JP2016063750A - 1α,25−ジヒドロキシビタミンD2の製造方法 - Google Patents
1α,25−ジヒドロキシビタミンD2の製造方法 Download PDFInfo
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- JP2016063750A JP2016063750A JP2014193073A JP2014193073A JP2016063750A JP 2016063750 A JP2016063750 A JP 2016063750A JP 2014193073 A JP2014193073 A JP 2014193073A JP 2014193073 A JP2014193073 A JP 2014193073A JP 2016063750 A JP2016063750 A JP 2016063750A
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- amino acid
- vitamin
- acid sequence
- dihydroxyvitamin
- arginine
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Abstract
【解決手段】特定のアミノ酸配列において特定の位置のアミノ酸が他のアミノ酸に置換された改変アミノ酸配列を有し、かつ0.25μMのビタミンD2水酸化酵素の存在下で、10μM 25−ヒドロキシビタミンD2と1mM NADPHとを30℃、20分間反応させた場合の1α,25−ジヒドロキシビタミンD2への変換率は0.30%以上であるところの活性を有するビタミンD2水酸化酵素、この酵素を発現する形質転換体、及びこの形質転換体を、25−ヒドロキシビタミンD2の存在下で培養して、1α,25−ジヒドロキシビタミンD2を得ることを含む、1α,25−ジヒドロキシビタミD2の製造方法。
【選択図】なし
Description
(a)配列表の配列番号1に記載のアミノ酸配列において、
239位のメチオニンがアラニン、セリン、グリシン、イソロイシン、バリン、フェニルアラニン、又はロイシンに置換された、改変アミノ酸配列
(b)前記(a)の改変アミノ酸配列において、73位のアルギニンがアラニン、バリン、ロイシン又はイソロイシンに、及び/又は84位のアルギニンがアラニン、バリン、ロイシン、イソロイシン、プロリン、フェニルアラニン、メチオニン、トリプトファン、グリシン、システイン、グルタミン、アスパラギン、セリン、トレオニン、チロシン、アスパラギン酸又はグルタミン酸に置換された、改変アミノ酸配列
(c)前記(a)または(b)の改変アミノ酸配列において、前記239位のメチオニン、73位のアルギニン及び84位のアルギニンの置換の他さらに、1から9個のアミノ酸の欠失、置換、逆位、付加及び挿入からなる群から選ばれる少なくとも1種の修飾を有するアミノ酸配列
(d)前記(a)または(b)の改変アミノ酸配列と90%以上の同一性を有する、前記239位のメチオニンのアルギニンの置換、又は前記239位のメチオニン、73位のアルギニン及び/若しくは84位のアルギニンの置換を含むアミノ酸配列
(e)0.25μMのビタミンD2水酸化酵素の存在下で、10μM 25−ヒドロキシビタミンD2と1mM NADPHとを30℃、20分間反応させた場合の1α,25−ジヒドロキシビタミンD2への変換率は0.30%以上である。
(A)ビタミンD2水酸化酵素
ビタミンD2水酸化酵素として、改変アミノ酸配列(a)を有する25−ヒドロキシビタミンD2水酸化酵素が挙げられる。改変アミノ酸配列(a)を有するビタミンD2水酸化酵素は、239位のメチオニンを置換したアミノ酸の種類によって異なるが、0.25μMのビタミンD2水酸化酵素の存在下で、10μM 25−ヒドロキシビタミンD2と1mM NADPHとを30℃、20分間反応させた場合の1α,25−ジヒドロキシビタミンD2への変換率は0.30%以上である活性を有する。上記条件での1α,25−ジヒドロキシビタミンD2への変換率は、239位のメチオニンを置換したアミノ酸の種類によって異なるが、好ましくは1.0%以上である。
ビタミンD2水酸化酵素をコードする核酸は、例えば、改変アミノ酸配列(a)、(b)、(c)、(d)のいずれかをコードする核酸が挙げられる。ただし、改変アミノ酸配列(a)または(b)をコードする核酸の発現産物は、0.25μMのビタミンD2水酸化酵素の存在下で、10μM 25−ヒドロキシビタミンD2と1mM NADPHとを30℃、20分間反応させた場合の1α,25−ジヒドロキシビタミンD2への変換率は0.30%以上、好ましくは1.0%以上である活性を有することが好ましい。また、改変アミノ酸配列(c)または(d)をコードする核酸は、上記条件での1α,25−ジヒドロキシビタミンD2への変換率が0.30%以上である活性を有するものであり、上記条件での1α,25−ジヒドロキシビタミンD2への変換率が1.0%以上である活性を有するものが好ましい。
