JP5199995B2 - ガラクトース転移活性を有するβ−ガラクトシダーゼ - Google Patents
ガラクトース転移活性を有するβ−ガラクトシダーゼ Download PDFInfo
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- JP5199995B2 JP5199995B2 JP2009502198A JP2009502198A JP5199995B2 JP 5199995 B2 JP5199995 B2 JP 5199995B2 JP 2009502198 A JP2009502198 A JP 2009502198A JP 2009502198 A JP2009502198 A JP 2009502198A JP 5199995 B2 JP5199995 B2 JP 5199995B2
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- galactosidase
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Description
材料及び方法
本試験において使用した全ての試薬および培地はSigma(ドーセット、英国)、Invitrogen(ペーズリー、英国)、Oxoid(ベージングストーク、英国)、Qiagen(ウェストサセックス、英国)及びPromega(サウサンプトン、英国)から入手した。
ビフィドバクテリウム・ビフィダム株(NCIMB 41171)は、Microbankチューブ中の低温ビーズ上で−70℃にて維持した。後の実験のため、該株を、Wilkinson Chalgren(WC)アガー(Oxoid、英国)及びTPY培地(トリプチケースファイトン酵母エキス培地)上で回復させ、嫌気的条件下(CO2およびN2がそれぞれ80%および20%)にて37℃で48時間増殖させた。グラム染色によりコロニーの形態、およびコンタミネーションがないことを確認した。
本試験で用いた大腸菌(Escherichia coli)DH5a株は、常法に従い、Luria Bertani(LB)アガーまたはLBブロス中で37℃の好気的条件下でインキュベートし(Sambrook J.and Russell W.D.(2001).Molecular Cloning:A Laboratory Manual.Cold Spring Harbor Laboratory Press、New York)、必要な場合には、抗生物質(100μg/mlのアンピシリン及び/または15μg/mlのクロラムフェニコール)及び40μlの2% X−β−Gal、7μlの20% IPTG(イソプロピル−β−D−チオガラクトシド)を、事前に作製した90mmアガープレート表面上に塗付することにより補充した。
以下の方法により、ビフィドバクテリウム・ビフィダム株(NCIMB 41171)からゲノムDNAを単離した。当該方法において、染色体DNAは、100mlのWC嫌気性菌培養液から回収した細胞ペレットより調製した。細胞を10mlのTESバッファー(10mM Tris−HCl、10mM EDTA、10mM NaCl、pH8)に再懸濁し、200μlのリゾチーム/ムタノリシン混合液(4:1、リゾチーム10 mg/ml、ムタノリシン1 mg/ml)で37℃にて30分間処理した。次いで、該細胞を、200μlのプロテイナーゼK(20 mg/ml)および200μlのRNase(いずれも10 mg/ml)で処理し、65℃で1時間インキュベートした。最後に該細胞を、2mlの10%SDSで処理して、65℃で15分間インキュベートした。12mlのフェノール/クロロホルムを添加し、水相が中間相から容易に分離できるようになるまで抽出を繰り返した。イソプロパノールを用いてゲノムDNAを沈殿させ、10mM Tris−HCl、1mM EDTA(pH8)に再懸濁した。次いで、該ゲノムDNAを制限酵素で処理し、同一の酵素で処理しアルカリホスファターゼ処理を施したpSP72にライゲーションした。B.ビフィダムのゲノムDNAの制限酵素処理は、EcoRI、PstI、BamHI、SmaI及びKpnIを用いて行った。ライゲーションミックスを用いて大腸菌 DH5aを形質転換し、β−ガラクトシダーゼ陽性クローンを、X−Gal含有プレート上の青色のコロニーとして同定した。
