JP2004173538A - Pyrroloquinolinequinone dependent glucose dehydrogenase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a modified pyrroloquinolinequinone-dependent glucose dehydrogenase having low reactivity with maltose, galactose, etc. <P>SOLUTION: The modified pyrroloquinolinequinone-dependent glucose dehydrogenase has an amino acid sequence of a pyrroloquinolinequinone-dependent glucose dehydrogenase derived from Acinetobacter calcoaceticus provided that one or more amino acids in each of the region corresponding to the 1st region composed of the 326th amino acid to the 354th amino acid and the region corresponding to the 2nd region composed of the 278th amino acid to the 320th amino acid of the sequence are replaced with other amino acids. <P>COPYRIGHT: (C)2004,JPO

Description

【００５２】
［実施例６］ ＰＱＱＧＤＨのＣ末端ヒスチジンタグの付加
先ずＰＱＱＧＤＨ遺伝子の下流にヒスチジンタグを導入し、続いてその直後に終始コドンを付加するプライマーを用いてＰＣＲを行った。実施例４で得られたｐＵＫ−ＧＤＨＢ（Ｓ）を鋳型として、次のプライマーを用いたＰＣＲを行った。
フォワードプライマー：５’−ＴＣＡＣＡＴＧＴＴＣＴＴＴＣＣＴＧＣＧＴＴＡＴＣ−３’（配列番号９）
リバースプライマー：５’−ＡＴＧＧＴＧＡＴＧＧＴＧＡＴＧＧＴＧＣＴＴＡＧＣＣＴＴＡＴＡＧＧＴＧＡＡＣＴＴＡＡＴＧＡＧＡ−３’（配列番号１０、下線で示したのが６×ヒスチジンタグの配列）
ＰＣＲは実施例４と同様の組成及び条件にて行った。
続いて増幅遺伝子を鋳型としてヒスチジンタグの下流に翻訳終止コドンを導入するため次のプライマーを用いたＰＣＲを行った。
フォワードプライマー：５’−ＴＣＡＣＡＴＧＴＴＣＴＴＴＣＣＴＧＣＧＴＴＡＴＣ−３’（配列番号９）
リバースプライマー：５’−ＧＣＧＧＣＣＧＣＣＴＧＣＡＧＣＴＡＴＴＡＡＴＧＧＴＧＡＴＧＧＴＧＣＴＴＡＧＣＣＴＴＡ−３’（配列番号１１）
ＰＣＲは実施例４と同様の溶液組成及び条件にて行った。
【００５３】
ＰＱＱＧＤＨ遺伝子下流にヒスチジンタグ、終止コドンを付加して得られた増幅遺伝子をＥｃｏＲＩとＰｓｔＩで切断した。切断した遺伝子断片を発現ベクターに連結し、このプラスミドで大腸菌ＪＭ１０９株を形質転換した。尚、得られた発現プラスミドをｐＵＫ−ＧＤＨＢ（Ｓ）Ｃ−Ｈｉｓとした。形質転換体を３０℃、１００μｇ／ｍＬアンピシリンと６μＭ ＰＱＱを含む１００ｍＬのＬＢ培地で一晩培養したところＰＱＱＧＤＨの産生が認められた。尚、このＰＱＱＧＤＨの分子量をアミノ酸配列から計算したところ約５０，０００であり、アシネトバクター・カルコアセティカスＩＦＯ １２５５２の分子量とほぼ一致した。さらに対照として実施例５で得られた野生型のＰＱＱＧＤＨと一般的性質を比較したところ同等であることが認められた。
【００５４】
［実施例７］ 変異ＰＱＱＧＤＨ遺伝子ライブラリーの構築
実施例６で得られたプラスミドｐＵＫ−ＧＤＨＢ（Ｓ）Ｃ−Ｈｉｓを鋳型とし、次のプライマーを用いてエラープローンＰＣＲ法を行い、変異の挿入を行った。
フォワードプライマー：５’−ＴＣＡＣＡＴＧＴＴＣＴＴＴＣＣＴＧＣＧＴＴＡＴＣ−３’（配列番号９）
リバースプライマー：５’−ＧＧＣＧＣＧＴＡＣＴＡＴＧＧＴＴＧＣＴＴＴＧＡ−３’（配列番号１２）
変異導入の条件は以下の通りである。ＰＣＲは以下の表３に示す組成の溶液中で行い、９４℃５分間の反応、次に９４℃１分間、５０℃１分間、７０℃２分間の反応を２５サイクル、最後に７２℃７分間の反応を行う条件とした。
【００５５】
【表３】
１００ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．３） ０．５ μＬ
５００ｍＭ ＫＣｌ ５ μＬ
１ｍｇ／ｍＬ ＢＳＡ ２．５ μＬ
（１５〜１００ｍＭ）ＭｇＣｌ_２ ５ μＬ
１ｍＭ ＭｎＣｌ_２ １．２５〜３．７５ μＬ
１０ｍＭ ＡＴＰ ０．５〜１．５５ μＬ
１０ｍＭ ＴＴＰ ０．５〜５．０ μＬ
１０ｍＭ ＧＴＰ １．０〜５．０ μＬ
１０ｍＭ ＣＴＰ ０．５〜５．０ μＬ
フォワードプライマー（２０ｐｍｏｌ／μＬ） ２．５ μＬ
リバースプライマー（２０ｐｍｏｌ／μＬ） ２．５ μＬ
Ｄｉｍｅｔｈｙｌｓｕｌｆｏｘｉｄｅ ０〜７．５ μＬ
０．２ｎｇ／μＬ ｔｅｍｐｌａｔｅ ５ μＬ
ｒＴａｑ Ｐｏｌｙｍｅｒａｓｅ ０．５ μＬ
Ｈ_２Ｏで５０ μＬに調整
【００５６】
変異の導入された遺伝子増幅断片をＥｃｏＲＩとＰｓｔＩで切断し、ｐＵＫ−ＧＤＨＢ（Ｓ）の野生型遺伝子と入れ換えた。このようにして得られたプラスミドで大腸菌ＪＭ１０９を形質転換して変異遺伝子ライブラリーを構築した。
【００５７】
得られたコロニーを９６穴の培養用深溝マイクロプレート中の１００μｇ／ｍＬのアンピシリン、５μＭのＰＱＱそして１ｍＭの塩化カルシウムを含む６００μＬのＣｉｒｃｌｅ Ｇｒｏｗ Ｍｅｄｉｕｍ（ストラタジーン社）に接種し、３７℃で一晩２００ｒｐｍの振とう条件で培養した。その後、菌体を遠心分離（３０００ｒｐｍ， ４℃， ３０ｍｉｎ）で回収し、３倍に希釈された溶菌液（５０ｍＭ トリス緩衝液ｐＨ８．０， ２ｍＭ ＥＤＴＡ， １ｍｇ／ｍＬリゾチーム， １５μＭ ＰＱＱ）６００μＬに再懸濁した。その後３７℃で１時間振とうし、３０μＬの２０ｍＭ塩化カルシウム溶液を添加した。ライセートをフィルタープレート（コーニング社製）に移し、遠心分離した（２０００ｒｐｍ， ４℃， ５ｍｉｎ）。得られた上清を以下のスクリーニングに用いた。
【００５８】
スクリーニングに用いる９６穴マイクロプレートには予め以下の方法で処理を施しておいた。即ち、まず９６穴マイクロプレート（コースター社製）に１５ｎｇ／μＬのＰｒｏｔｅｉｎ Ａ／Ｇ液を１００μＬ添加し３７℃で約２時間定置した。次にＰＢＳ溶液で３回洗浄しブロッキング溶液を１５０μＬ添加し、３７℃で約２時間定置し、スクリーニング用マイクロプイレートとした。予め用意した８０μＬの０．１ｎｇ／μＬのマウスＩｇＧ１抗５×ヒスチジンタグモノクローナル抗体の入った９６穴マイクロプレートに上記の遠心上清を添加、混和し１００μＬのブロッキング液をＰＢＳ溶液で洗浄除去したスクリーニング用マイクロプレートに添加し、２５℃で１時間定置した。その後ＰＢＳで３回洗浄し１４０μＬのＰＱＱＧＤＨアッセイ溶液（４ｍＭ １−ｍｅｔｈｏｘｙ ＰＭＳ ３．５ｍＬ， １ｍＭ ＸＴＴ溶液 ３．５ｍＬ， ２０ｍＭ マルトースもしくはグルコース溶液 ３．５ｍＬ， ４０ｍＭ ＭＯＰＳ−ＮａＯＨ ｐＨ７．０ ３．５ｍＬの混合液）を添加し、４１０ｎｍ の吸光度を１５分間室温で測定した。
【００５９】
第一段階のスクリーニングはランダム一塩基置換ライブラリーに対してマルトースを基質としたスクリーニングを実施し、酵素活性が野生型の約８０％以下に低下した変異体を発現するクローンを同定した。同定された変異株に対して次にグルコースを基質としたスクリーニングを実施し、グルコースへの反応性に比しマルトースへの反応性の低下が大きい変異体を発現するクローンを得た。これら変異ＰＱＱＧＤＨ遺伝子の塩基配列を決定したところ７５番目のグリシンがアルギニン、２９５番目のグリシンがグルタミン酸、３４２番目のメチオニンがバリン、３５１番目のアラニンがスレオニン、１６８番目のグルタミンがヒスチジン、１６９番目のロイシンがグルタミン、３４７番目のトリプトファンがアルギニンに変異していることが明らかとなった。以下の表４に示されるように、本発明で得られた改変型ＰＱＱＧＤＨはマルトースへの特異性が野生型に比し何れも低下していた。グルコースを基質としたときの野生型ＰＱＱＧＤＨの活性を１００とし、これに対する相対値としてマルトースに対する反応性を示した。尚、対照として実施例６で得られたヒスチジンタグを付加した野生型のＰＱＱＧＤＨを用いた。
表４のアミノ酸置換の欄における数字はアミノ酸番号を、数字の左側の記号は野生型におけるアミノ酸の種類を、数字の右側は変異後のアミノ酸の種類をそれぞれ表す。従って、例えばＧｌｙ７５Ａｒｇは第７５番アミノ酸のグリシンがアルギニンに置換された改変体を意味する。
【００６０】
【表４】

【００６１】
［実施例８］ 野生型ＰＱＱＧＤＨ遺伝子への部位特異的変異の導入と及び改変型ＰＱＱＧＤＨの基質特異性の検討
ＱｕｉｋＣｈａｎｇｅ Ｓｉｔｅ−ｄｉｒｅｃｔｅｄ Ｍｕｔａｇｅｎｅｓｉｓ ｋｉｔ（ストラタジーン社）を用いて、配列番号４で示されるアシネトバクター・カルコアセティカス由来ＰＱＱＧＤＨの構造遺伝子に部位特異的変異の導入を行った。実施例７で変異が導入された部位、即ち、７５番目のグリシン、２９５番目のグリシン、３４２番目のメチオニン、３５１番目のアラニン、１６８番目のグルタミン、１６９番目のロイシン、３４７番目のトリプトファンの各部位のアミノ酸をコードする塩基配列がランダムに２０種類のアミノ酸をコードするように置換される条件とした。各部位を２０種類のアミノ酸に置換するプライマーの配列を表５に示す。尚、表５において各配列名の語尾に記したＦ及びＲはそれぞれフォワード及びリバースを表す。
【００６２】
【表５】
配列名：配列
ＧＤＨＢ／Ｇ１００−Ｆ：５’−ＧＴＡＡＡＴＧＡＴＧＣＴＧＡＴＮＮＮＣＡＡＡＡＣＧＧＴＴＴＡＴＴＧＧＧ−３’（３５ｍｅｒ、配列番号１３）
ＧＤＨＢ／Ｇ１００−Ｒ：５’−ＣＣＣＡＡＴＡＡＡＣＣＧＴＴＴＴＧＮＮＮＡＴＣＡＧＣＡＴＣＡＴＴＴＡＣ−３’（３５ｍｅｒ、配列番号１４）
ＧＤＨＢ／Ｇ３２０−Ｆ：５’−ＣＡＡＡＴＴＡＡＡＧＡＴＴＴＡＮＮＮＣＡＡＡＡＴＧＧＴＴＴＡＡＡＡＧＴＧＧＣ−３’（３８ｍｅｒ、配列番号１５）
ＧＤＨＢ／Ｇ３２０−Ｒ：５’−ＧＣＣＡＣＴＴＴＴＡＡＡＣＣＡＴＴＴＴＧＮＮＮＴＡＡＡＴＣＴＴＴＡＡＴＴＴＧ−３’（３８ｍｅｒ、配列番号１６）
ＧＤＨＢ／Ｍ３６７−Ｆ：５’−ＣＣＡＡＣＣＴｇＴｇｇｇｇＡＴＮＮＮＡＣＣＴＡＣＡＴＴＴｇＣＴｇｇ−３’（３３ｍｅｒ、配列番号１７）
ＧＤＨＢ／Ｍ３６７−Ｒ：５’−ＣＣＡｇＣＡＡＡＴｇＴＡｇｇＴＮＮＮＡＴＣＣＣＣＡＣＡｇｇＴＴｇｇ−３’（３３ｍｅｒ、配列番号１８）
ＧＤＨＢ／Ａ３７６−Ｆ：５’−ｇＣＣＡＡＣｇｇＴＴＮＮＮＣＣｇＴＣＡＴＣＴｇＣＴＴＡＴｇＴＣＴＡ−３’（３３ｍｅｒ、配列番号１９）
ＧＤＨＢ／Ａ３７６−Ｒ：５’−ＴＡｇＡＣＡＴＡＡｇＣＡｇＡＴｇＡＣｇｇＮＮＮＡＡＣＣｇＴＴｇｇＣ−３’（３３ｍｅｒ、配列番号２０）
ＧＤＨＢ／Ｑ１９３−Ｆ：５’−ＧＡＴＣＡＧＧＧＧＣＧＴＡＡＣＮＮＮＣＴＧＧＣＴＴＡＴＴＴＡＴＴＣ−３’（３３ｍｅｒ、配列番号２１）
ＧＤＨＢ／Ｑ１９３−Ｒ：５’−ＧＡＡＴＡＡＡＴＡＡＧＣＣＡＧＮＮＮＧＴＴＡＣＧＣＣＣＣＴＧＡＴＣ−３’（３３ｍｅｒ、配列番号２２）
ＧＤＨＢ／Ｌ１９４−Ｆ：５’−ＧＧＧＧＣＧＴＡＡＣＣＡＧＮＮＮＧＣＴＴＡＴＴＴＡＴＴＣＴＴＡＣＣ−３’（３３ｍｅｒ、配列番号２３）
ＧＤＨＢ／Ｌ１９４−Ｒ：５’−ＧＧＴＡＡＧＡＡＴＡＡＡＴＡＡＧＣＮＮＮＣＴＧＧＴＴＡＣＧＣＣＣＣ−３’（３３ｍｅｒ、配列番号２４）
