JP4927332B2 - 酵母表面提示ベクターを利用して改良型酵素活性を有するリパーゼをスクリーニングする方法及びそのリパーゼ - Google Patents
酵母表面提示ベクターを利用して改良型酵素活性を有するリパーゼをスクリーニングする方法及びそのリパーゼ Download PDFInfo
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Description
本発明は、酵母表面提示ベクターを利用して、改良型酵素活性を有するリパーゼをスクリーニングする方法、及びその産物に関するものである。より詳細には、1)表面提示ベクターに遺伝子をクローニングする工程、2)変異誘発PCRにより工程1の変異リパーゼ遺伝子ライブラリーを作成する工程、3)工程2の変異遺伝子ライブラリー及び表面提示ベクターで宿主細胞を形質転換させる工程、及び4)形質転換された宿主細胞の表面に提示された変異リパーゼの活性を測定し、改善されたリパーゼ活性を有する変異リパーゼを選択する工程を含む、変異リパーゼのスクリーニング方法、及びそのような方法により作成された変異リパーゼに関する。
リパーゼ(lipase)は、カルボキシリックエステル水酸化酵素(carboxylic ester hydrolase)として食品産業、微細化合物合成、界面活性剤事業等に多く利用されている酵素である。多くの微生物由来のリパーゼ中でカンジダ・アンタークティカ(Candida antarctica)由来のリパーゼB(以下「CALB」と略称する)は、317個のアミノ酸で構成されていてα/β水酸化酵素の形態をしている。
本発明は、1)リパーゼ遺伝子を表面提示ベクターにクローニングする工程;2)前記工程1の表面提示ベクターのリパーゼ遺伝子を鋳型に変異誘発PCRを行い、変異リパーゼ遺伝子ライブラリーを作成する工程;3)前記工程2により増幅された変異遺伝子を表面提示ベクターと共に宿主細胞に形質転換させる工程;及び4)前記工程3の形質転換体表面に発現した変異リパーゼの活性を測定して活性が優れた変異リパーゼをスクリーニングする工程を含む、変異リパーゼスクリーニング方法を提供する。
1)リパーゼ遺伝子を表面提示ベクターにクローニングする工程;
2)前記工程1の表面提示ベクターのリパーゼ遺伝子を鋳型に変異誘発PCRを行い、変異遺伝子ライブラリーを作成する工程;
3)前記工程2により増幅された変異遺伝子を表面提示ベクターと共に宿主細胞に形質転換させる工程;及び
4)前記工程3の形質転換体表面に発現した変異リパーゼの活性を測定して活性が優れた変異リパーゼをスクリーニングする工程を含む、変異リパーゼスクリーニング方法に関するものである。
実際の、及び現状での本発明の好ましい態様を以下の実施例に示すように例示する。
本発明者らは、CALBをハンゼヌラ・ポリモルファ(H.polymorpha)の表面に発現させるかまたはタンパク質を細胞外に分泌させるベクターを構築した。
本発明者らは、CALBの変異株ライブラリーを構築するためにサッカロミセス・セレビシエ(S.cerevisiae)で報告されたことがあるインビボ組換え方法(Abecassisら、Nucleic.Acids.Res.,2000年、第28巻、E88頁)をハンゼヌラ・ポリモルファに応用してCALBライブラリーを構築した。
本発明者らは、CALBの変異株ライブラリーを構築するために、変異誘発PCR(error-prone PCR)方法とインビボ組換え方法を利用した。
本発明者らは、前記実施例3の過程で作成されたCALB変異株ライブラリーから高いリパーゼ活性を有するCALB変異株を選別しようとした。
本発明者らは、前記実施例4で最終的に獲得した3種の変異株から変異遺伝子を分析した。
本発明者らは、前記実施例4で選別したCALB変異株を利用してリパーゼの酵素活性を測定した。
本発明者らは、変異CALBの特性をより正確に分析するために野生型CALB及び変異CALBを分離精製した。
本発明者らは、変異タンパク質の特性変化が直接的にアミノ酸の変化によるものであるかどうか確認するために部位特異的変異誘発を利用した変異特性を分析した。
本発明者らは、CALBの最適培養条件を確立するために培養温度を変化させてCALBの活性を測定した。
酵素の最適な生産のためにサッカロミセス・セレビシエに変異CALBを発現した。
酵母を利用して改良CALBを大量生産するために5L発酵槽を利用して流加式培養することによりCALBの生産を最適化した。
前記で詳しく見たように、本発明の変異リパーゼをスクリーニングする方法を利用すると、高い活性の変異リパーゼを生産する形質転換体を容易に作成でき、前記形質転換体から作成した変異リパーゼは、形質転換された細胞の表面に固定化され、再生産が可能なだけではなく大量生産が可能であるため、食品産業、微細化合物合成及び界面活性剤事業等に有用に使用できる。
Claims (13)
- 配列番号:14に記載のアミノ酸配列からなるガンジダ・アンタークティカ リパーゼBにおいて、配列番号:14に記載のアミノ酸配列における219番目のロイシンがグルタミンに、及び/または278番目のロイシンがプロリンに置換されている、活性が増加した変異リパーゼタンパク質。
- 請求項1記載の変異リパーゼタンパク質をコードする遺伝子。
- 請求項2記載の遺伝子を含む発現ベクター。
- プロモーター遺伝子、分泌シグナル配列遺伝子、請求項2記載の遺伝子、ターミネーター遺伝子及び/または細胞壁を構成する表面提示媒介遺伝子を含む、請求項3記載の発現ベクター。
- プロモーター遺伝子が、GAPDH (glyceraldehyde-3-phosphate dehydrogenase)である、請求項4記載の発現ベクター。