本試験において、クローニングと発現にはpSP72ベクターを用いた(Promega、英国)(Krieg、P.A.and Melton、 D.A.(1987). In vitro RNA synthesis with SP6 RNA polymerase.Methods in Enzymology.155:397−415)。
原核生物のDNA内に高頻度に現れる6個のヌクレオチドから成る配列を認識する6種類の制限酵素を用いて、ゲノムDNAを部分的に消化した。EcoRI、BamHI、PstI、KpnI、SmaI及びHindIIIは、それぞれ5’G/AATTC’3、5’G/GATCC’3、5’CTGCA/G’3 、5’GGTAC/C3’、5’CCC/GGG3’及び5’A/AGCTT3’の配列を特異的に認識するタイプII制限エンドヌクレアーゼであり、これらの配列内で2本鎖を切断し、EcoRI、BamHI及びHindIIIについては、それぞれ、4個のヌクレオチドAATT、GATC、AGCTから成る5’突出を生じさせ、PstI及びKpnIは、それぞれACGT、GTACから成る3’突出を生じさせ、SmaIは、平滑末端を生じさせる。
ゲノムDNAサンプルの制限酵素処理は全てを37℃で2時間インキュベートし、最後に65℃で20分間インキュベートして熱により不活性化させた。次いで、反応物を室温にて冷まし、適当量のローディングバッファーを加え、密封ガラスキャピラリーを用いて穏やかに混和した。次いで、この溶液を0.8%アガロースゲルのウェルにローディングし(4〜5ボルト/cmの電力供給で14〜16時間)、消化したDNAのサイズを、1kbpのDNAスタンダード(Promega、英国)のサイズを用いて推計した(Sambrook J.Molecular Cloning:A Laboratory Manual(2002))。
前記反応混合物及びアガロースゲルからの断片の精製は、Qiagen製(ウェストサセックス、英国)のQIAEX gel extraction kitを用いて行った。プロトコルは、製造元のマニュアルに詳細に記載されている。
Qiaex gel extraction kitを用いてDNA断片を精製した後、これらの断片をCIAP処理したpSP72ベクターにライゲーションした。ライゲーションの際には、表1に示すように、適切量のDNAを滅菌した0.5 mlマイクロチューブに移した。
PstI処理した染色体DNAのライゲーション混合液からは、スクリーニングを行った約2500個の形質転換体の中から13個のβ−ガラクトシダーゼ陽性クローンが得られ、BamHI処理の場合には(スクリーニングを行った約1500個の形質転換体の中から)7個の陽性クローン、EcoRIの場合には(スクリーニングを行った約1300個の形質転換体の中から)3個の陽性クローン、KpnIの場合には(スクリーニングを行った約2000個の形質転換体の中から)7個の陽性クローン、SmaIの場合には(スクリーニングを行った約1600個の形質転換体の中から)3個の陽性クローン、そしてHindIIIの場合には(スクリーニングを行った約1200個の形質転換体の中から)2個の陽性クローンが得られた。
異なる種類のβ−ガラクトシダーゼ遺伝子を同定するため、以下の表に従って、陽性クローンから単離したプラスミドを消化した。
DNA配列決定は、BigDye Terminator v.3.0 cycle sequencing kit(Applied Biosystems、米国)を用いて、Sangerのジデオキシチェーンターミネーション法により行い、キャピラリー電気泳動が組み込まれた、蛍光に基づくDNA解析システムであるABI Prism 3100を用いて解析を行った。