ＧＤＨＢ／Ｗ３７２−Ｆ：５’−ｇｇＡＴＡＴｇＡＣＣＴＡＣＡＴＴＴｇＣＮＮＮＣＣＡＡＣｇｇＴＴｇＣｇＣＣｇ−３’（３７ｍｅｒ、配列番号２５）
ＧＤＨＢ／Ｗ３７２−Ｒ：５’−ＣｇｇＣｇＣＡＡＣＣｇＴＴｇｇＮＮＮｇＣＡＡＡＴｇＴＡｇｇＴＣＡＴＡＴＣＣ−３’（３７ｍｅｒ、配列番号２６）
【００６３】
ＱｕｉｋＣｈａｎｇｅ Ｓｉｔｅ−ｄｉｒｅｃｔｅｄ Ｍｕｔａｇｅｎｅｓｉｓ ｋｉｔに付属する１μＬのＰｆｕＴｕｒｂｏ ＤＮＡポリメラーゼ（２．５Ｕ／μＬ）、１２５ｎｇのフォワードとリバースの各プライマー、ｄＮＴＰのミックス、１０ｎｇのｐＵＫ−ＧＤＨＢ（Ｓ）と１／１０量のＰＣＲバッファーとを混合した。反応条件は９５℃３０秒間、次に、９５℃３０秒間、５５℃１分間、及び６８℃１０分間を１６サイクルとした。反応終了後に鋳型ＤＮＡを除去するためにキットに付属の制限酵素ＤｐｎＩ（１０Ｕ／μＬ）を１μＬ添加し、３７℃で１時間インキュベートした。反応産物の１μＬを用いて大腸菌ＸＬ１−Ｂｌｕｅを形質転換しコロニーを得た。
【００６４】
次に、得られた形質転換体のコロニーを６００μＬのＣｉｒｃｌｅ Ｇｒｏｗ ｍｅｄｉｕｍ（ストラタジーン社）に接種し、実施例７と同様の方法でスクリーニングを行った。その結果選択された変異株の塩基配列を決定したところ、以下の表６に示されるようにアミノ酸置換が行われた各種の変異体が得られていることがわかった。一方、これらの各変異体をアンピシリン１００μｇ／ｍＬ、０．０１ｍＭのイソプロピルチオガラクトシド及び６μＭのＰＱＱを含む２ｍＬのＬＢ培地で３０℃、一晩培養した。培養後、菌体を遠心分離で回収し、６μＭのＰＱＱを含む１０ｍＭ ＭＯＰＳ緩衝液（ｐＨ７．０）５００μＬに再懸濁した。続いて懸濁溶液に２５０ｍｇのガラスビーズ（安井器械社製）を加え、マルチビーズショッカー（安井器械社製）（２０００ｒｐｍ， 運転６０秒間， インターバル３０秒間を７サイクル）によって菌体を破砕した。
【００６５】
菌体破砕後の溶液を遠心分離（１５０００ｒｐｍ， １０分間， ４℃）し、上清をＨｉｓ ＭｉｃｒｏＳｐｉｎ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｍｏｄｕｌｅ（アマシャム・ファルマシアバイオテク社製）を用いて精製した。その結果、２００μＬの精製酵素溶液が得られた。この精製酵素溶液にＰＱＱを５μＭとなるように添加し、そして１ｍＭ 塩化カルシウム存在下で１時間以上放置してホロ化を行った。
【００６６】
以上のようにして得られた各改変型ＰＱＱＧＤＨの基質特異性を次の方法で測定した。即ち、精製酵素溶液を５０μＬずつ分注した後、１３０μＬのアッセイ溶液（２０ｍＭ ＭＯＰＳ ｐＨ７．０に溶解された３．３ｍＭ １−ｍｅｔｈｏｘｙ ＰＭＳを６０μＬ ２０ｍＭ， １７１μＭ ＸＴＴを７０μＬ）及びＤ−グルコース溶液２０μＬを最終濃度５ｍＭになるように添加し、実施例５に示した方法に従いＰＱＱＧＤＨ活性を測定した。尚、対照として実施例６で得られたヒスチジンタグを付加した野生型のＰＱＱＧＤＨを用いた。測定結果を表６に示す。表６から明らかなように、野生型のＰＱＱＧＤＨに比較していずれの改変型ＰＱＱＧＤＨもマルトース及びガラクトースに対する反応性が低下しており、またラクトース及びガラクトースに対する反応性も同時に低下している改変体も得られた。尚、表６のアミノ酸置換の欄における数字はアミノ酸番号を、数字の左側の記号は野生型におけるアミノ酸の種類を、数字の右側は変異後のアミノ酸の種類をそれぞれ表す。従って、例えばＧｌｙ７５ Ｔｒｐは第７５番アミノ酸のグリシンがトリプシンに置換された改変体を意味する。
【００６７】
【表６】

【００６８】
［実施例９］ ミューテーションスクランブリング法を利用した多重変異の導入及び多重置換体の基質特異性の検討
実施例８で明らかにされた、基質特異性の向上に有効なアミノ酸置換の可能なすべての組み合わせを有する多重置換体ライブラリーをミューテーションスクランブリング法により作製することにより、各アミノ酸置換を単独で有する場合に比べてより高い基質特異性を有する改変体の取得を試みた。多重置換体ライブラリーの作製には既報のバイアスミューテーションスクランブリング法（第３９回日本生物物理学会年会、平成１３年１０月６日開催、演題番号１Ｐ０５７、ノバルティスファーマ社；Ｂｉｏｐｏｌｙｍｅｒ、６４号（２００２年）、Ｐ．９５−１０５）を利用した。バイアスミューテーションスクランブリング法はミューテーションスクランブリング法の改変法であり、組み合わせるアミノ酸置換の導入確率を自由に設定可能であるという特徴を有する。本実施例では、構造モデルからの推定によって基質結合部位の近傍に位置しており特に基質特異性への貢献度が高いと期待されるＧ７５， Ｇ２９５， Ｍ３４２， Ａ３５１については８０％の高確率で、構造的に特徴が認められると考えられるＱ１６８， Ｌ１６９， Ｗ３４７については５０％の確率で変異が導入されるような条件に設定した。具体的には、ＰＱＱＧＤＨ遺伝子全長を、末端が重複し且つ実施例８で明らかにされたアミノ酸置換を１〜３個含むような図４に示した５つの断片に分割し、各アミノ酸置換が上記の確率で導入されるように図５に示した各断片の混合比率でミューテーションスクランブリング法を施行してＰＱＱＧＤＨ遺伝子の再構築を行った。
【００６９】
ミューテーションスプラングリング法では、まず野生型遺伝子を鋳型としたＰＣＲを行い部分増幅産物を得た（図４）。ＰＣＲに使用した反応液の組成を表７に示す。プライマーにはＧ７５を挟みＱ１６８とＬ１６９に対応する配列を含むものを用いた。
【００７０】
【表７】
１０×ｂｕｆｆｅｒ ｆｏｒ ＡｃｃｕＴａｑ（シグマ社製） ２ μＬ
１０ｍＭ ｄＮＴＰ １ μＬ
フォワードプライマー（１０ｐｍｏｌ／μＬ） １．２ μＬ
リバースプライマー（１０ｐｍｏｌ／μＬ） １．２ μＬ
Ｔｅｍｐｌａｔｅ（１０ｎｇ／μＬ） ０．５ μＬ
ｄＨ_２Ｏ １３．９ μＬ
ＡｃｃｕＴａｑ（５ｕｎｉｔｓ／μＬ） ０．２ μＬ
Ｔｏｔａｌで２０ μＬ
【００７１】
フォワードプライマー：５’−ＴＣＡＣＡＴＧＴＴＣＴＴＴＣＣＴＧＣＧＴＴＡＴＣ−３’（配列番号９）
Ｓ−ｐｒｉｍｅｒ（Ｇ）−１０−ＱＬ：５’−ＡＡｇＡＡＴＡＡＡＴＡＡｇＣＣＡｇＴＴＧｇＴＴＡＣｇＣＣＣＣＴｇＡＴＣ−３’（配列番号２７）
反応条件は９８℃３０秒間、次に９４℃５秒間、６０℃２０秒間、及び６８℃９０秒間を３０サイクル、最後に６８℃１０分間とした。次に同じ反応条件でＧ７５に変異を有する改変型遺伝子を鋳型としてＰＣＲを行い、上記と同じ領域の部分増副産物を得た。ここで、表６からわかるようにＧ７５部位、Ｑ１６８部位、Ｌ１６９部位のアミノ酸置換の組み合わせは１６通り存在する。これらすべての組み合わせに対応する部分増副産物は、図４に示した８種類のプライマー（Ｓ−ｐｒｉｍｅｒ（Ｇ）−１０−ＱＬ〜Ｓ−ｐｒｉｍｅｒ（Ｇ）−１０−ＹＦ）を用い、野生型と改変型の２種の遺伝子を鋳型にした遺伝子増幅反応を行うことによって調製された。得られた部分増幅産物を図５の混合比で混合して断片１（断片１、図４を参照）を得た。断片２から断片５（断片２、断片３、断片４、断片５、図４を参照）は、断片１の場合と同様の要領で、図４に示したプライマーを利用して得られる遺伝子増幅産物を図５にそれぞれ示される比率で混合することにより調製された。
【００７２】
次に上述の各断片を等量ずつ混合し、以下の表８に示す溶液組成においてＰＣＲを行った。反応条件は９８℃３０秒間、次に９４℃５秒間、６０℃２０秒間、及び６８℃２分間を５サイクルとした。ＰＣＲ反応終了後、２．４μＬずつの上記プライマー（１０ ｐｍｏｌ／μＬ）（配列番号７及び８）を添加し、再度ＰＣＲを行った。このときの反応条件は９４℃５秒間、次に６０℃２０秒間、及び６８℃２分間を２５サイクル、最後に７２℃１０分間とした。
【００７３】
【表８】
混合された断片 Ｘ μＬ
１０×ｂｕｆｆｅｒ ｆｏｒ ＡｃｃｕＴａｑ（シグマ社製） ２ μＬ
１０ｍＭ ｄＮＴＰ １ μＬ
ｄＨ_２Ｏ （１４．４−Ｘ） μＬ
ＡｃｃｕＴａｑ（５ｕｎｉｔｓ／μＬ） ０．２ μＬ
Ｔｏｔａｌで１７．６ μＬ
【００７４】
以上の操作によって得られた改変型遺伝子のライブラリーをＥｃｏＲＩとＰｓｔＩで切断し、ｐＵＫ−ＧＤＨＢ（Ｓ）の野生型遺伝子と入れ替え、大腸菌を形質転換した。形質転換体のコロニーを実施例８に示した方法で培養した後、ＰＱＱＧＤＨ活性及び基質特異性を指標としてスクリーニングを行った。基質特異性の向上が見られたクローンについてはそれが保有する改変型ＰＱＱＧＤＨの塩基配列を決定した。その結果、図６の表に示すように、置換されるアミノ酸の組合わせが異なる複数の改変型ＰＱＱＧＤＨが得られたことが判明した。尚、図６の表においてＧ７５（ＧＧＡ）等の記号は、数字で特定されるアミノ酸位置が数字の左側のアルファベット一文字で特定されるアミノ酸であることを意味する。またカッコ内は当該アミノ酸をコードする塩基配列を表す。一方、Ｐｈｅ（ＴＴＴ）等はそれが位置する列の最上段に記載された記号（例えばＬ１９４（ＣＴＧ））で特定されるアミノ酸位置が当該アルファベット３文字で特定されるアミノ酸に置換されていることを意味する。またカッコ内は当該アミノ酸をコードする塩基配列を表す。
【００７５】
これらの改変型ＰＱＱＧＤＨを保有する各形質転換体を実施例７に示した方法と同様の方法で精製した後、各基質に対する反応性を測定した。測定結果を図６の表の右欄に示した。この表から明らかなように、取得された改変型ＰＱＱＧＤＨは野生型に比較していずれもマルトースに対する反応性が顕著に低下している。また、ラクトースに対する反応性についても同様の低下が認められる。さらにガラクトースに対する反応性についてはいずれの改変型ＰＱＱＧＤＨにおいても大きく低下し、一部のものではその反応性の低下は著しい。このように、マルトース、ラクトース、及びガラクトースに対する反応性が顕著に低下した、即ち基質特異性に極めて優れた改変型ＰＱＱＧＤＨを取得することができた。この結果から図６の表に示されるアミノ酸位置の変異を組み合わせることにより、単独の変異の場合に比較して更に基質特異性に優れた改変型ＰＱＱＧＤＨを作製することができることが分った。
【００７６】
ここで、特にＮＯ．１改変体ではマルトースに対する反応性が野生型に比べて１／９にまで低下しており、同様にラクトースに対する反応性では約１／２、ガラクトースに対する反応性では約１／９にまでそれぞれ低下している。また、Ｎｏ．６改変体においてもマルトースに対する反応性が野生型に比べて１／２以下に低下していると同時に、ラクトースに対する反応性も低下し、ガラクトースに対する反応性についても約１／４に低下している。同様に、Ｎｏ．７改変体においてもマルトースに対する反応性が野生型に比べて約１／５にまで低下していると同時に、ラクトースに対する反応性も約１／２にまで低下し、ガラクトースに対しては実質的に反応性がないことがわかる。以上のように基質特異性の向上が特に顕著であったＮｏ．１、Ｎｏ．６及びＮｏ．７改変体におけるアミノ酸置換部位を比較すれば、第３４２番アミノ酸及び第３５１番アミノ酸の置換が基質特異性の向上に特に重要であることが示唆される。次いで、第２９５番アミノ酸の置換が重要であり、また第１９４番アミノ酸の置換も基質特異性の向上に大きく関与することが予想される。
【００７７】
本発明は、上記発明の実施の形態の説明に何ら限定されるものではなく、特許請求の範囲の記載を逸脱せず、当業者が容易に想到できる範囲で種々の変形態様もこの発明に含まれる。
【００７８】
【発明の効果】
本発明で提供される改変型のピロロキノリンキノン依存性グルコース脱水素酵素はグルコースに対する基質特異性が高い。従って、かかる改変型の酵素を利用すれば高い精度でグルコースの定量が行える。
【００７９】
【配列表】

【図面の簡単な説明】