上記核酸は適当なベクターに挿入して使用することができる。ベクターの種類は特に限定されず、例えば、自立的に複製することが可能なベクター(例えばプラスミド等)でもよいし、又は宿主細胞に導入された際に宿主細胞のゲノムDNAに組み込まれ、ゲノムDNAと共に複製されるものであってもよい。好ましくは、ベクターは発現ベクターである。発現ベクターにおいて上記核酸は、転写に必要な要素(例えば、プロモータ等)が機能的に連結されている。プロモータは宿主細胞において転写活性を示すDNA配列であり、宿主の種類に応じて適宜選択することができる。
上記核酸又は上記組換えベクターを適当な宿主に導入することによって形質転換体を作製することができる。すなわち、形質転換体は、ビタミンD2水酸化酵素をコードする核酸を導入してなる、又はビタミンD水酸化酵素をコードする核酸が挿入された組換えベクターを含有する形質転換体である。
上記形質転換体を常法にしたがって適当な培地に接種し、NADPHなどの還元型補酵素を加えることなく、25−ヒドロキシビタミンD2の存在下、好ましくは25−ヒドロキシビタミンD2及び包摂化合物の存在下、より好ましくは25−ヒドロキシビタミンD2、包摂化合物及びビタミンD2水酸化酵素の発現を誘導する薬剤の存在下で培養することにより、1α,25−ジヒドロキシビタミンDを製造することができる。本発明の1α,25−ジヒドロキシビタミンD2の製造方法は、反応系に菌体を用いる方法であるため、反応系にNADPHを加えずに実施することができ、ビタミンD水酸化反応と同時に触媒である菌体を増殖することができ(生菌体法)、増殖した菌体に対して精製などの処理を施さなくてもよく、菌体の保存が容易であることから菌体を繰り返し反応に使用でき、反応時間を例えば、100時間以上としても実施可能であり、並びに通常用いられる培地を利用することにより安価に工業的規模で大量生産ができることから、酵素を用いる方法と比較して、圧倒的に簡便かつ効率よく、25−ヒドロキシビタミンD2から1α,25−ジヒドロキシビタミンD2を製造できる。
CYP105A1二重変異体(R73A/R73A)を大腸菌内で発現させるためのプラスミド(pKSNdl−R73A/R73A)は、後述する参考文献(1)及びに記載した方法にしたがって構築した。三重変異体をコードする遺伝子は、pKSNdl−R73A/R73Aを鋳型に表1に示したプライマーを利用し作製した。それぞれの変異体作製に使用したプライマーの塩基配列を表1に示した。構築したプラスミドを大腸菌JM109株に導入した。発現系におけるベクターと大腸菌宿主については特に限定されるものではない。
それぞれの変異体を発現する組換え大腸菌は50μg/mlアンピシリンを含むTB培地(非特許文献1)において37℃で培養し、菌体濃度(O.D.660nm) が0.5に達した時点でイソプロピル−チオーβ−D−ガラクトピラノシドを終濃度1mMになるまで、及びδ−アミノレブリン酸を終濃度5mMになるまで添加した。その後、40〜50時間13℃で培養し、遠心分離により菌体を回収し、超音波破砕装置を用いて菌体を破砕し、遠心分離により細胞質画分を調製 した。
野生型および変異体の定量は吸収スペクトルを測定し417nmにおける吸光度を求め、モル分子吸光係数110mM-1cm-1を用いて算出した。
活性は100mM Tris−HCl(pH7.4)、1mM EDTA緩衝液中で、以下のものを含む再構成系を用いて測定した。10μM 25―ヒドロキシビタミンD2(25(OH)D2);0.25μM シトクロムP450(二重変異体あるいは三重変異体を含む組換え大腸菌細胞質画分);0.1mg/ml ホウレン草由来フェレドキシン(Fdx)、0.1U/ml ホウレン草由来フェレドキシン還元酵素(Fdr)、1U/ml グルコース脱水素酵素;1% グルコース;0.1mg/ml カタラーゼ (活性測定条件A)。
カラム:YMC社製 YMC−Pack ODS−AM (内径4.6mm×長さ300mm);検出波長:265nm;流速:1.0ml/min;カラム温度:40oC、溶出条件:水/アセトニトリル系;20−100% アセトニトリル直線濃度勾配(25分間)の後、100%アセトニトリル(10分間)
三重変異体(R73A/R84A/M239A)および二重変異体(R73A/R84A)について、上記の活性測定法(活性測定条件A)により、反応0分、および120分間反応させ、得られた代謝物のHPLCクロマトグラムを図1aおよびbに示す。
カラム:Finepak SLL−5 (日本分光);検出波長:265nm;流速:1.0ml/min;カラム温度:40℃、溶出条件:ヘキサン/イソプロパノール;80:20
各変異体を用いて、活性測定条件Aにて反応を行った。反応20分後の代謝物を前述の方法により逆相HPLCにアプライし、M1(M1−aとM1−bの混合物)を分取後、さらに順相HPLC分析を行うことによって、M1−aとM1−bの面積値を算出した。得られた面積値から、M1中におけるM1−b(1α,25(OH)2D2)の割合を算出した(表2)。また、20分後の1α,25(OH)2D2への変換率と1分あたりの酵素活性(代謝回転数)を表2に示した。
