromatography. Analysis conditions are as follows.
Column; Develosil ODS-MG-5 4.6 × 250 mm (Nomura Chemical)
Column temperature: 40 ° C
Pump flow rate: 1.0 ml / min.
Detection; UV254 nm,
Eluent: 50 mM monopotassium phosphate: methanol = 8: 1 (V / V)
Reference Example 1 (Reproducibility test of Japanese Patent Laid-Open No. 1-60396)
Synthesis of deoxycytidine was examined according to Example 1 of JP-A-1-60396. That is, the seven deposited strains shown in Table 1 were ordered from 24 strains described in Example 1. 50 ml of a medium (pH 7.0) containing 0.5 g / dl of yeast extract, 1.0 g / dl of peptone, 1.0 g / dl of meat extract and 0.5 g / dl of NaCl was dispensed into a 500 ml shoulder flask and sterilized. To this medium, one platinum loop of the microorganisms shown in Table 1 which had been pre-cultured in a bouillon agar medium at 30 ° C. for 16 hours was inoculated, and cultured at 30 ° C. for 16 hours with shaking. Bacterial cells were separated from the obtained culture broth by centrifugation, washed with 0.05M phosphate buffer (pH 7.0), and further centrifuged to prepare washed cells.
[0060]
The washed cells were added to 10 ml of 0.05 M Tris buffer (pH 7.2) containing 20 mM 2′-deoxyribose-1-phosphate and 20 mM cytosine so as to be 5 g / dl, and then at 60 ° C. for 24 hours. Reacted. As a result of diluting the reaction solution and analyzing by HPLC, cytosine as a substrate was decomposed and almost disappeared, but deoxycytidine was not detected.
[0061]
[Table 1]