【図１】図１はアシネトバクター・カルコアセティカス（Ａｃｉｎｅｔｏｂａｃｔｅｒ ｃａｌｃｏａｃｅｔｉｃｕｓ）由来のピロロキノリンキノン依存性グルコース脱水素酵素をコードする遺伝子の配列（シグナル領域は含まない）及びアミノ酸配列（シグナル領域は含まない）を示した図である。
【図２】図２はアシネトバクター・カルコアセティカス（Ａｃｉｎｅｔｏｂａｃｔｅｒ ｃａｌｃｏａｃｅｔｉｃｕｓ）由来のピロロキノリンキノン依存性グルコース脱水素酵素をコードする遺伝子の配列（シグナル領域を含む）及びアミノ酸配列（シグナル領域を含む）を示した図である。
【図３】図３は実施例において使用される発現ベクターｐＵＫ−ＧＤＨＢ（Ｓ）の構造を示す図である。
【図４】図４は実施例におけるミューテションスクランブリングで用いたプライマー及び増幅部位を示した表である。
【図５】図５はミューテーションスプランブリングにおける各ＤＮＡ断片の混合比率を示した表である。設定バイアス率を０．８ ： Ｇ７５， Ｇ２９５， Ｍ３４２， Ａ３５１、０．５ ：Ｑ１６８， Ｌ１６９， Ｗ３４７とした。尚、バイアス率とは、あるアミノ酸置換がＭｕｔａｔｉｏｎ Ｓｃｒａｍｂｌｉｎｇ法によって導入される確率である。
【図６】図６は実施例のミューテーションスクランブリングによって得られた各種の改変型ＰＱＱＧＤＨのアミノ酸置換位置、及び基質特異性をまとめた表である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to pyrroloquinoline quinone dependent glucose dehydrogenase. Specifically, the present invention relates to a modified pyrroloquinoline quinone-dependent glucose dehydrogenase in which an amino acid in a specific region is substituted with another amino acid as compared with a wild-type enzyme. The modified pyrroloquinoline quinone-dependent glucose dehydrogenase of the present invention is used, for example, for measuring the amount of glucose in a clinical test or the like.
[0002]
[Prior art]
Pyrroloquinolinequinone-dependent β-D glucose dehydrogenase (EC 1.1.199, PQQGDH) catalyzes the reaction of oxidizing β-D glucose to gluconolactone in combination with the coenzyme pyrroloquinoline (PQQ). I do. Utilizing this property, PQQGDH is used for quantification of glucose in clinical tests, food analysis, monitoring of culture processes, and the like.
PQQGDH reported in the past includes Acinetobacter sp. M. D79.41 strain produced PQQGDH and its modified PQQGDH are known (for example, see Patent Documents 1 to 5 and Non-Patent Document 1). M. The substrate specificity of PQQGDH produced by strain D 79.41 was low, for example, it had a reactivity to maltose corresponding to about 90% of the reactivity to glucose.
[0003]
[Patent Document 1]
JP-A-2000-31588
[Patent Document 2]
JP 2000-350588 A
[Patent Document 3]
JP 2001-197888 A
[Patent Document 4]
JP 2001-346587 A
[Patent Document 5]
WO 02/34919 A1
[Non-patent document 1]
A-M Cleton-Jansen et al. Mol. Gen. Genet. , 217, 430 (1989).
[0004]
[Problems to be solved by the invention]
PQQGDH is required to have high substrate specificity in order to measure an accurate amount of glucose. For example, if PQQGDH having a low substrate specificity is used, for example, when diagnosing diabetes in a patient who has received intravenous infusion by blood transfusion, the measurement value obtained will be the amount of maltose in the transfusion in addition to the amount of glucose. It is a measured value, and an accurate blood glucose level cannot be obtained. Similarly, for example, when a measurement is performed on a patient with hepatic dysfunction, highly reliable measurement may not be performed due to the influence of galactose. In this way, PQQGDH having a low substrate specificity is greatly influenced by other carbohydrates such as maltose, so that an accurate glucose amount cannot be measured.
[0005]
In the above-mentioned modified PQQGDH reported in the past, the substrate specificity has been improved by substituting some amino acids of wild-type PQQGDH. However, the reactivity to maltose and galactose still remains considerably, and further improvement in substrate specificity is desired to enable more accurate measurement of glucose amount. In particular, PQQGDH, which has a sufficiently low reactivity to galactose, has not been found yet, and its provision is eagerly desired.
An object of the present invention is to provide a modified PQQGDH having high substrate specificity for glucose under the above background. In particular, it is an object of the present invention to provide a modified PQQGDH having extremely low reactivity to galactose in addition to low reactivity to maltose. In addition, an object of the present invention is to provide a polynucleotide, an expression vector, and a transformant that can be used for producing such a modified PQQGDH, and a method for producing a modified PQQGDH using these, and a glucose measurement kit. And
[0006]
[Means for Solving the Problems]
The present inventors have studied in detail the relationship between amino acid substitution in a specific region and a change in substrate specificity in PQQGDH produced by Acinetobacter calcoaceticus to achieve the above object. As a result, we succeeded in finding multiple regions that are deeply involved in substrate specificity. That is, as shown in the examples described below, the present inventors have shown that the substitution of the 351st amino acid in the wild-type PQQGDH of Acinetobacter calcoaceticus significantly reduces the reactivity to maltose and galactose. Was found to decrease. It was also found that the amino acid at position 342 is also deeply involved in substrate specificity. Here, since these amino acids are located in the secondary structure consisting of the 326nd amino acid residue to the 354th amino acid residue, it is considered that the modification of the structural portion can effectively improve the substrate specificity for glucose. it was thought.