三重変異体R73A/R84A/M239Aを放線菌内で発現させ、組換え菌体を用いて、25(OH)D2の1α位水酸化体を製造することを試みた。大腸菌発現用のプラスミドであるpKSNdl−R73A/R73A/M239AのNdeI−HindIII断片を切り出すことによって、CYP105A1変異体およびその下流にフェレドキシン(FDX1)遺伝子を含む断片を得た。得られた断片を、NdeI−HindIII処理を行ったロドコッカス属放線菌用プラスミドpTipQT2ベクター(後述参考文献(2)) に連結した(pTipQT2−R73A/R73A/M239A)。得られたプラスミドをロドコッカス・エリスロポリス JCM3201株に導入した。形質転換の方法は以下のとおりである。
pTipQT2−R73A/R84A/M239Aをロドコッカス・エリスロポリス JCM3201株に導入、生えてきたコロニーを10 μg/mLテトラサイクリン含有LB培地植菌し、30℃で24時間培養した。得られた培養液を同濃度のテトラサイクリンを含むLB培地に初期菌体濃度(O.D.600nm) が0.05になるように添加し、菌体濃度(O.D.600nm)が0.6−0.8になるまで30℃で培養した。その後、終濃度が1μg/mLになるようにチオストレプトンを添加し、30℃で24時間誘導した。その後、菌体懸濁液に、25―ヒドロキシビタミンD2(25(OH)D2)(終濃度 50μM)および3−ヒドロキシプロピル−β-シクロデキストリン(終濃度 0.2%)を添加し、200μLずつ96穴ディープウェルに移して0−24時間反応させた。反応させた培養液にそれぞれ800μLのクロロホルム:メタノール=3:1混合液を加え、激しく攪拌した後、クロロホルム層を回収、減圧乾固し、アセトニトリルに溶解した。この溶液を逆相HPLCにより分析条件Aで分析した。基質添加6時間後の菌体懸濁液から得られた代謝物のHPLCクロマトグラムを図5に示す。
(1) Sugimoto, H.et al., (2008), Biochemistry 47, 4017-4027
(2) Nakashima, et al., (2004), Applied and Environmental Microbiology 70, 5557-5567
Claims (8)
- 下記(a)若しくは(b)のアミノ酸配列を有するか、または下記(c)若しくは(d)のアミノ酸配列を有し、かつ下記(e)の活性を有するビタミンD2水酸化酵素。
(a)配列表の配列番号1に記載のアミノ酸配列において、
239位のメチオニンがアラニン、セリン、グリシン、イソロイシン、バリン、フェニルアラニン、又はロイシンに置換された、改変アミノ酸配列
(b)前記(a)の改変アミノ酸配列において、73位のアルギニンがアラニン、バリン、ロイシン又はイソロイシンに、及び/又は84位のアルギニンがアラニン、バリン、ロイシン、イソロイシン、プロリン、フェニルアラニン、メチオニン、トリプトファン、グリシン、システイン、グルタミン、アスパラギン、セリン、トレオニン、チロシン、アスパラギン酸又はグルタミン酸に置換された、改変アミノ酸配列
(c)前記(a)または(b)の改変アミノ酸配列において、前記239位のメチオニン、73位のアルギニン及び84位のアルギニンの置換に加えて、1から9個のアミノ酸の欠失、置換、逆位、付加及び挿入からなる群から選ばれる少なくとも1種の修飾を有するアミノ酸配列
(d)前記(a)または(b)の改変アミノ酸配列と90%以上の同一性を有する、前記239位のメチオニンのアルギニンの置換、又は前記239位のメチオニン、73位のアルギニン及び/若しくは84位のアルギニンの置換を含むアミノ酸配列
(e)0.25μMのビタミンD2水酸化酵素の存在下で、10μM 25−ヒドロキシビタミンD2と1mM NADPHとを30℃、20分間反応させた場合の1α,25−ジヒドロキシビタミンD2への変換率は0.30%以上である。 - 前記(a)の改変アミノ酸配列が、239位のメチオニンがアラニン、セリン、又はグリシンに置換された配列である、請求項1に記載の酵素。
- 前記(b)の改変アミノ酸配列が、73位のアルギニンがバリン、ロイシン又はイソロイシンに、及び84位のアルギニンがアラニン、バリン、ロイシン、イソロイシン、プロリン、フェニルアラニン、メチオニン、トリプトファン、グリシン又はシステインに置換された配列である、請求項1または2に記載の酵素。
- 請求項1〜3のいずれか1項に記載のビタミンD2水酸化酵素を発現する形質転換体。
- 前記形質転換体の宿主が、大腸菌又は放線菌である、請求項4に記載の形質転換体。
- 請求項4または5に記載の形質転換体を、25−ヒドロキシビタミンD2の存在下で培養して、1α,25−ジヒドロキシビタミンD2を調製することを含む、1α,25−ジヒドロキシビタミンD2の製造方法。
- 前記培養が、25−ヒドロキシビタミンD2及び包摂化合物の存在下で実施される、請求項6に記載の製造方法。
- 前記包摂化合物が、シクロデキストリン、ゼオライト、フラーレン、クラウンエーテル又はカリックスアレーンである、請求項7に記載の製造方法。
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