[0062]
Reference Example 2 (PNP cloning and expression strain preparation, control cell preparation)
E. coli chromosomal DNA was prepared as follows.
[0063]
Escherichia coli K-12 / XL-10 strain (Stratagene) was inoculated into 50 ml of LB medium, cultured at 37 ° C. overnight, collected, and lysed with a lysate containing 1 mg / ml of lysozyme. After the lysate was treated with phenol, DNA was precipitated by ethanol precipitation by an ordinary method. The resulting DNA precipitate was collected by winding it around a glass rod, washed, and used for PCR.
[0064]
The primer for PCR is based on the base sequence of the deoD gene (GenBank accession No. AE000508 (coding region is base number 11531-12250) encoding known purine nucleoside phosphorylase (hereinafter referred to as PNP) of Escherichia coli. The designed oligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 1 and 2 (synthesized by consigning to Hokkaido System Science Co., Ltd.) were used. It has EcoRI and HindIII restriction enzyme recognition sequences.
[0065]
Using 0.1 ml of PCR reaction solution containing 6 ng / μl of the above-mentioned Escherichia coli chromosomal DNA completely digested with restriction enzyme HindIII and 3 μM of each primer, denaturation: 96 ° C., 1 minute, annealing: 55 ° C., 1 minute, extension reaction : PCR was carried out under the conditions of 30 cycles of 74 ° C. for 1 minute.
[0066]
The reaction product and plasmid pUC18 (Takara Shuzo Co., Ltd.) were digested with EcoRI and HindIII and ligated using Ligation High (Toyobo Co., Ltd.), and then the resulting recombinant plasmid was used to produce Escherichia coli. DH5α was transformed. The transformed strain was cultured on an LB agar medium containing 50 μg / ml of ampicillin (Am) and X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside), and was Am-resistant and white colony. A transformed strain was obtained.
[0067]
A plasmid was extracted from the transformant thus obtained, and the plasmid into which the target DNA fragment was inserted was named pUC-PNP73. The base sequence of the DNA fragment introduced into pUC-PNP73 was confirmed according to the usual base sequence determination method. The obtained base sequence is shown in SEQ ID NO: 3, and the amino acid sequence translated from the base sequence is shown in SEQ ID NO: 4. The subunit of this enzyme has a molecular weight of about 26000, and is known to be active as a hexamer. The optimum temperature of this enzyme is about 70 ° C., and the optimum pH is about 7.0 to 7.5. The transformant thus obtained was named Escherichia coli MT-10905.
[0068]
Escherichia coli MT-10905 strain was cultured with shaking at 37 ° C. overnight in 100 mL of LB medium containing 50 μg / ml of Am. The culture solution was centrifuged at 13,000 rpm for 10 minutes, and the obtained bacterial cells were suspended in 20 mL of 100 mM Tris-HCl buffer (pH 8.0). The suspension was centrifuged again at 13000 rpm for 10 min, and the resulting cells were treated with 2 mL of 100 mM Tris-HCl buffer (pH 8.0), 10 mM 2′-deoxyribose 1-phosphate di (monocyclohexylammonium) salt ( Suspended in SIGMA) and stored frozen at -20 ° C.
[0069]
Plasmid pUC18 (Takara Shuzo Co., Ltd.) was used to transform Escherichia coli DH5α. The transformed strain was named pUC18 / DH5α. The pUC18 / DH5α strain was cultured with shaking at 37 ° C. overnight in 100 mL of LB medium containing 50 μg / ml of Am. The culture solution was centrifuged at 13,000 rpm for 10 minutes, and the obtained bacterial cells were suspended in 20 mL of 100 mM Tris-HCl buffer (pH 8.0). The suspension was centrifuged again at 13000 rpm for 10 min, and the resulting cells were treated with 2 mL of 100 mM Tris-HCl buffer (pH 8.0), 10 mM 2′-deoxyribose 1-phosphate di (monocyclohexylammonium) salt ( Suspended in SIGMA) and stored frozen at -20 ° C.
[0070]
Comparative Example 1 (Deoxycytidine synthesis in E. coli recombined with PNP)
20 mM 2'-deoxyribose 1-phosphate di (monocyclohexylammonium) salt (manufactured by SIGMA), 20 mM cytosine (manufactured by Wako Pure Chemicals, special grade), 100 mM Tris-HCl buffer (pH 8.0), Reference Example 2 1.0 ml of the reaction solution consisting of 0.1 ml of the cell suspension of the pUC18 / DH5α strain obtained in the above was reacted at 50 ° C. for 20 hours. A comparative example was prepared by keeping the same temperature without adding a substrate. When the reaction solution was diluted and analyzed, deoxycytidine was not detected. FIG. 1A shows a comparative example, and FIG. 1B shows an HPLC analysis chart of the reaction solution.
[0071]
Comparative Example 2 (deoxycytidine synthesis of E. coli without PNP recombination)
20 mM 2'-deoxyribose 1-phosphate di (monocyclohexylammonium) salt (manufactured by SIGMA), 20 mM cytosine (manufactured by Wako Pure Chemicals, special grade), 100 mM Tris-HCl buffer (pH 8.0), Reference Example 2 The reaction solution (1.0 ml) consisting of 0.1 mL of the cell suspension of MT-10905 obtained in the above was reacted at 50 ° C. for 20 hours. A comparative example was prepared by keeping the same temperature without adding a substrate. When the reaction solution was diluted and analyzed, 10 mM deoxyuridine and 3 mM uracil were produced, but deoxycytidine was not detected. FIG. 2 (A) shows a comparative example, and FIG. 2 (B) shows an HPLC analysis chart of the reaction solution.
[0072]
Example 1 (Organic solvent treatment)
Methanol (MeOH) was added to the suspension of MT-10905 strain of Reference Example 2 at a concentration as shown in Table 2 and kept at 30 ° C. for 1 hour. Hereinafter, cytidine deaminase is expressed as cdd. The activity of cdd was measured by adding a cell suspension to 1.0 ml of a reaction solution of 100 mM Tris-HCl buffer (pH 8.0) and 20 mM cytidine and reacting at 50 ° C. for 2 hours. The above-mentioned cell suspension was not added with MeOH, but kept at 30 ° C. for 1 hour, and was shown as a relative value with 100%.
[0073]
The activity of PNP is 20 mM 2'-deoxyribose 1-phosphate di (monocyclohexylammonium) salt (manufactured by SIGMA), 5.4 mM adenine (manufactured by Wako Pure Chemicals, special grade), 100 mM Tris-HCl buffer (pH 8. 0) Measurement was performed by adding the cells to 1.0 ml of the reaction solution and reacting at 50 ° C. for 15 minutes. The reaction was carried out using the amount of bacterial cells in which the amount of deoxyadenosine produced was 2 mM or less. The above-mentioned cell suspension, which was kept at 30 ° C. for 1 hour without adding MeOH, was shown as a relative value as 100%. As shown in Table 2, the PNP activity was hardly decreased, but the cdd activity was almost inactivated.
[0074]
[Table 2]