Also, the 295th amino acid residue located in the secondary structure consisting of the 278th amino acid residue to the 320th amino acid residue was similarly shown to be greatly involved in the reactivity to maltose and galactose. Partial modification was considered to be extremely effective in improving the substrate specificity for glucose. Similarly, since the substitution of the 169th amino acid residue improved the substrate specificity, the modification of the secondary structure portion consisting of the 162nd amino acid residue to the 197th amino acid residue, in which the substitution was performed, was also carried out. It was considered effective for improvement.
In addition, several other amino acid residues were suggested to be involved in substrate specificity.
Here, proteins having the same function generally have similar structures in the active site and the region involved in substrate specificity. Therefore, it was expected that the above information obtained on the relationship between specific amino acid substitution and substrate specificity in PQQGDH derived from Acinetobacter calcoaceticus could be applied to PQQGDH derived from other microorganisms.
The present invention has been completed based on the above findings, and provides the following configurations.
[1] On the basis of the amino acid sequence of the corresponding wild-type enzyme, the first consisting of amino acids 326 to 354 of pyrroloquinoline quinone-dependent glucose dehydrogenase derived from Acinetobacter calcoaceticus A modified pyrroloquinoline quinone-dependent glucose dehydrogenase having an amino acid sequence in which one or more amino acids are substituted in a region corresponding to the region.
[2] The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to [1], wherein two or more amino acids are substituted in a region corresponding to the first region.
[3] The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to [1], wherein amino acids corresponding to amino acids 342 and 351 in the first region are each substituted with another amino acid. .
[4] one or more amino acids in a region corresponding to the second region consisting of amino acids 278 to 320 in pyrroloquinoline quinone-dependent glucose dehydrogenase derived from Acinetobacter calcoaceticus; The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to any one of [1] to [3], which is substituted with another amino acid.
[5] The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to [4], wherein the amino acid corresponding to the 295th amino acid in the second region is substituted with another amino acid.
[6] one or more amino acids in a region corresponding to the third region consisting of amino acids 162 to 197 in pyrroloquinoline quinone-dependent glucose dehydrogenase derived from Acinetobacter calcoaceticus; The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to any one of [1] to [5], which is substituted with another amino acid.
[7] selected from the group consisting of amino acids corresponding to amino acids 75, 168, and 347 in a pyrroloquinoline quinone-dependent glucose dehydrogenase derived from Acinetobacter calcoaceticus; The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to any one of [1] to [6], wherein one or more amino acids are substituted with another amino acid.
[8] The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to any one of [1] to [7], which is derived from Acinetobacter calcoaceticus.
[9] In the amino acid sequence of SEQ ID NO: 1, a modified pyrroloquinoline quinone having an amino acid sequence in which one or more amino acids are substituted with another amino acid in a first region consisting of amino acids 326 to 354. Dependent glucose dehydrogenase.
[10] The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to [9], wherein two or more amino acids are substituted in the first region.
[11] The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to [9], wherein amino acid 342 and amino acid 351 in the first region are each substituted with another amino acid.
[12] Any of [9] to [11], wherein one or more amino acids are substituted with another amino acid in the second region consisting of amino acids 278 to 320 of the amino acid sequence of SEQ ID NO: 1. 2. The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to item 1.
[13] The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to [12], wherein the 295th amino acid in the second region is substituted with another amino acid.
[14] any of [9] to [13], wherein one or more amino acids are substituted with another amino acid in the third region consisting of amino acids 162 to 197 of the amino acid sequence of SEQ ID NO: 1 2. The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to item 1.
[15] one or more amino acids selected from the group consisting of amino acids 75, 168, and 347 of the amino acid sequence of SEQ ID NO: 1 are substituted with other amino acids; The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to any one of [14].
[16] A modified pyrroloquinoline quinone-dependent glucose dehydrogenase having a reactivity to maltose of 40% or less and a reactivity to galactose of 10% or less based on the reactivity to glucose.
[17] An isolated polynucleotide encoding the modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to any one of [1] to [16].
[18] A vector containing the polynucleotide according to [17].
[19] A transformant having the polynucleotide of [17].
[20] a step of culturing the transformant according to [19] in a state where the polynucleotide can be expressed; and
A method for producing a modified pyrroloquinoline quinone-dependent glucose dehydrogenase, comprising the step of separating the expression product of the polynucleotide.
[21] A glucose measurement kit comprising the modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to any one of [1] to [16].
[0007]
BEST MODE FOR CARRYING OUT THE INVENTION
According to the present invention, there is provided a modified PQQGDH in which the amino acid sequence of a specific region is substituted as compared with the corresponding wild-type enzyme. The modified PQQGDH of the present invention is typically produced or designed by substituting some amino acids with other amino acids in naturally occurring PQQGDH, that is, wild-type PQQGDH. Thus, the wild-type PQQGDH, which is the basis of (derived from) the modified PQQGDH of the present invention, is referred to herein as “corresponding wild-type enzyme”. Examples of the wild-type enzyme in the present invention include Acinetobacter calcoaceticus, Acinetobacter baumannii, Pseudomonas aeruginosa, Pseudomonas pudumonas, Pseudomonas pudumonas and Pseudomonas pudumonas. Oxidizing bacteria, such as Burkholderia cepacia, Gluconobacter oxydans, Acetobacter aceti, and Agrobacterium radiobacter. ter), Escherichia coli, Klebsiella, Eero Jeans (Klebsiella aerogenes) or the like can be exemplified PQQGDH produced.
[0008]
In the present specification, “corresponding to” when used for amino acid residues or regions means that the functions resulting from the structure are equivalent between the proteins (enzymes) to be compared, particularly maltose and galactose. , Meaning that they have the same function for reactivity to carbohydrates other than glucose. For example, in PQQGDH derived from a microorganism other than Acinetobacter calcoaceticus, a secondary structure region having PQQGDH derived from Acinetobacter calcoaceticus is compared with the primary structure and considering the homology of the secondary structure. A region reasonably considered to have the same function can be referred to as "corresponding to the secondary structure region of PQQGDH derived from Acinetobacter calcoaceticus".
In the present specification, the position of an amino acid is specified by the number when the aspartic acid at the N-terminus is 1 and the numbering is performed in order from the N-terminus to the C-terminus.
[0009]
In the modified PQQGDH of the present invention, a pyrroloquinoline quinone-dependent glucose dehydrogenase derived from Acinetobacter calcoaceticus based on the amino acid sequence of the corresponding wild-type enzyme is referred to as “AC PQQGDH”. In the region corresponding to the first region consisting of amino acids 326 to 354 of the above, one or more amino acids are substituted with other amino acids. A. In the present specification. C. The amino acid sequence of PQQGDH is the sequence specified by SEQ ID NO: 1. This sequence is the amino acid sequence of PQQGDH produced by Acinetobacter calcoaceticus IFO 12552 strain, as shown in the Examples below.
[0010]
In addition to the above amino acid substitutions, C. It is preferable that one or more amino acids are substituted with another amino acid in a region corresponding to the second region consisting of amino acids 278 to 320 in PQQGDH.
Further, A.I. C. It is preferable that one or more amino acids are substituted with another amino acid in a region corresponding to the third region consisting of amino acids 162 to 197 in PQQGDH.
In addition, A.I. C. It is preferable that one or more amino acids selected from the group consisting of amino acids corresponding to amino acids 75, 168, and 347 in PQQGDH are substituted with other amino acids.
[0011]
The number of amino acids substituted in each region is not particularly limited. However, it is preferable that two or more amino acids are substituted in the region corresponding to the first region.
The position of the amino acid substituted in each region is not particularly limited. For example, in the region corresponding to the first region, the amino acid corresponds to the 342nd amino acid and / or the 351st amino acid in the first region. It is preferred that the amino acid at the position is substituted. Similarly, in the region corresponding to the second region, it is preferable that the amino acid at the position corresponding to the 295th amino acid in the second region is substituted, and in the region corresponding to the third region, the third region It is preferable that the amino acid at the position corresponding to the 169th amino acid is substituted.
[0012]
The type of amino acid after substitution is not particularly limited. For example, tryptophan at amino acid position 75, histidine at amino acid position 168, phenylalanine at amino acid position 169, and glutamic acid or aspartic acid at amino acid position 295. , Tyrosine, phenylalanine, etc., are proline at amino acid position 342, arginine at amino acid position 347, and threonine at amino acid position 351.
[0013]
The modified PQQGDH of the present invention is characterized in that the selectivity for glucose is improved as compared with the wild-type enzyme. That is, the modified PQQGDH of the present invention has a characteristic that its reactivity with maltose and lactose is lower than that of the wild-type enzyme, and the reactivity with galactose is particularly low. Expressed as a relative value with the reactivity to glucose as 100%, the modified PQQGDH of the present invention preferably has a reactivity to maltose of 40% or less, more preferably 30% or less, and still more preferably 20% or less. It is. Similarly, the reaction with lactose is preferably at most 50%, more preferably at most 40%. The reactivity with respect to galactose is preferably 20% or less, more preferably 10% or less, and further preferably substantially 0%. In the present specification, the term “substrate specificity” simply means this selectivity for glucose.
[0014]
In the modified PQQGDH of the present invention, some amino acids may be further modified in addition to the above amino acid substitutions. As used herein, the term "partial amino acid modification" refers to the deletion or substitution of one or several amino acids constituting an amino acid sequence, or the addition or insertion of one or several amino acids, or a combination thereof. However, such a modification can be performed as long as the activity as PQQGDH (that is, reactivity to glucose) is maintained in principle. However, even if the PQQGDH activity is slightly reduced, the reactivity to other substrates (for example, maltose or galactose) is also reduced and the substrate specificity for glucose is improved, or if the substrate specificity is slightly reduced. The above modification is also permitted if there is no problem in using glucose for measuring the amount of glucose.
The modification of some amino acids as described above is preferably performed within a range where the reactivity to maltose, galactose, and the like is kept low.
The position of the amino acid used for the modification here is not particularly limited, and the modification may be performed at a plurality of positions. Here, the term “plurality” refers to, for example, a number corresponding to 10% or less of all amino acids, and preferably a number corresponding to 5% or less of all amino acids. More preferably, it is a number corresponding to within 1% of all amino acids.
[0015]
The modified PQQGDH of the present invention is obtained by first obtaining a gene encoding wild-type PQQGDH, then modifying it to construct a polynucleotide encoding the modified PQQGDH, and finally converting the polynucleotide into an appropriate expression system. It can also be made by expressing. Hereinafter, the manufacturing method will be described. Once the sequence is designed, a polynucleotide (gene) encoding the modified PQQGDH of the present invention by chemical synthesis using deoxynucleotide triphosphates (dATP, dTTP, dGTP, dCTP) as a material based on the sequence information Can be prepared.
[0016]
<Acquisition of Gene Encoding Wild-Type PQQGDH>
First, a gene encoding PQQGDH is obtained from a PQQGDH-producing bacterium. PQQGDH-producing bacteria include, for example, Acinetobacter calcoaceticus, Acinetobacter baumannii, Pseudomonas aeruginosa, Pseudomonas pusoudosa, Pseudomonas pusoud esuda usadusonadusonadusonadusonadusonas. Oxidizing bacteria such as Burkholderia cepacia, Gluconobacter oxydans, Acetobacter aceti, and Agrobacterium radiobacter. ter), Escherichia coli, mention may be made of intestinal bacteria such as Klebsiella, Eero Jeans (Klebsiella aerogenes). Among them, Acinetobacter calcoaceticus is preferably used as a PQQGDH-producing bacterium.
