JP5748661B2 - ジフルクトース・ジアンヒドリドiii合成酵素 - Google Patents
ジフルクトース・ジアンヒドリドiii合成酵素 Download PDFInfo
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- JP5748661B2 JP5748661B2 JP2011519772A JP2011519772A JP5748661B2 JP 5748661 B2 JP5748661 B2 JP 5748661B2 JP 2011519772 A JP2011519772 A JP 2011519772A JP 2011519772 A JP2011519772 A JP 2011519772A JP 5748661 B2 JP5748661 B2 JP 5748661B2
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- Prior art keywords
- dfaiii
- synthase
- inulin
- enzyme
- strain
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- 229920001202 Inulin Polymers 0.000 claims description 67
- JYJIGFIDKWBXDU-MNNPPOADSA-N inulin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(OC[C@]2(OC[C@]3(OC[C@]4(OC[C@]5(OC[C@]6(OC[C@]7(OC[C@]8(OC[C@]9(OC[C@]%10(OC[C@]%11(OC[C@]%12(OC[C@]%13(OC[C@]%14(OC[C@]%15(OC[C@]%16(OC[C@]%17(OC[C@]%18(OC[C@]%19(OC[C@]%20(OC[C@]%21(OC[C@]%22(OC[C@]%23(OC[C@]%24(OC[C@]%25(OC[C@]%26(OC[C@]%27(OC[C@]%28(OC[C@]%29(OC[C@]%30(OC[C@]%31(OC[C@]%32(OC[C@]%33(OC[C@]%34(OC[C@]%35(OC[C@]%36(O[C@@H]%37[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O%37)O)[C@H]([C@H](O)[C@@H](CO)O%36)O)[C@H]([C@H](O)[C@@H](CO)O%35)O)[C@H]([C@H](O)[C@@H](CO)O%34)O)[C@H]([C@H](O)[C@@H](CO)O%33)O)[C@H]([C@H](O)[C@@H](CO)O%32)O)[C@H]([C@H](O)[C@@H](CO)O%31)O)[C@H]([C@H](O)[C@@H](CO)O%30)O)[C@H]([C@H](O)[C@@H](CO)O%29)O)[C@H]([C@H](O)[C@@H](CO)O%28)O)[C@H]([C@H](O)[C@@H](CO)O%27)O)[C@H]([C@H](O)[C@@H](CO)O%26)O)[C@H]([C@H](O)[C@@H](CO)O%25)O)[C@H]([C@H](O)[C@@H](CO)O%24)O)[C@H]([C@H](O)[C@@H](CO)O%23)O)[C@H]([C@H](O)[C@@H](CO)O%22)O)[C@H]([C@H](O)[C@@H](CO)O%21)O)[C@H]([C@H](O)[C@@H](CO)O%20)O)[C@H]([C@H](O)[C@@H](CO)O%19)O)[C@H]([C@H](O)[C@@H](CO)O%18)O)[C@H]([C@H](O)[C@@H](CO)O%17)O)[C@H]([C@H](O)[C@@H](CO)O%16)O)[C@H]([C@H](O)[C@@H](CO)O%15)O)[C@H]([C@H](O)[C@@H](CO)O%14)O)[C@H]([C@H](O)[C@@H](CO)O%13)O)[C@H]([C@H](O)[C@@H](CO)O%12)O)[C@H]([C@H](O)[C@@H](CO)O%11)O)[C@H]([C@H](O)[C@@H](CO)O%10)O)[C@H]([C@H](O)[C@@H](CO)O9)O)[C@H]([C@H](O)[C@@H](CO)O8)O)[C@H]([C@H](O)[C@@H](CO)O7)O)[C@H]([C@H](O)[C@@H](CO)O6)O)[C@H]([C@H](O)[C@@H](CO)O5)O)[C@H]([C@H](O)[C@@H](CO)O4)O)[C@H]([C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JYJIGFIDKWBXDU-MNNPPOADSA-N 0.000 claims description 67
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- KCXFHTAICRTXLI-UHFFFAOYSA-N propane-1-sulfonic acid Chemical compound CCCS(O)(=O)=O KCXFHTAICRTXLI-UHFFFAOYSA-N 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000001223 reverse osmosis Methods 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 229910021654 trace metal Inorganic materials 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 description 1
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- C12P19/12—Disaccharides
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01134—Difructose-anhydride synthase (3.2.1.134)
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Description
分子量:46,000〜48,000
至適温度:50〜60℃
熱安定性:80℃を超えると徐々に失活し始めるが、90℃で70%以上の残存活性を示す
至適pH:4〜6
pH安定性:pH3〜11で安定。
至適温度:50〜60℃、好ましくは55℃
熱安定性:80℃を超えると徐々に失活し始めるが、90℃で70%以上の残存活性を示す。
pH安定性:pH3〜11で安定。
まず、本発明のDFAIII合成酵素を産生するクルトバクテリウム属微生物、好ましくはクルトバクテリウムsp.D436-1株を適切な条件下で培養する。
至適温度: 40〜50℃
熱安定性: 40℃を越えると徐々に失活し始め、50℃で70%、60℃で40%の残存活性を示す。
pH安定性: pH6以上で安定。
のN末端アミノ酸配列を有する。