[0075]
Example 2 (Organic solvent treatment)
An organic solvent as shown in Table 3 was added to the suspension of MT-10905 strain of Reference Example 2 and kept at 30 ° C. for 1 hour. As shown in Table 3, the PNP activity hardly decreased, but the cdd activity was almost inactivated.
[0076]
[Table 3]

[0077]
Example 3 (heat treatment)
The suspension of MT-10905 strain of Reference Example 2 was kept warm at 60 ° C. Hereinafter, cytosine deaminase is expressed as cod. The cod activity was measured by adding a cell suspension to 100 mM Tris-HCl buffer (pH 8.0) and 20 mM cytosine and reacting at 50 ° C. for 2 hours. The amount of cytosine degradation when untreated cells were added was taken as 100%, and the residual activity was expressed as a relative value. As shown in Table 4, the activity of PNP hardly decreased, but the activity of cod was almost inactivated.
[0078]
[Table 4]

[0079]
Example 4 (heat treatment + organic solvent treatment)
The microbial cell of the heat-treated 20 hr obtained in Example 3 was subjected to MeOH treatment in the same manner as in Example 1. The relative values are shown when the cell activity of 20 hr treatment in Example 3 is taken as 100%. As shown in Table 5, the PNP activity was hardly decreased, but the cod and cdd activities were almost inactivated.
[0080]
[Table 5]