[0017]
The gene encoding PQQGDH can be extracted from the cells of PQQGDH-producing bacteria by a conventional method. The PQQGDH gene can also be prepared using a PCR method using the genomic DNA of PQQGDH as a template. On the other hand, the extracted gene may be once amplified using an amplification method such as a PCR method, and the amplified product may be subjected to subsequent operations. After the base sequence of the gene encoding PQQGDH has been identified, the target PQQGDH gene can also be prepared by a chemical technique. Hereinafter, as an example of a method for preparing the PQQGDH gene, a case where Acinetobacter calcoaceticus IFO 12552 strain is used as a starting material will be specifically described.
[0018]
First, after separating and purifying the chromosome of Acinetobacter calcoaceticus IFO 12552, a DNA fragment obtained by sonication, restriction enzyme treatment, and the like, and an expression vector prepared in a linear state were subjected to DNA ligase or the like. Utilization is used to bind and close both blunt ends or cohesive ends to construct a recombinant vector. Known Acinetobacter species L. M. Constructing a recombinant vector by amplifying the PQQGDH gene by polymerase chain reaction (PCR) using oligonucleotide primers designed based on the base sequence of the PQQGDH gene derived from the D79.41 strain and binding and closing the DNA ligase to construct a recombinant vector You can also. Next, the recombinant vector constructed by any of the above methods is transferred to a host microorganism capable of autonomously replicating it. Then, screening is performed using the marker specific to the expression vector and the PQQGDH enzyme activity as an index to obtain a host microorganism transformed with the recombinant vector containing the gene encoding PQQGDH.
[0019]
Next, the transformants selected as necessary are cultured, and then the cells are collected. The recombinant vector is separated and purified from the collected cells according to a conventional method. Thus, a recombinant vector containing the target PQQGDH gene is obtained. Isolation of the PQQGDH gene from such a recombinant vector can be performed by restriction enzyme treatment or the like.
[0020]
Here, the details of an example of the method for obtaining the PQQGDH gene from Acinetobacter calcoaceticus IFO 12552 will be described below. First, a culture solution obtained by stirring and culturing Acinetobacter calcoaceticus IFO 12552, for example, for 1 to 3 days is collected by centrifugation, and then the cells are lysed to obtain a lysate containing the PQQGDH gene. Prepare. As a lysis method, for example, treatment with a lytic enzyme such as lysozyme can be employed. If necessary, an enzyme treatment with a protease or the like or a treatment with a surfactant such as sodium lauryl sulfate (SDS) may be used in combination. Further, it may be combined with a physical crushing method such as freeze-thawing or French pressing.
[0021]
Separation and purification of DNA from the lysate obtained as described above can be performed according to a conventional method. For example, it can be performed by appropriately combining methods such as protein removal treatment by phenol treatment or protease treatment, ribonuclease treatment, and alcohol precipitation treatment.
[0022]
In order to obtain the PQQGDH gene from DNA isolated and purified from microorganisms, Acinetobacter sp. M. D. A gene amplification reaction is carried out using primers based on the nucleotide sequence of the 79.41 strain.
[0023]
It is also possible to clone the amplified PQQGDH gene into an appropriate vector. As a vector for cloning (cloning vector), a vector constructed for gene recombination from a phage or a plasmid capable of autonomous replication in a host microorganism is suitable. Examples of such phage include Lambda gt10 and Lambda gt11 using Escherichia coli as a host microorganism. Similarly, examples of plasmids include pBR322, pUC19, pBluescript, pTrc99, pGEM-T vector, and the like using Escherichia coli as a host microorganism.
[0024]
Cloning into a vector is performed by inserting the PQQGDH gene obtained by the above method into the cloning site of the vector. Such an operation can be performed by a known method using a restriction enzyme and DNA ligase.
[0025]
The host microorganism used for cloning is not particularly limited as long as the recombinant vector can stably and autonomously propagate therein and can express foreign genes. Generally, Escherichia coli W3110, Escherichia coli C600, Escherichia coli HB101, Escherichia coli JM109, Escherichia coli DH5α and the like can be used.
[0026]
As a method for transferring the recombinant vector to the host microorganism, for example, when the host microorganism is Escherichia coli, a competent cell method by calcium treatment, an electroporation method, or the like can be used.
In order to select a host microorganism into which the desired recombinant vector has been appropriately introduced, it is possible to search for a microorganism in which expression of a marker (eg, a drug resistance gene) of the vector and expression of GDH activity by addition of PQQ are simultaneously observed. Good. For example, a microorganism which grows on a selective medium based on a drug resistance marker and produces PQQGDH may be selected. A transformant selected by such a method can stably produce a large amount of PQQGDH by culturing it in a nutrient medium.
[0027]
The nucleotide sequence of the wild-type PQQGDH gene obtained by the above method was decoded by the dideoxy method described in Science, Vol. 214, 1205 (1981) (FIG. 1, SEQ ID NO: 2). The amino acid sequence of PQQGDH was deduced from the nucleotide sequence determined as described above (FIG. 1, SEQ ID NO: 1). The gene sequence (SEQ ID NO: 4) and the amino acid sequence (SEQ ID NO: 3) including the signal region are shown in FIG.
[0028]
The PQQGDH gene recovered from the recombinant vector having the PQQGDH gene can be easily modified using genetic engineering techniques. Specifically, the gene is modified using, for example, a site-specific mutagenesis method so that amino acid residues at a specific site include substitution, deletion, insertion, addition, or inversion.
[0029]
<Construction of polynucleotide encoding modified PQQGDH>
The gene encoding the modified PQQGDH of interest can be produced by mutating the PQQGDH gene obtained as described above so that a specific amino acid is substituted in the protein as an expression product. it can. Many methods for such site-specific nucleotide sequence substitution are known in the art (see, eg, Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New York), among which are suitable methods. Can be selected and used. The modified PQQGDH can also be encoded by inserting a random mutation into the PQQGDH gene and comparing the substrate specificities of the expression products of the mutants (variants) to select a gene having a preferable substrate specificity. Genes can be made. When such a random mutation is introduced, first, a mutation is randomly introduced into a target gene region using, for example, error-prone PCR to construct a modified PQQGDH gene library. Multiple substitutions having useful combinations of mutations obtained by random mutations have been published by Novartis Pharma in Bioscience and Industry vol. 59, No. 3 (2001), pp. 35-38. In this method, the modified enzyme can be easily and easily evaluated by procedures such as culturing the obtained colony, lysing cells using a lytic enzyme such as lysozyme, and measuring the enzyme activity.
[0030]
In order to efficiently screen the modified PQQGDH gene library, a base sequence encoding a histidine tag can be added to the PQQGDH gene in advance. By doing so, even if the amount of the produced enzyme varies due to the growth state, expression efficiency, and the like, it is possible to evaluate the enzyme activity using a fixed amount. Specifically, for example, a predetermined amount of an anti-histidine-tagged monoclonal antibody is adsorbed on the surface of a protein-adsorbing substrate such as a microplate, and a treatment for preventing nonspecific protein adsorption is performed. By doing so, a certain amount of PQQGDH contained in the lysate is immobilized on a substrate, and the enzyme activity can be evaluated. By performing the above operation in a 96-well microplate, the enzyme activity per unit amount of many variants can be efficiently evaluated at once.
Selection of a clone having the desired modified gene is performed, for example, as follows. After adding 1-methoxy-PMS and XTT to the lysate, the activity of PQQGDH was measured based on the absorbance at 410 nm after a certain period of time, and the reactivity to maltose was reduced to about 80% or less as compared with wild-type PQQGDH. First, a clone that has about 90% or more of the reactivity to glucose as compared with wild-type PQQGDH is selected. The mutation is confirmed by analyzing the nucleotide sequence of the clone obtained here.
[0031]
<Expression of modified PQQGDH gene>
For expression of the modified PQQGDH gene, an expression system using Escherichia coli as a host can be used. For example, first, the modified PQQGDH gene prepared by the above-described method is inserted into a vector (for example, a pUC vector or a pBluescript vector) using Escherichia coli as a host to construct an expression vector. The expression vector contains a promoter sequence, a replication origin, a terminator sequence, and the like necessary for expression of the modified PQQGDH in the host.
[0032]
As a method for introducing (transferring) the expression vector into the host microorganism, for example, when the host microorganism is Escherichia coli, a competent cell method or an electroporation method by calcium treatment can be used. Selection of the host microorganism into which the expression vector has been appropriately introduced into the host microorganism can be performed based on the presence or absence of a drug resistance marker possessed by the expression vector and the presence or absence of expression of GDH activity when PQQ is added. For example, the microorganism after the operation of introducing the expression vector is cultured in a selective medium that can grow only when it has the drug resistance marker, and subsequently, a modified PQQGDH is produced from the transformants that have grown. What you need to do is select.
[0033]
By culturing the transformant selected by the above operation under conditions where it can grow and proliferate well and the modified PQQGDH gene inserted into the expression vector can be expressed, a large amount of the modified PQQGDH can be stably obtained. Can be produced.
[0034]
As the nutrient source of the medium, those usually used for culturing microorganisms can be used. The carbon source may be any assimilable carbon compound, and for example, glucose, sucrose, lactose, maltose, lactose, molasses, pyruvic acid, and the like are used. The nitrogen source may be any available nitrogen compound, such as peptone, meat extract, yeast extract, casein hydrolyzate, and soybean meal alkaline extract. In addition, phosphates, carbonates, sulfates, salts such as magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, and specific vitamins are used as needed.
[0035]
The transformant is cultured under temperature conditions under which the transformant can grow and the modified PQQGDH is produced. For example, the culture temperature can be set within the range of 20 ° C to 30 ° C. The culturing time can be set in consideration of the growth characteristics of the transformant to be cultured, the modified PQQGDH production characteristics, or the required amount of modified PQQGDH production, for example, the yield of the modified PQQGDH reaches a maximum. Complete the culture at the appropriate time. The standard of the culture time is about 12 to 72 hours. The pH of the medium is adjusted so that the transformant grows and PQQGDH is produced. Preferably, the pH of the medium is about 6.0 to 9.0.
[0036]
The culture solution containing the cells producing the modified PQQGDH can be used as an enzyme solution as it is or after concentration, removal of impurities, etc., but generally, the modified PQQGDH is once obtained from the culture solution or the cells. to recover. If the modified PQQGDH to be produced is a secretory protein, it can be recovered from the culture solution, otherwise, it can be recovered from the cells. When recovering from the culture solution, for example, the culture supernatant is filtered, centrifuged to remove insolubles, and then concentrated under reduced pressure, membrane concentrated, salting out using ammonium sulfate or sodium sulfate, methanol, ethanol, acetone, or the like. Fractional sedimentation, dialysis, heat treatment, isoelectric point treatment, gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography, and other various chromatographies (eg, gels by Sephadex gel (Pharmacia Biotech), etc.) Filtration, modified by combining DEAE Sepharose CL-6B (Pharmacia Biotech), Octyl Sepharose CL-6B (Pharmacia Biotech), CM Sepharose CL-6B (Pharmacia Biotech) It is possible to get a QQGDH of purified product. On the other hand, when the cells are collected from the cells, the cells are collected by filtration, centrifugation, etc. of the culture solution, and then the cells are subjected to a pressure treatment, a mechanical method such as ultrasonic treatment, or an enzymatic method such as lysozyme. After destruction by the method, a purified product of the modified PQQGDH can be obtained by performing separation and purification in the same manner as described above. In addition, PQQGDH can be solubilized by adding a chelating agent such as EDTA and a surfactant, if necessary, and separated and collected as an aqueous solution.
