Purification and Characterization of an Intracellular Chymotrypsin-Like Serine Protease from Thermoplasma volcanium

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An intracellular serine protease produced by <I>Thermoplasma (Tp.) volcanium</I> was purified using a combination of ammonium sulfate fractionation, ion exchange, and α-casein agarose affinity chromatography. This enzyme exhibited the highest activity and stability at pH 7.0, and at 50 °C. The purifed enzyme hydrolyzed synthetic peptides preferentially at the carboxy terminus of phenylalanine or leucine and was almost completely inhibited by PMSF, TPCK, and chymostatin, similarly to a chymotrypsin-like serine protease. Kinetic analysis of the <I>Tp. volcanium</I> protease reaction performed using <I>N</I>-succinyl-<small>L</small>-phenylalanine-<I>p</I>-nitroanilide as substrate revealed a <I>K</I><SUB>m</SUB> value of 2.2 m<small>M</small> and a <I>V</I><SUB>max</SUB> value of 0.045 μmol<SUP>−1</SUP> ml<SUP>−1</SUP> min<SUP>−1</SUP>. Peptide hydrolyzing activity was enhanced by >2-fold in the presence of Ca<SUP>2+</SUP> and Mg<SUP>2+</SUP> at 2–12 m<small>M</small> concentration. The serine protease is a monomer with a molecular weight of 42 kDa as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and zymogram activity staining.

収録刊行物

  • Bioscience, biotechnology, and biochemistry  

    Bioscience, biotechnology, and biochemistry 70(1), 126-134, 2006-01-23 

    Japan Society for Bioscience, Biotechnology, and Agrochemistry

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各種コード

  • NII論文ID(NAID)
    10018535276
  • NII書誌ID(NCID)
    AA10824164
  • 本文言語コード
    ENG
  • 資料種別
    ART
  • ISSN
    09168451
  • NDL 記事登録ID
    7791436
  • NDL 雑誌分類
    ZR7(科学技術--農林水産--農産) // ZR2(科学技術--生物学--生化学) // ZP1(科学技術--化学・化学工業)
  • NDL 請求記号
    Z53-G223
  • データ提供元
    CJP書誌  NDL  J-STAGE 
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