Glycosylation Status and Conformational Stability of Recombinant Stereum purpureum Endopolygalacturonase Ⅳs Produced in an Aspergillus oryzae Expression System Glycosylation Status and Conformational Stability of Recombinant Stereum purpureum Endopolygalacturonase IVs Produced in an Aspergillus oryzae Expression System

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<I>Stereum purpureum </I>endopolygalacturonase (EndoPG) IV was expressed in <I>Aspergillus oryzae</I>. Recombinant forms of EndoPG IV (rEndoPGs IV<SUB>0</SUB>, IV<SUB>1</SUB>, and IV<SUB>2</SUB>) were purified from the culture filtrate by ammonium sulfate precipitation and two-step column chromatography with DE52 and hydroxyapatite. Each of these three recombinants was shown to be homogeneous by SDS-PAGE. Since it was predicted that the differences between them would be based on the number of sugar chains, the changes in molecular mass after enzymatic cleavage of the <I>N</I>-linked oligosaccharide with EndoH were determined by MALDI-TOF MS. From the data, the numbers of <I>N</I>-linked oligosaccharides were estimated to be 0, 1, and 2 for rEndoPG IV<SUB>0</SUB>, IV<SUB>1</SUB>, and IV<SUB>2, </SUB>respectively. These sugar chains appeared to be a mixture of GlcNAc<SUB>2</SUB>Hexose<SUB>5-10 </SUB>and to include galactose as well as mannose. Mass analyses of IV<SUB>0, </SUB>IV<SUB>1 </SUB>and IV<SUB>2</SUB> after deglycosylation with EndoH demonstrated a hexose carbohydrate, suggesting the presence of an <I>O</I>-linked oligosaccharide that is lacking in native EndoPG IVs. The thermal denaturation curves of rEndoPG IV<SUB>0 </SUB>and IV<SUB>1 </SUB>based on CD analysis indicated <I>T</I><SUB>m</SUB> values of 60 and 59 °C, with a one-step thermal denaturation curve, similar to that of native EndoPG IV.

収録刊行物

  • Journal of applied glycoscience

    Journal of applied glycoscience 59(1), 37-42, 2012-02-20

    日本応用糖質科学会

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各種コード

  • NII論文ID(NAID)
    10030138355
  • NII書誌ID(NCID)
    AA11809133
  • 本文言語コード
    ENG
  • 資料種別
    ART
  • ISSN
    13447882
  • NDL 記事登録ID
    023567050
  • NDL 請求記号
    Z78-A693
  • データ提供元
    CJP書誌  NDL  J-STAGE 
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