クルトバクテリウムsp.D436-1株をイヌリン1〜2%(w/v)、ペプトン1%、酵母エキス0.5%、リン酸1カリウム0.1%を含むpH7〜8の液体培地で培養後、遠心分離により除菌し、限外ろ過膜によって濃縮し、Phenyl-toyopearl 650M疎水クロマトグラフィー、DEAE-toyopearl 650M陰イオン交換カラムクロマトグラフィなどの各種精製工程を経てSDSゲル電気泳動的に均一に精製した。
溶離液: 蒸留水
流量: 0.5 ml/分
カラム温度: 50℃
検出器: 示差屈折計
注入量: 10μL
その結果、クルトバクテリウムsp.D436-1株が産生するDFAIII合成酵素の酵素活性は、117 u/mlであり、これまでに知られている同類の酵素と比べて非常に高いものであることがわかった。
クルトバクテリウムsp.D436-1株が産生するDFAIII合成酵素液1mLをエッペンドルフチューブに採り、37、40、50、60、70、80、90℃において30分処理した後、水冷して常温に戻した。熱処理した酵素液は以下に示す酵素活性測定法に従って残存酵素の活性を測定し、熱処理を行わなかったものに対する相対活性を求めた。結果を図1に示す。
スクロースからイヌリンを合成するイヌリン合成酵素(WO 02/00865に記載されているBacillus (バチラス) sp.217C-11株 (FERM BP-7450)が産生するイヌリン合成酵素、以下「IPE」と略す)、クルトバクテリウムsp.D436-1株が産生するDFAIII合成酵素(以下「DPE」と略す)を組み合わせることにより、スクロースからDFAIIIを製造した。
スクロースからIPEを用いてイヌリンを調製した溶液を、固定化DPEで処理することによってDFAIIIを製造した。
FERM BP-7450
Claims (8)
- イヌリンをDFAIIIに変換する活性を有し、80℃での残存活性が90%以上であり、以下の理化学的性質を有する、クルトバクテリウム属に属する微生物由来のDFAIII合成酵素:
分子量:46,000〜48,000
至適温度:50〜60℃
熱安定性:80℃を超えると徐々に失活し始めるが、90℃で70%以上の残存活性を示す
至適pH:4〜6
pH安定性:pH3〜11で安定。 - 配列番号1のN末端アミノ酸配列を有する、請求項1に記載のDFAIII合成酵素。
- クルトバクテリウム属に属する微生物が、クルトバクテリウム(Curtobacterium)sp.D436-1株(NITE BP-764)である、請求項1又は2に記載のDFAIII合成酵素。
- DFAIIIを製造する方法であって、請求項1〜3のいずれか1項に記載のDFAIII合成酵素をイヌリンに接触させることを含む、前記方法。
- 請求項1〜3のいずれか1項に記載のDFAIII合成酵素を産生する微生物の培養液又は培養菌体もしくはその処理物をイヌリンに接触させることを含む、請求項4に記載の方法。
- DFAIIIを製造する方法であって、スクロースに、スクロースをイヌリンに変換する酵素と、請求項1〜3のいずれか1項に記載のDFAIII合成酵素とを作用させることを含む、前記方法。
- スクロースをイヌリンに変換する酵素が、Bacillus (バチラス) sp.217C-11株 (FERM BP-7450)が産生するイヌリン合成酵素である、請求項6に記載の方法。
- クルトバクテリウム(Curtobacterium)sp.D436-1株(NITE BP-764)、または請求項1〜3のいずれか1項に記載のDFAIII合成酵素を産生するその変異株。
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Citations (4)
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JPH01285195A (ja) * | 1988-05-13 | 1989-11-16 | Natl Food Res Inst | ジフルクトース・ジアンヒドリドの製造方法 |
WO2002000865A1 (fr) * | 2000-06-28 | 2002-01-03 | Fuji Nihon Seito Corporation | Nouvelle synthase d'inuline et procede de production d'insuline par cette derniere |
JP2003512045A (ja) * | 1999-10-19 | 2003-04-02 | コリア リサーチ インスティテュート オブ バイオサイエンス アンド バイオテクノロジー | 蔗糖(fructose)および関連酵素からジフルクトースジアンハイドライIV(difructosedianhydrideIV)の酵素的生産およびこれを暗号化する遺伝子(genes) |
JP2005132774A (ja) * | 2003-10-30 | 2005-05-26 | Nippon Beet Sugar Mfg Co Ltd | ジフルクトース・ジアンヒドリドiiiの精製方法 |
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Publication number | Priority date | Publication date | Assignee | Title |
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JPH01285195A (ja) * | 1988-05-13 | 1989-11-16 | Natl Food Res Inst | ジフルクトース・ジアンヒドリドの製造方法 |
JP2003512045A (ja) * | 1999-10-19 | 2003-04-02 | コリア リサーチ インスティテュート オブ バイオサイエンス アンド バイオテクノロジー | 蔗糖(fructose)および関連酵素からジフルクトースジアンハイドライIV(difructosedianhydrideIV)の酵素的生産およびこれを暗号化する遺伝子(genes) |
WO2002000865A1 (fr) * | 2000-06-28 | 2002-01-03 | Fuji Nihon Seito Corporation | Nouvelle synthase d'inuline et procede de production d'insuline par cette derniere |
JP2005132774A (ja) * | 2003-10-30 | 2005-05-26 | Nippon Beet Sugar Mfg Co Ltd | ジフルクトース・ジアンヒドリドiiiの精製方法 |
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JPN6010039037; MINAMIDA K. et al.: 'Effects of difructose anhydride III (DFA III) administration on bile acids and growth of DFA III-ass' Journal of Bioscience and Bioengineering Vol.99,No.6, 2005, p.548-554 * |
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