The purified enzyme is preferably purified to such an extent that it shows a single band in electrophoresis (SDS-PAGE).
[0037]
The purified enzyme obtained as described above can be distributed in a powder form by, for example, freeze-drying, vacuum drying, spray drying, or the like. At this time, the purified enzyme may be dissolved in a phosphate buffer, Tris-HCl buffer or GOOD buffer in advance. A buffer suitable for the buffer used here is GOOD buffer, and PIPES, MES or MOPS buffer is particularly preferred.
[0038]
The modified PQQGDH of the present invention has an action of catalyzing a reaction of oxidizing glucose to produce δ-gluconolactone using PQQ as a coenzyme. Such enzyme activity can be determined by the amount of PQQ reduced by oxidation of glucose by PQQGDH by a color reaction of a redox reagent. As the coloring reagent, for example, PMS-DCIP, 1-methyoxy-PMS-XTT, potassium ferricyanide and the like can be used.
In the present invention, the PQQGDH activity is measured under the following conditions using the following reagents in principle.
<Reagent>
10 mM MOPS (pH 7.0)
1 mM 1-methodoxy PMS (phenazine methosulfate)
0.25 mM XTT
5 mM glucose
<Measurement conditions>
After preliminarily heating 140 μl of the above reagent mixture at 25 ° C. for about 5 minutes, 0.1 ml of the enzyme solution is added and mixed gently. Thereafter, recording is performed for 15 minutes using a spectrophotometer controlled at 25 ° C. with water as a control, and the change in absorbance at 410 nm per minute is measured from the linear portion. In the blind test, distilled water is added to the reagent mixture instead of the enzyme solution, and the change in absorbance is measured in the same manner. The amount of enzyme of 1/2 μmol of formazan produced per minute under the above conditions is defined as 1 unit (U).
[0039]
The selectivity of the modified PQQGDH of the present invention for glucose is determined by measuring enzyme activity in the same manner as described above using various sugars such as lactose, maltose, galactose, sucrose, and xylose as substrates, and using glucose as a substrate. It can be evaluated by examining the relative activity to the activity.
[0040]
A kit for measuring glucose can be constructed using the modified PQQGDH of the present invention. That is, another aspect of the present invention provides a glucose measurement kit containing the modified PQQGDH. Such a kit can contain, in addition to the modified PQQGDH, a solution such as a buffer solution required for measurement, a glucose solution as a standard, and the like.
[0041]
Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.
[Example 1] Isolation of chromosomal DNA
Chromosomal DNA of Acinetobacter calcoaceticus IFO 12552 strain was isolated by the following method. The strain was cultured in 10 mL of LB medium at 30 ° C. with shaking overnight, and then collected by centrifugation (15000 rpm, 10 minutes). Chromosomal DNA was extracted and purified from the obtained cells using Dneasy tissue kit (Qiagen), and dissolved in TE buffer.
[0042]
Example 2 Preparation of DNA Fragment Containing Gene Encoding PQQGDH and Construction of Recombinant Vector Containing the DNA Fragment
Using the chromosomal DNA obtained in Example 1 as a template, the DNA region containing the target PQQGDH gene was amplified by polymerase chain reaction (PCR) using the following primers. The primers used herein are Acinetobacter sp. M. It was designed based on the base sequence of soluble PQQGDH derived from D 79.41 strain (AM Cleton-Jansen et al., Mol. Gen. Genet., 217, 430 (1989)).
Forward primer: 5'-ACAAATCATATAGAGAACTCG-3 '(SEQ ID NO: 5)
Reverse primer: 5'-TTACTTAGCCTTATAGGTGAACTTAATGAGAGATCCTGGGG-3 '(SEQ ID NO: 6)
The PCR was performed in a solution having the composition shown in Table 1 below, and the reaction was performed at 94 ° C. for 2 minutes, followed by 30 cycles of reactions at 94 ° C. for 30 seconds, 48 ° C. for 30 seconds, and 72 ° C. for 2 minutes. The reaction was carried out for minutes.
[0043]
[Table 1]
TAKARA LA-taq 0.5 μL
10-fold buffer 5 μL
25 mM MgCl ₂ 5 μL
dNTP mix (2.5 mM) 8 μL
Forward primer (10 pmol / μL) 1 μL
Reverse primer (10 pmol / μL) 1 μL
template
H ₂ Adjust to 50 μL with O
[0044]
The obtained amplified gene fragment was ligated to pGEM-T Easy vector (Qiagen), and Escherichia coli JM109 strain was transformed with this plasmid. The obtained plasmid was designated as pTGEM-GDHB.
[0045]
[Example 3] Determination of base sequence of soluble PQQGDH of Acinetobacter calcoaceticus IFO 12552
The nucleotide sequence of the gene fragment inserted into the plasmid pTGEM-GDHB obtained in Example 2 was determined using a BigDye Terminator Sequencing Kit (manufactured by Applied Biosystems). The determined base sequence and amino acid sequence are as shown in SEQ ID NO: 2 and SEQ ID NO: 1, respectively. The base sequence and the amino acid sequence including the signal region are shown in SEQ ID NO: 4 and SEQ ID NO: 3, respectively. The molecular weight of the protein calculated from the amino acid sequence was about 50,000, which was almost identical to the molecular weight of soluble PQQGDH of Acinetobacter calcoaceticus.
[0046]
[Example 4] Construction of expression vector containing PQQGDH gene
Using the plasmid pTGEM-GHDB obtained in Example 2 as a template, PCR was performed using the following primers.
Forward primer: 5'-GCGGCCGCGAATTCATGAATAAACATTTATTGGCTAAAAATTCTTTAT-3 '(SEQ ID NO: 7)
Reverse primer: 5'-GCGGCCGCCTGCAGCTATTACTTAGCCTTATAGGTGAACTTAATGAGAGATCCTGGGG-3 '(SEQ ID NO: 8)
The PCR was performed in a solution having the composition shown in Table 2 below, and the reaction was performed at 94 ° C. for 2 minutes, followed by 30 cycles of reactions at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 2 minutes. The reaction was carried out for minutes.
[0047]
[Table 2]
Sigma KlenTaq 0.5 μL
10-fold buffer 5 μL
dNTP mix (10 mM) 2.5 μL
Forward primer (10 pmol / μL) 2 μL
Reverse primer (10 pmol / μL) 2 μL
template
H ₂ Adjust to 50 μL with O
[0048]
The obtained amplified gene was cut with restriction enzymes EcoRI and PstI. The cleaved gene fragment was ligated to an expression vector, and Escherichia coli JM109 strain was transformed with this plasmid. The obtained expression plasmid was designated as pUK-GDHB (S) (FIG. 3). When the transformant was cultured overnight at 30 ° C. in 100 mL of LB medium containing 100 μg / mL ampicillin and 6 μM PQQ, production of PQQGDH was observed. The molecular weight of PQQGDH calculated from the amino acid sequence was about 50,000, which was almost the same as the molecular weight of Acinetobacter calcoaceticus IFO 12552.
[0049]
Example 5 Production of Acinetobacter calcoaceticus PQQGDH Gene Product
One liter of the LB medium was dispensed into a 500-mL flask by 50 mL, and autoclaved at 121 ° C. for 20 minutes. After cooling, a 100 mg / mL ampicillin solution which had been separately sterile-filtered and 50 μL of isopropylgalactopyranoside were added, and a colony having the PQQGDH expression vector pUK-GDHB (S) was inoculated. Then, shaking culture was performed at 30 ° C. for 16 hours. The PQQGDH activity at the end of the culture was about 1 U / mL.
[0050]
The cells recovered by centrifuging the culture after the culture were suspended in 10 mM Tris-HCL (pH 7.5). Next, the cell suspension was subjected to ultrasonic crushing and then centrifuged again to obtain a supernatant as a crude enzyme solution. This crude enzyme solution was treated with a cationic flocculant, desalted and concentrated, and then separated and purified by DEAE Sepharose (Pharmacia Biotech) and CM Sepharose (Pharmacia Biotech) column chromatography to obtain a purified enzyme.
[0051]
PQQGDH obtained by the above method showed a substantially single band in electrophoresis, and the specific activity at this time was about 1600 U / mg. The properties of the obtained PQQGDH are shown below.

[0052]
[Example 6] Addition of C-terminal histidine tag of PQQGDH
First, a histidine tag was introduced downstream of the PQQGDH gene, and then immediately after that, PCR was performed using a primer to which a stop codon was added. Using pUK-GDHB (S) obtained in Example 4 as a template, PCR was performed using the following primers.
Forward primer: 5'-TCACATGTTCTTTCTCGCGTTATC-3 '(SEQ ID NO: 9)
Reverse primer: 5'- ATGGTGATGGGTGATGGGTG CTTAGCCTTATAGGTGAACTTAATGAGA-3 ′ (SEQ ID NO: 10, the underlined is the sequence of the 6 × histidine tag)
PCR was performed under the same composition and conditions as in Example 4.
Subsequently, PCR was performed using the following primers to introduce a translation termination codon downstream of the histidine tag using the amplified gene as a template.
Forward primer: 5'-TCACATGTTCTTTCTCGCGTTATC-3 '(SEQ ID NO: 9)
Reverse primer: 5'-GCGGCCGCCTGCAGCTATTAATGGTGATGGTGCTTAGGCCTTA-3 '(SEQ ID NO: 11)
PCR was performed with the same solution composition and conditions as in Example 4.
[0053]
The amplified gene obtained by adding a histidine tag and a stop codon downstream of the PQQGDH gene was digested with EcoRI and PstI. The cleaved gene fragment was ligated to an expression vector, and Escherichia coli JM109 strain was transformed with this plasmid. In addition, the obtained expression plasmid was set to pUK-GDHB (S) C-His. When the transformant was cultured overnight at 30 ° C. in 100 mL of LB medium containing 100 μg / mL ampicillin and 6 μM PQQ, production of PQQGDH was observed. The molecular weight of PQQGDH calculated from the amino acid sequence was about 50,000, which was almost the same as the molecular weight of Acinetobacter calcoaceticus IFO 12552. Furthermore, when the general properties were compared with the wild-type PQQGDH obtained in Example 5 as a control, it was found that they were equivalent.
[0054]
[Example 7] Construction of mutant PQQGDH gene library
Using the plasmid pUK-GDHB (S) C-His obtained in Example 6 as a template, error-prone PCR was performed using the following primers to insert a mutation.
Forward primer: 5'-TCACATGTTCTTTCTCGCGTTATC-3 '(SEQ ID NO: 9)
Reverse primer: 5'-GGCGCGTACTATGGTTGCTTTGA-3 '(SEQ ID NO: 12)
The conditions for mutagenesis are as follows. The PCR was carried out in a solution having the composition shown in Table 3 below. The reaction was performed at 94 ° C. for 5 minutes, followed by 25 cycles of reactions at 94 ° C. for 1 minute, 50 ° C. for 1 minute, and 70 ° C. for 2 minutes, and finally at 72 ° C. for 7 minutes. The conditions for carrying out the reaction were used.
