JP5066380B2 - ポリオール脱水素酵素の製造方法 - Google Patents
ポリオール脱水素酵素の製造方法 Download PDFInfo
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- JP5066380B2 JP5066380B2 JP2007090013A JP2007090013A JP5066380B2 JP 5066380 B2 JP5066380 B2 JP 5066380B2 JP 2007090013 A JP2007090013 A JP 2007090013A JP 2007090013 A JP2007090013 A JP 2007090013A JP 5066380 B2 JP5066380 B2 JP 5066380B2
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- RRAMGCGOFNQTLD-UHFFFAOYSA-N hexamethylene diisocyanate Chemical compound O=C=NCCCCCCN=C=O RRAMGCGOFNQTLD-UHFFFAOYSA-N 0.000 description 1
- FBPFZTCFMRRESA-UHFFFAOYSA-N hexane-1,2,3,4,5,6-hexol Chemical compound OCC(O)C(O)C(O)C(O)CO FBPFZTCFMRRESA-UHFFFAOYSA-N 0.000 description 1
- 150000002463 imidates Chemical class 0.000 description 1
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- 229910052751 metal Inorganic materials 0.000 description 1
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- 230000035772 mutation Effects 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- FSVCQIDHPKZJSO-UHFFFAOYSA-L nitro blue tetrazolium dichloride Chemical compound [Cl-].[Cl-].COC1=CC(C=2C=C(OC)C(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC(=CC=2)[N+]([O-])=O)=CC=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=C([N+]([O-])=O)C=C1 FSVCQIDHPKZJSO-UHFFFAOYSA-L 0.000 description 1
- HEGSGKPQLMEBJL-RKQHYHRCSA-N octyl beta-D-glucopyranoside Chemical compound CCCCCCCCO[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HEGSGKPQLMEBJL-RKQHYHRCSA-N 0.000 description 1
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- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
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- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 1
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- 239000001488 sodium phosphate Substances 0.000 description 1
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- DVKJHBMWWAPEIU-UHFFFAOYSA-N toluene 2,4-diisocyanate Chemical compound CC1=CC=C(N=C=O)C=C1N=C=O DVKJHBMWWAPEIU-UHFFFAOYSA-N 0.000 description 1
- 230000002463 transducing effect Effects 0.000 description 1
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- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
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Images
Landscapes
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
PQQ依存性PDHの酵素活性は、50μM DCIP、0.2mM PMS(フェナジンメトサルフェート)、450mM グリセロールを含んだ0.1g/100mL トライトンX−100を含む10mM リン酸緩衝液pH 7.0中に、酵素溶液を加え、酵素と基質の反応をDCIPの600nmの吸光度変化によって追跡し、その吸光度の減少速度を酵素の反応速度とした。1分間に1μmolのDCIPが還元される酵素活性を1単位(U)とした。なお、DCIPのpH 7.0におけるミリモル吸光係数は16.3 mM−1とした。
ソルビトール 2g/100mL、酵母エキス 0.3g/100mL、肉エキス 0.3g/100mL、コーンスティープリカー 0.3g/100mL、ポリペプトン 1g/100mL、尿素 0.1g/100mL、KH2PO4 0.1g/100mL、MgSO4・7H2O 0.02g/100mL、CaCl2・2H2O 0.1g/100mL、pH7.0からなる培地100mLを調製し、500mL容の坂口フラスコに該培地80mLを移し、121℃、20分間オートクレーブ処理した。
ストレプトマイシンの代わりに0.5M ジヒドロストレプトマイシン水溶液(pH8.0)を加えて架橋反応を停止後(得られたPDHを架橋DSMPDHとした(サンプル3))、凍結乾燥を行った(サンプル4)こと以外は、実施例1と同様に行った。
実施例1で得た修飾SMPDH及び実施例2で得た修飾DSMPDHの熱安定性を検討するため、サンプル1及び3を50℃で、10分間、ウォ−ターバスによりインキュベートした後、残存する酵素活性を測定した。同様に、ストレプトマイシンの代わりに0.5M トリス塩酸緩衝液(pH8.0)を加えて架橋反応を停止させた試料(サンプル5)および架橋処理していないグルコノバクター・オキシダンス由来PDHの残存する酵素活性を測定した。結果を表1に示す。
実施例1および2で得た修飾PDHの保存安定性を検討するため、50℃で、1時間保存し、残存する酵素活性を測定した。結果を表2に示す。また、比較としてストレプトマイシンの代わりに0.5M トリス塩酸緩衝液(pH8.0)を加えて架橋反応を停止後、凍結乾燥させた試料(サンプル6)を用いて同様に残存する酵素活性を測定した結果を併せて表2に示す。さらに、架橋処理していないグルコノバクター・オキシダンス由来PDHを用いて同様に残存する酵素活性を測定した結果(未架橋PDH)を併せて表2に示す。
50μM DCIP、0.2mM PMS、実施例1の酵素溶液(0.3U)を含んだ0.1g/100mLトライトンX−100を含む10mMリン酸緩衝液pH7.0中に、終濃度が100、200、300、400、500μMになるようにグリセロールを加え、DCIPの600nmにおける吸光度の減少を測定した。結果を図1に示す。100〜500μMグリセロールまで直線性がよく、定量可能であることが示された。
Claims (4)
- 補欠分子族としてピロロキノリンキノンを含むポリオール脱水素酵素をジアルデヒド化合物で架橋反応させる工程(1)と、
ストレプトマイシンおよびジヒドロストレプトマイシンの少なくとも一方で架橋反応を停止して酵素溶液を得る工程(2)と、
前記酵素溶液を凍結乾燥して粉末酵素を得る工程(3)と、
を含む、ポリオール脱水素酵素の製造方法。 - 前記ジアルデヒド化合物がグルタルアルデヒドである、請求項1に記載の製造方法。
- 前記ポリオール脱水素酵素は、グリセロールを基質とする、請求項1または2に記載の製造方法。
- 前記工程(1)は、界面活性剤の存在下で架橋反応を行う、請求項1〜3のいずれか1項に記載の製造方法。
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