[0081]
Example 5 (Synthesis of deoxycytidine by a recombinant PNP-expressing bacterium treated in Example 4)
20 mM 2'-deoxyribose 1-phosphate di (monocyclohexylammonium) salt (manufactured by SIGMA), 20 mM cytosine (manufactured by Wako Pure Chemicals, special grade), 100 mM Tris-HCl buffer (pH 8.0), Example 4 The reaction solution (1.0 ml) consisting of 0.1 ml of the bacterial cell solution treated with 50% MeOH obtained in (1) was reacted at 50 ° C. for 20 hours. When the reaction solution was diluted and then analyzed, 10.7 mM deoxycytidine, 0.2 mM deoxyuridine, and 0.1 mM uracil were produced. The reaction yield at that time was 53%. FIG. 2C shows an HPLC analysis chart of the reaction solution.
[0082]
Example 6 (Synthesis of deoxycytidine by Escherichia coli treated with Example 4)
20 mM 2'-deoxyribose 1-phosphate di (monocyclohexylammonium) salt (manufactured by SIGMA), 20 mM cytosine (manufactured by Wako Pure Chemicals, special grade), 100 mM Tris-HCl buffer (pH 8.0), Reference Example 2 The cell suspension of pUC18 / DH5α obtained in 1 above was treated with 50% MeOH in the same manner as in Example 4 and then reacted with 1.0 ml of the reaction solution at 50 ° C. for 20 hours. I let you. When the reaction solution was diluted and then analyzed, 0.14 mM deoxycytidine and 0.17 mM deoxyuridine were produced. The reaction yield at that time was 0.7%. FIG. 1C shows an HPLC analysis chart of the reaction solution.
[0083]
Example 7 (Synthesis of cytidine by recombinant PNP-expressing bacterium treated in Example 4)
20 mM ribose 1-phosphate di (cyclohexylammonium) salt (manufactured by SIGMA), 20 mM cytosine (manufactured by Wako Pure Chemicals, special grade), 100 mM Tris-HCl buffer (pH 8.0), 50 obtained in Example 4 1.0 ml of a reaction solution consisting of 0.1 ml of a cell suspension treated with% MeOH was reacted at 50 ° C. for 20 hours. When the reaction solution was diluted and analyzed, 5 mM cytidine, 0.1 mM uridine, and 0.1 mM uracil were produced. The reaction yield at that time was 25%.
[0084]
Example 8 (PNP purification and synthesis of deoxycytidine by purified PNP)
The cells obtained in Example 4 were suspended in 10 mL of 10 mM Tris-HCl buffer (pH 7.5), and the cells were crushed with an ultrasonic crusher. Next, the crude enzyme solution was adjusted by centrifugation, added to a DEAE-Toyopearl (Tosoh) 3 cm × 10 cm column equilibrated with 50 mM Tris-HCl buffer (pH 7.5), and eluted with a linear gradient from 50 mM NaCl to 500 mM NaCl. The active fraction was collected. The eluate was recovered as a precipitate with 70% ammonium sulfate saturation and dialyzed against 10 mM Tris-HCl buffer (pH 7.5). Add dialysate to a hydroxyapatite column (3 cm x 15 cm) equilibrated with 10 mM Tris-HCl buffer (pH 7.5), and add 10 mM Tris-HCl buffer (pH 7.5) to 50 mM Tris-HCl buffer (pH 7.5). The active fraction was collected by elution. The enzyme solution is recovered as a precipitate with 70% ammonium sulfate saturation, dissolved in 1 mL of 10 mM Tris-HCl buffer (pH 7.5), and dialyzed against 10 mM Tris-HCl buffer (pH 7.5) to obtain 2 mL of purified PNP. It was. The purified PNP thus obtained was confirmed to be a single band by SDS-polyacrylamide electrophoresis. The results are shown in FIG. 20 mM 2′-deoxyribose 1-phosphate di (monocyclohexylammonium) salt (manufactured by SIGMA), 20 mM cytosine (manufactured by Wako Pure Chemicals, special grade), 100 mM Tris-HCl buffer (pH 8.0), purified enzyme 0 1.0 ml of the reaction solution consisting of 1 ml was reacted at 50 ° C. for 20 hours. When the reaction solution was diluted and analyzed, 11.5 mM deoxycytidine was produced, and the reaction yield at that time was 57.5%.
[0085]
Example 9 (Synthesis of azacitidine by recombinant PNP-expressing bacterium treated in Example 4)
20 mM ribose 1-phosphate di (cyclohexylammonium) salt (manufactured by SIGMA), 20 mM azacytosine (manufactured by SIGMA), 100 mM Tris-HCl buffer (pH 8.0), 50% MeOH treated in Example 4 1.0 ml of the reaction solution consisting of 0.1 ml of the cell suspension was reacted at 50 ° C. for 20 hours. When the reaction solution was diluted and analyzed, 3.2 mM azacitidine was produced. The reaction yield at that time was 16%.