[0055]
[Table 3]
0.5 mM 100 mM Tris-HCl (pH 8.3)
500 mM KCl 5 μL
1 mg / mL BSA 2.5 μL
(15-100 mM) MgCl ₂ 5 μL
1 mM MnCl ₂ 1.25 to 3.75 μL
10 mM ATP 0.5 to 1.55 μL
10 mM TTP 0.5-5.0 μL
10 mM GTP 1.0-5.0 μL
10 mM CTP 0.5-5.0 μL
Forward primer (20 pmol / μL) 2.5 μL
Reverse primer (20 pmol / μL) 2.5 μL
Dimethylsulfoxide 0-7.5 μL
0.2 ng / μL template 5 μL
rTaq Polymerase 0.5 μL
H ₂ Adjust to 50 μL with O
[0056]
The amplified gene fragment into which the mutation was introduced was cut with EcoRI and PstI, and replaced with the wild-type gene of pUK-GDHB (S). Escherichia coli JM109 was transformed with the thus obtained plasmid to construct a mutant gene library.
[0057]
The obtained colony was inoculated into 600 μL of Circle Grow Medium (Stratagene) containing 100 μg / mL of ampicillin, 5 μM of PQQ and 1 mM of calcium chloride in a 96-well deep groove microplate for culture, and overnight at 37 ° C. The cells were cultured under shaking conditions of 200 rpm. Thereafter, the cells were collected by centrifugation (3000 rpm, 4 ° C., 30 min), and reconstituted in 600 μL of a 3-fold diluted lysate (50 mM Tris buffer, pH 8.0, 2 mM EDTA, 1 mg / mL lysozyme, 15 μM PQQ). Suspended. Thereafter, the mixture was shaken at 37 ° C. for 1 hour, and 30 μL of a 20 mM calcium chloride solution was added. The lysate was transferred to a filter plate (manufactured by Corning) and centrifuged (2000 rpm, 4 ° C., 5 min). The obtained supernatant was used for the following screening.
[0058]
The 96-well microplate used for screening was previously treated by the following method. That is, first, 100 μL of 15 ng / μL Protein A / G solution was added to a 96-well microplate (manufactured by Coaster), and the mixture was incubated at 37 ° C. for about 2 hours. Next, the plate was washed three times with a PBS solution, 150 μL of a blocking solution was added, and the mixture was allowed to stand at 37 ° C. for about 2 hours to obtain a screening micropirate. The above-mentioned centrifuged supernatant was added to a 96-well microplate containing a previously prepared 80 μL of 0.1 ng / μL mouse IgG1 anti-5 × histidine-tagged monoclonal antibody, mixed, and 100 μL of the blocking solution was washed and removed with a PBS solution. The plate was added to a microplate for incubation, and incubated at 25 ° C. for 1 hour. After that, the plate was washed three times with PBS and mixed with 140 μL of a PQQGDH assay solution (3.5 mL of 4 mM 1-methodyl PMS, 3.5 mL of 1 mM XTT solution, 3.5 mL of 20 mM maltose or glucose solution, 3.5 mL of 40 mM MOPS-NaOH pH 7.0). Liquid) was added and the absorbance at 410 nm was measured for 15 minutes at room temperature.
[0059]
In the first stage screening, a random single nucleotide substitution library was screened using maltose as a substrate, and clones expressing mutants in which the enzyme activity was reduced to about 80% or less of the wild type were identified. Next, the identified mutant was screened using glucose as a substrate, and a clone expressing a mutant having a large decrease in the reactivity to maltose compared to the reactivity to glucose was obtained. When the nucleotide sequences of these mutant PQQGDH genes were determined, glycine at position 75 was arginine, glycine at position 295 was glutamic acid, methionine at position 342 was valine, alanine at position 351 was threonine, glutamine at position 168 was histidine, and leucine at position 169. It was found that glutamine and tryptophan at position 347 were mutated to arginine. As shown in Table 4 below, all of the modified PQQGDH obtained in the present invention had reduced specificity to maltose as compared to the wild type. The activity of wild-type PQQGDH when glucose was used as a substrate was defined as 100, and the reactivity to maltose was shown as a relative value to this. As a control, the histidine-tagged wild-type PQQGDH obtained in Example 6 was used.
The numbers in the column of amino acid substitution in Table 4 indicate the amino acid number, the symbol on the left side of the number indicates the type of amino acid in the wild type, and the right side of the number indicates the type of amino acid after mutation. Therefore, for example, Gly75Arg means a variant in which glycine at the 75th amino acid is substituted with arginine.
[0060]
[Table 4]

[0061]
[Example 8] Introduction of site-specific mutation into wild-type PQQGDH gene and examination of substrate specificity of modified PQQGDH
Using the QuikChange Site-directed Mutagenesis kit (Stratagene), a site-specific mutation was introduced into the structural gene of PQQGDH derived from Acinetobacter calcoaceticus represented by SEQ ID NO: 4. The site where the mutation was introduced in Example 7, that is, each site of 75th glycine, 295th glycine, 342th methionine, 351th alanine, 168th glutamine, 169th leucine, 347th tryptophan The conditions were such that the nucleotide sequence encoding the amino acid was randomly substituted so as to encode 20 types of amino acids. Table 5 shows the sequences of the primers that substitute each site with 20 types of amino acids. In Table 5, F and R at the end of each sequence name indicate forward and reverse, respectively.
[0062]
[Table 5]
Array name: Array
GDHB / G100-F: 5'-GTAAATGATGCTGATNNNCAAAACGGTTTTATTGGG-3 '(35mer, SEQ ID NO: 13)
GDHB / G100-R: 5'-CCCAATAAAACCGTTTTGNNNATCAGCATCATTACTAC-3 '(35mer, SEQ ID NO: 14)
GDHB / G320-F: 5'-CAAAATTAAAGATTTANNNNCAAATGGTTTAAAAGTGGC-3 '(38mer, SEQ ID NO: 15)
GDHB / G320-R: 5′-GCCACTTTTAAAACCATTTTGNNNNTAATCTTTAATTTG-3 ′ (38mer, SEQ ID NO: 16)
GDHB / M367-F: 5'-CCAACCTgTggggATNNNNACCTACATTTgCTgg-3 '(33mer, SEQ ID NO: 17)
GDHB / M367-R: 5'-CCAgCAAATgTAggTNNNATCCCCACAggTTgg-3 '(33mer, SEQ ID NO: 18)
GDHB / A376-F: 5'-gCCAACggTTNNNNCCgTCATCTgCTTATgTCTA-3 '(33mer, SEQ ID NO: 19)
GDHB / A376-R: 5'-TAgACATAAgCAgATgACggNNNNACCgTTggC-3 '(33mer, SEQ ID NO: 20)
GDHB / Q193-F: 5'-GATCAGGGGCGTAACNNNCTGGCTTATTTTATTC-3 '(33mer, SEQ ID NO: 21)
GDHB / Q193-R: 5'-GAATAAATAAGCCAGNNNGTTTACGCCCCTGATC-3 '(33mer, SEQ ID NO: 22)
GDHB / L194-F: 5'-GGGGCGTAACCAGNNNGCTTTATTTATTCTTACC-3 '(33mer, SEQ ID NO: 23)
GDHB / L194-R: 5'-GGTAAGAATAAAATAAGCNNNCTGGTTACGCCCC-3 '(33mer, SEQ ID NO: 24)
GDHB / W372-F: 5′-ggATATgACCTACATTTgCNNNCCAACggTTgCgCCg-3 ′ (37mer, SEQ ID NO: 25)
GDHB / W372-R: 5'-CggCgCAACCgTTggNNNNgCAAATgTAggTCATATCC-3 '(37mer, SEQ ID NO: 26)
[0063]
1 μL of PfuTurbo DNA polymerase (2.5 U / μL) attached to the QuikChange Site-directed Mutagenesis kit, 125 ng of each forward and reverse primer, dNTP mix, 10 ng of pUK-GDHB (S) and 1/10 volume PCR The buffer was mixed. The reaction conditions were 95 ° C for 30 seconds, followed by 16 cycles of 95 ° C for 30 seconds, 55 ° C for 1 minute, and 68 ° C for 10 minutes. After completion of the reaction, 1 μL of a restriction enzyme DpnI (10 U / μL) attached to the kit was added to remove the template DNA, and the mixture was incubated at 37 ° C. for 1 hour. Escherichia coli XL1-Blue was transformed with 1 μL of the reaction product to obtain a colony.
[0064]
Next, the obtained transformant colonies were inoculated into 600 μL of Circle Grow medium (Stratagene) and screened in the same manner as in Example 7. As a result, the nucleotide sequence of the selected mutant strain was determined, and it was found that various mutants having amino acid substitutions were obtained as shown in Table 6 below. On the other hand, each of these mutants was cultured overnight at 30 ° C. in 2 mL of LB medium containing 100 μg / mL of ampicillin, 0.01 mM of isopropylthiogalactoside and 6 μM of PQQ. After the culture, the cells were collected by centrifugation, and resuspended in 500 μL of 10 mM MOPS buffer (pH 7.0) containing 6 μM PQQ. Subsequently, 250 mg of glass beads (manufactured by Yasui Kikai Co., Ltd.) was added to the suspension solution, and the cells were disrupted with a multi-beads shocker (manufactured by Yasui Kikai Co., Ltd.) (2000 rpm, operation 60 seconds, interval 30 seconds, 7 cycles).
[0065]
The solution after the disruption of the cells was centrifuged (15000 rpm, 10 minutes, 4 ° C.), and the supernatant was purified using His MicroSpin Purification Module (Amersham Pharmacia Biotech). As a result, 200 μL of a purified enzyme solution was obtained. PQQ was added to this purified enzyme solution to a concentration of 5 μM, and left for 1 hour or more in the presence of 1 mM calcium chloride to perform holation.
[0066]
The substrate specificity of each modified PQQGDH obtained as described above was measured by the following method. Specifically, after dispensing 50 μL of the purified enzyme solution, 130 μL of the assay solution (60 μL of 20 mM MOPS pH 7.0 dissolved in 1-methodoxy PMS, 70 μL of 20 mM and 171 μM XTT, and 20 μL of D-glucose solution) were added. PQQGDH activity was measured according to the method described in Example 5 to a final concentration of 5 mM. As a control, the histidine-tagged wild-type PQQGDH obtained in Example 6 was used. Table 6 shows the measurement results. As is clear from Table 6, any modified PQQGDH has reduced reactivity to maltose and galactose as compared to wild-type PQQGDH, and some modified PQQGDH also has reduced reactivity to lactose and galactose at the same time. Obtained. The numbers in the column of amino acid substitution in Table 6 indicate the amino acid number, the symbol on the left side of the number indicates the type of amino acid in the wild type, and the right side of the number indicates the type of amino acid after mutation. Therefore, for example, Gly75 Trp means a variant in which glycine at the 75th amino acid is replaced with trypsin.
[0067]
[Table 6]

[0068]
[Example 9] Introduction of multiple mutations using mutation scrambling method and examination of substrate specificity of multiple substitution products
By preparing a multi-substituted library having all possible combinations of amino acid substitutions effective for improving the substrate specificity, which was clarified in Example 8, by a mutation scrambling method, each amino acid substitution was independently performed. An attempt was made to obtain a variant having a higher substrate specificity as compared with the case of having the same. For the preparation of the multiple substitution library, the previously described bias mutation scrambling method (The 39th Annual Meeting of the Biophysical Society of Japan, held on October 6, 2001, abstract number 1P057, Novartis Pharma; Biopolymer, No. 64 ( 2002), pp. 95-105). The bias mutation scrambling method is a modification of the mutation scrambling method, and has a feature that the introduction probability of the combined amino acid substitution can be freely set. In this example, G75, G295, M342, and A351, which are located near the substrate binding site based on the estimation from the structural model and are expected to contribute to the substrate specificity in particular, have a high probability of 80%. For Q168, L169, and W347, which are considered to have structural features, conditions were set such that mutations were introduced with a probability of 50%. Specifically, the entire length of the PQQGDH gene was divided into the five fragments shown in FIG. 4 having overlapping ends and containing 1 to 3 amino acid substitutions identified in Example 8, and each amino acid substitution was as described above. Mutation scrambling was performed at the mixing ratio of each fragment shown in FIG. 5 so that the PQQGDH gene was introduced at the probability of reconstructing the PQQGDH gene.
