JP2012044992A - 植物バイオマスの加工方法 - Google Patents
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Abstract
【解決手段】リグノセルロース分解タンパク質を含み、且つリグノセルロース分解タンパク質の内部にインテインが融合されており、インテインが50℃以上の温度において、熱によるcis−スプライシングを誘発するものである、修飾タンパク質を含む、組換え植物、又は組換え植物の部分を取得する工程と、組換え植物、又は組換え植物の部分の温度を50℃以上に上昇させることにより、前記修飾タンパク質のスプライシングを誘導する工程とを含む方法により、植物バイオマスを加工する。
【選択図】図1
Description
ら再生されるであろう。
5’−ACAGAATGGGGAACGAGCGATGCTAGCATTTTACCGGAAGAATGGGTTC−3’ [SEQ ID NO:1]
5’−CGTGTCTGCTCCGTTTACCGCTTTTTTTAATTGGACGAATTTGTGCGTGA−3’ [SEQ ID NO:2]
5’−AGCATTCAGACCTCCCATTTCATACGAAAAGAGGAAATAGATAGATTTTC−3’ [SEQ ID NO:3]
5’−CGTGTCTGCTCCGTTTACCGCTTTTTTTAATTGGACGAATTTGTGCGTGA−3’ [SEQ ID NO:4]
5’−CTTTGGATTCCTCTATGCACATAATTCCGGAAACGGCGGTGTCTACCTCG−3’ [SEQ ID NO:7]
5’−CGTGTCTGCTCCGTTTACCGCTTTTTTTAATTGGACGAATTTGTGCGTGA−3’ [SEQ ID NO:8]
5’−ACAGAATGGGGAACGAGCGATGCTAGCATTTTACCGGAAGAATGGGTTC−3’ [SEQ ID NO:9]
5’−CGAGGTAGACACCGCCGTTTCCGGAATTATGTGCATAGAGGAATCCAAAG−3’ [SEQ ID NO:10]
5’−AGCATTCAGACCTCCCATTTCATACGAAAAGAGGAAATAGATAGATTTTC−3’ [SEQ ID NO:11]
5’−GAACCCATTCTTCCGGTAAAATGCTAGCATCGCTCGTTCCCCATTCTGTG−3’ [SEQ ID NO:12]
5’−ATGGCCTTCAAGCAGCTCGTCGCAG−3’ [SEQ ID NO:13]
5’−TTAAGCACCCGGCGGCGGGGGGCTG−3’ [SEQ ID NO:14]
例1にて述べられているようにPCR反応に使われる。拡大についで、結果として生じるPCR成果物は、例1に記述したように分子量標準を伴って、アガローゼゲル上のゲル電気泳動を使って分析される。エンチジウム臭をともなったゲル臭化物の付着した後、1567basepair(bp)バンドはメスによってゲルから切り離される、上記に記述したようにそしてゲルから精浄化さる。ゲルからの破片を清浄した後、破片はAusubel, et al., 1998に述べられているように、直接の確認のために制限消化、または配列をすることによって分析される。
5’−CTTTGGATTCCTCTATGCACATAATTCTCGCCCGCGACTCCCGCACCGCT−3’ [SEQ ID NO:15]
5’−TAAGCACCCGGCGGCGGGGGGCTGGAAGAGGAATATGTCAGCTGGGGGC−3’ [SEQ ID NO:16]
5’−ATGGCCTTCAAGCAGCTCGTCGCAGCGATTTCCCTCGCACTCTCGCTCAC−3’ [SEQ ID NO:17]
5’−GAACCCATTCTTCCGGTAAAATGCTGTGTGGTCGGTCTGGATGCGGATCT−3’ [SEQ ID NO:18]
5’−AGATCCGCATCCAGACCGACCACACAGCATTTTACCGGAAGAATGGGTTC−3’ [SEQ ID NO:19]
5’−GCGGTGCGGGAGTCGCGGGCGAGAATTATGTGCATAGAGGAATCCAAAG−3’ [SEQ ID NO:20]
5’−CTTTGGATTCCTCTATGCACATAATTTCCTTCGACACCATCTCCACCAGCA−3’ [SEQ ID NO:21]
5’−CTAAGCAAAACACTCCGCCCAATCACCGCCAGATCTGGCAAAGCTCAACC−3’ [SEQ ID NO:22]
5’−AGTGACCTACCTCATGGACATGTGCAGCATTTTACCGGAAGAATGGGTTC−3’ [SEQ ID NO:23]
5’−GCTGGTGGAGATGGTGTCGAAGGAATTATGTGCATAGAGGAATCCAAAG−3’ [SEQ ID NO:24]
5’−ATGGGTGTCTCTGCCGTTCTACTTCCTTTGTACCTCCTGTCCGGAGTATG−3’ [SEQ ID NO:25]
5’−GAACCCATTCTTCCGGTAAAATGCTGCACATGTCCATGAGGTAGGTCACT−3’ [SEQ ID NO:26]
Claims (17)