[0086]
Example 10 (Synthesis of fluorocytidine by recombinant PNP-expressing bacterium treated in Example 4)
20 mM ribose 1-phosphate di (cyclohexylammonium) salt (manufactured by SIGMA), 20 mM 5-fluorocytosine (manufactured by SIGMA), 100 mM Tris-HCl buffer (pH 8.0), 50% obtained in Example 4 1.0 ml of a reaction solution consisting of 0.1 ml of MeOH-treated cell suspension was reacted at 50 ° C. for 20 hours. When the reaction solution was diluted and then analyzed, 2.4 mM 5-fluorocytidine was produced. The reaction yield at that time was 12%.
[0087]
Example 11 (synthesis of methylcytidine by recombinant PNP-expressing bacterium treated in Example 4)
20 mM ribose 1-phosphate di (cyclohexylammonium) salt (manufactured by SIGMA), 20 mM 5-methylcytosine (manufactured by SIGMA), 100 mM Tris-HCl buffer (pH 8.0), 50% obtained in Example 4 1.0 ml of a reaction solution consisting of 0.1 ml of MeOH-treated cell suspension was reacted at 50 ° C. for 20 hours. When the reaction solution was diluted and then analyzed, 2.1 mM 5-methylcytidine was produced. The reaction yield at that time was 10.5%.
[0088]
Example 12 (Deoxycytidine was synthesized by a bacterial cell in which PNP was incorporated into a degradation activity deficient strain)
(1) Preparation of E. coli-Bacillus subtilis shuttle vectors pPNP04 and pPNP05 for expressing E. coli PNP in Bacillus subtilis
Escherichia coli purine nucleoside phosphorylase gene was amplified by PCR using pUC-PNP73 as a template and the synthetic oligonucleotide primers of SEQ ID NO: 5 and SEQ ID NO: 6 to obtain a 0.8 kb DNA fragment A. Next, the plasmid pNP150 (FERM BP-425) described in JP-A-60-210986 is used as a template by PCR using the synthetic oligonucleotide primers of SEQ ID NO: 7 and SEQ ID NO: 8 (B. amyloliquefaciens ( A region including a transcriptional promoter derived from a neutral protease (Bacillus amyloliquefaciens) and a translational regulatory site was amplified to obtain a DNA fragment B of 1.0 kb. Subsequently, annealing is performed using the overlapping regions of DNA fragment A and DNA fragment B, and this product fragment is used as a template to amplify by PCR using synthetic oligonucleotide primers of SEQ ID NO: 6 and SEQ ID NO: 7. An 8 kb DNA fragment C was obtained. This DNA fragment C was digested with restriction enzymes EcoRI and HindIII to obtain an insert fragment. As vector DNA, Bacillus subtilis / E. Coli shuttle vectors pRB373 and pRB374 were obtained from Bacillus Genetic Stock Center (BGSC; OH), and each was digested with restriction enzymes EcoRI and HindIII to prepare vector fragments. This vector fragment was ligated in the presence of an excess amount of the above-mentioned insert fragment to prepare E. coli purine nucleoside phosphorylase expression shuttle vectors pPNP04 and pPNP05.
(2) Introduction of pPNP04 and pPNP05 into Bacillus subtilis, and synthesis of deoxycytidine using the transformant
As Bacillus subtilis, Bacillus subtilis 1A479 strain and Bacillus subtilis 1A480 strain having no cytidine deaminase activity were obtained from Bacillus Genetic Stock Center (BGSC; OH).
[0089]
Transformation with the plasmids pPNP04 and pPNP05 obtained in (1) was performed according to Chang's protoplast method (Chang. S and Cohen, SN; Mol. Gen. Genet. 168, 111 (1978)). Transformants 1A480 (pPNP04), 1A480 (pPNP05) and 1A479 (pPNP04) and 1A479 (pPNP05) were obtained.
[0090]
Next, deoxycytidine synthesis activity was evaluated as follows for the 1A480 (pPNP04) strain.
[0091]