P2のオープンリーディングフレーム(ORF)の位置は、NCBI(ウェブアドレスhttp://www.ncbi.nlm.nih.gov/gorf/gorf.html)のORF finderを用いて特定された。細菌の遺伝暗号を用い、フレームの長さを100bpと定めた。ヌクレオチド配列を、可能な6種類のフレーム全てについて翻訳し、β−ガラクトシダーゼと推定される配列をコードする738個のアミノ酸から成るオープンリーディングフレームを同定した(翻訳結果を図2に示す)。
ビフィドバクテリウム・ビフィダムNCIMB41171から単離してクローニングしたβ−ガラクトシダーゼ酵素を用いた、大腸菌宿主(DH5a株)内における合成
他に記載のない限り、以下に記載する合成は、細胞透過性を増加させ、その細胞膜を破壊することにより細胞を生存不能にするために、大腸菌試料(10、000gの遠心により回収)を2000ppmの濃度のトルエンで処理した大腸菌DH5a宿主細胞を用いて行った。該大腸菌試料は、実施例1「大腸菌株」に記載の通りに調製した。
β−ガラクトシダーゼを用いた合成は、初期ラクトース濃度を40%(w/w)とした基質で行った。合成溶液は、1g/lのTween80(ポリオキシエチレン(20)ソルビタンモノオレエート)を添加したpH6.0の0.1Mリン酸バッファー中に調整した。合成は、150rpmの振とう水浴中で40℃にて行った。特定の酵素試料の異なるpH値での活性測定(基質としてo−ニトロフェニル−β−D−ガラクトピラノシドを使用)に基づいて、特定の酵素の最適pHを選択した。
Claims (15)
- 配列番号2に示されるアミノ酸配列を有するタンパク質をコードする配列を有するDNA。
- 配列番号1に示される配列を有する、請求項1に記載のDNA。
- 請求項1または請求項2に記載のDNAによってコードされるβ−ガラクトシダーゼ酵素。
- 配列番号2に定められる配列を有するβ−ガラクトシダーゼ。
- 請求項1または請求項2に記載のDNAを含む組換えベクター。
- 発現ベクターである、請求項5に記載のベクター。
- 請求項1または請求項2に記載のDNAを含む宿主細胞。
- 請求項5または請求項6に記載のベクターを含む宿主細胞。
- 細菌細胞、酵母細胞または真菌細胞である、請求項7または請求項8に記載の宿主細胞。
- ビフィドバクテリウム属、ラクトコッカス属、ラクトバチルス属、エシェリキア属、バチルス属、及びアスペルギルス属から成る群より選択される微生物の細胞である、請求項9に記載の宿主細胞。
- ビフィドバクテリウム・ビフィダムの細胞、バチルス・スブチリスの細胞、バチルス・サーキュランスの細胞及びアスペルギルス・ニガーの細胞から成る群から選択される、請求項10に記載の宿主細胞。
- オリゴ糖混合物の製造のための、請求項3または請求項4に記載の酵素、または請求項7〜請求項11のうちいずれか1項に記載の細胞、の使用。
- 液乳、乾燥粉乳、乳幼児用ミルク、粉ミルク、アイスクリーム、ヨーグルト、チーズ、発酵乳製品、乳製品、フルーツジュース、飲料、乳児食、シリアル、パン、ビスケット、菓子類、ケーキ、食品サプリメント、栄養サプリメント、プロバイオティック食品、プレバイオティック食品、動物飼料、家禽飼料及び薬物から成る群より選択される製品の一部となるオリゴ糖混合物の製造のための、請求項3または請求項4に記載の酵素、または請求項7〜請求項11のうちいずれか1項に記載の細胞、の使用。
- 液乳、乾燥粉乳、乳幼児用ミルク、粉ミルク、アイスクリーム、ヨーグルト、チーズ、発酵乳製品、乳製品、フルーツジュース、飲料、乳児食、シリアル、パン、ビスケット、菓子類、ケーキ、食品サプリメント、栄養サプリメント、プロバイオティック食品、プレバイオティック食品、動物飼料、家禽飼料及び薬物から成る群より選択される製品の製造のための、請求項7〜請求項11のうちいずれか1項に記載の細胞、の使用。
- 請求項7〜請求項11のうちいずれか1項に記載の宿主細胞を、請求項3または請求項4に記載の酵素を発現させる条件下で、適切な培養培地中で培養すること、および、生じた酵素を前記培養物から回収することを含む、請求項3または請求項4に記載の酵素の製造方法。
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