- 分泌シグナル配列をコードする遺伝子が、AMY(alpha-amylase)またはキラートキシン(killer toxin)である、請求項4記載の発現ベクター。
- 細胞壁を構成する表面提示媒介遺伝子が、CWP1(cell wall protein 1)である、請求項4記載の発現ベクター。
- 請求項3記載の発現ベクターが導入されている形質転換体
- Korean Collection for Type Cultureにアクセッション番号:KCTC 10320BPでアクセッションされた、請求項8記載の形質転換体。
- Korean Collection for Type Cultureにアクセッション番号:KCTC 10321BPでアクセッションされた、請求項8記載の形質転換体。
- 請求項8記載の形質転換体を培養して請求項1記載の変異リパーゼタンパク質を生産する方法。
- 形質転換体がハンセヌラの場合、培養温度が25℃〜35℃である、請求項11記載の方法。
- 形質転換体がサッカロミセスの場合、培養温度が20℃〜28℃である、請求項11記載の方法。
Applications Claiming Priority (3)
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KR10-2002-0055575 | 2002-09-13 | ||
KR10-2002-0055575A KR100475133B1 (ko) | 2002-09-13 | 2002-09-13 | 효모 표면 발현 벡터를 이용하여 변이 리파제를스크리닝하는 방법 및 신규 변이 리파제 |
PCT/KR2003/001820 WO2004024954A1 (en) | 2002-09-13 | 2003-09-04 | Method for screening of a lipase having improved enzymatic activity using yeast surface display vector and the lipase |
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JP2005538711A JP2005538711A (ja) | 2005-12-22 |
JP4927332B2 true JP4927332B2 (ja) | 2012-05-09 |
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EP (1) | EP1546405B1 (ja) |
JP (1) | JP4927332B2 (ja) |
KR (1) | KR100475133B1 (ja) |
CN (1) | CN100595283C (ja) |
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JP5438259B2 (ja) * | 2006-04-13 | 2014-03-12 | Bio−energy株式会社 | カンジダ・アンタークティカ由来リパーゼbを細胞表層に提示する酵母 |
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US8759044B2 (en) | 2011-03-23 | 2014-06-24 | Butamax Advanced Biofuels Llc | In situ expression of lipase for enzymatic production of alcohol esters during fermentation |
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WO2002012509A1 (en) * | 2000-07-26 | 2002-02-14 | Korea Research Institute Of Bioscience And Biotechnology | Novel cell wall anchor proteins derived from yeast, genes thereof and cell surface expression systems using the same |
Also Published As
Publication number | Publication date |
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US20060275760A1 (en) | 2006-12-07 |
EP1546405A1 (en) | 2005-06-29 |
CN100595283C (zh) | 2010-03-24 |
EP1546405B1 (en) | 2012-02-29 |
CN1681942A (zh) | 2005-10-12 |
KR100475133B1 (ko) | 2005-03-10 |
KR20040024074A (ko) | 2004-03-20 |
AU2003261629A1 (en) | 2004-04-30 |
JP2005538711A (ja) | 2005-12-22 |
EP1546405A4 (en) | 2006-01-18 |
US7736643B2 (en) | 2010-06-15 |
ATE547517T1 (de) | 2012-03-15 |
WO2004024954A1 (en) | 2004-03-25 |
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