1A480 (pPNP04) was cultured at 35 ° C. for 17 hours in 20 mL of L-concentration L medium. The bacterial cells obtained by centrifuging 1.5 mL of this culture solution were stored frozen at -20 ° C. Reaction solution comprising 24 mM 2′-deoxyribose 1-phosphate di (monocyclohexylammonium) salt (manufactured by SIGMA), 20 mM cytosine (manufactured by Wako Pure Chemicals, special grade), 100 mM Tris-HCl buffer (pH 8.0) 1 ml was added to the frozen cells and shaken at 50 ° C. Sampling was performed after 1.5 hours and 18 hours, diluted 20 times with water, centrifuged to remove the bacterial cells, and then the supernatant was analyzed by HPLC. In 18 hours, 6.4 to 7.0 mM of deoxycytidine was accumulated, and no degradation product of substrate and product was observed.
[0092]
As a result of measuring deoxycytidine synthesis for 1A480 (pPNP05), 1A479 (pPNP04) and 1A479 (pPNP05), it was found that the deoxycytidine synthesis ability was almost the same as that of the 1A480 (pPNP04) strain.
[0093]
Example 13 (Acquisition of amino acid substitution product retaining cytosine nucleoside phosphorylase activity)
Using the plasmid DNA of pUC-PNP73 obtained in Reference Example 2 as a template, a mutation was introduced using QuickChange Site-Directed Mutagenesis Kit manufactured by STRATAGENE. Hereinafter, it is simply called a kit. In the following examples, the principle and operation method of the kit were basically followed.
[0094]
10 ml of LB liquid medium was prepared in a 30 ml test tube, and sterilized by autoclaving at 121 ° C. for 20 minutes. Ampicillin was added to this medium to a final concentration of 100 μg / ml, and then the MT-10905 strain obtained in Reference Example 2 was inoculated in the ear and cultured at 37 ° C./300 rpm for about 20 hours. . After 1 ml of the culture broth was collected into an appropriate centrifuge tube, the cells were separated by centrifugation (15000 rpm × 5 minutes). Subsequently, plasmid DNA of pUC-PNP73 was prepared from the cells by alkaline SDS extraction.
[0095]
SEQ ID NOs: 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, using plasmid DNA of pUC-PNP73 as a template 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, and 45 and 46 were used as primers for PCR. The composition of the PCR reaction solution is shown in Table 6. Amplification conditions are shown in Table 7.
[0096]
4 units of restriction enzyme DpnI was added to the PCR amplification reaction solution and incubated at 37 ° C. for 1 hr. 1 μL of this reaction solution was added to 100 μL of E. coli K-12DH5α competent cell (Toyobo) and kept in ice for 30 minutes. After soaking in a constant temperature bath at 42 ° C. for 30 seconds, 0.9 mL of NZY + medium attached to the competent cell was added and shaken at 37 ° C. for 1 hour. The culture solution was smeared on a medium obtained by adding ampicillin to LB agar medium so as to be 100 μg / mL, and incubated at 37 ° C. for 20 hours to form colonies.
[0097]
Five clones arbitrarily selected from the colonies were inoculated in each ear, and cultured with shaking at 37 ° C. overnight in 100 mL of LB medium containing 50 μg / ml of Am. The culture solution was centrifuged at 13,000 rpm for 10 minutes, and the obtained bacterial cells were suspended in 20 mL of 100 mM Tris-HCl buffer (pH 8.0). The suspension was centrifuged again at 13000 rpm for 10 min, and the resulting cells were treated with 2 mL of 100 mM Tris-HCl buffer (pH 8.0), 10 mM 2′-deoxyribose 1-phosphate di (monocyclohexylammonium) salt ( The product was suspended in SIGMA), and the cells were treated in the same manner as in Example 4, and deoxycytidine synthesis reaction was carried out in the same manner as in Example 5. As a result of measuring deoxycytidine by HPLC analysis, production of deoxycytidine was detected in 4 out of 5 clones, and it was confirmed that cytosine nucleoside phosphorylase activity was retained.
[0098]
The cells of the 4 clones were separated from the remaining 1 ml of the culture broth used for the measurement of cytosine nucleoside phosphorylase activity, and plasmid DNA of each clone was prepared by alkaline SDS extraction. The base sequence of the DNA fragment was confirmed according to the usual base sequence determination method. The activity was measured by the same method as in Example 1. A list of the results is shown in Table 8.
[0099]
[Table 6]