[0069]
In the mutation sprangling method, PCR was first performed using a wild-type gene as a template to obtain a partially amplified product (FIG. 4). Table 7 shows the composition of the reaction solution used for PCR. Primers containing sequences corresponding to Q168 and L169 with G75 interposed therebetween were used.
[0070]
[Table 7]
10 μbuffer for AccuTaq (manufactured by Sigma) 2 μL
1 μL of 10 mM dNTP
Forward primer (10 pmol / μL) 1.2 μL
Reverse primer (10 pmol / μL) 1.2 μL
Template (10 ng / μL) 0.5 μL
dH ₂ O 13.9 μL
AccuTaq (5 units / μL) 0.2 μL
20 μL in total
[0071]
Forward primer: 5'-TCACATGTTCTTTCTCGCGTTATC-3 '(SEQ ID NO: 9)
S-primer (G) -10-QL: 5'-AAgAATAAAATAAgCCAgTTGgTTACgCCCCTgATC-3 '(SEQ ID NO: 27)
The reaction conditions were 98 ° C. for 30 seconds, then 30 cycles of 94 ° C. for 5 seconds, 60 ° C. for 20 seconds, and 68 ° C. for 90 seconds, and finally to 68 ° C. for 10 minutes. Next, PCR was performed under the same reaction conditions using a modified gene having a mutation in G75 as a template to obtain a partially amplified by-product in the same region as above. Here, as can be seen from Table 6, there are 16 combinations of amino acid substitutions at the G75 site, the Q168 site, and the L169 site. The partial amplification by-products corresponding to all these combinations were obtained by using the eight types of primers (S-primer (G) -10-QL to S-primer (G) -10-YF) shown in FIG. It was prepared by performing a gene amplification reaction using two modified genes as templates. The obtained partial amplification products were mixed at the mixing ratio shown in FIG. 5 to obtain fragment 1 (fragment 1, see FIG. 4). Fragment 2 to Fragment 5 (Fragment 2, Fragment 3, Fragment 4, Fragment 5; see FIG. 4) were obtained in the same manner as Fragment 1 by using the primers shown in FIG. Was prepared by mixing at the ratios shown in FIG.
[0072]
Next, the above fragments were mixed in equal amounts, and PCR was carried out in a solution composition shown in Table 8 below. The reaction conditions were 98 ° C. for 30 seconds, followed by 5 cycles of 94 ° C. for 5 seconds, 60 ° C. for 20 seconds, and 68 ° C. for 2 minutes. After the completion of the PCR reaction, 2.4 μL of each of the primers (10 pmol / μL) (SEQ ID NOs: 7 and 8) was added, and PCR was performed again. The reaction conditions at this time were 94 ° C. for 5 seconds, then 60 ° C. for 20 seconds, and 68 ° C. for 2 minutes for 25 cycles, and finally 72 ° C. for 10 minutes.
[0073]
[Table 8]
X μL of mixed fragments
10 μbuffer for AccuTaq (manufactured by Sigma) 2 μL
1 μL of 10 mM dNTP
dH ₂ O (14.4-X) μL
AccuTaq (5 units / μL) 0.2 μL
17.6 μL in Total
[0074]
The library of the modified gene obtained by the above operation was cut with EcoRI and PstI, replaced with the wild-type gene of pUK-GDHB (S), and transformed into Escherichia coli. After the transformant colonies were cultured by the method described in Example 8, screening was performed using PQQGDH activity and substrate specificity as indices. For clones showing improved substrate specificity, the nucleotide sequence of the modified PQQGDH possessed by the clone was determined. As a result, as shown in the table of FIG. 6, it was found that a plurality of modified PQQGDHs having different combinations of amino acids to be substituted were obtained. In the table of FIG. 6, symbols such as G75 (GGA) mean that the amino acid position specified by the numeral is the amino acid specified by one alphabetic character on the left side of the numeral. The parentheses indicate the nucleotide sequence encoding the amino acid. On the other hand, for Phe (TTT) and the like, the amino acid position specified by the symbol (for example, L194 (CTG)) described at the top of the row in which it is located is replaced by the amino acid specified by the three letters of the alphabet Means The parentheses indicate the nucleotide sequence encoding the amino acid.
[0075]
Each of the transformants having the modified PQQGDH was purified by a method similar to that shown in Example 7, and then the reactivity to each substrate was measured. The measurement results are shown in the right column of the table in FIG. As is clear from this table, the obtained modified PQQGDH has significantly reduced reactivity to maltose as compared with the wild type. A similar decrease is also observed in the reactivity to lactose. Furthermore, the reactivity to galactose is greatly reduced in any of the modified PQQGDHs, and the reactivity is remarkably reduced in some of them. As described above, it was possible to obtain a modified PQQGDH having significantly reduced reactivity to maltose, lactose, and galactose, that is, extremely excellent substrate specificity. From this result, it was found that by combining the mutations at the amino acid positions shown in the table of FIG. 6, a modified PQQGDH having more excellent substrate specificity can be produced as compared with the case of a single mutation.
[0076]
Here, in particular, NO. In one variant, the reactivity to maltose is reduced to 1/9 as compared to the wild type, and similarly, the reactivity to lactose is reduced to about 1/2 and the reactivity to galactose is reduced to about 1/9. ing. No. In the six variants, the reactivity to maltose is reduced to 以下 or less as compared with the wild type, and at the same time, the reactivity to lactose is reduced, and the reactivity to galactose is also reduced to about ４. . Similarly, no. Also in the 7 variant, the reactivity to maltose is reduced to about 1/5 compared to the wild type, and at the same time, the reactivity to lactose is also reduced to about 1/2, and the reactivity to galactose is substantially reduced. It turns out that there is no reactivity. As described above, no. 1, No. 6 and no. Comparison of the amino acid substitution sites in the seven variants suggests that the substitution of amino acids 342 and 351 is particularly important for improving the substrate specificity. Next, the substitution at the 295th amino acid is important, and the substitution at the 194th amino acid is expected to greatly contribute to the improvement of the substrate specificity.
[0077]
The present invention is not limited to the description of the embodiment of the present invention at all, and does not depart from the scope of the claims, and includes various modifications within a range that can be easily conceived by those skilled in the art. It is.
[0078]
【The invention's effect】
The modified pyrroloquinoline quinone-dependent glucose dehydrogenase provided by the present invention has high substrate specificity for glucose. Therefore, if such a modified enzyme is used, glucose can be quantified with high accuracy.
[0079]
[Sequence list]

[Brief description of the drawings]
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the sequence (not including a signal region) and the amino acid sequence (not including a signal region) of a gene encoding a pyrroloquinoline quinone-dependent glucose dehydrogenase derived from Acinetobacter calcoaceticus. FIG.
FIG. 2 shows a sequence (including a signal region) and an amino acid sequence (including a signal region) of a gene encoding a pyrroloquinoline quinone-dependent glucose dehydrogenase derived from Acinetobacter calcoaceticus. FIG.
FIG. 3 is a diagram showing the structure of an expression vector pUK-GDHB (S) used in Examples.
FIG. 4 is a table showing primers and amplification sites used in mutation scrambling in Examples.
FIG. 5 is a table showing the mixing ratio of each DNA fragment in mutation sprambling. The set bias rates were 0.8: G75, G295, M342, A351, 0.5: Q168, L169, W347. In addition, the bias rate is a probability that a certain amino acid substitution is introduced by the Mutation Scramble method.
FIG. 6 is a table summarizing amino acid substitution positions and substrate specificities of various modified PQQGDHs obtained by mutation scrambling in Examples.

Claims (21)

Based on the amino acid sequence of the corresponding wild-type enzyme, this corresponds to the first region consisting of amino acids 326 to 354 in the pyrroloquinoline quinone-dependent glucose dehydrogenase derived from Acinetobacter calcoaceticus. A modified pyrroloquinoline quinone-dependent glucose dehydrogenase having an amino acid sequence in which one or more amino acids are substituted in a region where the amino acid is substituted. The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to claim 1, wherein two or more amino acids are substituted in a region corresponding to the first region. The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to claim 1, wherein amino acids corresponding to amino acids 342 and 351 in the first region are substituted with other amino acids, respectively. In the pyrroloquinoline quinone-dependent glucose dehydrogenase derived from Acinetobacter calcoaceticus, one or more amino acids are other amino acids in a region corresponding to the second region consisting of amino acids 278 to 320. The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to any one of claims 1 to 3, which is substituted with: The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to claim 4, wherein the amino acid corresponding to the 295th amino acid in the second region is substituted with another amino acid. In a region corresponding to the third region consisting of amino acids 162 to 197 in a pyrroloquinoline quinone-dependent glucose dehydrogenase derived from Acinetobacter calcoaceticus, one or more amino acids are other amino acids The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to any one of claims 1 to 5, which is substituted with: One selected from the group consisting of amino acids corresponding to amino acids 75, 168, and 347 in a pyrroloquinoline quinone-dependent glucose dehydrogenase derived from Acinetobacter calcoaceticus. The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to any one of claims 1 to 6, wherein the amino acid is substituted with another amino acid. The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to any one of claims 1 to 7, which is derived from Acinetobacter calcoaceticus. In the amino acid sequence of SEQ ID NO: 1, a modified pyrroloquinoline quinone-dependent glucose having an amino acid sequence in which one or more amino acids are substituted with another amino acid in a first region consisting of amino acids 326 to 354. Dehydrogenase. The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to claim 9, wherein two or more amino acids are substituted in the first region. The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to claim 9, wherein amino acids 342 and 351 in the first region are each substituted with another amino acid. The modified form according to any one of claims 9 to 11, wherein one or more amino acids are substituted with another amino acid in the second region consisting of amino acids 278 to 320 of the amino acid sequence of SEQ ID NO: 1. Pyrroloquinoline quinone-dependent glucose dehydrogenase. The modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to claim 12, wherein the 295th amino acid in the second region is substituted with another amino acid. The modified form according to any one of claims 9 to 13, wherein at least one amino acid is substituted with another amino acid in the third region consisting of amino acids 162 to 197 of the amino acid sequence of SEQ ID NO: 1. Pyrroloquinoline quinone-dependent glucose dehydrogenase. 15. The amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid selected from the group consisting of amino acids 75, 168, and 347 is substituted with another amino acid. A modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to any one of the above. A modified pyrroloquinoline quinone-dependent glucose dehydrogenase having a reactivity to maltose of 40% or less and a reactivity to galactose of 10% or less based on the reactivity to glucose. An isolated polynucleotide encoding the modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to any one of claims 1 to 16. A vector comprising the polynucleotide according to claim 17. A transformant having the polynucleotide according to claim 17. A step of culturing the transformant according to claim 19 in a state in which the polynucleotide can be expressed, and a step of separating an expression product of the polynucleotide, wherein the step of culturing the modified pyrroloquinoline quinone-dependent glucose dehydrogenase comprises Production method. A glucose measurement kit comprising the modified pyrroloquinoline quinone-dependent glucose dehydrogenase according to any one of claims 1 to 16.

Images