- リグノセルロース分解タンパク質を含み、且つ前記リグノセルロース分解タンパク質の内部にインテインが融合されており、前記インテインが50℃以上の温度において、熱によるcis−スプライシングを誘発するものである、修飾タンパク質を含む、組換え植物、又は組換え植物の部分を取得する工程;及び
前記組換え植物、又は前記組換え植物の部分の温度を50℃以上に上昇させることにより、前記修飾タンパク質のスプライシングを誘導する工程、
を含む、植物バイオマスの加工方法。 - さらに、前記組換え植物、又は前記組換え植物の部分が、酵母に接触される、請求項1記載の方法。
- 前記リグノセルロース分解タンパク質が、リグニンの分解、セルロースの分解、及びヘミセルロースの分解からなる群より選択される反応を触媒する、請求項1記載の方法。
- 前記リグノセルロース分解タンパク質が、E.C.3.2.1.4に分類されるセルラーゼ、E.C.3.2.1.91に分類されるエクソセロビオヒドロラーゼ(exocellobiohydrolases)、E.C.3.2.1.21に分類されるグルコシダーゼ、エンドセルラーゼ、エクソセルラーゼ、キシラナーゼ、ヘミ−セルラーゼ、リグニナーゼ、及びリグニンペルオキシダーゼからなる群より選択される酵素である、請求項1記載の方法。
- 前記リグノセルロース分解タンパク質が、セルラーゼである、請求項1記載の方法。
- 前記インテインが、Pyrococcus spp由来のインテイン(PSP Pol.)である、請求項1記載の方法。
- 前記インテインがPsp polインテインの変異体である、請求項1記載の方法。
- 前記インテインが、配列番号27における第1839〜3449塩基の配列を有する核酸にコードされるアミノ酸配列を有するPsp polインテインである、請求項1記載の方法。
- さらに、他の植物基質、化学基質、自治体の廃棄物、副産物による製造品、酵素、原核細胞、真核細胞、からなる群より選択される少なくとも1種を、前記取得工程、及び前記修飾タンパク質のスプライシングを誘導する工程のうち1以上の工程の、前、途中、又は後に添加する工程を含む、請求項1記載の方法。
- さらに、非組換え植物を、前記取得工程、及び前記修飾タンパク質のスプライシングを誘導する工程のうち1以上の工程の、前、途中、又は後に添加する工程を含む、請求項1記載の方法。
- 前記組換え植物、又は前記組換え植物の部分が、第2インテインと融合された標的タンパク質を含む第2組換えタンパク質を含有し、
前記第2インテインが、温度を50℃以上に上昇させることにより、熱による前記第2組換えタンパク質のcis−スプライシングを誘発するものであり、
さらに、前記組換え植物、又は前記組換え植物の部分の温度を50℃以上に上昇させることにより、前記第2組換えタンパク質のスプライシングを誘導する工程を含む、請求項1記載の方法。 - 前記第2インテインと融合された標的タンパク質が、糖を転化する酵素、又は酵素断片であり、
さらに、第2組換えタンパク質のスプライシングを誘導する工程を含む、請求項11記載の方法。 - 前記第2インテインと融合された標的タンパク質が、エタノールを生産する反応を触媒する酵素、又は酵素断片であり、
さらに、第2組換えタンパク質のスプライシングを誘導する工程を含む、請求項11記載の方法。 - 前記第2インテインと融合された標的タンパク質が、エタノール生産における中間体を生産する反応を触媒する、酵素、又は酵素断片であり、
さらに、第2組換えタンパク質のスプライシングを誘導する工程を含む、請求項12記載の方法。 - 前記第2インテインと融合された標的タンパク質が、ブタンジオールの生産、エタノールの生産、フェノールの生産、グリセロールの生産、エチレンの生産、プロピレンの生産、トルエンの生産、エチルベンゼンの生産、スチレンの生産、キシレンの生産、エチレングリコールの生産、ブタジエンの生産、ホルムアルデヒドの生産、イソプロパノールの生産、アセトンの生産、ブタンジオールの生産、メタノールの生産、プロパノールの生産、ブタノールの生産、プロパンジオールの生産、ビタミンの生産、メタンの生産、エタンの生産、プロパンの生産、ブタンの生産、ペンタンの生産、ヘキサンの生産、ヘプタンの生産、オクタンの生産、ベンゼンの生産、デキストリンの分解、ペクチンの分解、脂質の分解、タンパク質の分解、キチン質の分解、及び糖化からなる群より選択される反応を触媒する、酵素、又は酵素断片であり、
さらに、第2組換えタンパク質のスプライシングを誘導する工程を含む、請求項11記載の方法。 - さらに、他の植物基質、化学基質、自治体の廃棄物、副産物による製造品、酵素、原核細胞、真核細胞、からなる群より選択される少なくとも1種を、前記取得工程、前記修飾タンパク質のスプライシングを誘導する工程、及び前記第2修飾タンパク質のスプライシングを誘導する工程のうち1以上の工程の、前、途中、又は後に添加する工程を含む、請求項11記載の方法。
- さら、非組換え植物を、前記取得工程、前記修飾タンパク質のスプライシングを誘導する工程、及び前記第2修飾タンパク質のスプライシングを誘導する工程のうち1以上の工程の、前、途中、又は後に添加する工程を含む、請求項11記載の方法。
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