[0100]
[Table 7]

[0101]
[Table 8]

[0102]
Reference Example 3 (Transformation to Escherichia coli K-12 W3110 strain)
The plasmid pUC-PNP73 obtained in Reference Example 2 was transformed into E. coli K-12 W3110 strain (ATCC 27325) according to a conventional method. The obtained transformant was named MT-10948. The activity of the transformant cultured in the same manner as in Reference Example 2 was twice that of the transformant into E. coli K-12 DH5α strain.
[0103]
Example 14 (Removal of reaction inhibitor: precipitation with polar solvent)
MT-10948 cells cultured in the same manner as in Reference Example 2 were crushed with an ultrasonic crusher. The same volume of acetone as the crushed liquid was added, and the precipitate was removed by centrifugation. Next, half of the acetone was added to the centrifugal supernatant and the precipitate was collected by centrifugation. The recovered precipitate was dried with a vacuum dryer. The dried precipitate was dissolved in 2 mL of 100 mM Tris-HCl buffer (pH 8.0), 10 mM of 2′-deoxyribose 1-phosphate di (monocyclohexylammonium) salt (manufactured by SIGMA) and treated in the same manner as in Example 4. did. 2 mL of the enzyme solution fractionated with acetone was added to cytosine (6.96 g), 2′-deoxyribose 1-phosphate diammonium salt (18.7 g) and magnesium hydroxide (6.2 g) in water (105.3 g). In addition, the reaction was carried out at 50 ° C. for 24 hours. After completion of the reaction, HPLC analysis revealed that 11.4 g (80%) of 2′-deoxycytidine was obtained. As a comparative example, 2 mL of a bacterial cell solution before acetone treatment was reacted in the same manner as described above to obtain 8.5 g (60%) of 2′-deoxycytidine.
[0104]
Example 15 (Removal of reaction inhibitor: precipitation with ammonium sulfate)
MT-10948 cells cultured in the same manner as in Reference Example 2 were crushed with an ultrasonic crusher. The pulverized ammonium sulfate was slowly added to the crushed liquid and added to ammonium sulfate saturation of 40%. The mixture was stirred slowly in ice water for 1 hour, and the precipitate was removed by centrifugation. Centrifugated ammonium sulfate was added to ammonium sulfate saturated to 70%, and the mixture was slowly stirred in ice water for 1 hour, and a precipitate was obtained by centrifugation. The precipitate was dissolved in 2 mL of 100 mM Tris-HCl buffer (pH 8.0) and 10 mM of 2′-deoxyribose 1-phosphate di (monocyclohexylammonium) salt (manufactured by SIGMA). When treated in the same manner as in Example 4 and reacted in the same manner as in Example 14, 11.0 g (80%) of 2′-deoxycytidine was obtained.
[0105]
Example 16 (Removal of reaction inhibitor: purified enzyme is the enzyme of Example 8)
The same reaction as in Example 14 was performed using 2 mL of the enzyme solution obtained in the same manner as in Example 8. As a result, 11.0 g (80%) of 2′-deoxycytidine was obtained.
[0106]
Example 17 (Effect of phoA deficient strain)
The same reaction as in Example 14 was performed using 2 ml of the bacterial cells obtained in Example 4. As a result, 11.0 g (80%) of 2′-deoxycytidine was obtained. On the other hand, when the MT-10948 strain was treated in the same manner as in Example 4, the activity was detected to be twice that of MT-10905. However, when the same reaction as in Example 14 was performed, 8.5 g of 2′-deoxycytidine ( Only 60%).
[0107]
Example 18
6.96 g (62.6 mmol) of cytosine, 18.7 g (75.4 mmol) of 2′-deoxyribose 1-phosphate diammonium salt, 7.58 g (132 mmol) of magnesium hydroxide, frozen cells prepared in Reference Example 3 (2.0 g) was added to a mixed solution of water (96.4 g) and cyclohexane 4.8 g, and the mixture was reacted at 45 ° C. for 18 hours while controlling the pH of the reaction solution to 8.8 with acetic acid. After completion of the reaction, analysis by HPLC gave 10.03 g (70.5 mol% / cytosine) of the target product, 2′-deoxycytidine. At this time, uracil as a by-product was 0.73 g (10.4 mol% / cytosine) and 2'-deoxyuridine was 1.80 g (12.6 mol% / cytosine).
[0108]
Example 19
Tables 9 and 10 show the results when 2'-deoxycytidine was synthesized under the same conditions as in Example 18 except that the solvent used for cell treatment and the solvent added during the reaction were changed.
[0109]
[Table 9]

[0110]
[Table 10]

[0111]
Example 20
Synthesis of 2'-deoxycytidine
20 g of pure basic anion exchange resin (MP500 lebatide: exchange capacity 1.1 eq / L) 20 ml (total exchange capacity 24 mmol) and 2-deoxyribose 1-phosphate di (ammonium) salt (4.96 g, 20 mmol) And stirred for 30 minutes at room temperature. After 30 minutes, an enzyme solution (0.5 ml) and cytosine (2.11 g, 19 mmol) prepared in the same manner as in Example 4 were added, and the mixture was reacted at 50 ° C. for 10 hours with stirring. As a result of analyzing the reaction mass by HPLC after 10 hours, the desired 2'-deoxycytidine was obtained with a reaction yield of 80%.
[0112]
【The invention's effect】
Production of nucleoside compounds using nucleoside phosphorylase is possible to produce nucleoside compounds in a position and stereospecific manner under mild conditions, and establishment of an industrial production method is desired. A method for synthesizing a typical cytosine nucleoside compound was not known.
[0113]
According to the present invention, it is possible to produce a cytosine nucleoside compound from pentose-1-phosphate and cytosine or a cytosine derivative using a nucleoside phosphorylase having reactivity to cytosine. At that time, a cytosine nucleoside compound can be efficiently produced by providing a method for specifically reducing the degradation activity of the substrate or product.
[0114]
[Sequence Listing]

