Thermoanaerobium brockii由来耐熱性トレハロースホスホリラーゼの精製とその諸性質 Purification and Characterization of Thermostable Trehalose Phosphorylase from Thermoanaerobium brockii

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Abstract

好熱嫌気性菌Thermoanaerobium brockii ATCC35047株由来耐熱性トレハロースホスホリラーゼをDEAE-トヨパール,ブチルトヨパール,ウルトロゲルAcA44の各種カラムクロマトクラフィーにより電気泳動的に単一にまで精製した.本酵素の分子量はゲル濾過で190,000,SDS-ポリアクリルアミドゲル電気泳動で88,000であった.加リン酸分解の至適pHは7.0-7.5,合成の至適pHは6.0-7.0であった.至適温度は分解合成とも70℃ であった.本酵素はpH6.0-9.0,60℃ 以下で安定であった.本酵素活性はCu2+,Hg<SUP>2+</SUP>,Mg<SUP>2+</SUP>,Mn<SUP>2+</SUP>,Pb<SUP>2+</SUP>,Zn<SUP>2+</SUP>の各金属イオンで阻害された.本酵素のトレハロース,無機リン酸,グルコース,β-グルコース1-リン酸に対する.Km値はそれぞれ0.97mM,0.57mM,2.4mM,0.75mMであった.本酵素はこれまで報告されているトレハロースホスホリラーゼの中で耐熱性が最も高かった.

A thermostable trehalose phosphorylase (EC 2.4.1.64) from a thermophilic anaerobe, Thermoanaero bium brockii ATCC 35047, was purified to an electrophoretically homogeneous state by successive column chromatographies on DEAE-Toyopearl 6505, Butyl-Toyopearl 650 M and Ultrogel AcA 44 . The molecular weight of the enzyme was estimated to be 190, 000 Da by gel filtration, and 88, 000 Da by SDS-polyacrylamide gel electrophoresis. The enzyme showed the highest activity in the range of pH 7.0 to 7.5 for phosphorolysis, and pH 6.0 to 7.0 for synthesis . The optimum temperature of the enzyme was 70°C in both directions. The enzyme was stable from pH 6.0 to 9.0, and up to 60°C . This result showed that our trehalose phosphorylase was the most thermostable in those reported to date . Activity was inhibited by Cue, Hg<SUP>2+</SUP>, Mg<SUP>2+</SUP>, Mn<SUP>2+</SUP>, Pb<SUP>2+</SUP>, and Zn<SUP>2+</SUP> The Kms for trehalose, Pi, D-glucose and R-D-glucose 1-phosphate were 0.97, 0.57, 2.4 and 0.75 mM, respectively.

Journal

  • Journal of Applied Glycoscience

    Journal of Applied Glycoscience 46(4), 399-405, 1999-12-01

    The Japanese Society of Applied Glycoscience

References:  19

Cited by:  8

Codes

  • NII Article ID (NAID)
    10008259332
  • NII NACSIS-CAT ID (NCID)
    AN10453916
  • Text Lang
    ENG
  • Article Type
    Journal Article
  • ISSN
    13403494
  • NDL Article ID
    4976502
  • NDL Source Classification
    ZP24(科学技術--化学・化学工業--糖・澱粉)
  • NDL Call No.
    Z17-15
  • Data Source
    CJP  CJPref  NDL  J-STAGE  JASI 
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