[Brief description of the drawings]
1A is an HPLC analysis chart of Comparative Example 1, FIG. 1B is an HPLC analysis chart of a reaction solution of Comparative Example 1, and FIG. 1C is an HPLC analysis chart of Example 6; is there.
2A is an HPLC analysis chart of a comparative example in Comparative Example 2, FIG. 2B is an HPLC analysis chart of a reaction solution of Comparative Example 2, and FIG. 2C is an HPLC analysis chart of Example 5. It is an analysis chart.
3 is a diagram showing the results of SDS-polyacrylamide electrophoresis in Example 8. FIG.

Claims (14)

In the method for producing a cytosine nucleoside compound,
A step of obtaining a cytosine nucleoside compound by reacting a phosphorylated saccharide with cytosine or a cytosine derivative in the presence of an enzyme having cytosine nucleoside phosphorylase activity;
A method for producing a cytosine nucleoside compound, wherein the enzyme has purine nucleoside phosphorylase activity.   The production method according to claim 1, wherein the enzyme is derived from Escherichia coli. The cytosine derivative is represented by the following formula (I):

(In the above formula (I), X represents a carbon atom or a nitrogen atom, and Y represents a hydrogen atom, a halogen atom or a lower alkyl group.)
The production method according to claim 1, wherein the compound is represented by the formula:   The phosphorylated saccharide is ribose-1-phosphate, 2-deoxyribose-1-phosphate, 2 ′, 3′-dideoxyribose 1-phosphate, or dioxolane sugar phosphate. The manufacturing method as described.   The manufacturing method according to any one of claims 1 to 4, wherein the enzyme is supplied as a microbial cell having cytosine nucleoside phosphorylase activity, or an enzyme preparation obtained from the microbial cell or a culture solution thereof.   6. The production method according to claim 5, wherein the microorganism has cytosine nucleoside phosphorylase activity and does not have cytosine deaminase activity and / or cytidine deaminase activity and / or phosphatase activity.   The production method according to claim 6, wherein cytosine deaminase activity and / or cytidine deaminase activity of the bacterial cell or the enzyme preparation is inactivated or decreased by the reduction treatment.   The microbial cells subjected to the reduction treatment may contact the microbial cells having cytosine nucleoside phosphorylase activity with water containing an organic solvent to selectively deactivate or reduce cytosine deaminase activity and cytidine deaminase activity. The production method according to claim 7, which is obtained. The production method according to claim 6, wherein the substance that inhibits cytosine nucleoside phosphorylase activity is removed from the cells or enzyme preparation thereof by any of the following means a) to c).
a) obtaining cytosine nucleoside phosphorylase from the enzyme preparation as a precipitate with a polar solvent,
b) obtaining cytosine nucleoside phosphorylase as a precipitate from the enzyme preparation by salting out;
c) Cytosine nucleoside phosphorylase is separated from the enzyme preparation by an appropriate carrier such as a resin. For the amino acid sequence set forth in SEQ ID NO: 4 , the following (1) to (12):
(1) The 10th methionine is replaced with alanine,
(2) Replacing the 42nd arginine with leucine,
(3) Substitute 54th lysine with serine,
(4) replacing 67th isoleucine with leucine,
(5) Substituting serine for 157th alanine,
(6) Substituting 178th glycine with alanine,
(7) Substituting 179th valine with threonine,
(8) Replacing 183rd alanine with serine,
(9) Substituting 199th threonine with alanine,
(10) Replacing 210th histidine with leucine,
(11) Substituting 228th isoleucine with serine, and
(12) Replacing 233rd valine with leucine
A cytosine nucleoside phosphorylase obtained by performing any one of the substitutions .   A recombinant plasmid comprising a gene encoding the cytosine nucleoside phosphorylase according to claim 10.   A transformed strain carrying the recombinant plasmid according to claim 11.   A method for producing cytosine nucleoside phosphorylase, comprising culturing the transformant according to claim 12, and recovering cytosine nucleoside phosphorylase from the transformant obtained by culturing, a culture solution, and a processed product thereof.   The enzyme preparation according to any one of claims 6 to 8, comprising an enzyme having purine nucleoside phosphorylase activity.

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